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1. Modifying an existing classification To modify an existing classification 1 If required replace the current image by loading a different plate see Loading plates into the instrument on page 6 and capturing a new image see Capturing an image on page 7 2 Press to display the Modify Previous Classification page of the Colony Classification Wizard Colcany jai ifinten Wear E Modify Proviows Classification How do you wart to modily lhe classibcalon D Rewew Existing Gassification D Add Now Sub Classitecation Remove Classification 64 Protos Systems User Manual The Classification tab Note If there are no sub classifications the final item in the list is Repeat Color Classification instead of Remove Classification 3 Press e Review Existing Classification to change the classification details you can also adjust the Reject Small Particles and colony splitter settings for a color classification provided it has no sub classifications e Add New Sub Classification to add a new sub classification e Remove Classification to remove a sub classification e Repeat Color Classification to replace the existing color classification with a new one 4 Press Next to go on to the next step which depends on the selection you made in Step 3 For details see e the next section Reviewing an existing classification e Adding a new sub classification on the next page e Removing a sub classifica
2. to abort the new batch you will be asked to confirm that you want to do this Managing batches Exporting batch data 94 To export data about a batch to an XML file 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu 4 Expat Daka as AM ta ores Baich Management i About Protos 4 pi Eal F realeas Protos Systems User Manual Managing batches 2 Press 44 Export Data os AN to display the Export Data dialog box this is a standard Windows Save As dialog box 3 Use the Export Data dialog box to select a location and enter a filename for the exported batch details 4 Press Save to save the batch details file If required you can move the XML file to another PC and use it as the basis for a new batch on that PC see Creating a new batch based on an existing batch or an exported batch file on page 20 Viewing batch details To view details about the current batch 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu ie E WEN Dalas xA p zelngs E Managemeni rit HT 2 Press P ey Balch Wotaals re 5 ie to oe aa or ad Dark Coon to display the Batch Management submenu 3 Press Protos Systems User Manual 95 Creating a new batch to display the Open Batch dialog box Open Batch The selected batch is SP test SpiralPlate
3. to display the image controls 2 Press to display a dialog box Ten 40 Chick balch or ddubon lo ww plate image Pate ID EE Protos Systems User Manual 13 Capturing images The box on the left hand side lists the batch images beginning with the batch setup image which is followed by the measurement images listed in order of their plate identifiers and in the case of dilution series dilutions 3 To view a measurement image click on the plate identifier or in the case of dilution series the dilution line within the plate identifier entry To view the batch setup image click on the batch identifier the top line 4 To export all the batch images a Press to display the Browse for Folder dialog box b Select the folder you want to export the images to if required press Make New Folder to add a new folder to the selected folder c Press OK to save the batch images to the selected folder The batch setup image will be saved using the name of the batch the measurement images will be saved using the image identifier if the same identifier has been used for more than one measurement for example with a dilution series batch _2 _3 etc will be added to the identifier for the second third etc images An xml file containing information about the images will also be saved in the folder 5 To export a single batch image a Select the required image se
4. I Larie baich gre sure You wish to dalete this batch If you have selected more than one batch you will be asked about each of them in turn 8 Press Yes to delete the batch es Protos Systems User Manual Managing batches You will be asked if you want to delete any images saved with the batch es B Baich Dato z 2 Do you wish jo delete the image files 9 Press Yes to delete the image files press No to leave them in the folder defined by the Image path seeSystem settings on page 136 for how to view or change the Image path 10 Repeat Steps 5 to 9 if you want to delete any further batches 11 Press Cancel in the Delete Batch dialog box to return to the main Protos 3 window Note Although deleted batches can no longer be viewed in Protos 3 they are not actually removed from the Protos 3 database and can be viewed there if required for audit purposes Protos Systems User Manual 99 Creating a new batch 100 Protos Systems User Manual Using Measurement Mode to count and detect colonies Once you have created and accepted a new batch see Creating a new batch on page 15 and Accepting the batch design on page 93 the general procedure for detecting and counting colonies on plates is 1 Open or select the batch see the next section Opening and selecting batches 2 Load the first plate into Protos 3 see Loading plates into the instrument on page 6 3 Capture an image see Capturing
5. a Harara a If you want to import a csv file containing a dilution series press Yes and go to Step b If you do not want to import a dilution series press No the default dilution will be used for each of the imported plate identifiers Go to Step 5 If you want to abort the procedure for importing plate identifiers completely press Cancel 80 Protos Systems User Manual The Configuration tab b If you pressed Yes in the previous step controls will be displayed for you to import a second csv file containing the dilution series to open a standard Windows Open dialog box d Use the Open dialog box to locate and select the required CSV file e Press Open to open the selected file The contents of the file will be shown in the preview box Ploase verify the dilution senes before clicking OK to marge the plate IDs Protos Systems User Manual 81 Creating a new batch f Check the preview box to make sure the contents are correct If the contents are correct press OK to show the combined plate identifiers and dilutions Ure ee eral Saunne for Pane Ii y Click ihe Open button fo select a CSV file to impart The contents of the file will be shown below plate csv Dilution 1 10 piate 1 100 plate 1 1000 a ka m g piaig b i 5 The contents of the file will be shown in the preview box see example above check the preview box to make sure
6. e If small colonies are being wrongly rejected try decreasing the Reject Small Particles setting 5 If the colonies have indistinct fringes around them they may be interpreted as separate colonies To remove the fringes from colonies if they are causing problems Check Remove Fringes Around Colonies 6 To choose whether to include or reject colonies that the frame passes through Check Complete Colonies Across Frame to include the colonies uncheck it to reject them 7 f there is a problem with overlapping colonies being counted as a single colony Click on the Split check button next to the colony type GF 112 colonies Split For example e Split unchecked Protos Systems User Manual 51 Creating a new batch Note Note 52 e Split checked 8 When you are happy that the color classification is as good as possible press Next to display the Classification Details page see the next section Classification Details The settings for the Reject Small Particles slider and colony splitter you make in the Review Color Classification page will be used to perform the initial counts for plates when you are working in Measurement mode However you will be able to vary these settings for individual plates by editing the measurement after you have performed the count see Rejecting small particles in a result on page 128 and Splitting colonies in a result on page 129 for details Image and colony marker disp
7. radio button for the number of size sub classifications you want to add or if you want to add more than four click on More and use the arrow buttons to set the required number Protos Systems User Manual 5 Creating a new batch 6 Press Next to carry out an automatic size classification and display the results in the Review Size Classification page for example Dae Dfi dor i Renew Sire Classification Classeicabon Flesuits Here are fhe oohonies found within the same image which match fiese sire tanges diameters in mm The Review Size Classification page allows you to review and adjust the automatic size classification results 7 Examine the marked colonies on the image to check whether the size classification is as required It may be easier to see the results if you zoom in on areas of the image as in the example above you can zoom the image in the same way as in the main window see Zooming the image on page 8 It may also help to change the way the colonies are marked by pressing the display button labeled All Colony Markers in the picture 58 Protos Systems User Manual The Classification tab 8 If required adjust the boundaries between the different size classifications a If the number of size ranges is larger than the space available a scroll bar will appear so that you can display the required controls for example If necessary scroll to the classification you want to adjust b
8. Adjust the upper or lower size boundary for the classification by typing in the edit box or clicking on the up or down arrow buttons attached to the right hand side of the edit box The lower upper boundary of the adjacent classification will be adjusted accordingly and the results of the new classification shown 9 When you are satisfied with the classification press Next to display the Classification Details page showing the new classification tree Protos Systems User Manual 59 Creating a new batch 60 10 Use the classification tree controls in the Classification Details page in the same way as for color classification see Classification Details on page 53 to specify for each sub classification e whether it corresponds to colonies or debris you cannot specify that it is part of the background and for a colony sub classification if you are not going to sub classify it any further e the name to be used to identify it e the marker used to identify it on the image e the color used for the marker When you have completed the classification details press Finish to return to the main Protos 3 window which will show the updated classification tree Shape classification The Add Shape Classification button becomes enabled once you have performed a color classification see Color classification on page 35 You can use a shape classification to distinguish differently shaped colonies as different ty
9. Chromogenic Media batches on page 69 24 Protos Systems User Manual The Classification tab Classification colony counting batches To display the classification controls in the Batch Designer Press the Classification tab In order to use the Classification tab you will need a sample image to work with 1 Insert a typical plate from the batch into Protos 3 see Loading plates into the instrument on page 6 This plate should have the full range of colony background and debris colors appearing on plates in the batch 2 Capture the image see Capturing an image on page 7 The Classification tab in the Batch Designer for counting colonies allows you to e set the frame see Frame on the next page e configure a simple total plate count see Total Plate Count on page 30 e view details of the current classification and set the properties of the markers used to identify detected colonies on the image see The Classification panel on page 33 Protos Systems User Manual 25 Creating a new batch e create a color classification see Color classification on page 35 create a size sub classification see Size classification on page 56 e create a shape sub classification see Shape classification on page 60 modify an existing classification see Changing an existing classification on page 63 Note By default if you base a new batch on an existing batch it will use th
10. Note When you are working in Measurement mode you can use a barcode reader in conjunction with a custom plate list to match each plate you are measuring to its identifier dilution in the plate list see Using a barcode reader for plate identifiers on page 76 84 Protos Systems User Manual The Measure tab The Measure tab ipso be Measurement last results li ij The dilution control does not appear for Chromogenic Media batches and has different forms for Dilution Series and Spiral batches The Count Restrictions button does not appear for Spiral or Chromogenic Media batches The Test Measure Plate button is permanently disabled for Chromogenic Media batches Notes To display the measure controls in the Batch Designer Press the Measure tab ane The Measure tab in the Batch Designer allows you to e enter text to act as a template for use with Auto incrementing identifiers see Selecting auto incrementing plate identifiers on page 74 set the dilution ratio when relevant see Dilution on the next page set a count limit for Pour Plate and Dilution Series batches see Setting Count Restrictions on page 90 for all but Chromogenic Media batches carry out a test count of the current plate see Test Measure Plate on page 91 so that you can check the batch settings if you are using a color classification see Color classification on page 35 you must carry out a test measurement before you c
11. You will find out about e Displaying information about the program version you are using see About Protos 3 below e Using the Settings dialog box to configure the program see System settings on the next page e Changing the color of the frame see Display properties on page 138 e Specifying the location of the Protos 3 database on the PC or a network see Database location on page 139 e Calibrating the instrument see Calibration on page 141 About Protos 3 To display information about the version of Protos 3 you are running 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu SS me ooo pei Selings E Baich Manage nani m a r E About Protos F TEPEE ME Caul Folosi 2 Press a About Protos Protos Systems User Manual 135 Configuring Protos 3 to display the About Protos 3 dialog box Abgul PbS KX Product Priza eet eae L026 3 L026 Fp Demesa vami 1DE E a PD Leomen Dema Gr Fha Protos J J Agee Profoas Wira Oo a8h J L sia Puio lakie I Cope GI Frapbta 3 Press Save Details to display a standard Windows Save As dialog box if you want to save the program version details in a text file 4 Press Close to close the dialog box System settings To view or change the system settings 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3
12. around the marker showing it is selected for example marker A is selected in the picture Drag the marker until it is at the center of its gray patch Repeat Steps 3 and 4 for any other markers that are out of position Press to save the new marker positions and return to the main Protos 3 screen Camera measurements 144 Protos 3 is fully calibrated in the factory and in normal circumstances will require no further calibration after delivery However in exceptional circumstances you may need to recalibrate the camera measurements which enable the instrument to measure distances on the image accurately Protos Systems User Manual Calibration To recalibrate the camera measurements 1 Insert an object of known size into Protos 3 see Loading plates into the instrument on page 6 2 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu G al Qo Expo Lata as All Lee 115 ee e c Management i 3 Choose Calibrate Camera Measurements from the Settings gt Calibration submenu to display the Calibrate Camera for Measurements dialog box This dialog box is also displayed when you calibrate an imported image and is used in exactly the same way see Calibrating the image on page 11 for detailed instructions Protos Systems User Manual 145 Configuring Protos 3 146 Protos Systems User Manual Index A About Protos 3 135 136 Accep
13. e Marker positions on page 143 e Camera measurements on page 144 Color balance Protos 3 is fully calibrated in the factory and in normal circumstances will require no further calibration after delivery However in exceptional circumstances you may need to recalibrate the color balance Protos Systems User Manual 141 Configuring Protos 3 To recalibrate the color balance 1 Insert a neutral test sheet into Protos 3 see Loading plates into the instrument on page 6 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu 3 Choose Calibrate Color Balance from the Settings Calibration submenu to display the Calibrate Color dialog box 4 Compare the color of the test sheet image on the screen with the actual color of the test 142 sheet If the image has a color cast compared with the test sheet adjust the color sliders accordingly For example if the image is too yellow increase the Blue setting or decrease the Red and Green settings The image will not reflect the new settings yet see the next step Protos Systems User Manual Calibration to check the effect of the changes 7 Repeat Steps 4 6 until the color balance of the image matches the color balance of the test card 8 Press to save the new color balance settings and close the dialog box If required during the above procedure To undo all the changes you have made si
14. menu Export Data as AMI ee r e E Managemeni AE akos pi Eal F realeas 136 Protos Systems User Manual System settings 2 Choose System Settings from the Settings submenu to display the Settings dialog box Sebreyy x Company Name Save images fo Save images to file Image Path C ProgramDoatalSynbiosss Protos Dats Export Path C ProgramDatalSynbiesss Protos Oat Min Audit Reason Length 3 Authentication Method Time Out Minutes a Display Exponential Numb 7 Flip Scanner image Note The Min Audit Reason Length Authentication Method Time Out and Flip Scanner Image settings are not used in Protos 3 3 Enter the name of your organization under Company Name The Company Name is included in reports see Compiling reports on page 133 4 Inthe Save Images to box choose whether to Save Images to file when you carry out a measurement choose Do not save images if you do not want the images saved Note You will only be able to edit results after you have performed a measurement if the image is saved with the results see Editing plate measurements on page 120 for details 5 Set the Image Path for the folder used to save images by typing the path directly into the box or press f to display a standard Windows Browse For Folder dialog box so that you can select the required folder Protos Systems User Manual 137 Configuring Protos 3 6 Set the Export Path f
15. on page 126 Editing Chromogenic Media batch results Manually changing detection results To change a detection result manually for a Chromogenic Media batch 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Click on the Detected check boxes in the results table Results Detected L monocytogenes to manually change the setting Results Detected Ei 3 Repeat Step 2 for any other colony types 4 Save the change to the result see Saving changes to results on page 125 Protos Systems User Manual 131 Working with results The result will be updated to reflect the changes made and an M flag will appear in the Flags column showing you have manually edited the plate Changing the Probability Threshold To change the Probability Threshold used for a Chromogenic Media batch result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Adjust the Probability Threshold by clicking on the or buttons or dragging the slider left or right When you make an adjustment Protos 3 will automatically carry out a remeasurement so that you can see the effects of the change 3 Save the change to the result see Saving changes to results on page 125 The result will be updated to reflect the changes made and an M flag will appear in the Flags column showing you have manually edited the plate Setting the results for all co
16. see Excluding regions during color classification on the facing page for instructions Otherwise if you have selected Detect amp Remove Grid the wizard will display the Detect amp Remove Grid page see Detecting and removing a grid on page 43 for instructions Otherwise if you have not selected Automatic background correction the wizard will display the Background Correction page see Manual background correction on page 44 for instructions Otherwise ie you have not selected Exclude Regions or Detect amp Remove Grid and you have selected Automatic background correction e If you have selected manual color classification the wizard will display the Manual Color Classification page see Manual color classification on page 47 for instructions e Otherwise ie you have selected automatic color classification the automatic colony color classification will be carried out immediately and the wizard will then display the Review Color Classification page showing the results see Review Color Classification on page 50 Protos Systems User Manual The Classification tab Excluding regions during color classification The Exclude Regions page in the Colony Classification Wizard is displayed if Exclude Regions is selected when you press Next in the Configure Color Classification page see Color classification on page 35 H part of fis plale is obscured by a Label of other contaminabor Click Exclude Repon
17. 36 The Configure Color Classification page will be displayed in the wizard Colao Dhsslficeihen Wired Configure Gwar Classification Sous image Conmectaon O Erdut Regions O Detect Rergre Grig E Automate Backyound Coreachon Automatic Classiicaton How marty dieen ootors of colony of debas are visible in his image w ooo 2 Ouah D F OORoTs More Mimun Ghreas D o Chits Gobo m red alep If there is no current image an image will be automatically captured when you press the Color Classification button If the image shown in the wizard is too light or too dark press Cancel to close the wizard and adjust the exposure accordingly see Setting the exposure on page 7 then press the Color Classification button again to restart the wizard 3 If required you can move and or resize the frame shown on the image using the same techniques as in Adjusting the position and size of frames on page 27 This may be useful in restricting the area used for color classification see Step 5 for another way of limiting the area of the plate used for color classification The frame in the Colony Classification Wizard defines the area used for color classification adjusting the color classification frame has no effect on the size or position of the main colony counting frame 4 If required you can zoom the image in the same way as in the main window see Zooming the image on page 8 If one o
18. 95 Showing or hiding the Light and Dark Colonies button for Total Plate Counts 96 Delene Dat AOS n sania duces bisa EAR E A 97 Using Measurement Mode to count and detect colonies 101 Opening and selecting batches 0 2 2 c cece cece cece c ccc ccc cece cece ceeceeeeeeeeees 101 Adjusting frame settings in Measurement mode 22 222222 e eee eee eee eeeeeeeeee 103 Entering plate identifiers and dilutions 2 000000 0002 eee eee eee ees 103 Auto incrementing plate identifiers 22 22 cece cece ence cence ee eee eee ee eeenenees 104 Manually entered plate identifiers 00 0000222 e ccc cece ccc ccceeeeeeeeeeeeeeceees 104 Changing the dilution set for a plate 20 00 2 o ccc ccc cece cece ccc cece eee eecccceceeeeeeeesees 105 External source CSV file or Custom plate list 22 0 0000 022222 e cece ee 106 Using a barcode reader for identifiers and dilutions 200000cc cece cece e eee eee 108 Counting colonies on Pour Plate Spiral and Dilution Series plates 22220220 222222 108 Counting the colonies on a plate automatically 00 eee cece 109 EOMMG SC ON S ais Sea en aa Satie aaa a a sedan setae eee eesbaete 110 Test measurement and adjusting settings 0 2 22 110 Manual colony counting eee zs eased beso ae tiets b tei ncn se eda nee Ot Mietededueiamantnael ete 112 Entering a Zero COUN we ics twinned cog gece w
