Plaques Assay
(Plaques are clear zones formed in a lawn of cells due to lysis by phages)
E. coli lambda phage
The phage recognizes specific receptor on the surface of the host cell: maltose binding protein for the purpose of adsorption. Hence, the host E.coli is grown in a medium containing maltose and magnesium which further facilitates the process of adsorption.
Duration of experiment
Day 1 : 2 hours (Preparation of media and Revival of Host)
Day 2 : 15 minutes (Inoculation of media)
Day 3 : 5 hours (Preparation of plating cells and Phage titration)
Day 4 : 45 minutes (Observation and Interpretation)
Materials........................Quantity
Host................... a single lyophilized vial
Phage Lysate....................... 50 μl
20% Maltose (sterile)....... 1.5 ml
1 M MgSO4 (sterile).......... 1.5 ml
SM Buffer........................... 30 ml
LB Broth............................... 25 g
1.5 ml vials..............................10
1x25ml vial...............................1
Materials Required
Equipment : Centrifuge, Incubator, Spectrophotometer,Shaker (37°C).
Glassware : Conical flasks, Petriplates,Measuring cylinder, Test tubes.
Reagent : Distilled water.
Other Requirements: Capped centrifuge tubes,Crushed ice, Tips, Micropipettes.
• All microbiological operations should be done strictly under aseptic conditions.
• Revive the strain as soon as the lyophilized vial is opened.
• Prepare plating cells before each experiment.
• Use sterile water to dilute MgSO4.
• Ensure that no moisture is present on the surface of hard agar plates.
Preparation of LB Agar/broth (1 litre): Dissolve 25 g of media in 800 ml of distilled water. Adjust the pH to 7.0 with 5N NaOH (if necessary) and make up the volume to 1000 ml, sterilize by autoclaving.
For LB agar, add 1.5% agar and autoclave.
Preparation of soft agar: To prepare soft agar, add 0.8% of agar to LB broth. Boil to dissolve the agar. Aliquot 5 ml into test tubes and autoclave. Keep soft agar in molten state in a water bath or oven, adjusted to 42-45°C, just before use.
Preparation of 10 mM MgSO4: Dilute MgSO4 (1M), 100 times with sterile water, under aseptic conditions to get a working concentration of 10 mM MgSO4.
Following aliquots of media are required per experiment (excludes preparation of media for revival of strain):
• LB Broth: 5 ml + 25 ml
• LB Hard Agar: 100 ml
• LB Soft Agar: 30 ml (6 x 5 ml)
Note: Prepare 25 ml of LB broth in a 100 ml conical flask.
Procedure
Day 1
Revival of Host.
1. Break open the lyophilized vial and resuspend the sample by adding 0.1 ml of LB broth.
2. Streak a loopful (each) of this suspension on to two LB plates.
3. Incubate the plates overnight at 37ºC.
Day2
4. Inoculate single colony from revived plate in 5 ml of LB broth with 0.2% Maltose and 10mM MgSO4.
5. Incubate at 37ºC in a shaker, overnight .
Day 3
Preparation of plating cells.
6. To 25 ml of LB broth, add 0.2 % Maltose and 10 mMMgSO4. Inoculate the broth with 1% (0.25 ml) of overnight grown culture. Incubate at 37° C till the 0.D.reaches 0.6 at A600, (approximately 2-3 hours).
7. Chill on ice for 10 minutes.
8. Centrifuge the cells at 5000 rpm for 10 minutes in sterile centrifuge tubes. Discard supernatant.
9. Resuspend the cell pellet gently in 5-6 ml of 10 mMMgSO4 and store at 4ºC.
10. Pipette 1ml of SM buffer each into six serially labeled 1.5 ml vials.
11. Transfer 10 μl of stock lysate into vial # 1 for diluting it to 10^-2. Mix and transfer 10 μl of 10^–2 dilution to vial # 2. (i.e. 10^-4 dilution). Repeat the same till the last dilution of 10^-12.
Note: Mix the phage lysate dilutions thoroughly by vortexing or inverting the vial.
Change the tip after every dilution.
12. Label six LB agar plates and six sterile vials as 10^-2,10^-4.... 10^-12.
13. Pipette 100 μl of plating cells into each one of the vials.Add 10 μl of the respective phage dilution, mix gently and keep at 37°C for 15 minutes for adsorption of the phage onto host cell.
Note: Do not vortex.
14. Pipette out the content of vial labeled as 10^-2 into a test tube containing 5 ml of soft agar and swirl to mix. Care must be taken to ensure that the temperature of soft agar does not exceed 45°C (as the host will die) or fall below 40°C (as soft agar will solidify).
Note: Change the tip for each dilution.
15. Pour the mix (phage-plating cells with soft agar)immediately on the respective LB agar plate and let the agar solidify.
16. Repeat steps 14 & 15 for each dilution.
17. Close the lids and incubate at 37°C for 16-18 hours.
Calculation of Phage Titre value
Phage Titre value= Plaque Number*Reciprocal of Diluition*Reciprocal of Volume in ml
e.g. if there is a mean number of 50 plaques with 0,1ml a 10^-6 diluition there are : 50*10^6*10=50*10^8 p.f.u. ml^-1.
Note: On every dilution, a decrease in number of phage particles by 100 fold is observed. For example, if 1000 plaques are seen at 10^-8 dilution, the number of plaques that may be observed at 10^-10 will be about 10.
