Detection of Bacteriophage
The following tests are based on the principle that bacteriophage specific to the culture in use will be present in high numbers in the cheese whey. Therefore, by monitoring whey for the presence of phage "a dead vat" on subsequent days can be avoided.
Culture Activity Test
The culture activity test described above can be used to detect the presence of phage in cheese whey. Prepare 300 ml of reconstituted skim milk and place 99 ml in each of three beakers. Add 1 ml of whey to Beaker 1 (100 x dilution), then transfer 1 ml from Beaker 1 to Beaker 2 (10,000 x dilution) and finally, transfer 1 ml from Beaker 3 to Beaker 4 to make a 1 million times dilution. Add culture and monitor pH as described in section 3.8.
Bromocresol Purple (BCP) Phage Inhibition Test
This test is quite simple to perform, and produces more accurate results than the culture activity test.
1. Prepare Materials
- BCP stock solution (1 g/100 ml water)
- Test tubes containing 9.9 ml sterile BCP-milk (5 ml BCP stock solution/litre milk)
- 30-32C water bath or heating block
- 1 ml graduated pipettes
- Membrane filter (0.45 u) -- optional
- Disposable syringe -- optional
- Clinical centrifuge -- optional
- Whey sample for phage testing
- Freshly grown culture, frozen syringe, or frozen can of each strain
2. Add Whey to BCP Milk and Make Dilutions
Transfer 0.1 ml of fresh (or filter-sterilized) whey to the first dilution tube (10-2) and mix well. Transfer 0.1 ml from the first to the second dilution tube and mix well. Repeat process for the third dilution tube. (If unfiltered whey is used, a control tube containing BCP milk and whey only, must be prepared. This control tube tests for the presence of active culture in the whey that could mask phage inhibition of a strain.) Whey samples should be refrigerated immediately after collection and held cold until tested for phage.
3. Add Culture to Control and Whey Dilution Tubes
Cheese culture (0.2 ml) is added to whey dilution tubes and to a control tube for each strain. If you are using direct-to-the-vat culture, dilute 1 ml of culture in 9 ml of milk and then add 0.2 ml of the mixture to the dilution tubes. The control tube contains only BCP milk and culture---NO whey. The control tube serves to show starter strain inhibition by colour comparison with the other tubes.
Incubate Tubes and Interpret Results. Incubate both control and dilution tubes for 6 hours at 30-32C. Compare the colour of the whey dilution tubes to that of the control tube. Ignore coagulation. An uninhibited culture will produce sufficient acid to turn the BCP dye from blue to yellow. Strains should be removed from the culture blend when full inhibition persists at the 10-6 dilution level. The following system should be used to record phage inhibition:
0 = No inhibition at any dilution
1 = Partial inhibition at 10-2 dilution
2 = Full inhibition at 10-2 dilution
3 = Partial inhibition at 10-4 dilution
4 = Full inhibition at 10-4 dilution
5 = Partial inhibition at 10-6 dilution
6 = Full inhibition at 10-6 dilution