19. All Colony Markers to display the detected colonies on the image For more detailed information about the precise areas identified as colonies you can also select All Colony Outlines or All Colony Regions instead If required you can zoom the image to check the colony detection see Zooming the image on page 8 If required you can change the symbol and color used to mark the colonies for details see the next section The Classification panel Protos Systems User Manual 31 Creating a new batch 3 To adjust the settings used when counting colonies using a Total Plate Count if the button below the image is labeled All Colony Markers All Colony Outlines or All Colony Regions the detected colonies will be shown on the image when you make the adjustments a Press to display the Total Plate Count controls again see picture in Step 1 b Click on the Split Touching Colonies check button to specify whether to use the colony splitter for colonies or not For example e Split Touching Colonies unchecked e Split Touching Colonies checked c In most cases the automatic sensitivity setting will give excellent results but if the colonies are not being detected correctly you can experiment by adjusting the sensitivity manually until you get an optimal result To adjust the detection sensitivity Drag the Sensitivity slider click the arrow buttons at the ends of the slider to adjust the
20. Detection the Line spacing controls will become enabled Minimum Maximum Line Ba 17G T7 t seen 4 ry Protos Systems User Manual 43 Creating a new batch 2 Set Minimum and Maximum limits for the grid Line Spacing in the search for the grid by typing directly into the edit boxes or by clicking on the up or down arrow buttons attached to the right hand side of the edit boxes As you adjust the figures the Colony Classification Wizard will carry out a new search for the grid 3 Repeat Step 2 using narrower search criteria until the grid has been detected successfully 4 Press Next to proceed to the next step in the color classification procedure The next step in the color classification procedure depends on selections you made in the Configure Color Classification page see Color classification on page 35 e If you did not select Automatic background correction the wizard will display the Background Correction page see Manual background correction below for instructions e Otherwise ie you selected Automatic background correction e If you selected manual color classification the wizard will display the Manual Color Classification page see Manual color classification on page 47 for instructions e Otherwise ie you selected automatic color classification the automatic colony color classification will be carried out immediately and the wizard will then display the Review Color Classification page s
21. Lower 70 Exclude From Mean As this example shows if there is more than one colony type the count limits 99 and 20 in this case are applied to each of them separately Test Measure Plate If you are using a color classification see Color classification on page 35 you will not be allowed to accept the new batch see Accepting the batch design on page 93 until you have carried out a separate test measurement Notes The Total Plate Count procedure see Total Plate Count on page 30 includes a test measurement so you are not required to carry out another one if you are using that instead of a color classification The Test Measure Plate button is permanently disabled for Chromogenic Media batches To carry out a test measurement 1 Press the Measure tab GUNGES PY to display the measure controls 2 Press Note If you are using an uncalibrated imported image a warning dialog box will be displayed asking you to calibrate the image see Calibrating the image on page 11 for details The results of the measurement will be shown in the Measurement test results panel If the button below the image is labeled All Colony Markers the detected colonies will also be marked on the image using the colors and markers selected for each colony type if the button is labeled No Colony Markers press the button and select All Colony Markers to display the detected colonies on the image Protos Systems User Manual 91 Creat
22. Manual 53 Creating a new batch 2 Select Colony Debris or Background as required If necessary you can assign more than one color to Debris or Background The button will show the selected classification type Debris Background or the name of the colony type Note The following instructions show you how to change the classification details for a colony type produced by the color classification However if you intend to use one of the other classifications size or shape see Adding sub classifications on the facing page as a sub classification for this colony type you can omit this step as any settings you make here will be overridden by the sub classification settings To change the classification details for a colony type 1 Press the colony classification button to display the classification details panel next to the classification menu 2 Edit the Colony Name as required for example to show the name of the organism forming the colonies 3 If you do not want to change the colony marker or color press the Return key or press 4 Select the shape of colony marker you want to use from the Colony Marker panel Note The classification menu and panel will close after you have selected the colony marker so you will need to go back to Step 1 if you want to carry out any more changes 5 To change the color used for the colony marker press the Color button to display the Standard Colors control 6 Cli
23. a series of plates e using Measurement mode to actually detect count the colonies on the plates and record the results The next section also introduces you to the idea of a batch which is used to hold the settings made in the Batch Designer and then to store the results produced in Measurement mode The final two sections of this Getting Started chapter give instructions for starting Protos 3 see Starting Protos 3 on the facing page and then for closing Protos 3 see Closing Protos 3 on page 4 The remaining chapters are e Capturing images starting on page 5 which shows you how to load plates into Protos 3 and capture an image of the plate These procedures are used in both the Batch Designer and Measurement mode e Creating a new batch starting on page 15 which shows you how to use the Batch Designer to create a new batch and specify how colonies should be detected counted Some of these procedures can also be applied in Measurement mode e Using Measurement Mode to count and detect colonies starting on page 101 which shows you how to detect count the colonies on a series of plates e Working with results starting on page 119 which shows you how to view and edit the results of your measurements and how to create reports using the results e Configuring Protos 3 starting on page 135 which shows you how to configure Protos 3 in a variety of ways Using Protos 3 overview Protos 3 uses batches to
24. also open a previously created batch if there is no batch open currently or if you want to open another batch in addition to any currently open batches you can have several batches open at the same time see later in this section for how to select which open batch to work with To open a batch in Measurement mode 1 Press Protos Systems User Manual 101 Using Measurement Mode to count and detect colonies to display the Open Batch dialog box listing the most recent batches up to 10 will be listed Open Basch The celected batch DG test Oluen Serres 4 batches found SP tost 02 122014 1255 PP test My 02 12 2014 12 54 CM test oavi22014 1253 ii the balch you are looking tor is nol listed please enter all or part of the name or loave empty lo show all batches and click the search button amen la ij u ee _ SS NN SS I SS SSmrEIIEmE Open batches are marked with a in the Available column 2 If the required batch is not in the list of most recent batches a Type the name or a part of the name of the required batch into the Search criteria box b Press Only batches with names that contain the text you entered will be listed 3 Select the required batch in the list 4 Press Open to open the batch If there is more than one batch currently open in Protos 3 you can select which one to work on at any time unless you are in the process of creati
25. automatically detecting and counting colonies Once the system has been set up all you have to do to detect count the colonies on a series of plates is to put each plate on the plate holder and press a button The detection and counting of each plate takes no more than a few seconds and the results are shown instantly in a table Protos 3 allows you to enter plate identifiers and dilutions manually to use auto incrementing plate identifiers or to enter lists of plate identifiers and dilution series within Protos 3 from a CSV file If you use auto incrementing plate identifiers or a plate list Protos 3 will prompt you for which plate to insert next The plate identifiers and dilutions are then automatically assigned to the results and recorded You can also read plate identifiers and dilutions using a barcode reader When you are counting colonies you can use Protos 3 e to distinguish between different types of colony according to their color size and or shape and to distinguish between colonies and debris or other artifacts e to apply sophisticated algorithms to distinguish touching colonies e to add or remove colonies manually after an automatic count you can also perform a completely manual count if required e to define exclude regions on the plate if there are any problem areas on an individual plate where colonies cannot be distinguished for some reason the missing area is then automatically allowed for in calcula
26. cases you should be able to carry out an automatic colony count by simply pressing a button for details see Counting the colonies on a plate automatically on the facing page However some plates or batches may require individual attention In particular you can e Create exclude regions to avoid areas of the plate that might cause problems see Exclude regions on page 110 e Carry out a test measurement and adjust the colony detection settings see Test measurement and adjusting settings on page 110 e Carry out a manual colony count see Manual colony counting on page 112 e Enter a zero result for a blank plate see Entering a zero count on page 113 Note Some of the above procedures can only be applied to certain batch types all exceptions are noted in the instructions All the above colony counting procedures use the Measure tab Next Plate ID Testi 108 Protos Systems User Manual Counting colonies on Pour Plate Spiral and Dilution Series plates The picture shows the Measure tab in Measurement Mode for a Dilution Series batch some Note of the controls do not apply to some batch types and are hidden or appear in a different form To display the Measure tab in Measurement mode Press aINSES P amp Counting the colonies on a plate automatically To count the colonies on a plate automatically Press The results will be added to the Results table see Working with re
27. color samples added Press Remove All Color Samples Note This removes the color sample s from the currently selected color channel if the channel you want to remove the sample from is not currently selected click on it first to select it 4 Repeat Steps 2 3 as required to select a representative sample covering the full range of the background color as it appears in the image 5 Identify the next color in the image for which you want to define a color channel it may be a colony color a debris color or another background color you will specify which it is in the Classification Details page later in the wizard see Classification Details on page 53 6 Either if an undefined color channel already exists Click on the Click here to select this colony item for the channel to select it Or if there are currently no undefined color channels Press Add New Colony to create a new channel the new channel will be selected 48 Protos Systems User Manual The Classification tab The selected channel will be highlighted in orange and labeled Click on image to sample colony color see pictures in Step 1 If required you can zoom the image in the same way as in the main window see Zooming the image on page 8 Zooming the image may help you click accurately on colonies or debris when you are selecting color samples in Step 8 In the image click on areas of the image colony debris or background that have the
28. how to customize the batch settings in the Batch Designer Protos Systems User Manual Setting up batches Note You will not be able to edit the settings made in the Plate Configuration dialog box when the original batch was created see Creating a completely new batch on page 16 for details of the settings made in the Plate Configuration dialog box Setting up batches Once you have created a new batch see Creating the batch on page 16 it will be loaded into the Batch Designer for final set up and testing before you can use it to carry out and record measurements While the Batch Designer is active e The message area at the bottom of the Protos 3 window contains an edit box allowing you to enter a name for the batch see Giving the batch a name on the next page Designing Batch e The Discard New Batch button appears near the top left corner of the Protos 3 window Press Discard New Batch to abort the new batch you will be asked to confirm that you want to do this e The Accept New Batch button appears near the top left corner of the Protos 3 window Initially the button will be disabled When you have completed the steps required to set up the batch the button will become enabled See Accepting the batch design on page 93 for further details The steps you will need to follow before you can start carrying out measurements depend on the type of plate you are using and on whether you based the new bat
29. in combination with a plate list See Using a barcode reader for plate identifiers on page 76 for details Manually entered plate identifiers 72 To set up a batch to use manually entered plate identifiers and where relevant dilutions Press uoReINGyLOD go Protos Systems User Manual The Configuration tab to display the Configuration tab Plate i0 jim Use Barcodes ih Auto increment ra Plate Lisi i cana Lacey 2 If the new batch is based on an existing batch there will be a set of Parent Batch controls below the Plate List controls If the parent batch did not use manually entered plate identifiers uncheck the Parent Batch check box es see The Configuration tab on page 70 for details 3 If the icon in the Plate List box is a go straight to Step 4 Otherwise an external source of plate identifiers has been selected for the batch and the Auto Increment control will be disabled To enable the Auto Increment control and deselect the external source a Press Protos Systems User Manual 73 Creating a new batch to display the Use External Source for Plate ID dialog box Lhe oe herr pourte for Plate iD I oh a oe Please select a source for baich plate IDs b Press 4 If Auto increment is selected in the Plate ID box click the check box to deselect it 5 If you want to read the manuall
30. of all the colonies on the plate all you need to do in most cases is just specify whether the colonies are either lighter than the background or darker than the background or that there are both lighter and darker colonies that you do not want to distinguish from each other However for difficult cases or if you want to distinguish between different types of colony you Can create a colony classification according to the color of the colonies you can also add size and or shape classifications if required The color classification is the primary classification and is used to distinguish between colonies and the plate background medium and any debris or other artifacts and between different types of colony if you want to distinguish between them For most batches the classification can be performed completely automatically using wizards all you need to do is choose how many classifications you want and specify how the results should be interpreted for example whether a particular color represents a type of colony background or debris However you can also customize the detection settings to handle problem cases Accepting a batch Taking The detection parameters for Chromogenic Media batches are set automatically but for the other types of batch you can perform test measurements and experiment with and change the settings used to detect and classify the colonies while you are still working in the Batch Designer Then once
31. page 69 e The Configuration tab on page 70 for setting up how plate identifiers will be assigned to plates when they are measured e The Measure tab on page 85 including e Plate ID on page 86 e Dilution on page 86 e Setting Count Restrictions on page 90 e Test Measure Plate on page 91 e The Results tab on page 92 for choosing what to display in the Results table Once you have completed all the settings you will need to accept the batch so that you can use it for measuring plates see Accepting the batch design on page 93 The sections described above show you how to use the Batch Designer but many of the operations covered may also be carried out when you are working in Measurement mode The final sections of this chapter describe a number of batch management operations for details see e Exporting batch data on page 94 e Viewing batch details on page 95 e Showing or hiding the Light and Dark Colonies button for Total Plate Counts on page 96 e Deleting batches on page 97 Protos Systems User Manual 15 Creating a new batch Creating the batch Note Before you can start detecting counting colonies on plates you need to set up and create a batch to define how the detection counts should be carried out and to hold the results You can create e A completely new batch from scratch see Creating a completely new batch below e A new batch based on an existing batch or an exported batch
32. paini f Protos Systems User Manual 11 Capturing images 3 Move the pointer to the other end of the feature as you do this a line will be drawn back to the starting point for example lait Cae Se eee Now Ghck imap a fe ond of ten panera Z 4 Click to mark the other end of the feature The calibration edit box and units button will be displayed Now enter the length of the Jing batween the two points in ihe units speciied or change lio a different unit of measurement a a 5 If required to change the units a Press the units button to display the units menu Centimeters cm Millimeters rm Microns urn Uncalibrated uic b Select the required units from the menu 12 Protos Systems User Manual Viewing batch images 6 Type the length of the known feature into the calibration edit box 7 Press to set the calibration and return to the main Protos 3 window Viewing batch images By default see System settings on page 136 when you create a new batch the image used to set it up see Setting up batches on page 23 is saved with the batch and then after you have accepted the batch design each time you take a measurement in Measurement mode the image is also saved with the result in the batch To view the batch setup image and images used for measurements 1 Press the Image tab
33. plate for a rectangular plate enter the Dimensions Protos Systems User Manual 17 Creating a new batch iv To choose the units for the Diameter or Dimensions press the corresponding units button to display the units menu i A Millimeters ren Centimeters om and select the required unit v Enter the Sample volume you will be using vi To choose the units for the Sample volume press the units button to display the units menu ie Merolters pl ililiters rL and select the required unit vii Go to Step d c For spiral plates i Click on the Spiral Plates tab to display the Spiral Plates page Piate Loniqquralert Standard Plates SpiralPtales Colony Parametors sHiral Plate Diamalar WASP 90mm y Spiral Volume meane Sel lhe threshold under whch all colonies ar counted per wedge ii Select the Spiral Plate Diameter type you will be using iii Click on the button corresponding to the Spiral Volume you will be using the options available depend on the selected Spiral Plate Diameter type iv If you are using a two sector counting frame and the number of colonies counted 18 Protos Systems User Manual Creating the batch within the first sector is less than a set threshold the whole frame will be used instead To Set the threshold under which all colonies will be counted type in a value directly or press the or button to increase or decrease the curr
34. selected automatic color classification the automatic color classification will be carried out immediately and the wizard will then display the Review Color Classification page showing the results see Review Color Classification on page 50 46 Protos Systems User Manual The Classification tab Manual color classification If you selected Choose colors in next step or Choose colors in later step in the first page of the wizard see step 9 in Color classification on page 35 the Manual Color Classification page is displayed in the Colony Classification Wizard Select Gach Colony or backround channel below then cick on Ihe image ip Sample odors lice here b salad baap You use the Manual Color Classification page to enable Protos 3 to distinguish the colonies you want to count from the background and any debris you want ignored You do this by identifying the colors of the distinct colony types the debris and the background appearing in the image and creating a color channel for each of them You then define the color of each color channel by taking samples of the corresponding color from the image At this stage one of the color channels is identified as a background color and the other channels as colony colors However the Classification Details page later in the wizard see Classification Details on page 53 will allow you to redefine each of the colony colors independently as a colony type debris or background co
35. slider by a single step or press anywhere on the slider bar to set the slider to that position as you adjust the setting the control shows the sensitivity set setings Sensiinaty 89 7 oe a Heyeact Small FParbdes 0 Simm a _o _ amp 7 Spit Toucheng Colonies To set the Sensitivity back to AUTOMATIC drag the slider back to the right hand end in fact this corresponds to a manual sensitivity setting at the middle of the scale 32 Protos Systems User Manual The Classification tab d To set a lower limit to the size colonies must be in order to be counted you can use this for example to prevent small particles of debris being counted as colonies Drag the Reject Small Particles slider click the arrow buttons at the ends of the slider to adjust the slider by a single step or press anywhere on the slider bar to set the slider to that position as you adjust the setting the control shows the minimum colony size set setings Sensimih AUTOMA TK Heyect Small Parades 0 584 4 Spat Touching Colonies To switch small particle filtering off drag the slider to the left hand end Note The Sensitivity Reject Small Particles and Split Touching Colonies settings you make here will be used to perform the initial counts for plates when you are working in Measurement mode However you will be able to vary these settings for individual plates by carrying out a test count in Measurement mode see T
36. than cick on fe image lo draw around fhe region fen double click fo complete Click Exclude Region again to exclude further repons Click Remove Lasi Exdude Ropon to remove modudo reqpons LH Barna peo On occasions you may want to avoid counting colonies on part of the plate used for color classification because of some problem with it You can do this by creating one or more exclude regions which will be ignored when Protos 3 classifies the colonies To define an exclude region on the classification image 1 Press to select it Note You may find it helpful to zoom the image before placing the exclude region on the image see Zooming the image on page 8 Protos Systems User Manual 39 Creating a new batch 2 Move the pointer to the position you want to place the first vertex of the exclude region il 3 Click to place the first corner of the exclude region 4 Move the pointer to the position you want to place the a line will be dragged out from the first vertex AM 5 Click to add the new vertex for the exclude region 40 Protos Systems User Manual The Classification tab 6 Repeat Steps 4 and 5 as required to place any further vertices until you have placed the penultimate one the included area is shown as you drag the mouse Ay OOO 7 Move the pointer to the final vertex of the exclude region 8 Double click to add the vertex and compl
37. the contents are correct see the subsection following these instructions for the format to use for the CSV file 6 If the contents are correct press OK to close the Use External Source for Plate ID dialog box and load the plate list into the batch The Plate ID box on the Measure tab see The Measure tab on page 85 will show that the plate identifiers have been taken from an external source Plate ID External Source Note When you are working in Measurement mode you can use a barcode reader in conjunction with a CSV list to match each plate you are measuring to its identifier dilution in the CSV list see Using a barcode reader for plate identifiers on page 76 CSV file format Each line of the file should consist of two strings separated by a comma Identifier Dilution where Identifier is the plate identifier Dilution is text describing the dilution if the string is not recognized or no dilution is given No Dilution is used 82 Protos Systems User Manual The Configuration tab The order of the lines in the file defines the order of the plates in the list For example Testl No Dilution TestZ Leo Test3 l in 1000 Test4 1 in 10 6 Note Protos 3 does not require you to use unique identifiers dilutions Creating a plate list To create a plate list containing a list of plate identifiers and dilutions 1 Press in the Use External Source for Plate ID dialog box see Using an external s
