Colony Counting
Contents
1. Quantity One 4 6 7 Microsoft PowerPoint 2 10 07 AM ES Colony Counting Save Count Data to File Excel other gt Step 1 Define Region and Count Ek For best results adjust the Gel Doc zoom lens so that Define Counting Region Drag the cursor from center to edge of dish image __ Sensitivity ait Averaging 471 6 3 6 5 7 Plaque Step z Adjust Results White Blue Colony court Adjusted count n Count vs Peak density STEP 4 Select New Batch Enter or edit filename utoff Ww Fite Blue Step 3 Toolsoptions Ignore region x Show data area x Mark white colonies x Mark blue colonies Erase Colony White 152 Blue 0 Step 4 Save To Batch File x Batch mode Mew batch Open batch Save Count Load LT 7 Batch File Becky CFP Neg 03 batcheount 1 xls iJ amp Eu ale Count Operator 2010 07 09 lO0hr 10 11 41 Count Comment Close Reset start E a x fa Et Quantity One 4 6 7 Microsoft PowerPoint 2 zi D 10 11 AM Colony Counting Viewers Counters at FV Key Features Even glare free illumination Light is soread uniformly over the entire culture plate Colonies are bright and easily distinguished Adjustable dish holder for centering both round dishes and square culture2. Darkfield BioRad UMAX GS 800 Scanner Viewers and Bio Rad Quality One Software Colony Counting SOP and Description Prepared by Bob Morrison FVCC Instrumentation Specialist August 2008 July 2010 P 1 FVCC BioTech Morrison 7 15 2010 age AS Colony Counting Using GS800 Scanner Quality One Software TIENE Turn on Scanner and wait for both lights to show steady green this is the ready state Turn on Host computer logon as Operator SMET Select Quality One to scan acquire and image nternet C pue s expert JUantity One E 2 obe 10 1LUUU Y os JUantity mi Bit JL ESL Link to BioRad Quantity One 1 D Analysis Software Users Manual pdf FVCC BioTech Morrison 7 15 2010 Page 2 Quantity One 4 6 7 File Edit View I Salt Co lony C ountin g Quality One Select Scanner ul Select Scanner s Open 12 oom et 3 Transform Volume Rect Tool P B5 Valume Free hand Tool uantity One X ES 4 Volume Contour Tool E E emn Select Scanner Select Tool Print Image 3 Gel Doc EQ ee 22 9 Volume Analysis Report a ChemiDoc EQ ChemiDoc XES 1 Choose Select Scanner on the T Volume Quick Guide menu 2 Select GS 800 as scanner type Fluor 8 Fluor S Fluor
3. plates Adjustable focusing rod Lens rotates a full 360 Built in tilt leg Optional 1 5X auxiliary lens fits over standard lens increasing magnification to 3X Footprint 12 5in wide x 14 in high x 12 in depth Quebec Darkfield Model 3330 1 An adjustable dish holder for centering round dishes with diameters up to 100mm square culture plates up to 100mm x 100mm 2 An optional 1 5X auxiliary lens fits over the standard lens increasing magnification to 3 0X 3 The adjustable focusing rod allows the 1 5X standard lens to be raised or lowered The lens also rotates a full 360 for ready access to culture plates 4 A built in tilt leg may be mounted in the front or rear of the instrument allowing a convenient tilt angle or it may be locked flat to the instrument base 9 A white ruled Wolffheugel counting plate is included 6 Internal standard light bulb 110V plug connection On Off switch Quebec Darkfield Model 3325 3327 FVCC BioTech Morrison 7 15 2010 Page 12 Counter Automated Invitrogen Countess The Countess Automated Cell Counter uses trypan blue staining combined with a sophisticated image analysis algorithm to produce accurate cell and viability counts in just 30 seconds The algorithm also measures average cell size of live dead and total cells to give you all the data you need to proceed with your experiments The measurement range extends from 1 x 104 to 1 x 107 cells ml with an optim
4. specimen 5 Select ACQUIRE to capture the image Hit STOP if image preview fails to appear Q cqui t Stop Options Save After Scan mes Make Backup Highlight Saturated Options H Help E O K X iv S quantity one 4 6 7 me o SR 10 03AM Quant Colony Counting Scanner Examine Acquired Image Step Select Application see ck Select mum ray film Fg pen V2 Zoom Box Ba ite SERI Transform PoE eee E Volume Rect Tool Volume Free hand Tool Volume Contour T aal Select Tool Print Image M Le D D Le b D D D E Volume Analysis Report Keyboard commands Ctrl click copy a volume Shift click rotate a volume box DaAcaure t Stop Options Save After Scan Hide Grid Backup Copy 43 15 17 18 4 23 25 27 29 Highlight Saturated Pixels Options H Help start I I x El i O Quantity one 4 6 7 lic Microsoft PowerPoint 7 v P 10 04 am x Quantity One 4 6 7 Edit View Image Lane Band Match volume Analysis Re
