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Introduction: As you saw from the demonstration plates, many different types of bacteria, along with different types of bacteriophage can be easily isolated from waste water. Other environmental sources can also yield a variety of viruses, depending upon the appropriate target cells and enrichment methods. Regardless of isolation method, it is frequently important to quantitate the concentration or amount of virus obtained. This is fairly easy to do for lytic viruses. The situation is more difficult with non-lytic viruses.

The plaque-forming method for bacteriophage used in this exercise is a general method which can be applied, with modification, to animal and plant viruses as well. For plant viruses, samples are applied to leaves instead of agar plates. For animal viruses, samples are added to cell cultures. In all cases, holes or plaques form where cells have lysed due to the activity of a lytic virus. Each plaque represents one virus particle capable of an infection cycle which occurs first in one cell, then repeats in neighboring cells. The titer is determined by counting the number of plaque-forming units. First, the number of plaques on a plate are counted. That number is then multiplied by the reciprocal of the amount of sample added to the plate and multiplied by the reciprocal of the dilution factor. For example, 85 plaques on a plate which received 0.1 ml sample from a 10-4 dilution tube is equal to

85 x 10 x 104 = 8.5 x 106 pfu/ml.

For bacteriophage, a second method is sometimes used called the broth-clearing assay. A dilution series of phage in broth is set up, then inoculated with target bacteria. An endpoint is determined based on the highest dilution producing lysis of bacteria and clearing the broth culture. This method can be used in parallel with the plaque-forming assay for a given phage, then used later alone as a quick check on concentrations of samples.

Basic methods: Rationale

1. Sterile technique is critical in this type of work. You must prevent contaminating bacteria and fungi from getting into your samples and plates. All materials are initially sterilized, including tubes of diluent, solid and liquid media, and pipet tips. While working with these materials, care needs to be taken to avoid contamination. Tubes and plates are kept covered, except for brief periods to manipulate them. Pipet tips are aseptically mounted on the pipettors and care is used in avoiding touching anything with your pipet tip but your desired sample.

2. Proper micropipet use is important in order to get the appropriate quantities consistently. Practice holding the pipettor and pushing the plunger until you are comfortable. The plunger can be pushed to two positions. The first position is to draw up a measured sample. The second, farthest, position is to dispense the contents. Be sure to carefully seat the pipet tip on the pipettor. Otherwise you may have leaks and inconsistent volumes as a result.

3. Labeling petri plates first is important in this exercise. Plates are labeled on the bottom, not the top. Include plate number, initials, and date.

4. Biohazard discard of pipet tips will be in the provided beakers. Used agar tubes and dilution tubes are placed in the provided racks. If there is a spill, let me know. Spills need to be surrounded with bleach, then covered with paper towels. The contaminated towels need to be discarded in biohazard waste.

5. Organization in setting up and in doing this exercise is necessary to get good results. Read the entire exercise. Working in pairs helps. Decide who does what, especially when transferring bacteria and diluted phage to the top agar tubes. Reasonable speed is necessary to avoid overheating the samples. Time is also of the essence when pouring the top agar onto a petri plate to facilitate a smooth and even layer.

EXERCISE: Titration of Bacteriophage: Work in pairs

Materials: Per pair

1 rack to hold eppendorf tubes
6 eppendorf tubes with 0.9 ml sterile saline
1 P-100 pipettor set to "100" [100 ml or 0.1 ml]

6 melted top agar tubes [3 ml 0.4% TSA each]: in water bath
6 TSA plates
1 stock tube of assigned bacteria [in log phase growth]

Materials: Per four

1 marking pen
1 box sterile pipet tips
1 discard beaker for used pipet tips
1 sample tube of assigned phage
1 heat block, calibrated to 50ºC

Record assigned bacteria:

Record assigned phage:




1. Label the eppendorf dilution tubes and the petri plates "1" through "6" by writing on the bottom of the plates. On the plates, also label with date and initials for later identification.

2. Aseptically mount a pipet tip on the P-100 pipettor, as demonstrated, by pushing the pipettor tip into the top of the disposable tip in the box and lifting it free of the box without touching any surface with the tip.  

3. Aseptically add 100 ml [0.1 ml] phage suspension to tube 1 containing 0.9 ml saline. Blow out tip into tube 1, then mix by carefully aspirating up and down three times with the pipet. Discard tip and mount a new pipet tip. Transfer 100 ml from tube 1 to tube 2 and mix well. Transfer 100 ml from tube 2 to tube 3 and mix well. Change pipet tips.

4. Repeat the transfer steps through tube 5. Add nothing to tube 6- this is your control tube.

Why are the pipet tips changed between dilution steps?


What dilution exists in each dilution tube, relative to your sample tube?

Tube 1aaaa

Tube 2aaaa

Tube 3aaaa

Tube 4aaaa

Tube 5aaaa






What purpose does the control, tube 6, serve?


5. Transfer 6 top agar tubes from the water bath to the heat block. Heat block temperature should remain between 48-50ºC.

6. With a fresh pipet tip, transfer 100 ml bacteria suspensions to each of the 6 top agar tubes. Roll the tubes briefly between your palms, as demonstrated, to mix, then return to the heat block. Discard tip.

7. Starting with tube 6, aseptically transfer 100 ml to a top agar tube. Mix tube by rolling between your palms. Quickly pour the inoculated top agar over the surface of petri plate 6, tilting the plate to spread evenly. Let it sit upright [lid up] on the counter to solidify.  

8. You may use the same pipet tip to continue transferring 100 ml of each successive dilution tube to a top agar tube, provided it remains uncontaminated. [If sterility is broken or if in doubt, change tips.] After each transfer of dilution sample, mix and pour top agar over the surface of the appropriately labeled plate.

9. When all plates have solidified, they may be stacked and taped as a bundle. Be sure the plates are labeled clearly as to date, names, etc. Invert your plates and incubate at 35ºC in D234.

10. Plaques should be visible within a few hours. You may check your plates in the morning in D234. To count, select a plate with between 30 and 300 plaques. Count the number of plaques, then multiply by 10, then multiply by the reciprocal of the dilution of that plate to give the number of plaque forming units per milliliter. If you see distinct differences of plaques on a plate, you may choose to count the types separately. To do so, use different colors of pens to facilitate your counting. Please return your plates to the tray. They will be removed from the incubator about 12 noon and stored for next week's discussion. A sign will be posted to indicate their location in case you want to check them later.  

Why is the number of plaques multiplied by 10?


Record your results in pfu/ml:


Show your calculations:





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