Titration

Methodology

1. Mark five sterile universal bottles with the times of the culture in incubation; 0, 10, 30, 50 and 70 minutes.

2. Label the bottom of 5 petri plates A-E. Label 21 test tubes A1, A2, A3, B1, B2, B3, C1, C2, C3, C4, C5, D1, D2, D3, D4, D5, E1, E2, E3, E4 and E5.

3. Fill the A and B tests tubes with 4.95ml, 4.95 ml and 4.5 ml of diluent and fill 5 tests tubes of C, D and E with diluent as shown below.

4. At the given time intervals collect the flask of culture from the incubation and transfer 3ml into the labelled sterile universal bottle for that time.

5. Use a fresh pipet to transfer 0.05ml of the culture from the sterile universal bottle into A1 (and discard the used tip into the yellow sharps bin provided), transfer another 0.05ml from A1 into A2 and finally transfer 0.5 ml from A2 into A3. Mix well using the vortex mixer before sampling.

6. For samples C, D and E the same sampling is done for tests tubes 1, 2 and 3 in addition to two further transfers of samples; 0.5ml from 3 to 4 and a final 0.5ml from 4 to 5. As shown in the diagram below.

7. Divide the nutrient agar plate into six section and using a 20 µl pipette and starting with the highest dilution (10-5 for A and B, 10-7 for samples C, D and E), plate out duplicate samples of dilution indicated in the diagram on below by carefully placing 20 µl on each section as indicated in the diagram.

8. The remainder of each culture in the sterile universal bottles is transferred into plastic cuvette and the optical density is measured at 540 nm using a spectrophotometer, making sure to use a nutrient broth cuvette provided to calibrate. Discard all the contaminated cuvettes into the yellow sharp bin provided.

Things to remember when doing the Viable Count:

* Make sure all transfer of sample is some near the flame to avoid contamination

* When placing dilution into agar plate make sure you hold the pipette vertically and high enough above to prevent the...