How to determine the total number of aerobic bacteria present in a sample

Total Viable Count (TVC)

Total viable count is a method to determine the total number of aerobic bacteria present in a particular water sample or deposit. This is an empirical measurement because occuring singly, in pairs, chains, clusters, or packets and no single growth medium or set of physical and chemical conditions can satisfy the physiological requirement of all bacteria in a water sample.

 

Summary Of Method

The standard plate count method for determining total viable count consists of taking a suitable volume of sample or a dilution of the sample and transferring it to a petriplate. The number of sample dilutions required is determined by the type of sample being analysed. Nutrient agar is then poured into the plate. The agar and sample are mixed and incubated at 37OC till the agar has solidified. After incubation is over, the bacterial colonies on the plate are counted. Multiplying the number of colonies by dilution factor yields actual total viable count per ml of the original undiluted water sample.

 

Sample Requirements

Water samples should be collected in sterile, glass bottles, containers prepared by the microbiology laboratory. The deposit samples may also be collected in sterile containers. In each case samples should be sent to microbiology lab soon after collection and as early as possible. No preservatives should be added to the sample

Equipment

Incubator

Autoclave

Pipettes

Dilution bottles and tube containing sterile buffered water. Petriplates

Nutrient Agar Butts

Glassware is sterilised at 121OC at lbs / inch2 for 20 minutes in an autoclave.

 

Procedure

1. With a sterile 1 ml pipett, transfer aseptically, 1 ml of the thoroughly shaken sample to sterile tube containing 9 ml saline solution. Shake thoroughly. This is 1 / 10 dilution.

2. With another sterile 1 ml pipette, transfer 1 ml from the 1 / 10 dilution to another sterile tube containing 9 ml saline solution. Shake thoroughly. This is 1 / 100 dilution.

3. Similarly carryout serial 10-fold dilutions depending on the microbial load in the sample. For recirculating water sample, generally 1 / 10,0000 i.e. 100-5 dilutions are carried out.

4. Transfer aseptically 1 ml of diluted samples (of every dilution viz 10-3, 10-4 & 10-5) in separate sterilised petri dishes. Prepare a duplicate set in similar way.

5. To each petriplate containing water sample, add 15 ml nutrient agar (which is previously melted and cooled to @ 40 – 45oC). Thoroughly mix water sample and agar by swirling the dish.

6. After the agar has solidified, invert the plates & place in an incubator at 37oC for 48 hours.

7. Count the bacterial colonies in the plates using a colony counter. The final plate count is then multiplied by the dilution factor.

8. Record the final result of Total Viable Count as number of organisms / ml.

 

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