38. to taking measurements if you wish you can still make further changes to the setup if required before pressing the Accept New Batch button If you have given the batch the same name as an existing batch you will be asked to confirm that you want to same the batch with the same name press Yes if you do press No to close the dialog box so that you can give the batch a different name Protos 3 does not use the name internally to identify and distinguish batches so if you do save a batch with the same name as an existing one you will not overwrite or destroy the original data If you wish you can create and use multiple batches with the same name though there is an obvious risk of confusion Note however that Protos 3 checks the name of the new batch against all the batch names in the database including deleted ones see Deleting batches on page 97 so a reported name clash may not be problematic or obvious If this is the first Chromogenic Media batch you have created in this session you will be reminded that you should use the black plate holder press OK to continue The next chapter in the Manual Using Measurement Mode to count and detect colonies shows you how to use Protos to carry out colony counts and detection measurements the chapter following that Working with results on page 119 shows you how to view and edit the results and then compile a report If you change your mind about creating the new batch Press
39. won eanby wo iA The Use Barcodes check box is disabled if there is no barcode reader attached to the PC Protos Systems User Manual The Configuration tab To display the configuration controls Press the Configuration tab _ r yeniyi we The Configuration tab in the Batch Designer allows you to specify how plate identifiers and where relevant dilutions should be assigned to plates in measurement mode If the batch you are creating is based on another batch the Configuration tab shows the settings selected in the parent batch Plate iD Plate List Parent Batch hese are the proparhes thal are selin thea parent baich They can also be inhented by the child batch Parentis using a plate list heel Q Parent is linked with LIMS 3 5 S Parent can use barcodes S Parentis using auto increment Note LIMS links are not supported by Protos 3 The green check marks show the settings selected in the parent batch if the parent batch uses manually entered parent plate identifiers the Parent is using auto increment will have a red cross next to it but its check box will be enabled To use the same configuration tab settings as in the parent batch Leave the Parent Batch check boxes unchanged If the Parent is using a plate list you will need to create a new plate list for the new batch Note see Selecting auto incrementing plate identifiers on page 74 for
40. zoom the image Press Protos Systems User Manual Copying the image to the clipboard When it is zoomed the button shows the scaling factor To zoom out Press To return to fitting the image to the image pane Press Note The zoom controls are also displayed with the image in some dialog boxes you use them in exactly the same way as in the main Protos 3 window Copying the image to the clipboard To copy the current captured image in the Protos 3 window to the clipboard 1 Press the Image tab Ke E to display the image controls 2 Press See also Viewing batch images on page 13 for how to export previous images from a batch Exporting the image to a file To export the current captured image in the Protos 3 window to a file 1 Press the Image tab Ke E to display the image controls Protos Systems User Manual 9 Capturing images 2 Press to display the Export Image dialog box this is a standard Windows Save As dialog box 3 Use the Export Image dialog box to e select an image format from the Save as type drop down list e select a folder to hold the image e enter a file name for the image 4 Press Save to save the file See also Viewing batch images on page 13 for how to export previous images from a batch Importing an image from a file To import an image from a file into the Protos 3 window 1 Press the Image tab eo E to display
41. Laboratory Equipment Manufacturer www mrelab com CE f Net amp Protos 3 User Manual Contents Getting started 1 WME OS HOn uee pegs dherche esis oes ep tyes asus alee Dada bbe ean ete ate ee eee 1 Ste ture of the Manual 5 25214265050052005 cose ooo a set Seid Saeed eS hha tats 1 Usine Protos a OVEIWIOW 01 5 00 vas aside en tot asa se pete ae avai dauabal se huiee a totale 2 E AO E EEEE AA E E E AE A ES EE E A A E 2 FRC C SUIS AAC Meese nann e r S E 3 Takin measure mente asecartcckien ele daaa EE a EA a 3 AOLO ar E E EA EE E E A enese oeGeeeee 3 CIOE POO sr O E N O ose aliases lene 4 Capturing images 5 Tanela S aN enaa a E T E 5 Loading plates into the instrument 222 2 22222222 e cece cece cece cece cece cece cece cece cece cece eee eee 6 CaplUMine ANIMAS Cah t02 eee e a sue tade tia dddce ute aaa oeae 7 Selle Neve xX POSING ee nash deahauisumddensaeasmonoausinin AEE 7 ZOOMMWAETNOMMMAC Cs ccdaacusvusded dats nico sonasanteudont a E N 8 Copying the image to the clipboard 0 0 000002 2 2 ccc cece ccc eee eee eect eee eeeeeeee 9 Expone the Imace TO a Tileney 0ie is eve ie onal eee ad eee yas 9 Importing an image from a file 2cect
42. Mies COMMS UNA on LAD apanan Sessile siete saa Steecad a aa a a Ea EE EA O 70 Manually entered plate identifiers 00 00000222 eee cece ee ccceeeeceeeeeeeeeeeeeeeees 72 Selecting auto incrementing plate identifiers 0 222222 c cece eee cece eee ee ee eens 74 Using a barcode reader for plate identifiers 2 22 0 0002 o cece 76 Using an external source for plate identifiers 2 2 00 2 220s 77 Protos Systems User Manual Contents Contents Using a CSV list of identifiers 000 000 eee eee ee eee cece cece eeeeeeeeeee 79 Ceanne a CCH GU haat cea tacaceenost asa ta ts Nalacnttacaine saatencs teeta deete coat 83 TIME Mesure taD ssaka O epuannaiaite EEAS 85 AE E E E EREE Soc ETEA AE E IP AEE E A E EET EE TE aed aoe TAAT AEE 86 DOE EEA EE EE EA A EEE E EE EE AE EEE EA OE ETA 86 Pour Plate Bate hes arses E AN 87 Dilution Series and Spiral Plate batches 0000 00000 87 Setting COUNE RESEIGHONS 32 ctictcteci ote se baste eat oes e eat e thee eS Leo 90 Test Measure cd lt a PC Tee Se Soe NU RACE Re TE ae TOES ONS VCE EE Rte eee a Er 91 TR eSa o anette ee Ree Seen Ray Am aT CET SMC Cet oem eee ere Ree Mn ie Tees ee eee 92 Accepting the Dalen ESIGN sd ee cia ceinnsnit s hen atewd or E E Ea r ae e Taaa 93 Manas ine AUC NE S oc ris tis eta tapos e a N E A ET Ao A 94 FxpOniNe DaN dala euroren a a a EEE eeeet i 94 Viewing batch details cate att henc en a a cette eet ee a gee N
43. Protos 3 3 When you have finished viewing the audit history press Cancel to close the dialog box Editing colony counting results Manually adding and removing colonies To add colonies to or remove colonies from the detected colonies for a result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 If you want to remove all colony markers currently on the image so that you start the count from scratch press If you want to remove colonies press Protos Systems User Manual 127 Working with results 128 4 If you have used a color size and or shape classification to define more than one colony type the first type will be selected by default To select another type to add or remove click on its row in the Results box at the bottom of the Edit Review tab avoid clicking on the actual result text for example 88 colonies in the following picture Results If you have chosen to add colonies click on the image at the point you want to add the colony you cannot add colonies outside the frame If you have chosen to remove colonies click on the colony markers you want to remove The count will be updated in the Results box as you click in the image 6 Repeat Steps 4 5 for any other colony types 7 Save the change to the result see Saving changes to results on page 125 The count s will be updated in the result together with any values dependin
44. Title Value Batch Name SP test Batch ID 19 Plate Type Spiral Plate ls based on Batch False Image Source Imported Image Use Barcode False Cancel 4 To copy one or more of the detail rows a Select the rows you want to copy e Click in a single row to select it e Ctrl click or shift click to extend the selection in the usual way e Press Select all to select all the rows b Press Copy to copy the data in the selected rows to the clipboard so that you can use it in another application 5 Press Cancel to return to the main Protos 3 window Showing or hiding the Light and Dark Colonies button for Total Plate Counts To choose whether to show the Light and Dark Colonies button for Total Plate Counts see Total Plate Count on page 30 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu IE Foal Protoss 96 Protos Systems User Manual 2 Managing batches Press v B Delete Fetch H kals ine a ar aan he So Lor aad Dark onli to display the Batch Management submenu Press to change between showing and hiding the Light and Dark Colonies for Total Plate Counts Deleting batches To delete one or more batches from the database 1 If a batch you want to delete is currently open close it see Closing batches on page 117 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu e Expa
45. age Press For a colony counting batch to reset the result to the original measurement value before any changes were made to it and saved using the Edit Review tab Press Note Any changes made to the plate identifier dilution or comment in the Editing Plate panel will not be reset Deleting results To delete the result s for a plate 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Press 3 Save the change to the result see Saving changes to results on the previous page The result will be deleted from the Results table Note Although the result is deleted from the Results table and can no longer be viewed in Protos 3 it is not removed from the Protos 3 database and can be viewed there if required for audit purposes Viewing the audit history of changes When you make any changes to a result Protos 3 makes an entry in the audit history for that plate To view the audit history for a plate in the Results table 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Press 126 Protos Systems User Manual Editing plate measurements to display the Audit History dialog box Putt resi Date Acton Element Changed id Value Hew Value User Audit Reason B01 125459 PM UPDATED Counl A 716 G70 arire B21 1244 09 PM UPDATED Diluten Ba 1 10 andrew Note The Audit Reason column is always blank in
46. ails page see Classification Details on page 53 Otherwise proceed to the next step 50 Protos Systems User Manual The Classification tab 3 If colors are being wrongly classified for example if colonies are not being detected because they are being treated as background or if colonies and debris with different colors are not being distinguished accurately e If you performed an automatic color classification try pressing Back until you get to the Configure Color Classification page and try increasing or decreasing the number of colors you selected or try using a manual color classification instead see Manual color classification on page 47 e If you performed an automatic background correction try pressing Back until you get to the Configure Color Classification page and use a manual background correction instead see Manual background correction on page 44 4 Protos 3 applies a size filter to remove the effect of small colored regions of the image that may be wrongly interpreted as colonies You can use the Reject Small Particles slider bar to set the level of size filtering used To adjust the Reject Small Particles slider Drag the slider click the arrow buttons at the ends of the slider to adjust the slider by a single step or press anywhere on the slider bar to set the slider to that position e If small debris particles are wrongly detected as colonies try increasing the Reject Small Particles setting
47. ake sure there is a barcode reader attached to the PC see the note in Step 2 2 Press Ly ucgen ga to display the Configuration tab Plate ID jm Use Barcodes Te Auto Increment gt ri Flate Last La woqranby wo 25 Note The Use Barcodes check box is disabled if there is no barcode reader attached to the PC 3 Select Use Barcodes See Using a barcode reader for identifiers and dilutions on page 108 for more on using the barcode reader to input plate identifiers dilutions Using an external source for plate identifiers You can use the following external sources for plate identifiers e a CSV file e a list created in Protos 3 Protos Systems User Manual 11 Creating a new batch To specify an external source for plate identifiers 1 Press to display the Configuration tab Piate iD Wi Use Barcodes Auto increment jim 50 Ba te i Plate List bp Pane Lice i 2 Press 78 Protos Systems User Manual The Configuration tab to display the Use External Source for Plate ID dialog box Loe eternal source for Plate iD x a el eee a emner Please select a source for baich plate IDs 3 Press the button for the required option The following sections describe each of the options in turn e Using a CSV list of identifiers below e Creating a plate list on page 83 If one of the options is already selected and you want to go back to u
48. an accept the batch for Measurement mode Protos Systems User Manual 85 Creating a new batch Plate ID _____ PJate D ___________ The Plate ID control on the Measure tab in the Batch Designer allows you to specify a template for auto incrementing plate identifiers see Selecting auto incrementing plate identifiers on page 74 for details Note If you choose to use auto incrementing plate identifiers you will not be able to accept the batch design until you have entered a template for the identifiers see Accepting the batch design on page 93 However if you have chosen to use an imported plate list or a plate list created in Protos 3 see Using an external source for plate identifiers on page 77 the Plate ID control will show ______ Piai iD ______ External Source Dilution Notes Dilutions are not relevant for Chromogenic Media batches 86 If you have chosen to use a CSV file or a custom plate list as the source of the plate identifiers for use in Measurement mode see Using an external source for plate identifiers on page 77 you can include individual dilutions with each plate identifier see the previous section Plate ID f no dilution is supplied in the lists No dilution is assumed To specify the dilution to use in calculating results Press the Measure tab to display the dilution controls The dilution controls used for Dilution Series and Spiral Plate batches are different from t
49. an image on page 7 4 Adjust the measurement frame s if required see Adjusting frame settings in Measurement mode on page 103 5 Enter a plate identifier if it has not been entered automatically or change an automatically entered identifier and dilution if required see Entering plate identifiers and dilutions on page 103 6 Carry out the measurement see Counting colonies on Pour Plate Spiral and Dilution Series plates on page 108 and Detecting colonies on Chromogenic Media plates on page 113 7 Repeat Steps 2 6 for all the other plates in the batch 8 Check the results and edit them if necessary then create a report for the batch see the following chapter Working with results on page 119 9 Close the batch see Closing batches on page 117 Steps 2 and 3 are also required when creating and setting up the batch the cross references take you back to earlier sections in the Manual Step 8 is described in the next chapter Working with results on page 119 Instructions for the remaining steps are given in this chapter though much of Step 4 is common to creating and setting up the batch and is covered by references to the Creating a new batch chapter Opening and selecting batches If you have just created and accepted a new batch see Creating a new batch on page 15 and Accepting the batch design on page 93 it will be automatically open and selected in Measurement mode However you can
50. assification tree with the selected sub classification removed you cannot change any of the details in the tree Press Finish to confirm the change and return to the main Protos 3 window e The color classification A dialog box will be displayed Cedar Clas aifenanean ear This action will remove all classifications Do you want to configure a new color classiicaton Click Yos to repeal color classification Click No lo continue without classifications Click Cancel to stay on this page j P j a hi Press Yes if you want to replace the existing classification and its sub classifications with a new color classification You will be taken to the Configure Color Classification page so that you can create the new color classification see Color classification on page 35 for details Press No if you want to remove the existing classification and its sub classifications without replacing it with a new color classification The Classification Details page will be displayed with an empty classification tree If you then press Finish in the Classification Details page another warning dialog box will be displayed Clasur ian Creat This action will generate an emply classiicaton which cannot be used for measurement Do you want to continue l Click Yes to Fintsh with no classification Click No to stay on this page e Press Yes if you want to continue with an empty classification this will take you ba
51. ate Count 30 33 96 97 U Undoing changes to results 126 V Version 135 136 Viewing batch details 95 96 Viewing batch images 13 14 X XML 94 95 Z Zero count 113 Zeroing a result 130 Zooming the image 8 9 Protos Systems User Manual Index iil
52. ation you selected in Step 3 and are exactly the same as the corresponding step in the procedures for adding sub classifications using the classification buttons in the Classification tab for details see e Size classification on page 56 e Shape classification on page 60 Removing a sub classification Note The Remove Classification command is only available if the color classification already has one or more sub classifications The next step after you select Remove Classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard see Modifying an existing classification on page 64 is to select which classification to remove using the classification tree in the Remove Sub Classification page Which set of classifications should be removed Select below or click here to Gear selechon 1 Click in the tree to select which classification to remove Note You select a classification by clicking on one of the results of the classification For example if you click on B1 the size sub classification will be selected and both B1 and B2 will be highlighted if you click on A the color classification will be selected and the whole tree will be selected Protos Systems User Manual 67 68 Creating a new batch 2 Press Next 3 What happens next depends on whether in Step 1 you selected e A sub classification The Classification Details page will be displayed showing the new cl
53. blem area is not counted To define an exclude region on an image 1 Press You may find it helpful to zoom the image before placing the exclude region on the image see Zooming the image on page 8 2 You define the exclude region in the same way as when you add exclude regions in the Colony Classification wizard see Excluding regions during color classification on page 39 for detailed instructions To remove the last exclude region you added to the image you can repeat the procedure to remove each of the regions in turn Press Test measurement and adjusting settings 110 If required you can carry out a test measurement to check the colony detection for an individual plate without recording the result and if the batch was based on a Total Plate Count see Total Plate Count on page 30 you will be able to modify some of the batch colony detection settings if there are any problems To carry out a test colony count and adjust the detection settings 1 Press Protos Systems User Manual Counting colonies on Pour Plate Spiral and Dilution Series plates The result of the test measurement will appear in the Last Measurement Results panel and if the batch is based on a Total Plate Count the Adjust button will be enabled The result of the test measurement will not be added to the Results table see The Results table on page 119 If the button below the image is labeled All Co
54. ch on an existing batch However in both cases you will need to give the new batch a name see the next section Giving the batch a name Protos Systems User Manual 23 Creating a new batch The other settings you will need to make when you are creating a completely new batch or the settings you can edit if the new batch is based on an existing one are grouped on a series of tabs see the following sections for details e The Classification tab below in particular see e Classification colony counting batches on the facing page e Classification Chromogenic Media batches on page 69 e The Measure tab on page 85 e The Configuration tab on page 70 e The Results tab on page 92 Giving the batch a name After you have created a new batch see Creating the batch on page 16 you must give it a name before you can accept the design see Accepting the batch design on page 93 To give a name to a batch Type the name into the Designing Batch box at the bottom of the Batch Designer screen Designing Batch See Setting up batches on the previous page for a list of other steps in setting up a new batch The Classification tab You use the Classification tab to specify how colonies are automatically detected by Protos 3 The content of the tab and the procedures required depend on the type of batch for details see e Classification colony counting batches on the facing page e Classification
55. ck on the required color in the Standard Colors control When you have completed the classification details press Finish to complete the color classification and return to the main Protos 3 window the color classification will be shown in the Classification panel see The Classification panel on page 33 54 Protos Systems User Manual Notes Note The Classification tab Adding sub classifications Once you have added a color classification to the batch the other classification buttons become enabled the first step in adding a sub classification is to choose where to add the sub classification see the next section Choosing where to add the sub classification You cannot add a color sub classification if you press the Color Classification button to create a new color classification you will be warned that if you continue the current color classification and all of its sub classifications will be replaced by the new color classification You can add a Size classification see on the next page or Shape classification see on page 60 as a sub classification to any existing colony type Choosing where to add the sub classification When you press one of the sub classification buttons the appropriate wizard opens However if there is already more than one colony type the first step in all of these wizards is the same for example Cae Sle calc e Wied Add New Size Sub Classificalion Where should the new size
56. ck to the situation you were in when you first created the batch see Creating the batch on page 16 you will not be able to accept the batch until you have created a color classification or set a Total Plate Count see Accepting the batch design on page 93 e Press No if you have changed your mind about removing the color classification You will be returned to the Classification Details page where you can press Cancel to stop the classification removal procedure altogether or press Back to return to Step 1 Protos Systems User Manual The Classification tab Repeating a color classification Note The Repeat Color Classification command is only available if the color classification has no sub classifications The next step after you select Repeat Color Classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard see Modifying an existing classification on page 64 is to create the new color classification using the Configure Color Classification page see Color classification on page 35 for details The new color classification will replace the existing one Classification Chromogenic Media batches LOWES SEL To display the classification controls in the Batch Designer Press the Classification tab OES Ser 6 In order to use the Classification tab you will need a sample image to work with 1 Insert a typical plate from the batch into Protos 3 see Load
