1. Results of serial dilution No lysis discrete plaques web plate complete lysis want this for counting (20-200 plaques) want this for lysate collection

2. Objective and Rationale  Objective  To deduce the concentration and volume of phage stock needed to produce a perfect web pattern for high titer lysate preparation  Rationale  Students need to know how to get a web pattern biologically and mathematically

3. What is a web plate? 3-4 rounds of purification High titer lysate 5-web plate infection Electron microscopy DNA purification & restriction digest You will predict the tip- dip/serial dilution conditions needed -intuition -math (empirical testing) You will predict the tip- dip/serial dilution conditions needed -intuition -math (empirical testing)

4. Results of serial dilution No lysis discrete plaques web plate complete lysis want this for counting & titer (20-200 plaques) want this for lysate collection

5. Two approaches  Two ways to do obtain a web plate:  Empirical: Based on plaque size (radius)  Calculate the number of plaques needed to cover a plate -problems: plaques sizes vary, lysates lose titer over time, cultures vary and change over time  Intuitive: Based on previous titers  Because you’ve titered the phage stock already, or done multiple rounds of serial dilution, you should have a good idea of which dilution will give you a web pattern -experiment with concentrations above and below your known value to obtain your ideal.

6. Overview of calculations  Calculate your phage titer (‘pseudo titer’ if from purification plate) titer (pfu/ml phage stock ) = (# plaques/#  l) X (1000  l/ml) X dilution factor  Calculate the pfu needed to cover the plate: pfu maxweb =Area(mm 2 ) plate /Area(mm 2 ) plaque  Calculate the amount to pipette: ( pfu maxweb )/(pfu/mL phage stock )=Volume(mL) phage stock converting your answer to µL gives the volume and dilution on which to base your empirical assay  Determine the range of pfu to be tested by assaying either: two 5-fold dilutions above and below calculated volume (big plaques) or two 2-fold dilutions above and below calculated volume(small plaques) Tame pg 9 in PDF Part A. Purify the Phage: The Phage-Titer Assay Tame pg 21 in PDF Part B. Make Phage Stocks: Empirical Testing of Phage Lysates In your resource guide:

7. Step 1: PFU maxweb calculation  Calculate the pfu needed to cover the plate: pfu maxweb =Area(mm 2 ) plate /Area(mm 2 ) plaque 2 mm plaque diameter 85 mm plate diameter pfu maxweb = ?? pfu needed for max web

8. Step 2: Phage titer calculation  Calculate the phage titer: Titer(pfu/ml)= (pfu/#ul) X (1000 ul/1ml) X dilution factor 50 pfu counted 10  l = volume tested 10 -7 dilution of phage stock Titer = ?? pfu/ml Part 1, Tame, 18

9. Step 3: Calculate volume needed for web plate  Convert desired pfu to a volume, in  l: ( pfu maxweb )/(pfu/mL phage stock )=Volume(mL) phage stock pfu maxweb = 1806 pfu needed for max web Titer = 5 X 10 10 pfu/ml Volume (ml) phage stock = (1806 pfu) / (5 X 10 10 pfu/ml) = ?? ml

10. Step 4: Calculate volume needed for web plate  Calculate volume for two 2X/5X higher and lower pfu maxweb : Samplepfu desiredpfu to volume conversionVolume to pipette ÷4÷4450 ÷2÷2903 pfu maxweb 18060.00000003612 ml 36  l (10 -6 dilution) 2X3612 4X7224 step 1 step 3 step 4 10-100  l range

11. Potential results This plate is just right! Over-estimated lysate volume Underestimated lysate volume Can go from right to left over a few hours

12. Empirical calculations summary  Calculate your phage titer (‘pseudo titer’ if from purification plate) titer (pfu/ml phage stock ) = (# plaques/#  l) X (1000  l/ml) X dilution factor  Calculate the pfu needed to cover the plate: pfu maxweb =Area(mm 2 ) plate /Area(mm 2 ) plaque  Calculate the amount to pipette: ( pfu maxweb )/(pfu/mL phage stock )=Volume(mL) phage stock Note: you will need to convert your answer to µL to give a pipettable volume  Determine the range of pfu to be tested by assaying either: two 5-fold dilutions above and below calculated volume (big plaques) or two 2-fold dilutions above and below calculated volume(small plaques) Tame pg 9 in PDF Part A. Purify the Phage: The Phage-Titer Assay Tame pg 25 in PDF Part B. Make Phage Stocks: The 5-Plate Phage Infection and Harvest In your resource guide:

13. Empirical test table Samplepfu desiredpfu to volume conversionVolume to pipette ÷4 or 25 ÷2 or 5 pfu maxweb 2X or 5X 4X or 25X 10-100  l range Dilution Total vol needed Sum the amount needed at each dilution so you can adjust your serial dilution