Total Viable Count is a quantitative idea about the presence of microorganisms such as bacteria, yeast and mold in a sample. It counts the number of colonies produced by a very dilute suspension of bacteria on an agar plate and to observe the differential staining behaviour of the living bacteria. This involves counting the colonies produced by viable cells under favourable growth conditions. Some techniques needed before the viable count, like pour plate method, spread plate method and most probable number method. The viable count is very specidic, as it represents the number of colony forming units (/g) or (/ml) of the sample.
B) VIABLE COUNTS
Two methods of getting viable counts are available
i. Spread plate method.
ii. Most probable number (MPN) method.
(I) SPREAD PLATE METHOD (LAWN CULTURE)
Dilution series prepared in (A)
Sterile 1ml pipette
6 nutrient agar (NA) plates
1 glass hockey stick
1 beaker of alcohol
All steps should be done using aseptic technique
The culture labeled 10-8 was gently mixed. 0.1ml of this dilution was aseptically transferred onto the center of a NA plate.
Bend end of the glass spreader was dipped in alcohol, then it was sterilized by flaming and allowed to cool down.
The glass hockey stick was used to spread the sample over the surface of the plate. It was an even distribution.
This procedure was repeated using a second NA plate. These plates were labeled as 10-8. So, duplicated plate was counted.
Steps 1,2 and 3 was repeated for dilution 10-7 and 10-6 in order. 6 plates was prepared and 2 at each of the dilution 10-8, 10-7 and 10-6.
The plates were incubated at 32oC for about 2 days.
The number of colonies on the plate was counted. Ideally, the plate should have over 30-300 colonies, more than this is difficult to count because the colonies often overlap, fewer than this leads to statistical inaccuracies.
The number of viable calls per ml in the original culture was calculated.
(II) MOST PROBABLE NUMBER (MPN) TECHNIQUE
If for some reason solid medium cannot be used for culturing the bacteria, viable counts can be carried out using liquid media, by means the Most Probably Number (MPN) Technique. The sample to be counted is diluted until small aliquots contain an estimated one viable cell each. If the dilution is correct, some aliquots will contain a viable cell, and other will not, so that after incubation, growth will occur in some tubes and not in others. If the sample has not been diluted sufficiently, all aliquots will contain viable cells and all inoculated tubes will show growth on incubation. If the sample has been diluted too much, none of the inoculated tubes will show growth. MPN method can be carried out by a 3 tube or 5 tube methods. Three successive tenfold-diluted samples are inoculated. After incubation the inoculated tubes showing positive growth are recorded. With reference to the MPN table the most probable numbers of bacterial sample analysed can be obtained.
Figure 1: Observation of the plate 1 labeled 10-8
Figure 2: Observation of the plate 2 labeled 10-8
Figure 3: Observation of the plate 1 labeled 10-7
Figure 4: Observation of the plate 2 labeled 10-7
Figure 5: Observation of the plate 1 labeled 10-6
Figure 6: Observation of the plate 2 labeled 10-6
The most probable number (MPN) for 3 tubes method in this experiment are
Number of viable cells (/ml) =
The spread plate method is a technique which used to grow and isolate colonies of bacteria. Nutrient was provided when bacteria is transferred to agar plate. The diluted liquid contain bacteria is applied to agar plate and the sterilized hockey stick will spread and dilutes the amount of bacteria in each section of the agar plate continuously, so that the isolated colonies can be easily studied. When the...
Please join StudyMode to read the full document