57. d and distinguished correctly if they are not adjust the Probability Threshold by clicking on the or buttons or dragging the slider left or right When you make an adjustment Protos 3 will automatically carry out a test measurement so that you can see the effects of the change Closing batches To close one or more open batches 1 Press Protos Systems User Manual 117 Using Measurement Mode to count and detect colonies to display the Review Open Batches dialog box Pee pen bate hes Curent Batch sees Other Open Batches Click to Select The currently selected batch is listed with a check box in the Current Batch box and there are buttons and check boxes for the Other Open Batches in the box below it 2 Either to close a selection of one or more batches a Click in the check boxes for the batches you want to close to select them b Press Or to close all the open batches Press The Review Open Batches dialog box will close and either the selected batches or all the batches will be closed 118 Protos Systems User Manual Working with results This chapter shows you how to view and process the results produced by Protos 3 and then compile a report based on them see Using Measurement Mode to count and detect colonies starting on page 101 for how to carry out the measurements This chapter has three main subsections e The Results table below e Editing p
58. ded and repeat Step 2 if the result is still not satisfactory Note You may find it helpful to zoom the image see Zooming the image on page 8 4 Save the change to the result see Saving changes to results on page 125 The count s will be updated in the result together with any values depending on the count s M and E flags will appear in the Flags column showing you have manually edited the plate Changing the Sensitivity setting for a result Note This section is only relevant for colony counting batches based on a Total Plate Count see Total Plate Count on page 30 The Sensitivity control is hidden for batches based on a color classification When you create a batch based on a Total Plate Count you can adjust the detection Sensitivity see Total Plate Count on page 30 After you have accepted the batch you can also change the Sensitivity setting in Measurement mode before taking a measurement see Test measurement and adjusting settings on page 110 This section shows you how to change the Sensitivity setting for an individual plate after it has been measured To change the detection Sensitivity setting for an individual plate result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Drag the Sees lrwiby 710 I l p slider click the arrow buttons at the ends of the slider to adjust the slider by a single step or press anywhe
59. define the settings used to detect colonies on plates and to store the results of the measurements you make using those settings This means that before you can use Protos 3 to take any measurements you must have an open batch You can do this by opening an existing batch or by creating a new batch You can create a completely new batch or base a new batch on an existing one either to use the same settings or as a starting point for defining new settings Protos 3 automatically saves your results in the current batch so once you have created and accepted a batch design you do not need to take any action to save it again For colony counting batches each batch contains the settings and results for detecting and counting the colonies on a series of plates that are of the same type Pour Plate Spiral Plate Dilution Series have the same types of colony and use the same medium For Chromogenic Media batches you can select the medium used for each plate separately when you make the measurements so a batch can contain plates using different media and containing different colony types Creating a batch For Chromogenic Media batches the detection parameters are set automatically according to the type of medium which you only specify after you have created the batch and can select individually for each plate in the batch 2 Protos Systems User Manual Starting Protos 3 For a colony counting batch if you are just interested in a total count
60. e 101 Creating a new batch 16 23 from existing Batch 20 23 from exported batch 20 23 D Database location 139 140 Deleting batches 97 99 Detecting colonies Chromogenic Media 101 outline procedure 101 Dilution 86 90 changing 105 106 editing 123 124 selecting 105 106 Dilution series batch 87 90 frame type 26 27 Discard New Batch 23 94 E Excel 134 Exclude region adding to a result 124 125 clearing from a result 124 125 during color classification 39 42 in measurement mode 110 Index i Index Exponential number format 138 Export Data as XML 94 95 Exporting batch data 94 95 Exporting images 9 10 Exposure setting 7 8 F Frame Color 138 139 Frames 69 70 Chromogenic Media circular 69 70 partial plate 69 70 rectangular 69 70 colony counting 26 adjusting 27 103 124 annulus 26 changing type 26 27 103 choosing type 26 27 circular 26 27 color 138 139 moving 28 103 partial plate 26 27 rectangular 26 27 reshaping 28 29 103 resizing 28 29 103 rotating 29 103 spiral 26 27 two sector 26 types 26 whole frame 26 G Grid detecting and removing 43 44 H Hide Light and Dark Colonies 96 97 Image files 137 Image tab 5 6 Images calibrating 11 13 capturing 5 7 copying 9 exporting 9 10 importing 10 zooming 8 9 Importing images 10 Limit count per frame 90 Loading plates 6 7 M Marker positions 143 144 Measure tab Batch Designer 85 92 Index ti Measurement mode 103 108 N Network 139 140 New batch batc
61. e Step 2 b Press to display a Standard Windows Save As dialog box c Use the Save As dialog box to select a folder and enter a filename for saving the image d Press Save to save the selected image Note See Exporting the image to a file on page 9 for how to save the current image to a file 6 Press Close to close the dialog box and return to the main Protos 3 window 14 Protos Systems User Manual Creating a new batch Note This chapter shows you how to create and set up a new batch for detecting counting colonies It also describes a number of batch management procedures for use once you have created the batch The process of creating a new batch is divided into two main stages which are covered in the following main sections e Creating the batch see the next section This section shows you how you can create e A completely new batch from scratch see Creating a completely new batch on the next page e A new batch based on an existing batch or an exported batch file see Creating a new batch based on an existing batch or an exported batch file on page 20 e Setting up batches on page 23 This section describes e Giving the batch a name on page 24 e The Classification tab on page 24 for specifying how colonies are detected this section is divided into two major subsections e Classification colony counting batches on page 25 e Classification Chromogenic Media batches on
62. e defines the order of the dilutions in the list For example No Dilution ere L an 1000 1 in 10 6 Note Protos 3 does not require you to use unique dilutions Setting Count Restrictions Note The Count Restrictions button does not appear for Spiral Plate or Chromogenic Media batches To place restrictions on the count result 1 Press the Measure tab T a to display the measure controls including the Count Restrictions button 2 Press the Count Restrictions button to display the Count Restrictions controls F COUNT Restrictions Count Limits E irt cout per trame Ipper Count f Lower Cool r Exclude from mean 3 Check Limit count per frame to enable count restrictions 4 Either type the required limits directly into the Upper Count and Lower Count edit boxes or press the arrow buttons to increase or decrease the values 5 Check Exclude from mean if you want any results violating the count restrictions to be ignored when calculating the mean results leave it unchecked to include all results in mean calculations 90 Protos Systems User Manual The Measure tab When count restrictions have been set and you carry out a test measurement in the Batch Designer see Test Measure Plate below or perform a measurement in Measurement mode the Measurement test results or Last measurement results box respectively will show whether the limit has been breached for example Last measurement results Upper 99
63. e example at the beginning of this section there are two colony types A and B Protos Systems User Manual 119 Working with results If there is more than one result with the same plate identifier for example the results for a Dilution Series they will appear as separate plate results within the same identifier item and below the Mean result test Mean Mean Count ml 262 Plates Plate Name Dilution Count Frame Count ml test 1 5 59 279 test 1 2 122 231 test 1 1 290 2 5 You can select a row in the Results table by clicking in it and move to the next or previous line by pressing the down or up arrow key on the keyboard To select the next or previous plate identifier in the Results table there may be several plates with the same plate identifier in a Dilution Series batch for example F Press es or EM on the right hand edge of the Results table Editing plate measurements Notes 120 Protos 3 allows you to edit measurements in a variety of ways using the Edit Review tab You can only edit results if the image was saved with the result see System settings on page 136 for how to choose to save images with results If the image was not saved with the result a dialog box will be displayed Bic caved image 7 Thee 1s no Saved Image for this Hale unable to Gontnue with mansal eiil Press OK to continue To edit a measurement 1 Select the measurement you want to edit by clicki
64. e picture to create a detailed report for the current batch e Summary of all Batches to create a summary report covering all the batches in the Protos 3 database e Audit trail of all batches to create an audit report covering all the batches in the Protos 3 database e Audit trail of the current batch to create an audit report for the current batch 3 To limit the information given for each result in the current batch report to the columns currently shown in the Results table the default Leave Restrict report by batch headings checked Protos Systems User Manual 133 Working with results To include all the data for each result including columns not selected for the Results table see The Results tab on page 92 Click Restrict report by batch headings so that it is not checked Note The Restrict report by batch headings setting has no effect on summary or audit trail reports 4 Check or uncheck Report Results Oldest to Newest to set the ordering of results in the report 5 Press Microsoft Excel will open with a worksheet containing the selected report To compile a results report for an individual plate 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Press Microsoft Excel will open with a worksheet containing the plate report 134 Protos Systems User Manual Configuring Protos 3 This chapter shows you how to configure Protos 3 in a variety of ways
65. e same total plate count or classification settings as the existing batch though you can change them if required If you are creating a completely new batch you will not be able to accept it for taking measurements until you have defined a total plate count or color classification Frame The following sections tell you about e Choosing the type of frame to use see the next section e Adjusting the position and size of frames on the facing page Choosing the type of frame to use The frame defines the area within which colonies will be counted The choice of frames depends on the type of batch e Pour Plate and Dilution Series the frame can be circular rectangular or a single sector of a circle occupying 1 10 1 8 1 4 or 1 2 of the total area e Spiral Plate e Whole Frame Whole Frame the frame is circular e Whole Frame Annulus Frame the frame consists of two concentric circles e Two Sector a series of concentric circles with two opposite highlighted sectors For example To change the type of frame used to define the region for counting colonies 1 Press the frame button to display the frame menu 2 Choose the required option see the following sections for details of the options available for each type of batch e Pour Plate and Dilution Series batches on the facing page 26 Protos Systems User Manual Note Note Notes The Classification tab e Spiral Plate batches below You ca
66. e text you entered will be listed d Select the required batch in the list e Press Protos Systems User Manual 21 Creating a new batch 22 f a If you chose an existing batch that is currently in use you will be warned z Haik h m wie The selected balch 1s currently in use and may be beng altered fj at you sure you wish lo continue Press Yes to create the new batch anyway press No to close the warning dialog box and return to Step c to choose another batch The new batch will be created see the paragraph following Step 3 for what to do next If you want the new batch to have the same settings as an exported batch see Exporting batch data on page 94 for how to export batches Press to display the Create from Exported Batch dialog box eer Create romSported Faun a Fiease select a batch fle to umpari to display the Please select a batch file to import dialog box this is a standard Windows Open dialog box Use the dialog box to locate and select the required exported batch file Press Open to select the file and return to the Create from Exported Batch dialog box Press to create the new batch The new batch will be created and loaded into the Batch Designer with the same settings as the selected existing batch or exported batch file You will be able to edit some of these settings in the Batch Designer see Setting up batches on the facing page for
67. ement is not selected in the Plate ID box click the check box to select it 5 Press the Measure tab aunseep amp to display the measure controls see The Measure tab on page 85 6 Inthe Plate ID box type in text to act as a basis for the identifier If the text includes a sequence of digits these will be used as the starting point for the auto incrementing if it includes more than one sequence the rightmost sequence will be used as the starting point if there are no digits in the text auto incrementing digits will be added to the end of the text For example Template First identifier Second identifier Third identifier abc abc1 abc2 abc3 abc18 abc19 abc20 abc21 abc 1 8def abc 1 9def abc 20def abc 21def 66abc 1 8def 66abc 1 9def 66abc 20def 66abc 21 def Using a barcode reader for plate identifiers Note Depending on how the barcode reader is programmed the barcode reader may or may not be able to read dilution information 76 Protos Systems User Manual The Configuration tab When you are working in Measurement mode you can use a barcode reader to read the identifier dilution from the plate in combination with an external source of plate identifiers see Using an external source for plate identifiers below or to input manual identifiers see Manually entered plate identifiers on page 104 If you intend to use a barcode reader to read plate identifiers and dilutions where relevant see the note above 1 M
68. ent value Note You will not be able to edit the settings made in the Plate Configuration dialog box after you have created the batch in the next step d If you wish to restrict the maximum detected size of colonies for example to reduce the possibility of debris being detected as colonies i Click on the Colony Parameters tab to display the Colony Parameters page re P i 1 1 a is har a PUP Li Au UE Standard Plates Spiral Plates Colony Parameters bed Macc miu Colony SA Restid Haganum Detected Sizo Learmeler rr ii Check Restrict Maximum Detected Size iii Enter the maximum Diameter for a detected colony iv Press OK to confirm the settings close the dialog box and return to the Launch Batch Designer dialog box 3 To create the new batch press the button for the type of batch required You can create batches for the following plate types e Chromogenic Media e Dilution Series e Pour Plate e Spiral Plate The Batch Designer will be launched for the selected plate type so that you can complete the process of setting up the new batch see Setting up batches on page 23 for details Protos Systems User Manual 19 Creating a new batch Creating a new batch based on an existing batch or an exported batch file Note See Creating a completely new batch on page 16 for how to create a completely new batch from scratch To create a new batch based on an existing batch or an exported batch file 1 Pr
69. equired Results with a selected check box will be included in the Results table unselected results will not be included The order of the results in the list is the order they will appear in the Results table To change whether a result will be included in the Results table 1 Click on the result to select it 2 Click on the result again to select deselect the check box 3 To carry out the change when working in Measurement mode press To move a result up or down the list to change the order of results in the Results table 1 Click on the result to select it 2 To move the result up the list press To move the result down the list press 3 To carry out the change when working in Measurement mode press Accepting the batch design To confirm that you have completed the setup procedure and want to proceed to taking measurements Press Protos Systems User Manual 93 Creating a new batch Notes The button will be disabled until you have carried out all steps required for setting up the batch However even if the button is enabled you will prevented from accepting the batch if you have not given the batch a name see Giving the batch a name on page 24 or if you are using auto incrementing plate identifiers see Selecting auto incrementing plate identifiers on page 74 you have not set a template plate id see Plate ID on page 86 The Accept New Batch button is enabled to show that you can proceed
70. es see Review Color Classification on page 50 To add a size classification to the batch so that you can distinguish between colonies or between colonies and debris according to their sizes 1 If the current image does not have the full range of sizes appearing on plates in the batch you should replace it by loading a plate having the full distribution see Loading plates into the instrument on page 6 and capturing a new image see Capturing an image on page 7 2 Press to start up the Size Classification Wizard If there is already more than one colony type in the colony classification the Add New Size Sub Classification page will be displayed go to Step 3 otherwise if there is currently only one type of colony in the colony classification the wizard will skip straight to the Configure Size Classification page go to Step 5 3 In the Add New Size Sub Classification page click on the existing colony type that you want to subdivide with the new size classification see Adding sub classifications on the previous page for more details Protos Systems User Manual The Classification tab 4 Press Next to display the Configure Size Classification page for example Configure Sire Chssiication Aulomabc Classaiicabon How mary Si2e range Chassifcabors ane ee Ti O Smering D Jarti 4 arstanges O More ae 5 Inthe Configure Size Classification page see picture in previous step click on the
71. espond to colonies debris or background To change the colony marker or color 1 Move the pointer over the colony item to display the colony button O Debris O Background 2 Press the colony button to display the marker controls 3 Either Select the shape of colony marker you want to use from the marker controls Protos Systems User Manual The Classification tab Or a Press Color to display the Standard Colors control Standard Colors b Click on the required color in the Standard Colors control Color classification In many cases you can use the Total Plate Count see Total Plate Count on page 30 to make accurate and reliable counts of the colonies on the plates in the batch However you can use the more powerful features offered by the color classification tool for difficult cases or if you want to make separate counts of differently colored colonies or to use size and shape to distinguish colonies see Adding sub classifications on page 55 Note You can only perform size or shape classifications after you have performed a color classification To carry out a color classification procedure 1 Capture the image of a typical plate from the batch see Loading plates into the instrument on page 6 and Capturing an image on page 7 2 Press to start the Colony Classification Wizard for color classification Protos Systems User Manual 35 Creating a new batch Note Note
72. ess to display the Launch Batch Designer dialog box Lawnch Gatch Deria Please select the type of batch you want to create Go to Step 2 if you want to create a new batch from an existing batch go to Step 3 if you want to create a new batch from an exported batch file Note The plate configuration will be inherited from the existing batch and if you make any settings using the Plate Configuration button they will be ignored 2 If you want the new batch to have the same settings as an existing batch a Press 20 Protos Systems User Manual Creating the batch to display the Base on Existing Batch dialog box Fart Gr aa ees Fate x Batch Name Number Batch Name Plate Type J Test 1 e2eac002iseM Show templates only Note Any batches that are currently open will be marked with 38 in the Available column You will not be prevented from choosing to base the new batch on one of these but you will be warned when you confirm the selection see Step f b Select Show templates only to hide any batches in the list that were based on an existing batch or an exported batch file c If there are a large number of existing batches the batch you want to use as a basis for the new batch may not be shown in the list To search for an existing batch i Type the name or a part of the name of the required batch into the Batch Name box ii Press Only batches with names that contain th
73. est measurement and adjusting settings on page 110 or by editing the result after you have carried out a count see Rejecting small particles in a result on page 128 Changing the Sensitivity setting for a result on page 129 and Splitting colonies in a result on page 129 4 Press to hide the Total Plate Count controls again The Classification panel The Classification panel appears below the frame button in the classification tab If you have selected Total Plate Count see Total Plate Count on page 30 it shows a key for the markers used to identify the detected colonies on the image q opa Ma Background Protos Systems User Manual 33 Creating a new batch 34 If you have set up a color classification see Color classification on the facing page it will show the classified colors and a key for the markers used to identify the detected colonies on the image BS O Ws O Background In this example the only classification is a color classification with two colony types A and B along with the Background classification items can also be classified as debris see Classification Details on page 53 If you add sub classifications see Adding sub classifications on page 55 the classification will appear as a tree for example As well as showing you the current classification the Classification panel allows you to change the colony marker and color but not the name or whether colors corr
74. ete the region Protos Systems User Manual 41 Creating a new batch To remove the last exclude region you added to the image Press You can repeat these procedures to add and remove as many exclude regions as required Note The regions defined here will only be excluded from the color classification process itself see Exclude regions on page 110 for how to define exclude regions for individual plates during the measurement process To proceed to the next step in the color classification procedure Press Next The next step in the color classification procedure depends on selections you made in the Configure Color Classification page see Color classification on page 35 e If you selected Detect amp Remove Grid the wizard will display the Detect amp Remove Grid page see Detecting and removing a grid on the facing page for instructions e Otherwise if you did not select Automatic background correction the wizard will display the Background Correction page see Manual background correction on page 44 for instructions e Otherwise ie you did not select Detect amp Remove Grid and selected Automatic background correction e If you selected manual color classification the wizard will display the Manual Color Classification page see Manual color classification on page 47 for instructions e Otherwise ie you selected automatic color classification the automatic colony color classification
