Bacteriophages can be recovered consistently from various environmental niches where these viruses may play a role in the natural control of bacteria. In the laboratory, an aqueous suspension of the source material (if not already liquid) is passed thorough a 0.45 µm membrane filter in order to produce a suspension of viruses free from contaminating bacteria and other cells. Procedures utilized in the phage enumeration experiment are then used to detect the viruses by their formation of plaques with one or more suitable bacterial host cultures.
Following is a procedure which utilizes direct plating for bacteriophages in the environment. This procedure is extended to include phage isolation (the picking of plaques and the culturing of phages in broth cultures of the host organism) and determination of the host range of the phage isolates (as done in the phage typing experiment).
Period 1
Materials
Young culture of Escherichia coli strain B or other host organism
Sewage or soil suspension, filtered through a 0.45µm membrane filter. ("Activated sludge filters easily and usually has abundant E. coli phage.)
1 dilution blank (9 ml)
3 tubes melted Top Agar (4 ml/tube; in 50°C water bath)
3 plates Bottom Agar
Micropipettes and sterile tips
Safety note: Gloves should be used if analyzing the sewage sample, as human viruses may be present in the filtered raw sewage!
Prepare a 1/10 dilution of the filtrate (i.e., the filtered sample). Inoculate the tubes of melted Top Agar (work as quickly as possible!) from the sample or dilution as follows:
0.5 ml of the undiluted sample.
0.1 ml of the undiluted sample.
0.1 ml of the diluted sample.
With a fresh pipette tip, inoculate 0.1 ml of the host culture into each tube of Top Agar.
Pour the contents of each Top Agar tube onto a separate plate of Bottom Agar and allow the plates to solidify before incubation at 30°C. If the next period is more than two days away, place the plates into the tray on stage for appropriate incubation.
Period 2
Materials
Young culture of host organism
4 tubes of Tryptone Yeast Extract (TYE) Broth (5 ml)
Micropipettes and sterile tips
Observe the plates for plaques. From the results of a countable plate, knowing the actual amount of original inoculum, the number of plaque-forming units (PFUs) per ml of the original sample can be calculated. (Keep in mind that this result will only apply to the phages able to lyse the particular host culture used.)
Look for variety in the different types of plaques present. Choose three well-isolated plaques.
Inoculate each of four tubes of TYE Broth with the appropriate host organism such that you see a slight turbidity in the broth.
With a sterile needle, inoculate each of the three chosen plaques into a separate tube of broth. The fourth broth tube will serve as a control to show normal growth of the uninfected host organism.
Place the tubes into the rack on stage such that they can be incubated for the short time needed to effect lysis by the phage of the developing broth cultures of host organisms. This is normally less than 24 hours for phage that grow on E. coli
Period 3
Materials
Dropper bottle of chloroform
Young broth cultures of 6-8 potential host strains
3-4 plates of Bottom Agar (well-dried)
6-8 sterile swabs
Micropipettes (preferably P20) and sterile tips
Do not inhale chloroform. Only use in a well-ventilated area
Observe each tube of TYE Broth, comparing the three tubes inoculated with plaques to the control tube. What we expect to work with now are lysates. For each plaque-inoculated culture that shows less turbidity than the control, proceed as follows.
Add a few drops of chloroform to each lysate. (Careful! See note above.) Vortex vigorously and frequently, and allow the chloroform to dissipate. What you are doing is killing off any remaining viable cells (including phage-resistant cells) in the lysate; the bacteriophages should remain active.
Following the procedure in the phage typing experiment, test your lysates for the ability to lyse the various strains of potential host cultures available. Make sure that the streaks of the test organisms are at least one inch wide and completely dried before you apply the drops of the lysates! Incubate the plates at 30°C until the next period.
Period 4
Observe the plates for plaques and tabulate your results.