Total Bacteria Count in Process Waters | Total Coliform Count in Water

The method determines the level of microbial contamination in water. Total bacteria count process water. Total coliform count method.

Principal of Total bacteria count: Water is added to an R2A agar specific for water samples. Following incubation at 22°C over 5 nights, the number of colonies is counted. Three alternative methods for routine analysis are acceptable.

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3. APPARATUS

3.1 Incubator at 22 ± 2°C

3.2 Autoclave

3.3 Sterile pipettes 1 and 0.1ml

3.4 Sterile Petri dishes (90mm diameter)

3.5 100 ml sterile bottles and caps

3.6 2000ml flask

3.7 Balance

3.8 Water bath at 45-50°C

For filtration method:

3.9 Sterile filter holder

3.10 Sterile membrane filters of maximum 0.45μm pore size

3.11 10ml Sterile distilled or purified water.

3.12 Vacuum flask

3.13 Vacuum pump

Millipore test kit method:

3.14 Millipore HPC100 25

4. REAGENTS

R2A Agar. Oxide CM096 or equivalent. Prepare and sterilize according to the manufacturer's instructions.

Total bacteria count in process waters

5. Procedure for Plate count method

5.1 Plate count method

Aseptic procedures to be used throughout. Pipette 1.0ml of water sample into each of two Petri dishes Pour 18-20 MLS of molten cooled agar (45-50°C) into each petri dish and swirl the dish to mix and allow to solidify. Incubate the Petri dishes inverted in the incubator at 22 ± 2°C over 5 nights. Examine the plates for growth and count the number of colonies present.

If no colonies are recovered record the results as 0 cfu/ml.

If the number of colonies is between 1 and 300, calculate the average number of colonies present on the two plates, and record the results as cfu/ml of sample.

If the average number of colonies is more than 300, record the result as >300 cfu/ml

Note:

For infeed and untreated waters where the counts may be expected to exceed 300 cfu/ml, use 0.1ml samples in place of 1.0ml and multiply the result by 10 to give the result as cfu/ml.

5.2 Filtration method

Add 1ml of water sample to 10ml of sterile water.

Filter all 11mls through the 0.45μm sterile membrane filter.

Aseptically remove the filter from the filtration unit and place it on the R2A agar plate.

Incubate the plate, without inverting, at 22°C ±2°C over 5 nights

Examine the filter for growth and count the number of colonies present.

If no colonies are recovered record the results as 0 cfu/ml.

If the number of colonies is between 1 and 300 records the results as cfu/ml of sample.

If the average number of colonies is more than 300, record the result as >300 cfu/ml

Note:

For infeed and untreated waters where the counts may be expected to exceed 300 cfu/ml, use 0.1ml samples in place of 1.0ml and multiply the result by 10 to give the result as cfu/ml. For water supplies where the normal total count is less than 5 cfu/ml, the sample size may be increased to provide increased sensitivity.

5.3 Millipore Total Count sampler MHPC100 25 Use according to the supplier's instructions. (This will automatically test a sample volume of 1ml.)

Incubate the sampler, at 22°C ±2°C over 5 nights

Examine the sampler for growth and count the number of colonies present.

If no colonies are recovered record the results as 0 cfu/ml.

If the number of colonies is between 1 and 300, and record the results as cfu/ml of sample.

If the average number of colonies is more than 300, record the result as >300 cfu/ml

Note: For infeed and untreated waters where the counts exceed 300 cfu/ml, this method is not appropriate and either the plate count or filtration method must be used.

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