75. fication tree 10 Use the classification tree controls in the Classification Details page in the same way as for color classification see Classification Details on page 53 to specify for each sub classification e whether it corresponds to colonies or debris you cannot specify that it is part of the background and for a colony sub classification if you are not going to sub classify it any further e the name to be used to identify it e the marker used to identify it on the image e the color used for the marker 11 When you have completed the classification details press Finish to return to the main Protos 3 window which will show the updated classification tree Changing an existing classification Once you have accepted a batch for use in Measurement mode see Accepting the batch design on page 93 you cannot make significant changes to the way colonies are detected and classified If you are using a Total Plate Count you will be able to carry out a test measurement and change the settings for the Reject Small Particles and Sensitivity sliders and the colony splitter before recording the result see Test measurement and adjusting settings on page 110 You can also edit a result by adjusting these settings after you have recorded it see Rejecting small particles in a result on page 128 Changing the Sensitivity setting for a result on page 129 and Splitting colonies in a result on page 129 for details You ca
76. file see Creating a new batch based on an existing batch or an exported batch file on page 20 Once you have created the new batch you will still need to set it up and test it before you can start using it to detect count colonies see Setting up batches on page 23 Creating a completely new batch Note 16 See Creating a new batch based on an existing batch or an exported batch file on page 20 for how to create a batch with the same settings as an existing or exported batch To create a completely new batch 1 Press to display the Launch Batch Designer dialog box Lawnch Gate Deeper Please setect the type of batch you want fo create Protos Systems User Manual Creating the batch 2 To set the plate configuration for a colony counting detecting batch a Press Plate Lonfquestor Standard Plates Spiral Plates Colony Parameters 7 gq Diameter Cah oe FLI LFA mi Dimensions BS Well Diameter sample volume Note The Well Diameter setting has no effect in Protos 3 If you are going to use the batch for spiral plates go to Step c otherwise go to Step b b For non spiral plates i Click on the Standard Plates tab if it is not already selected see picture in Step a ii Press the appropriate radio button to specify whether the plates you are using are circular or F E rectangular iii Fora circular plate enter the Diameter of the
77. g on the count s M and E flags will appear in the Flags column showing you have manually edited the plate Rejecting small particles in a result When you create a batch you can adjust the setting of the Reject Small Particles filter to remove the effect of small colored regions of the image that may be wrongly interpreted as colonies see Total Plate Count on page 30 and Review Color Classification on page 50 For a batch based on a Total Plate Count you can also change the Reject Small Particles filter in Measurement Mode before taking a measurement see Test measurement and adjusting settings on page 110 This section shows you how to change the Reject Small Particles setting for an individual plate after it has been measured In this case the batch can be based on a color classification or a Total Plate Count To change the Reject Small Particles filter setting for an individual plate result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 Drag the Reject Small Parbaes 0 hm slider click the arrow buttons at the ends of the slider to adjust the slider by a single step or press anywhere on the slider bar to set the slider to that position The plate will be remeasured with the new setting Protos Systems User Manual Editing plate measurements 3 Check that the real colonies are still being counted while the small particles you want to reject are being exclu
78. guish between some of them but not others for example if there are two colony colors that you want to count together but also some debris that you want to distinguish from the colonies In this case you could e Set the number of distinct colony types plus the number of distinct colors of debris to 2 and check whether the automatic color classification successfully distinguishes the colonies from the debris see Review Color Classification on page 50 if it does not you can press Back to return to the Configure Color Classification page to try one of the following alternatives e Set the number of colors for automatic color classification to be three and combine the results for the different colony colors later outside Protos 3 e Use manual color classification b Select the Automatic Classification radio button corresponding to the number of different colors to be classified If you want to enter a number greater than 3 select More and press the arrow buttons to set the required number Protos Systems User Manual 37 Creating a new batch 38 9 e To set the color classification manually Select the Choose colors in next step radio button the button is labeled Choose colors in later step if you have not selected the default options for other settings Press Next What happens next depends on the selections you have made If you have selected Exclude Regions the wizard will display the Exclude Regions page
79. h name 24 creating 15 23 overview 15 creating from existing batch 20 23 creating from exported batch 20 23 P Plate ID 72 84 86 103 108 auto incrementing 74 76 104 editing 123 124 entering 103 108 entering manually 72 74 104 105 external source 77 84 106 108 from CSV list 79 83 Plate list 83 84 Pour Plate batch frame type 26 27 Probability Threshold 117 132 Program details 135 136 Program version 135 136 Protos 3 closing 4 icon 4 overview 2 3 starting 3 4 R Rectangular plates plate dimensions 17 18 Report compiling 133 134 Resetting changes to results 126 Results 92 93 119 134 adjusting the frame 124 changing order 93 deleting 126 editing 120 129 129 132 including excluding 93 resetting changes 126 saving changes 125 undoing changes 126 zeroing 130 Results table 119 120 S Sample holder 6 7 Sample volume 18 Saving changes to a result 125 Sensitivity 32 33 111 adjusting in a result 129 Setting exposure 7 8 Protos Systems User Manual Index Shape classification 60 63 Show Light and Dark Colonies 96 97 Show X Plates 138 Size classification 56 60 Small particles 33 51 52 111 rejecting from a result 128 129 Spiral plate batch 87 90 count threshold 18 19 frame type 26 27 plate configuration 18 19 spiral plate diameter 18 19 spiral volume 18 19 Split Touching Colonies 32 33 Starting Protos 3 3 4 Sub classification adding 55 67 removing 67 68 System settings 136 138 T Test Measure Plate 91 92 Total Pl
80. hanging the Sensitivity setting for a result 0 000s 129 Splitting colonies ima res lt zi cireres osre aaa deaa a onian 129 ZETOING INMESCOUNL eran e a A a aa 130 Editing Chromogenic Media batch results 00000000000000000000000000000000000000001000100ani 131 Manually changing detection results 00 2 2222 e cece eee eee eee ees 131 Changing the Probability Threshold 202 2 c cece e cece cece ee eeeee eee 132 Setting the results for all colonies to Not Detected 22 2000 c cece cece eee eee 132 COMMONS FEDOS aae bania races ste dietem aad cistertaustian R O a ates eea 133 Configuring Protos 3 135 ADORF sn 3 seduces ated eae a hae mutes tata eee es an ese Bee te ne a 135 SV SIEM TSE UINE Seka s tt 2608s teat lie lee Cale eo ell e tn lake 136 Display properties 0 00000 eee eee eee eee eee eee eee cece cece c cece eeeeeeeeceeeeeeeees 138 Database lo dioe aireicetade Gu Gussinctan E ae oleae setae ant on hence inna Geen dt 139 Testim laMe easa cece naasimadas nani mecaosen E E EAE 140 AMO AON aa a tee etek teh E ER 141 G6 ole e 1 el een meer eee ent ee a rE en ene ne rc dt oT a ae Tee Pe tee ee 141 Marker POSINIOMS hax oi to eee sate tet at leicht eaten ak leit es Aue Rie t 143 Cameriere mens se beg e ihe ke ge ante a ee et a ho el hl et 144 Protos Systems User Manual Contents iii Getting started Introduction Protos 3 is a combined hardware and software system for
81. hose for other types of batch for details see e Pour Plate batches on the facing page e Dilution Series and Spiral Plate batches on the facing page Protos Systems User Manual The Measure tab Pour Plate batches Note See Dilution Series and Spiral Plate batches below for how to set the dilutions for Dilution Series and Spiral Plate batches dilutions are not relevant for Chromogenic Media batches To set the dilution for a Pour Plate batch 1 Press the button shows the current dilution to open the dilution editor Dilytinn Editer E Enler dilution acio 2 Use the dilution boxes to enter the dilution you can type directly into the boxes or use the and buttons to increase or decrease the values Note The left hand box is the sample proportion the right hand box is the total so 1 10 means one part sample in ten parts total volume This means that the right hand number must not be smaller than the left hand number 3 Press Update to confirm the dilution you have set and return to the main Protos 3 window Dilution Series and Spiral Plate batches Note See Pour Plate batches above for how to set the dilutions for Pour Plate batches dilutions are not relevant for Chromogenic Media batches Protos Systems User Manual 8 Creating a new batch To set the dilutions for a Dilution Series or Spiral Plate batch 1 Press the dilutions button to display the dilutions menu the button shows the curren
82. howing the results see Review Color Classification on page 50 Manual background correction In most cases the Protos 3 automatic background correction produces very good results and should always be used in the first instance In exceptional cases however you may find you can get better results by using manual background correction or no background correction at all Note You should only use manual background correction after you have tried using automatic background correction and found that it does not produce satisfactory results 44 Protos Systems User Manual The Classification tab If you chose not to carry out automatic background correction see Step 8 in Color classification on page 35 the Background Correction page is displayed in the Colony Classification Wizard Background Comechon B Use Background Comrechon To carry out a manual background correction 1 If you do not want to apply any background correction press the Use Background Correction check box to uncheck it and go to Step 5 Otherwise leave Use Background Correction checked and go on to the next step 2 If required you can zoom the image in the same way as in the main window see Zooming the image on page 8 3 Press the display button labeled Original Image in the example picture and choose Colormap Image so that the image is displayed in contrasting colors to show which areas of the image have been detected as the bac
83. ing a new batch 3 Check the image to make sure that the colonies have been correctly detected and distinguished from the background and any debris on the plate 4 lf there are any problems with the colony detection you will need to go back to change the batch settings using one of the procedures described earlier in Setting up batches starting on page 23 If you are happy with the measurement results you can proceed to accept the batch for Measurement mode see Accepting the batch design on the facing page The Results tab Notes 92 The Results tab allows you to specify what results should be included in the Results table and the order of the columns aE F Plate Name E Dilution E Colony Name E Count Frame Count ml Mean Count Frame E User E Flags El Created E Comrimens Motes O Frame Plate O Frame Area O Plate Area m Area Fraction so O Count Plate O Mean Diameter fo Mean Count ril rt a i a be The picture shows the Results tab in Measurement mode the Apply button does not appear in the Batch Designer The results included in the list depend on the type of plate used in the batch To display the results controls Press the Results tab smsoy m Protos Systems User Manual Accepting the batch design You can set a default selection of results in the Batch Designer but you can also make a different selection at any time in Measurement mode as r
84. ing plates into the instrument on page 6 This plate should have the full range of colony background and debris colors appearing on plates in the batch 2 Capture the image see Capturing an image on page 7 All you need to do on the Classification tab in the Batch Designer for Chromogenic Media batches is set the frame Protos Systems User Manual 69 Creating a new batch Note You can choose Circular Frame or Rectangular Frame or to use a frame covering a single sector only choose 1 2 1 4 1 8 or 1 10 from the Partial Plate Frame submenu Whole Plate Frans Ey Circular Frannie y Eectandular Franta JE Rectangular Fram Partial Piate Frame E Partial Plate Frame 1 4 Total Area Partial plate frames can be useful when you have plates that are heavily loaded with bacteria or if there is some problem with a plate and you just want to read results from an unaffected part This means that you are more likely to want to choose a partial plate frame for an individual plate while you are working in Measurement mode rather than for all the plates in a batch when you are creating it in the Batch Designer You can also avoid problematic areas on individual plates by placing Exclude regions over them see Exclude regions on page 110 The Configuration tab Note Note 70 The Configuration tab does not appear in Measurement mode Plate iD WH Use Barcodes Auto increment gt z ri Plate List he
85. instructions If you do not want to use the same configuration tab settings as in the parent batch Uncheck the Parent Batch check box es for the settings you do not want to use see the references below for how to set independent configuration tab settings for the current Protos Systems User Manual 71 Creating a new batch batch If you are creating a new batch from scratch or have chosen not to use the parent batch settings you can choose to use e Manual plate identifiers and dilutions You will need to enter an identifier manually for each batch and if required manually change the dilution See Manually entered plate identifiers below for how to choose this option e Auto incrementing plate identifiers and manual dilutions Automatic identifiers will be created for each plate as it is measured and if relevant you can manually change the dilution See Selecting auto incrementing plate identifiers on page 74 for details e A plate list of plate identifiers and optionally dilutions Each identifier and dilution in the list is assigned to each of the plates in turn as they are measured You can enter a plate list by hand or import it from a file See Using an external source for plate identifiers on page 77 for details e A barcode reader The barcode reader can be used to read identifiers and depending on the setup dilutions directly from a barcode on the plate to enter identifiers dilutions manually or
86. ions selected when the batch was created Protos Systems User Manual 103 Using Measurement Mode to count and detect colonies Note If several plates are given the same identifier for example with a Dilution Series they will be grouped together under the same item in the Results table see Working with results on page 119 for details For details see e the next section Auto incrementing plate identifiers e Manually entered plate identifiers below e External source CSV file or Custom plate list on page 106 e Using a barcode reader for identifiers and dilutions on page 108 Auto incrementing plate identifiers See Selecting auto incrementing plate identifiers on page 74 for details of how to set up a batch to use auto incrementing plate identifiers including the rules used to generate the new identifier When any type of batch with auto incrementing plate identifiers is first opened the template identifier set when the batch was created see Selecting auto incrementing plate identifiers on page 74 will be set automatically in the Next Plate Id box as the first identifier for the batch For example Next Plate ID tes plate 1 After each measurement e For non Dilution Series batches a new plate identifier will be set ready for the next plate by auto incrementing the previous identifier e For Dilution Series batches if there are still some dilutions on the next dilution list the plate identifier wi
87. is section the new settings will be applied to the batch until you close the batch If you wish to revert to the settings made when the batch was created either make a note of the values and re enter them using the above procedure or close the batch see Closing batches on page 117 and reopen it again see Opening and selecting batches on page 101 Manual colony counting Note You cannot carry out a manual colony count for a Spiral Plate batch or batches with more than one type of colony color size or shape classification To carry out a manual colony count 1 Press The button will become selected showing that a manual count is in progress 2 Click on the image to count each of the colonies in turn as you click the total in the Last measurement results panel will be incremented and the image will be marked to show that the colony has been counted 3 When the count is complete press to record the result in the results table Notes Once you have begun a manual count the only way to cancel it is to close Protos 3 You can only add to the total by clicking on the image you cannot decrease the count See also Manually adding and removing colonies on page 127 for how to edit a result in the results table by adding or removing colonies after the count has been made and recorded 112 Protos Systems User Manual Detecting colonies on Chromogenic Media plates Entering a zero count To enter a zero resul
88. it is the color of the parts of the spiral that are not included in the two sectors of a two sector frame and it is the color of the inner boundary of an annular frame 3 Either click on one of the Standard Colors in the palette to select it or press More Colors to display a color picker allowing you to choose from a wider range of Standard colors or to create your own Custom color To choose whether to show or hide tool tips showing the size of a colony when you hover over it 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu 2 Choose Show Colony Tool Tip or Show Colony Tool Tip from the Settings gt Display Properties submenu Database location If you are using several Protos 3 instruments connected across a network they can share a single Protos 3 database In order to do this you will need to specify the location of the shared database in each of the Protos 3 installations Alternatively you may have multiple databases on the local database server on your PC in which case you can use the following procedure to specify which one to use Protos Systems User Manual 139 Configuring Protos 3 To specify the location of the Protos 3 database 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu hs Exar Wit as Xl e sens ac Management About Protos 3 scl Proakas 2 Choose Edit Database location fro
89. justed frame 5 Save the change to the result see Saving changes to results on the facing page The measurements will be updated in the result together with any values depending on counts For colony counting batches M and E flags will appear in the Flags column showing you have manually edited the plate for Chromogenic Media batches there will just be an M flag Adding exclude regions to a result Note You cannot place exclude regions on Spiral Plate batch results If required you can add exclude regions to a plate image before you take a measurement from it see Exclude regions on page 110 However you can also add and remove exclude regions after you have performed the measurement when you remove exclude regions after the measurement you can clear exclude regions that were added before the measurement as well as those added to the result after the measurement To define exclude regions for a plate result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Press 124 Protos Systems User Manual Editing plate measurements to select it Note You may find it helpful to zoom the image before placing the exclude region on the image see Zooming the image on page 8 3 You define the exclude region in the same way as when you add exclude regions in the Colony Classification wizard see Excluding regions during color classification on page 39 The plate
90. kground and which as foreground features 4 Adjust the Detect and Reject sliders to optimize the background correction so that the areas shown as background non background features in the colormap image correspond as accurately as possible to the actual background non background in the image see following note You can drag the sliders press the arrow buttons to adjust the sliders by a single step or click anywhere on a slider bar to set the slider to that position Protos Systems User Manual 45 Creating a new batch Note You can view the effect of the background correction by pressing the display button labeled Original Image in the example picture You can view e Colormap Image this uses contrasting colors to show which areas of the image have been detected as the background and which as foreground features e Original image the uncorrected image e Corrected image the image after the background correction has been applied to the image 5 If you wish to revert to the automatic background correction settings Detect and Reject both set to 5 0 press 6 When you are satisfied with the background correction press Next What happens next depends on the selections you made earlier see Color classification on page 35 e If you selected manual color classification the wizard will display the Manual Color Classification page see the next section Manual color classification for instructions e Otherwise ie you
91. late measurements on the next page e Compiling reports on page 133 The Results table When Protos 3 has detected counted the colonies on a plate it adds the results to the top of the Results table at the bottom of the window for example Plate Name Dilution Flags Created Comments Notes Test 3 1 10 23 07 2009 09 52 05 Colony Name Count Frame Count ml B 61 210 A 14 40 Test 2 1 10 23 07 2009 09 49 57 Test 1 1 10 23 07 2009 09 47 45 Note The default choice of which columns are included and their order in the Results table is made when the batch is set up in the Batch Designer but you can change this at any time in Measurement mode see The Results tab on page 92 in Measurement mode you will need to press the Apply button to carry out any changes you make in the Results tab In the example there are results for three colony counting plates Test 3 Test 2 and Test 1 the bottom two have been closed so they only show summary information the top one is open and shows the detailed results To open or close a result Press the or button next to the plate name If required you can resize the Results table by dragging the handle at the top of the table Comments Notes You can also resize the table columns by dragging the dividing line between the headings Count Frame bCount ml For a colony counting result if there is more than one colony type the count will be shown in a separate row for each type In th
92. lay You can press the display button labeled Original Image All Colony Markers in the example picture and choose to display the image as the e Colormap Image this uses contrasting colors to show which areas of the image have been detected as the background and which as foreground features e Original Image the original uncorrected image e Corrected Image the image after the background correction has been applied to the image You can also press the display button to choose how to mark the detected colonies on the image Here colonies means non background parts of the image that have one of the classified colors which may include different types of colony different colors of the same type of colony or debris You will be able to specify whether each of these colony types should be counted as a colony in the Classification Details page later see Classification Details on the facing page You can choose to display e No Colony Markers e All Colony Markers each detected colony is identified with a symbol showing the type of colony color classification a key to the colony symbols is shown to the right of the image e All Colony Outlines the boundary of each detected colony is marked in the color of the symbol representing the type of colony e All Colony Regions the area of each detected colony is marked in the color of the symbol representing the type of colony Protos Systems User Manual The Cla