5. Colonies Adjusted coun If you know you have white and blue colonies in the image and there are two clear peaks on the histogram to the right ofthe background peak you can use the histogram to distinguish between these types of colonies Count vs Peak density 111 Results Vw ita Bius Colony count s3 1 2 Adjusted count 152 Cutoff White Blue White peak _Blue peak Step 3 Tools options Count ws Peak density T Ignore region x Show data area x Mark white colonies Make Colony Cutott _ whiterbiue Mark blue colonies Totals we division mark amp Erase Colony White 0 tae A Blue 75 Step 4 Save To Batch File Batch mode New batch batch Save Count Load Next Batch File Becky GFP Neg 03 batchcount 1 xls Fig o6 Using the Vvhite bBlue slider Drag the White Blue slider to the left until itis positioned between the two peaks The white colony data range is indicated by gold on the bar beneath the histogram and the blue colony data range 15 marked with blue Count Name Operator 2010 07 09 lOhr O3min 10 06 46 Count Comment As you drag the slider the numbers of white and blue colomes will change in the dialog and in the text box on the image Also on the image you should see the marked white colonies gold triangles change to blue colonies blue squa
6. S Mane VersaDoc PMI FX dF GeBier gh 7415 2010 a Quantity One 4 5 7 ica Microsoft PowerPoint Quantis One 4 6 7 Colony Counting Set t Light Preview Acquire Step Select Application Volume Analysis Report Bottom 19 Aight 20 8 Select 3 H m I d PERE EEE EEE Tuem 3 7 5 2 Zoom 2 Filter xjGree Blue white ETAS Transform Light Reflective Trynemissive 11 EIER Rect Tool Volume Free hand Tool BM nue step II Select Scan Area T Volume Contour Tool a Previ a M EE Iz Select Tool 2 Click and drag in diagram to set scan V Fn LEN Left 11 5 19 zl 2 H EN Keyboard commands Ctrl click copy a volume Shift click rotate a volume box Step 111 Select Resolution resolution E Ei E p i m lil NS MT HHE HE E E m Em E pz S elect Y resolution amp 3 5 Image size 4 17 Mb 1 Select image and then Photograph 2 Change Light from Reflective to Transmissive to best see your specimens Preview Scan and wait for image outline to appear Adjust crop box to contain just your
7. al range from 1 x 105 to 4 x 106 cells ml broader than that of a hemocytometer view technical notes for more comparison data The optimal cell size is between 5 um to 60 um view validated cell lines A handy dilution calculator even helps you determine how to prepare your sample for your next passage or experiment FVCC BioTech Morrison 7 15 2010 Example Only Presently Not at FV 3 1 4 Counters Automated Cel Count Homeo tonsa easured concentration 2 1O Cell suspension dilution cells ml The Countess Automated Cell Counter eliminates the tedium and subjectivity of manual cell counting Automated counting frees up your time reduces eye strain and minimizes subjective judgments that can lead to error It takes 3 simple steps 1 Mix 10 ul of sample with 10 ul of trypan blue and pipet into Countess chamber slide 2 Insert slide into the instrument 3 Press the Count cells button results are displayed in 30 seconds Page 13 Other Protocols or Notes e Future home of other or more details protocols FVCC BioTech Morrison 7 15 2010 Page 14
