Microbiology: Viable Counts

INTRODUCTION

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)

MATERIALS:

Dilution series prepared in (A)

Sterile 1ml pipette

6 nutrient agar (NA) plates

1 glass hockey stick

1 beaker of alcohol

PROCEDURE

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.

RESULTS

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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

P1

P2

P3

MPN

3

3

3

24.00

Number of viable cells (/ml) =

DISCUSSION

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 colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow.

Most probable number method [MPN]

No plate viable count:

The most probable number method is a way of determining approximate viable count by diluting cultures then growing the dilution cultures in broth tubes.

At the dilution at which broth becomes turbid or not with similar likelihood, the culture has been dilution to the point that the broth tubes were inoculated with on the order of only a single microorganism (turbid ) or fewer (not turbid ).

The concentration of the culture is then taken to be equal to the amount of dilution necessary to have reached this point.

Advantageous where broth is advantageous:

MPN is especially useful in situations where there is an advantage to using broth over solid medium .

For example, many organisms are not good at forming colonies, such as highly motile organisms.

"When samples contain too few organisms to give reliable measures of population size by the standard plate count method, as in food and water sanitation studies, or when organisms will not grow on agar, the most probable number method is used." (p. 143, Black, 1996)

Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 140-144.