93. lick on the radio button for the number of shape sub classifications you want to add or if you want to add more than four click on More and use the arrow buttons to set the required number Protos Systems User Manual 61 Creating a new batch 6 Press Next to carry out an automatic shape classification and display the results in the Review Shape Classification page for example age Curio wierd ge 62 Hore are the colonies found within the sample image a 143 colonies 3 colonics The Review Shape Classification page allows you to review the automatic shape classification results Examine the marked colonies on the image to check whether the shape classification is as required It may be easier to see the results if you zoom in on areas of the image as in the example above you can zoom the image in the same way as in the main window see Zooming the image on page 8 It may also help to change the way the colonies are marked by pressing the display button labeled All Colony Markers in the picture If the automatic shape classification has not produced the required results press Back to return to the Configure Shape Classification page and try selecting a different number of sub classifications see Step 5 Protos Systems User Manual Notes The Classification tab 9 When you are satisfied with the new classification press Next to display the Classification Details page showing the new classi
94. ll be unchanged and the next dilution in the dilution list will be set When there are no more dilutions in the list the plate identifier will be auto incremented and the first dilution in the dilution list will be set If required you can edit the identifier in the Next Plate Id box That identifier will then be used for the next measurement and will be auto incremented for subsequent measurements according to the normal auto incrementing rules If you enter a new identifier for a Dilution Series batch the measurement will be used as the first measurement of a new dilution series Manually entered plate identifiers 104 To enter a manual plate identifier Type the identifier into the Next Plate ID box Next Plate ID Protos Systems User Manual Entering plate identifiers and dilutions You can also use a barcode reader to enter a manual plate identifier and dilution if the barcode reader is programmed to read dilutions Changing the dilution set for a plate Note Dilutions are not relevant for Chromogenic Media batches If required you can change the dilution set for the current plate The procedure required depends on the type of batch Pour Plate batches To change the dilution for the current plate in a pour plate batch 1 Press the dilution button for example to open the Dilution Editor Dilution Edig E Emer dilution taci 2 Use the dilution boxes to enter the dilution you can type di
95. lonies to Not Detected To set the results for all the colonies on a plate to not detected 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Press All the Detected check boxes in the Results table will be unselected Results Detected al Enterococcus E coli Klebsiella Enterobacter Ciir Proteus Pseudomonas Slaph aureus Slaph saprophyticus gt 3 Save the change to the result see Saving changes to results on page 125 The result will be updated to reflect the changes made and an M flag will appear in the Flags column showing you have manually edited the plate 132 Protos Systems User Manual Compiling reports Compiling reports You can create reports for an individual plate see instructions at the end of this section or for the currently selected batch or all batches see the following instructions To compile a results report for the currently selected batch or all batches 1 Press in the Results tab to display the Protos 3 Reports dialog box ProtewOLs Rept Available Report Templates ag Pour Plate i summary of all Batches a Adit tral of all batches B Audit tral of the currant batch Restrict report by batch headings Report Results Oldest to Newest 2 Click on the template for e CSV Report to create a detailed report for the current batch in CSV format e Batch type Pour Plate in the exampl
96. lony Markers the detected colonies will also be marked on the image using the colors and markers selected for each colony type if the button is labeled No Colony Markers press the button and select All Colony Markers to display the detected colonies on the image 2 Check the image to make sure that the colonies have been correctly detected 3 If you are satisfied with the colony detection go straight to Step 4 Otherwise if there are problems with the colony detection and the batch is based on a Total Plate Count press Adjust to display the Settings menu matti 75 Sonsitnit AUTOMATIC i Herpec Small Pardes 0 Azmun e g W Spit Touching Colonies The controls are used in exactly the same way as when you are setting up a batch using a Total Plate Count see Total Plate Count on page 30 for details Note If required you will also be able to change the Sensitivity Reject Small Particles and Split Touching Colonies settings after you have performed and recorded the count see Rejecting small particles in a result on page 128 Changing the Sensitivity setting for a result on page 129 and Splitting colonies in a result on page 129 for details Protos Systems User Manual 111 Using Measurement Mode to count and detect colonies 4 To carry out the measurement and record the results in the Results table press Note If you make any changes to the settings using the procedures described in th
97. lor For example if the background has some patches of different colors you can assign one color to the background color channel and the other colors in the background to separate colony colors and then redefine these later as background colors in the Classification Details page You can add as many colony channels as required However you should bear in mind that when you perform a count the results will show a separate count for each colony channel you define This means that if the plates have colonies with different colors but you do not want to distinguish between them in the results you just want the total number of colonies irrespective of color you should define a single color channel for these colonies and sample all of the different colony colors to define that channel Protos Systems User Manual 47 Creating a new batch To carry out a manual color classification 1 Click on the Background color sample item at the bottom of the channel list to select it Click here to select background X The selected item will be highlighted in orange and the wording changed Click here to select this colony 2 Inthe image click on the background to sample its color When you click a circle showing a sample of the color you clicked on will appear in the color channel 3 If you click at the wrong point and want to remove the last color sample added Press Remove Last Color Sample If you want to remove all the
98. m the Settings submenu to display the Create Protos 3 Database Connection dialog box id raabe Freie Detahete Loni i Database Name Server Nan 3 Enter the names of the Protos 3 Database and Server 4 Press Connect A dialog box will be displayed warning you that Protos 3 will restart I Restart required A change lo tha database settings requires a restart of the system please click OK to continue 5 Press OK Protos 3 will close down and then restart automatically connected to the new database location Testing lamps Note The procedure described in this section is mainly used for demonstration purposes 140 Protos Systems User Manual Calibration To test the lamps by making them cycle through a variety of color patterns 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu P r z Export Data as XMI x Sengs system Settings E Edit Database Location LE ike Calibration lest Lamps KRed Green Blue Rainbow Slow Rainbow aay Stop 3 To end the demonstration and switch the lamps off choose Stop from the Settings Test Lamps submenu Calibration Protos 3 is fully calibrated in the factory and in normal circumstances will require no further calibration after delivery However in exceptional circumstances you may need to carry out some recalibration For details see e Color balance below
99. n also change the colony markers and colors in Measurement mode see The Classification panel on page 33 However you can change the way colonies are detected and classified while you are still working on the batch in the Batch Designer You cannot do this by editing the definition of an existing classification for example the color samples defining the background the boundary between two size classes or the number of shape classes but you can remove an existing sub classification and replace it with a new one with the required properties Protos Systems User Manual 63 Creating a new batch Note This section and the following ones are concerned with changing an existing classification but you can also replace a classification or Total Plate Count with a new Total Plate Count see Total Plate Count on page 30 The next section Modifying an existing classification describes how to change classifications using the Modify Existing Classification button However some of these operations can be carried out in other ways In particular you can e Add a new Size classification see on page 56 or Shape classification see on page 60 using the procedures described in previous sections e Create a new color classification replacing the existing one and any sub classifications it may have by pressing the Color Classification button see Removing a sub classification on page 67 for how to remove sub classifications individually
100. n change the type of frame in the Batch Designer or Measurement mode Pour Plate and Dilution Series batches You can choose Circular Frame or Rectangular Frame or to use a frame covering a single sector only choose 1 2 1 4 1 8 or 1 10 from the Partial Plate Frame submenu Rade Plate Frana ey Circular Frane mi Rectangular Frans FB Rectangular Frame Partial Fate Frame Partial Plate Frame 1 4 Total Area Partial plate frames can be useful when you have plates that are heavily loaded with bacteria or if there is some problem with a plate and you just want to read results from an unaffected part This means that you are more likely to want to choose a partial plate frame for an individual plate while you are working in Measurement mode rather than for all the plates in a batch when you are creating it in the Batch Designer You can also avoid counting in problematic areas of individual plates by placing Exclude regions over them see Exclude regions on page 110 Spiral Plate batches You can choose Whole Frame Annulus Frame or 2 Sector Ahas Frare wt Sle Frannie As a general rule if there has only been a small amount of colony growth you should use one of the Whole Frame options use Annulus Frame if the spiral plater does not reach the center of the plate use Whole Frame if it does reach the center otherwise use 2 Sector If you choose 2 Sector and the number of colonies counted in the first sector fall
101. nce you opened the dialog box Press To reset the settings to the values set in the factory Press Marker positions Each time you capture an image in Protos 3 the instrument carries out an automatic recalibration from a grayscale on the instrument stage In order to do this it must have an accurate record of the positions of the gray patches Protos 3 is fully calibrated in the factory and in normal circumstances will require no further calibration after delivery However in exceptional circumstances you may need to recalibrate the marker positions over the gray patches To recalibrate the marker positions 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu i 1 j m 8 i 1 SS 4 c Managemeni m TT m eee eee El Protos Protos Systems User Manual 143 Configuring Protos 3 2 Choose Calibrate Marker Positions from the Settings gt Calibration submenu to display the Calibrate Camera for Color Accuracy screen Circ on iy demi kig ii Be ire eee fio a rap ee Cee P r mee Praami The A B C and D markers on the left hand side of the screen should lie at the center of each of the gray patches with A on the top patch and D on the bottom patch If any of the markers are out of position press the radio button at the top right hand corner of the screen with the corresponding letter to select it A circle will appear
102. ng a new batch To select an open batch 1 Press 102 Protos Systems User Manual Adjusting frame settings in Measurement mode to display the Review Open Batches dialog box Poaceae Hati hs Current Batch gt P lest Cher Open Batches Click to Select The currently selected batch is listed with a check box in the Current Batch box and there are buttons and check boxes for the Other Open Batches in the box below it 2 Press the button for the required batch in the Other Open Batches box The Review Open Batches dialog box will close and the selected batch will be shown in Measurement mode Adjusting frame settings in Measurement mode If required you can make temporary adjustments to the frame settings in Measurement mode using essentially the same techniques as when you are setting up a batch for details see e Frame shape see Choosing the type of frame to use on page 26 e Frame size position and orientation see Adjusting the position and size of frames on page 27 Note Any changes you make to the settings in measurement mode will be lost when you close the batch the default settings set in the Classification tab see The Classification tab on page 24 when you created the batch will be reloaded the next time you reopen the batch Entering plate identifiers and dilutions Each plate in the batch must be given a plate identifier but the procedure required depends on the opt
103. ng in the plate s result in the Results table see The Results table on the previous page 2 Press the Edit Review tab asoy WP Iy Protos Systems User Manual Editing plate measurements to display the Editing Plate panel at the bottom of the Protos 3 window and the Edit Review controls in the Edit Review tab The controls included on the Edit Review tab depend on whether the selected batch is e a Pour Plate Spiral Plate or Dilution Series batch MEANY UPA O iiz 61 eolomes LJ Spit so colonies O Split Notes The Exclude Region Clear Exclusion and Adjust Frame controls are hidden for Spiral Plate batches For batches based on a Total Plate Count see Total Plate Count on page 30 a Sensitivity slider appears above the Reject Small Particles slider Protos Systems User Manual 121 Working with results 122 e a Chromogenic Media batch Probability Threshold g ana Results EnioroCOCCUS i Eol P Li Aiebsieva Enferabacter Cur m A roieus wf Seu las 4 SEN AUTES Sian sanraonyicus r P i so colonies O Split i a b imi The controls on the Edit Review tab and the Editing Plate panel allow you to carry out a wide range of editing operations which are described in the following sections All batch types except where noted e Editing the plate identifier comments and dilution on
104. nt criteria 1 Press the Classification tab in the Batch Designer see The Classification tab on page 24 2 To specify that the colonies to be counted are lighter than or darker than the background or that there are both lighter and darker colonies a Press Sethngs Sensitivitr AUTOMATIC Hejec Small Paroles 0 Shon _ gt _ A Spat Touching Colonies N Notes The Light and Dark Colonies button may be hidden see Showing or hiding the Light and Dark Colonies button for Total Plate Counts on page 96 If there is no current image an image will be automatically captured when you press the Total Plate Count button If the image shown in the viewer is too light or too dark press the Image tab and adjust the exposure as required see Setting the exposure on page 7 then go back to Step 1 30 Protos Systems User Manual The Classification tab b Press the appropriate button to choose whether to count e Light Colonies Only the number of colonies lighter than the background e Light and Dark Colonies the number of colonies lighter than the background plus the number of colonies darker than the background e Dark Colonies Only the number of colonies darker than the background The Total Plate Count controls will be hidden and the icon on the Total Plate Count will show the selected option c If the button below the image is labeled No Colony Markers press the button and select
105. nto the instrument To load a sample plate into the instrument 1 Open the curtain doors at the front of the instrument by sliding them into the instrument 2 If required change the sample holder fitted in the instrument e Two pillared plate holders should be fitted with the plate stop pillars to the rear e Three pillared plate holders should be fitted with the plate stop pillars to the left and rear 6 Protos Systems User Manual Capturing an image Note Protos 3 is supplied with five sample holders transparent translucent white and opaque black holders for circular plates translucent white and opaque black holders for rectangular plates Apart from the shape of plate the choice of which type to use depends on the properties of the samples you are using and generally speaking is best determined by experiment Note however that you must use a black opaque holder for Chromogenic Media batches 3 Place the sample plate on the holder and push it up against the plate stop pillars to fix its position For rectangular plates use a three pillared plate holder and push the plate against the two side pillars first then slide it back along the side pillars until it touches the rear pillar Note Protos 3 automatically displays a live image when you insert a new plate However if required you can choose to display a live image manually by checking the Live Image box on the The Image tab on page 5 4 Close the instrument doors Cap
106. nto the instrument on the next page Capturing an image on page 7 Setting the exposure on page 7 Zooming the image on page 8 Copying the image to the clipboard on page 9 Exporting the image to a file on page 9 Importing an image from a file on page 10 Calibrating the image on page 11 Viewing batch images on page 13 The view batch images procedure is used to view images captured during the measurement process so is only available in Measurement mode All of the other procedures described in this chapter are used in both the Batch Designer and Measurement mode The Image tab To display the image controls Press the Image tab abewj a Protos Systems User Manual Capturing images You use the controls on the Image tab to capture and work with images Live Image a i Exposure 50 ms The View Batch Images button is enabled when you are working in Measurement mode see Using Protos 3 overview on page 2 for more information about the Batch Designer and Measurement mode For instructions on how to use the image controls see the following sections e Capturing an image on the facing page e Setting the exposure on the facing page e Copying the image to the clipboard on page 9 e Exporting the image to a file on page 9 e Importing an image from a file on page 10 e Viewing batch images on page 13 See also e Zooming the image on page 8 Loading plates i
107. o Markers No colony markers it will also be shown in the Last Measurement Results panel Last measurement results See Testing colony detection below for details of the highlighting of detected colonies on the image Testing colony detection To carry out a test colony detection for a Chromogenic Batch and adjust the detection threshold if required 1 Make sure the display is not set to Markers No colony markers 2 Press Protos Systems User Manual 115 Using Measurement Mode to count and detect colonies 116 The result of the test measurement will appear in the Last Measurement Results panel Next Plale ID test 1 l 7 Probability Throshotd kz T ii Last measurement results Klebsiella Enterobacter Cir Delocied E coli Detected seniors Hol Heledd The result of the test measurement will not be added to the Results table see The Results table on page 119 The detected colonies will also be marked on the image using different colors to distinguish the different colony types Protos Systems User Manual Closing batches If you click on one of the detected colony entries in the Last Measurement Results panel it will be highlighted in the color used to mark those colonies Klebsiella Enterobacter Cit_ Detected and the marking of the other colonies will be removed 3 Inspect the results carefully to determine whether the colonies are being detecte
108. o so that you can count the colonies manually press Clear Markers see Manually adding and removing colonies on page 127 If you want to delete a faulty result press Delete Plate see Deleting results on page 126 However the result would then be completely removed from the results table and you could not reset the change later if you change your mind about excluding the result see Undoing and resetting changes on page 126 To set the count s for a plate to zero 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 Protos Systems User Manual Editing plate measurements All other result editing controls will be disabled apart from Save Changes Undo All Reset if there are previously saved changes and the controls in the Editing Plate panel at the bottom of the Protos 3 window 3 Save the change to the result see Saving changes to results on page 125 The result will be zeroed in the Results table and M E and Z flags will appear in the Flags column showing you have manually edited the plate to zero the count Note When you zero a result that contributes to a mean for example in a dilution series the result will still contribute to the mean with count 0 If you want to remove the result s contribution to the mean you can change its plate identifier see Editing the plate identifier comments and dilution on page 123 or delete the result see Deleting results
109. on tab The frame boundary will turn orange showing that you can adjust its size For example 2 For circular frames drag the frame boundary to resize the frame For rectangular frames drag the top or bottom side to adjust the height drag the left or right side to adjust the width drag a corner if you want the frame to maintain the same shape aspect ratio when you adjust the size Rotating frames You can rotate 2 Sector Spiral Plate frames and Partial Plate frames any type To rotate a frame 1 Move the pointer to a point about half way between the center and edge of the frame An orange line will appear joining the pointer to the center For example 2 Drag the line around the center to rotate the frame Protos Systems User Manual 29 Creating a new batch Total Plate Count You can use the Total Plate Count button to set simple criteria for detecting and counting the total number of colonies on a plate according to their color see Color classification on page 35 for how to set more sophisticated color criteria including the ability to distinguish between different colony colors see also Adding sub classifications on page 55 for how to further distinguish between colonies according to their size and or shape Note If you press the Total Plate Count button after adding a color classification that classification and any sub classifications you have added will be removed To set simple Total Plate Cou
110. or the folder used to save exported batch details see Exporting batch data on page 94 by typing the path directly into the box or press f to display a standard Windows Browse For Folder dialog box so that you can select the required folder 7 Check Display Exponential Numb if you want to display results in exponential format 8 Set a value for Show X Plates to choose how many plates to include in the Results table see The Results table on page 119 9 Press to reset all settings to the values they had when you opened the dialog box 10 Press to confirm the new settings and close the dialog box Display properties To set the color of the frame or frame border see the notes in Step 2 of the following instructions for what this means 1 138 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu B gt Expo Data as AM oo SSS ae xy eth W ae a SS aS Balch Management Bhan Aboul Protoss T Coal Prolosd Protos Systems User Manual Notes Database location 2 Choose Frame Color or Frame Color border from the Settings gt Display Properties submenu to display the Color palette for example e em e m m e a r Expand Dhala cis K Mil Standand Colars Saas SSe888 More Colors The Zone Marker Color settings are not used in Protos 3 The border color only applies to spiral frames In particular