8. nt vs Peak density Cutoff amp White Blue TEP 1 DEFINE REGION and Basic Count Controls lo os Step 3 Tools options 2 Select Icon to Define Region at Center of Image Ignore region xjShowdataarea 3 Drag cursor to move Blue diameter circle Make Cory x Mark white colonies 4 Adjust sensitivity higher to find more colonies Mark blue colonies SN Erase Colony 9 4 Step 4 Save To Batch File Batch mode New batch Open batch Save Count Load Next so Va Batch File Becky GFP Neq 03_batchcount 1 xls Count Name Operator 2010 07 09 lOhr O3min l0 04 54 Count Comment Reset Help Start 9 f fx a 6f Quantity One 4 6 7 t3 Microsoft PowerPoint 9 2 SJ C 10 05 am Quantity One 4 6 7 Colony Counting Adjust Count vs Density m gt Step 1 Define Region and Count 1 If there is a clear peak on the left end of the Define Counting Region Tec e coros hon Conr to edge of dish image YA colony histogram It IS probably due to background intensity or noise in the image 2 Use Cutoff slide to adjust per notes below Count Sensitivity f l Averaging 1 3 5 Plaque Step 2 Adjust Results White Blue Colony count 0 75 Adjusted count r ae Count vs Peak density NN If background is being detected as colome
9. ports Window Help eggs gr Densitometer ERI 12 AlNtomation Manager EN S TS Colony Counting Scanner Select Analysis Colon Counting Colony 7aunting SARRA R X ray film Open mm Display 0 2 ZoomB m oom Box VE Gispla 3 Transform 1 17 Ready AgarosAg6 Plus Tal 4 Volume Rect Tool ef 5 V alume Free hand Tool b Vnlume Contour Toal 5 f Select Tool Print Image Volume Analysis Report le le le be le Le Le E keyboard commands Ctrl click copy a volume Shift click rotate a volume box Select Analysis Select Colony Counting from Drop Down Menu DaAcaure t Stop Options Save After Scan Hide Grid al 1 13 15 17 18 21 23 25 27 28 Highlight Saturated Pixels Options H Help start PN x Eh Quantity one 4 6 7 7 00 v P 10 04 am lic Microsoft PowerPoint Colony Counting Adjust Area of Colony Step 1 Define Region and Count e For best results adjust the Gel Doc zoom lens so that Define Counting Region the cursor from center to edge of dish image Count Sensitivity f l C1 C5 7 Plaque Step 2 Adjust Results White Colony count 0 0 Adjusted count Cou
10. res ote Ifthe blue colonies are not marked on the image check to make sure that the Mark Blue Colonies checkbox at the bottom ofthe dialogis checked Start M x a E v o 65 Quantity One 4 6 7 c3 Microsoft PowerPoint Quantity One 4 6 7 File Edit 1 x Colony Counting Adjust counted items ae For best results adjust the Gel Doc zoom lens z0 that gt Step 1 Define Region and Count Define Counting Region Drag the cursor from center to edge of dish image __ Sensitivity ait Averaging 471 6 3 6 5 i Step 2 Adjust White Blue Colony count 153 4 Marked proce Colonies Adjusted coun 77 Count ws Peak density lI ul Cutoff whiter Blue A Step 3 Toolsoptiorns Ignore region x Show data area x Mark white colonies amp Make Colony Mark blue colonies Totals Erase Colony white 153 Blue Step 4 Save To Batch File _ Batch mode Mew batch Open Batch save Count Load Batch File Becky GFP Neg O03 batchcount 1 xls Count Mame operator 2010 07 09 lOhr O2min l0 07 38 Count Comment Use STEP 3 to Adjust items in out of the counts 2 Erase colony then pick items from image that are NOT part of the colonies 3 Make Colony then pick items to be added as colonies yellow markers start G fa E lw
11. s you can use the histogram and the Cutoff slider to correct this Cutoff m Drag the Cutoff slider to the right until itis centered on the nght edge of the background peak Step 3 Tools options 7 Ignore region Show data area x Mark white colo 7 Make Colon Mark blue colon Erase Colony Step 4 Save To Batch File Batch mode New batch Open batch Save Count Load Ne Batch File Becky GFP Neg 03 batchcount 1 xls Cutoff mark Count Name Operator 2010 07 09 lOhr O3min 10 06 46 Count Comment Fig 6 5 Using the cutoff slider The yellow portion ofthe bar beneath the histogram marks the range ofimage data has been designated as background noise andis not being considered for colony start 3 f amp a t e quantity one 4 6 7 451 G counting purposes The gold portion of the bar marks white colony datarange e ML m i 4 0 pis LB x Quantity One 4 6 7 E 8 x ES Colony Counting Adjust White vs Blue 4 ee For best results adjust the el Doc zoom lens so that Step 1 Define Region and Count oo o Define Counting Region Drag the cursor from center to edge of dish image Count Sensitivity f l Averaging 1 3 C5 7 Plaque Step 2 Adjust Results White Blue Colony count 0 75 oes 5 White and Blue
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