111. ou need to do to compile a report on the results is press a few buttons Starting Protos 3 Note You can control Protos 3 using the touch sensitive screen a mouse or keyboard shortcuts See the Protos 3 Quick Guide for how to set up the hardware ready for use To turn on the Protos 3 system 1 Make sure the system is connected to a suitable mains power supply Protos Systems User Manual 3 Getting started 2 Switch on the system using the toggle switch on the rear of the unit The orange LED on the front of the unit will light 3 Switch on the built in PC using the push switch on the right hand side of the unit The green LED on the front of the unit will light To start the Protos 3 colony counting program Double click on the Protos 3 icon on the Windows Desktop oa a Protass The Protos 3 Application window will open You can either create a new batch see Creating a completely new batch on page 16 or open an existing batch if there is one see Opening and selecting batches on page 101 and then start detecting counting colonies immediately Closing Protos 3 To exit Protos 3 1 Press the Protos 3 button at the top left hand corner of the window to display the Protos 3 menu is 2 Press x Foot Protos 4 Protos Systems User Manual Capturing images This chapter shows you how to capture and work with images You will find out about The Image tab below Loading plates i
112. ource for plate identifiers on page 77 to display controls for creating a plate list Loe Pas eral Sauce Par Plate M Plate ID and dilubon Click Add to move bo list Plate ID list Chick OF to save with batch Pate ID Dilution Note Dilution settings are irrelevant for Chromogenic Media batches 2 Type the identifier for the first plate into the edit box at the top of the dialog box 3 Use the dilution boxes to enter the dilution you can type directly into the boxes or use the and buttons to increase or decrease the values Note The left hand box is the sample proportion the right hand box is the total so 1 10 means one part sample in ten parts total volume This means that the right hand number must not be smaller than the left hand number Protos Systems User Manual 83 Creating a new batch 4 Press m to add the identifier dilution to the plate list 5 Repeat Steps 2 4 for the remaining plates in the batch 6 If you change your mind about one of the plates in the list click on it in the list to select it and press 7 When you are satisfied that the list is complete and correct press OK to close the Use External Source for Plate ID dialog box and load the plate list into the batch The Plate ID box on the Measure tab see The Measure tab on the facing page will show that the plate identifiers have been taken from an external source Peb External Source
113. pes or classes of colony or to distinguish between colonies and unevenly shaped debris To add a shape classification to the batch so that you can distinguish between colonies and or debris according to their shape how close they are to being circular 1 If the current image does not have the full range of shapes appearing on plates in the batch you should replace it by loading a plate having the full distribution see Loading plates into the instrument on page 6 and capturing a new image see Capturing an image on page 7 Press to start up the Shape Classification Wizard If there is already more than one colony type in the colony classification the Add New Shape Sub Classification page will be displayed go to Step 3 otherwise if there is currently only one type of colony in the colony classification the wizard will skip straight to the Configure Shape Classification page go to Step 5 In the Add New Shape Sub Classification page click on the existing colony type that you want to subdivide with the new shape classification see Adding sub classifications on page 55 for more details Protos Systems User Manual The Classification tab 4 Press Next to display the Configure Shape Classification page for example fl st ape Deprem fiend Aubomate Classificaion 2 shape cassiicahons Od A mi mial O More 5 In the Configure Shape Classification page see picture in previous step c
114. r Agar Properties Exposure Time ms Pits Holder Colour Disch Gran 6 becteria ami yeasts Mo rth mhaba Oier rar e Morth inhabited b Select the supplier of the medium from the Suppliers drop down list c Select the medium from the Agar Selection drop down list 3 If required define one or more exclude regions to avoid problematic areas of the plate the procedure is the same as that used when you add exclude regions in the Colony Classification wizard see Excluding regions during color classification on page 39 for detailed instructions 4 In most cases the default sensitivity settings are suitable for carrying out colony 114 Protos Systems User Manual Detecting colonies on Chromogenic Media plates detection but it is a good idea to carry out a test measurement for the batch see Testing colony detection below 5 If you want to identify the colonies on the image and list the results in the Last measurement results panel make sure the display is not set to Markers No colony markers Note Ifthe display is set to Markers No colony markers ProtoCOL 3 will still detect and record the prescence of colonies in the results table but the detected colonies will not be marked on the image and the result will not appear in the Last measurement results panel 6 Press The results will be added to the Results table see Working with results on page 119 for details and if the display is not set t
115. r more parts of the plate you are using for the color classification has some problem such as a label or other artifacts you can create one or more exclude regions so that these areas are not included in the color classification If you want to create one or more exclude regions on the plate image before the color classification is carried out check Exclude Regions The Exclude Regions page of the wizard will be displayed when you click Next in the Configure Color Classification page see Excluding regions during color classification on page 39 for details Protos Systems User Manual The Classification tab 6 When you are using plates that have a grid pattern the grid lines may interfere with the detection of colonies that have a similar color to the grid To remove the effects of the grid on the count check Detect amp Remove Grid The Detect amp Remove Grid page of the wizard will be displayed before the color classification is carried out see Detecting and removing a grid on page 43 for details 7 By default Protos 3 automatically compensates for any irregularities in the illumination of the plate this is called background correction In most cases automatic background correction produces excellent results but you may find that if the plates in a batch have some unusual properties you can get better results by manually adjusting the background correction parameters or by disabling background correction completel
116. r used for the marker 3 Press Finish to close the Colony Classification Wizard and return to the main Protos 3 window which will show the updated classification details Adding a new sub classification Note The procedure described in this section is an alternative to the procedures described earlier in Size classification on page 56 and Shape classification on page 60 The next step after you select Add New Sub classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard see Modifying an existing classification on page 64 is to select where to create the new sub classification using the classification tree in the Add New Sub Classification page Whare should lhe new sub classitcaton be Select below or click hare to clear selection ok He We 1 Click on the existing colony type that you want to subdivide with the new classification see Adding sub classifications on page 55 for more details 66 Protos Systems User Manual The Classification tab 2 Press Next to display the Modify Previous Classification page which has buttons for selecting the type of classification to add Add New Sub Classification Which type of sub classification should be added See ee e Shape classification 3 Click on the radio button for the classification you want to add 4 Press Next to create the new sub classification 5 The remaining steps depend on which type of classific
117. re on the slider bar to set the slider to that position The plate will be remeasured with the new setting 3 Check that the real colonies are being counted and the background and any debris are being excluded and repeat Step 2 if the result is still not satisfactory Note You may find it helpful to zoom the image see Zooming the image on page 8 4 Save the change to the result see Saving changes to results on page 125 The count s will be updated in the result together with any values depending on the count s M and E flags will appear in the Flags column showing you have manually edited the plate Splitting colonies in a result When you create a batch you can choose whether to use the colony splitter to split overlapping colonies so that they are counted separately see Total Plate Count on page 30 and Review Color Classification on page 50 For a batch based on a Total Plate Count you can also change the colony splitter setting in Measurement Mode before taking a measurement see Test measurement and adjusting settings on page 110 Protos Systems User Manual 129 Working with results Note Notes 130 This section shows you how to change the splitter setting for an individual plate after it has been measured In this case the batch can be based on a color classification or a Total Plate Count To change the colony splitter setting s for an individual plate result 1 Select the Edit Re
118. re relevant a Press the dilution button for example i Dilution 1 3 to open the Dilution Editor Cilubon b Use the dilution boxes to enter the dilution you can type directly into the boxes or use the and buttons to increase or decrease the values Notes The left hand box is the sample proportion the right hand box is the total so 1 10 means one part sample in ten parts total volume This means that the right hand number must not be smaller than the left hand number Protos Systems User Manual 123 Working with results c Press OK to confirm the dilution you have set and return to the Editing Plate panel 5 Save the change to the result see Saving changes to results on the facing page The identifier comment and or dilution will be updated for the result and if you have changed the dilution values depending on the dilution will also be updated An E flag will appear in the Flags column showing the result has been edited Adjusting the frame for a result Note You cannot adjust the frame for a Spiral Plate batch To adjust the frame for a plate result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 2 Press 3 Adjust the frame using the same techniques as when you create a batch or before taking a measurement see Adjusting the position and size of frames on page 27 4 Press Somos again The plate will be remeasured using the ad
119. re relevant and if the barcode reader is programmed to read dilutions from the plate Protos 3 will give an error if the identifier dilution is not on its plate list for the batch Alternatively if you need to choose a different plate identifier dilution from the plate list 1 Press to display the Select next Plate ID to use dialog box Select next Plate ID to use Select a plate ID from the list below and click OK to continue Dilution Ce 1 1 4 i lea 1 1 a Cancel 2 Click in the row containing the required plate identifier dilution to select it 3 Press OK to close the dialog box the selected identifier will appear in the Next Plate Protos Systems User Manual 107 Using Measurement Mode to count and detect colonies Id box and the dilution will appear on the dilution button Using a barcode reader for identifiers and dilutions You can use a barcode reader to enter a manual plate identifier and dilution if the barcode reader is programmed to read dilutions or pick a plate identifier and dilution from the plate list created from an external source see Using an external source for plate identifiers on page 77 Counting colonies on Pour Plate Spiral and Dilution Series plates This section of the Manual describes how to count the colonies on a plate in Measurement mode for how to take measurements from Chromogenic Media plate see Detecting colonies on Chromogenic Media plates on page 113 In most
120. rectly into the boxes or use the and buttons to increase or decrease the values Note The left hand box is the sample proportion the right hand box is the total so 1 10 means one part sample in ten parts total volume This means that the right hand number must not be smaller than the left hand number 3 Press Update to confirm the dilution you have set and return to the main Protos 3 window Dilution Series and Spiral Plate batches To select one of the other dilutions in the series either Press one of the arrow buttons below the dilution button to select the next or previous dilution in the series for example Next Plate ID Test Protos Systems User Manual 105 Using Measurement Mode to count and detect colonies Or 1 Press the dilution button for example Next Plate ID rel 2 Select the required dilution from the Dilution List To edit the dilution list and choose a dilution from the new list 1 Press the dilution button for example 2 Press Edit Dilution List to display the Dilution List Editor 3 Use the Dilution List Editor to edit the dilution list see Dilution Series and Spiral Plate batches on page 87 for how to use this dialog box 4 Press the dilution button again and select the required dilution from the Dilution List Note The new dilution list will be used for future plates as well as the current one External source CSV file or Custom plate list When
121. required color you should select a representative sample covering the full range of the color as it appears for these objects in the image Note Remember that when you perform a count the results will show a separate count for each colony channel you define if you do not want the results to distinguish between different colored colonies you should sample all of the colony colors for a single color channel When you click a circle showing a sample of the color you clicked on will appear in the color channel See Step 3 if you want to remove the last sample added When you have added a representative number of samples to the channel e If you want to add another color channel go back to Step 5 e If you do not want to add another color channel go to Step 10 If after adding samples for all the colors appearing in the image there are any unused channels colony definitions or if you want to remove a channel you have added a Click on the Click here to select this colony item for the first channel you want to remove to select it you cannot remove the default background item b Press Remove Whole Colony c Repeat Steps a and b for any other channels you want to remove Press Next to display the Review Color Classification page see the next section Review Color Classification Next is disabled if there are any undefined channels see previous step Protos Systems User Manual 49 Creating a new batch Revie
122. s below the threshold under which all colonies are counted see Creating a completely new batch on page 16 Protos 3 will count the whole frame Adjusting the position and size of frames The following sections describe how to adjust the position and size of frames Moving frames on the next page Resizing frames on the next page Protos Systems User Manual 27 Creating a new batch Note 28 Rotating frames on the facing page You should load a plate from the batch into the instrument and capture an image before adjusting the frame see Loading plates into the instrument on page 6 and Capturing an image on page 7 Moving frames To move a frame 1 Move the pointer near to the center of the frame an orange circular drag handle will appear in the center of the frame For example 2 Drag the frame and drop it in the required position Resizing frames Generally frames should be made as large as possible within the area of the plate but avoiding the very edge as this may produce spurious results and allowing some margin for variation between plates You can adjust the size for individual plates in Measurement mode but it will save time if you can use a single setting for all the plates in a batch To resize a frame 1 Move the pointer near to the frame boundary for Partial Plate frames the frame boundary is the circle of which the frame forms a sector Protos Systems User Manual The Classificati
123. sing Auto incrementing identifiers or manual identifiers Press When you have selected an external source for plate identifiers the icon in the Plate List box in the Configuration tab will show A if you have chosen not to use an external source of plate identifiers Using a CSV list of identifiers To use a CSV file containing a list of plate identifiers and dilutions see the text following the instructions for the format to use for CSV files 1 Press Protos Systems User Manual 79 Creating a new batch in the Use External Source for Plate ID dialog box see Using an external source for plate identifiers on page 77 to display controls for opening the CSV file Lie Pas eral Sauce fer Phare M E Click the Open button to select a CSV file to import The contents of the file will be shown below Please verity the content of the file before clicking OK io save with batch m a ummm 2 Press L J to open a standard Windows Open dialog box 3 Use the Open dialog box to locate and select the required CSV file 4 Press Open to open the selected file If you are creating a Dilution Series batch and the CSV file does not contain any dilution information you will be asked if you want to import a dilution series otherwise go straight to Step 5 Beet Cilution Snes E The CSV file only contains plate IDs Would you like to import a Suey dilution series now
124. ssification tab Classification Details You use the Classification Details page to specify whether the detected colors and sizes or shapes for a size or shape sub classification correspond to colonies debris or background and if there is more than one type of colony color size or shape what organisms they correspond to Note The following instructions show you how to change the classification details for a colony type produced by a color classification However this page is also displayed for the other classifications size or shape see Adding sub classifications on page 55 and the same procedure with the obvious wording changes is also used for them Ceki Gai e G x Classification Details The left hand pane of the Classification Details page shows the image with the colony markers superimposed The right hand pane lists the classified colors in the image and shows whether they represent colonies debris or the background with a button allowing you to change the classification the button for a colony type is labeled with the colony name and marker otherwise it is labeled Debris or Background To change the classification for a color 1 Press the classification button to display the classification menu Back ground Note When the current classification is Colony the classification details panel is shown alongside the classification menu see example in next instructions Protos Systems User
125. sub classibcabon be acide Salect a colony marker below or cick here to Clear selechon iam a Wer The left hand panel shows the image with colony markers the right hand panel shows the current colony classification as a list The first step in adding a new classification is to specify which of the existing colony types you want to divide with the new classification The wizards will skip this step if there is currently only one colony type since in that case there is no choice to be made To choose where to add the new classification Click in the classification list on the existing colony that you want to divide Protos Systems User Manual 55 Creating a new batch Notes 56 Size classification The Add Size Classification button becomes enabled once you have performed a color classification see Color classification on page 35 You can use a size classification to distinguish between different sizes of a particular colony type or to set maximum and or minimum size limits for colonies of that type to be counted When you create a new batch you can specify a maximum size for colonies by choosing to Restrict Maximum Detected Size in the Plate Configuration dialog box see Creating a completely new batch on page 16 for details You can use the Reject Small Particles slider in the Review color classification page of the Color classification wizard to set a minimum size for colonies of all colony typ
126. sults on page 119 for details and shown in the Last Measurement Results panel i Last measurement results If you have created a classification with more than one colony type the results for each type will be shown F Last measurement resuils Ra Gi Eg ao For a Spiral Plate batch the Last Measurement Results will also show the concentration Last measurement results PA 108 Count mi 2 1600 See also Counting colonies on Pour Plate Spiral and Dilution Series plates on the previous page for a summary of how to modify the way automatic colony counts are performed and how to carry out colony counts using other methods Protos Systems User Manual 109 Using Measurement Mode to count and detect colonies Exclude regions Note Note Note The following instructions do not apply to Spiral Plate batches you cannot place exclude regions on the images in Spiral Plate batches On occasions you may want to avoid detecting counting colonies on part of a plate because of some problem with it One way of doing this is to create one or more exclude regions which will be ignored when Protos 3 detects counts the colonies where relevant the area of the exclude regions will be deducted from the area of the frame to ensure that results such as count ml are correct An alternative for some batch types might be to choose a Partial Plate frame and or to rotate the frame so that the pro
127. t dilution Next Plate ID Test ae Notes For a Dilution Series batch there are left and right arrow buttons below the dilutions button These can be used to change the dilution for a plate as an alternative to selecting the dilution from the dilutions menu though selecting the dilution in the Batch Designer has no effect for a Dilution Series batch However when you carry out a test measurement for a Spiral Plate batch in the Batch Designer the dilution selected in the dilutions menu does have an effect as the results show a count ml value see Test Measure Plate on page 91 2 Select Edit Dilution List from the menu to open the Dilution List Editor p 1 10 Bno 11 100000 Hindi 3 If required to import a list of dilutions see Dilutions file format after these instructions for details a Press Import to open a standard Windows Open dialog box b Use the Open dialog box to locate and select the required file c Press Open to open the selected file 88 Protos Systems User Manual The Measure tab The dilution list in the file will be added to the Dilution List Editor replacing any existing entries 4 To add a new dilution to the list a Click on the dilution at the point you want to add the new dilution b Press Add to open the Dilution Editor Emer dilution tachku c Use the dilution boxes to enter the dilution you can type directly into the boxes or use the and bu
128. t Data as XMI s Jens OR Sich Management Press TE SH REDE al to display the Batch Management submenu Press Protos Systems User Manual 97 Creating a new batch 98 to display the Delete Batch dialog box listing the most recent batches up to 10 will be listed Deitit each The teie ted batch ii af ben pre Fistel 4 batches found Piease seegi the batch and chock Delete to contin Number Batch Name Plate Type SP les a TAUIA 18S DS test SA 02 12 2014 12 55 PP test oanze014 12 54 CM tesi Ee O212 2014 12 53 Ifthe batch you are looking for is not listed please enter all or part of the name or leave empty to show all batches and click the search button Sr ir pel Li 7 Ley a CTI e If the required batch is not in the list of most recent batches a Type the name or a part of the name of the required batch into the Search criteria box b Press Only batches with names that contain the text you entered will be listed Select the required batch es in the list you will only be able to delete batches if they are ticked in the Available column open batches will not be available If required you can select more than one batch using the normal ctrl click and shift click techniques Press Delete to delete the batch es You will be asked to confirm that you want to delete the batch es
129. t New Batch 93 94 Audit history 126 127 B Background correction automatic vs manual 37 manual 44 46 using none 45 Barcode reader 72 74 for dilutions 108 for plate IDs 76 77 108 Batch closing 117 118 deleting 97 99 opening 101 103 selecting 102 Batch design accepting 93 94 exporting 94 95 Batch details viewing 95 96 Batch images viewing 13 14 Batch name entering 24 C Calibrating image 11 13 Calibrating the instrument 141 145 Camera measurements 144 145 Capturing an image 5 7 Chromogenic Media 69 70 94 110 113 117 131 132 detecting colonies 113 115 editing results manually 131 132 frame type 70 testing colony detection 115 117 Circular plate 17 18 diameter 17 18 Classification 24 Chromogenic Media 69 70 colony counting 25 26 26 69 adding a sub classification 55 67 changing 63 69 modifying 64 65 reviewing 65 66 Classification details 53 54 Protos Systems User Manual Classification panel 33 35 Colonies manually adding and removing 127 128 Colony markers 34 52 Colony outlines 52 Colony Parameters 19 Colony regions 52 Colony splitting 32 33 51 52 111 for an individual result 129 130 Color balance 141 143 Color classification 35 38 44 54 69 automatic 37 manual 37 47 49 repeating 69 reviewing 50 52 Colormap image 52 Comments adding editing 123 124 Company Name 137 Configuration tab 70 80 80 84 Copying images 9 Count restrictions 91 Counting colonies 101 108 113 automatically 109 outline procedur
130. t for a plate 1 Click on the drop down arrow button on the right hand end of the Zero Count button 2 Either e Press to add a new result to the Results table with a O count and a Z flag Alternatively just click on the left hand end of the Zero Count button in Step 1 e Press to add a new result to the Results table with a O count and a T flag Note In both cases the image will not be saved with the result even if Save images to file is selected in the Settings dialog box see System settings on page 136 See also Zeroing the count on page 130 for how to zero a result in the results table after the count has been made and recorded Detecting colonies on Chromogenic Media plates This section of the Manual describes how to detect the presence of specific colonies on a plate using a Chromogenic Media batch in Measurement mode To detect the presence of specific colonies on a Chromogenic Media plate 1 Press the Measure tab ansta Protos Systems User Manual 113 Using Measurement Mode to count and detect colonies To display the Measure controls pi h ex si ate ID test 1 a hs 2 ae 4 Probability Threshold Bir eal i a SE b 2 To choose the medium used in the plate a Click on the Agar Selection button to display the medium selector CHROMagar Agar Selection Acneatobacte
131. the batch is set to use an external source of plate identifiers see Using an external source for plate identifiers on page 77 Protos 3 creates a list of plate identifiers and if relevant dilutions from the external source 106 Protos Systems User Manual Note Entering plate identifiers and dilutions When you first open the batch the Next Plate ID box shows the first plate identifier on the list and where relevant the dilution control shows the dilution for example Next Plate ID test The Next Plate ID text box is disabled for a batch using an external source for its plate identifiers you cannot edit the identifiers After you have performed the measurement the identifier and dilution where relevant for that measurement will be removed from the plate list and the next identifier and dilution will be shown You can then repeat this procedure until the list is exhausted When the list is exhausted no more measurements can be made and an error message will be displayed if you try to perform a count If the plates are not arranged in the same order as the plate list you can choose another identifier dilution from the list see the following subsection Selecting a different plate from the plate list If the batch has been set up for use with a barcode reader see Using a barcode reader for plate identifiers on page 76 you can use the barcode reader to read the plate identifier and dilution whe
132. the facing page e Adjusting the frame for a result on page 124 not Spiral Plate batches e Adding exclude regions to a result on page 124 not Spiral Plate batches e Saving changes to results on page 125 e Undoing and resetting changes on page 126 e Deleting results on page 126 e Viewing the audit history of changes on page 126 Colony counting batch e Manually adding and removing colonies on page 127 e Rejecting small particles in a result on page 128 e Changing the Sensitivity setting for a result on page 129 Protos Systems User Manual Editing plate measurements e Splitting colonies in a result on page 129 e Zeroing the count on page 130 e Chromogenic Media batch e Manually changing detection results on page 131 e Changing the Probability Threshold on page 132 e Setting the results for all colonies to Not Detected on page 132 All batch types Editing the plate identifier comments and dilution To add or edit a comment and or edit the plate identifier and or where relevant edit the dilution for a result 1 Select the Edit Review tab for the plate result see Editing plate measurements on page 120 The Editing Plate panel will be displayed at the bottom of the Protos 3 window Com me Editing Plate plate 5 2 Edit the plate identifier in the left hand edit box as required 3 Add to edit the Comments in the right hand edit box as required 4 To edit the dilution whe
133. the image controls 2 Press to display the Import Image dialog box this is a standard Windows Open dialog box 3 Use the Import Image dialog box to locate the folder holding the required image and select the image 4 Press Open to open the file and load it into the Protos 3 window A warning dialog box will be displayed asking you to calibrate the image see the next section Calibrating the image for details 10 Protos Systems User Manual Importing an image from a file Calibrating the image Note When you import an image from a file see Importing an image from a file on the previous page or try to perform a measurement from an uncalibrated imported image a warning dialog box will be displayed asking if you want to calibrate the image for example F apar image 4 TRIS image should bo GCalbrind for Measurements before any Measurement is petonmed Do you want to do this mow Chek Yes to calibrate now Chek No to conbnue withoul calibration In order to create an accurate calibration you need to know the actual size of some feature in the image To calibrate the image 1 Press Yes in the warning dialog box to display the Calibrate Camera for Measurements dialog box 2 Choose some horizontal or vertical feature of known length in the image such as the plate itself and click on one end of it for example Do Cie to Seer Glick image at the start of a with ia good ampio This will ba
134. ting the total count for the plate All detection counting results are displayed and saved automatically and any manual changes you make to a result are marked against the individual result with coded flags By default an image of the plate is automatically saved with each result and can be reloaded at a later time for auditing Once you have completed the counts measurements you can compile a report showing the results This introduction has only mentioned a few of the features that make Protos 3 such a powerful but easy tool to use The rest of the Manual gives full instructions for using all of its many functions Structure of the Manual Note The cross references in this manual assume that it is printed or viewed with odd numbered pages on the right hand side If you are using Adobe Acrobat to read this manual on screen you can display two pages side by side by choosing Two Page View from the View Page Display submenu and you can then choose Show Cover Page in Two Page View from the same submenu to display the odd numbered pages on the right hand side The rest of this Manual is structured as follows e The next section Using Protos 3 overview in this Getting Started chapter provides a brief overview of the way you use Protos 3 to detect count colonies This involves two Protos Systems User Manual 1 Getting started main phases e using the Batch Designer to set up the system for detecting and counting colonies on
135. tion on page 67 e Repeating a color classification on page 69 Reviewing an existing classification The next step after you select Review Existing Classification and press Next in the Modify Previous Classification page see Modifying an existing classification on the previous page of the Colony Classification Wizard depends on whether the existing classification has any sub classifications 1 If the existing classification has any sub classifications the Classification Details page will be displayed immediately go straight to Step 2 If the existing classification has no sub classifications the Review Color Classification page of the Colony Classification Wizard is displayed again a If required adjust the Reject Small Particles Remove Fringes Around Colonies Complete Colonies Across Frame and colony splitter settings see Review Color Classification on page 50 for details b Press Next to display the Classification Details page Protos Systems User Manual 65 Creating a new batch 2 The Classification Details page shows the current classification tree You can use the classification tree in the same way as in the Colony Classification Wizard see Classification Details on page 53 to specify for each sub classification e whether it corresponds to debris background or a type of colony and for colony types e the name to be used to identify it e the marker used to identify it on the image e the colo
136. ttons to increase or decrease the values Note The left hand box is the sample proportion the right hand box is the total so 1 10 means one part sample in ten parts total volume This means that the right hand number must not be smaller than the left hand number d Press Add to close the Dilution Editor and add the dilution to the list 5 To change a dilution in the list a Click on the dilution in the list to select it b Press Edit to open the Dilution Editor c Use the dilution boxes to edit the dilution you can type directly into the boxes or use the and buttons to increase or decrease the values d Press Update to close the Dilution Editor and confirm the change to the dilution 6 To remove a dilution from the list a Click on the dilution in the list to select it b Press Remove A confirmation dialog box will be displayed c Press Yes to remove the dilution 7 To move a dilution up or down in the list in order to change the order of dilutions a Click on the dilution in the list to select it b Press the up or down arrow button 8 When you have finished editing the dilutions list press Close to return to the main Protos 3 window Protos Systems User Manual 89 Creating a new batch Dilutions file format Each line of the file should consist of a string representing the dilution if the string is not recognized or no dilution is given No Dilution is used The order of the lines in the fil
137. turing an image To capture an image in Protos 3 1 Press the Image tab i to display the image controls 2 Press The image will be captured and displayed in the Protos 3 window Note In fact three images will be captured with red green and blue illumination respectively and the results combined to form a color image this procedure produces more accurate results than can be obtained using a color camera If the image is too light or too dark you can adjust the exposure see the next section Setting the exposure and capture the image again Setting the exposure Note The exposure setting for Chromogenic Media batches is set automatically and cannot be changed For other batch types you can adjust the exposure in both the Batch Designer and Measurement Mode Protos Systems User Manual 7 Capturing images To set the exposure for capturing an image 1 Press the Image tab to display the image controls including the exposure controls Ba Exposure 50 ms We 2 The current exposure time is shown between the arrow buttons To change the exposure time press to decrease the exposure by a large step to decrease the exposure by a small step to increase the exposure by small step to increase the exposure by a large step Or Drag the handle in the exposure slider bar Zooming the image By default the image is scaled to fit the image pane in the Protos 3 window To
138. uscnte ore ech ais eued cui iat Selatan Seca tnd bl eetocadctetanadete 10 Calibrating the image ccswoduidotietccciwet tdncweamna taliomedastheeaeegoe ht tatiedatmiabsdeudvenietteasat 11 Viewing alCWINMA Ges aisean oss toa thee cere e Atal eehel Stan eut aetna 13 Creating a new batch 15 CE Ue ALC 4a c a E a Ssaaaupiciniaeaeeeenaaeceddlnass dort A E RA 16 Creating a completely new batch 2 2 2 202202 e eens 16 Creating a new batch based on an existing batch or an exported batch file 20 SCLUMG UDDA NES aeae shh Sau ak aie Uta eul ett ote acta aah ts delta lat ln sseaiiee 23 Giving the batch a MAME neaceacvicbaesedanesdidudeaaabiriedodap wus deinen edi Sedamunleiemcageeness 24 WCAG FASS ICA Ola A052 ocsut sue cien moat E a E Er a E EEES 24 Classification colony counting batches 222 22222222222 e eee eee eee eee 25 PAS oot asuinta ecuneniana aa ate solacensec lea od h bates cneuledeenasea 26 Total Plate CO NE Gi tnereea ebay te catece teets rrpaiet aes accede otters E dane ede ade eerie ae 30 The Classification panel she secci205 25 ieee huts Adit a A AAE 33 Colorelassihica NON aaan a S 35 Adding sub classifications 0 0 cece cece eee eee eee eee eee cece cece eeeeeeeeeeeeeee 55 Changing an existing classification lt secssieasi si scdoneiassnadandateissedcsesbersiesduondaiiact 63 Classification Chromogenic Media batches 2 2 222 220022222 c eee e cece eee cece eee 69
139. vacsbenaes qaweesodeigauls aeaecddeacsmotseumeeteseunavee seas 113 Detecting colonies on Chromogenic Media plates 2 0 0 0 22000 c ccc e ene e cece eee e seen ees 113 TEStIne COLONY detectoare aa aa eis aetanaseueawicasoesee 115 Closing DAU CNS Sossen ioga O E eee oven 117 Working with results 119 THERESES TADE riie EE E E E E 119 Editing plate measurements csiscccnsnssn a a a T EAER 120 AP ACG AND Greate ee eee ees ee oes eae oe ea ae a tee eet 123 Editing the plate identifier comments and dilution 2 20 2222 20020020222 eee ee 123 Adjusting the frame for a result 0 0000000 eee eee eee cece cece eeeeeeeeees 124 Adding exclude regions to a result 00 0 0 20 c cece ccc eee e cece eee eeeeeees 124 Saving changes to results i 5c 302 2suceudes ceed seekedcunmeamaeeitaineresdcdasadinbustaanueonas 125 Undoing and resetting changes opctici cen y bat ares dh seer dud shaban Y dbus wi peetivyud iow k 126 Delete resule ini fun a cocaaaeel sich sauntag kins neupiciniatet a e RSA 126 Viewing the audit history of changes 2 00 00000 c cece ee eee eee 126 Contents il Protos Systems User Manual Contents Editing colony counting results 02 022222222 e cece eee eee cee cece cece cece eee e eee eeeeeeeees 127 Manually adding and removing colonies 2222 22222 e cece cece cece eee eeeeeeeeeeeees 127 Rejecting small particles in a result 000000 0000000 eee eee eee eee 128 C
140. view tab for the plate result see Editing plate measurements on page 120 2 Click on the Split check box in the Results box at the bottom of the Edit Review tab to change the setting if you have used a color size and or shape classification to define more than one colony type there are separate check boxes for each type and you can change the Split setting for each of them independently Results O E 63 colonies mo 61colonies O Split q l H The plate will be remeasured with the new split setting 3 Check that overlapping colonies are being split satisfactorily and single colonies are not being split You may find it helpful to zoom the image when checking the effect of the colony splitter see Zooming the image on page 8 4 Save the change to the result see Saving changes to results on page 125 The count s will be updated in the result together with any values depending on the count s M and E flags will appear in the Flags column showing you have manually edited the plate Zeroing the count If the result for a plate is faulty in some way and you wish to exclude it you can zero the count If you zero a result it will still be shown in the Results table see The Results table on page 119 with count s equal to 0 This section shows you how to set the count s for a result to zero to record a zero result If you just want to remove all the colony markers from the image and set the counts to zer
141. w Color Classification The Review Color Classification page is displayed in the Colony Classification Wizard after you have carried out an automatic or manual color classification see Color classification on page 35 or Manual color classification on page 47 respectively fee Ca een eed EF Review Color Ciassihicaiin Classbcaton Results Here are the colonies tourd withen the sample mage which maich the clessificaten Use Rayoct Small Pardes to rad small particles Use the Split conius to saparsa colonies which louch each athi Reject Small Parides 0 10mm O Remove Fringes Around Colonies H Complete Colones Across Frame 4 colonies O Sper QE coon O Spat 2 Background To review the color classification 1 Examine the detected colonies and image carefully to check that e the colonies in the image are being detected successfully e different colors of colony are being distinguished successfully e the colonies are distinguished from debris with a different color Note It may help to change the way the image is displayed and or the way the colonies are marked see Image and colony marker display following these instructions Also it may be easier to see the results if you zoom in on areas of the image you can zoom the image in the same way as in the main window see Zooming the image on page 8 2 If you are happy that the color classification is as good as possible press Next to display the Classification Det
142. will be carried out immediately and the wizard will then display the Review Color Classification page showing the results see Review Color Classification on page 50 42 Protos Systems User Manual The Classification tab Detecting and removing a grid When you are using plates that have a grid pattern the grid lines may interfere with the detection of colonies that have a similar color to the grid The Detect amp Remove Grid page allows you to remove the effects of the grid The Detect amp Remove Grid page in the Colony Classification Wizard is displayed during color classification if Detect amp Remove Grid is selected in the Configure Color Classification page see Color classification on page 35 When the Detect amp Remove Grid page is displayed it performs an automatic grid detection and marks the detected grid on the image Detect amp Remove Grid Automatic Grid Detection Gnd willbe detected automatically Manual Grid Detection If the automatic grid detection has succeeded in locating the grid on the plate press Next to proceed to the next step in the color classification procedure see the end of this section for details Note It may be easier to see the results if you zoom in on areas of the image you can zoom the image in the same way as in the main window see Zooming the image on page 8 To narrow the grid search if the grid has not been correctly detected 1 Select Manual Grid
143. will be remeasured with the new exclude region in place 4 To remove the last exclude region you added to the image you can repeat the procedure to remove each of the regions in turn including any added before the measurement was made Press The plate will be remeasured with the last exclude region removed 5 Save the change to the result see Saving changes to results below The measurements will be updated in the result together with any values depending on counts For colony counting batches M E and G flags will appear in the Flags column showing you have manually edited the plate with an exclude region for Chromogenic Media batches there will just be a M and G flags Saving changes to results To save any changes you have made to a result on the Edit Review tab and or the Editing Plate panel see Fditing plate measurements on page 120 Press Note Any changes you have made to a result will be lost if you move away from the Edit Review tab before saving them If required you can view an audit history listing the changes you have made to the result see Viewing the audit history of changes on the next page Protos Systems User Manual 125 Working with results Undoing and resetting changes To undo all the changes made to a result on the Edit Review tab and the Editing Plate panel see Editing plate measurements on page 120 since the result was last saved see Saving changes to results on the previous p
144. y Note You should only use manual background correction after you have tried using automatic background correction and found that it does not produce satisfactory results To set the background correction manually or to disable background correction Press the Automatic background correction check box so that it becomes unchecked see Manual background correction on page 44 for how to carry out the manual background correction procedure To allow Protos 3 to carry out the background correction automatically Leave Automatic background correction checked 8 In this step you choose whether to make Protos 3 carry out an automatic color classification of the colonies or to carry out a manual color classification yourself e To make Protos 3 carry out an automatic color classification of the colonies a Decide on the number of colors to be classified on the plate excluding the background this is the number of distinct colony types plus the number of distinct colors of debris Note As an example suppose the plates have colonies with two different colors but no debris If you want the results to show separate totals for the two different colors of colony you should set the number of colony types to 2 but if you just want the total number of colonies irrespective of color the number of colony types should be set to 1 However the automatic color classification may have problems if there are more than two colors and you want to distin
145. y entered plate identifiers and dilutions depending on your setup from a barcode on the plate select Use Barcodes in the Plate ID box see Using a barcode reader for plate identifiers on page 76 for details Selecting auto incrementing plate identifiers Note This option is selected by default when you create a new batch from scratch To use auto incrementing plate identifiers 1 Press 1 1 F WoqeIN Gage 74 Protos Systems User Manual The Configuration tab to display the Configuration tab Plate i0 jim Use Barcodes ih Auto increment ra Plate Lisi i cana Lacey 2 If the new batch is based on an existing batch there will be a set of Parent Batch controls below the Plate List controls If the parent batch did not use auto incrementing plate identifiers uncheck the Parent Batch check box es see The Configuration tab on page 70 for details 3 If the icon in the Plate List box is a go straight to Step 4 Otherwise an external source of plate identifiers has been selected for the batch and the Auto Increment control will be disabled To enable the Auto Increment control and deselect the external source a Press Protos Systems User Manual 75 Creating a new batch to display the Use External Source for Plate ID dialog box Lhe baiemaal faute for Plate i Please select a source for baich plate IDs b Press 4 If Auto incr
146. you are satisfied with the design the next step is to accept the batch and switch Protos 3 to Measurement mode which you then use to detect count the colonies on each plate and record the results Once you have accepted the batch most of the batch settings including the definitions of any color size and shape classifications are fixed and cannot be changed However there are a few settings that can be changed in Measurement mode measurements Once the batch has been accepted for Measurement mode and in the case of a Chromogenic Media batch the medium selected the process of detecting counting the colonies is very straightforward put a plate in the plate holder press a button and move on to the next plate In most cases this is all you will need to do but if there are any problems with difficult plates you can make manual adjustments before carrying out the measurement For example you can add exclude regions carry out a test measurement and optimize the colony detection settings or perform a manual colony count After you have carried out a measurement the results are recorded in a results table However if required you can edit the individual results in a number of ways For example you can add exclude regions and adjust the colony detection settings before carrying out another automatic colony detection and recount or you can add and remove colonies manually Finally once you have taken all the measurements all y

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