Determine the Total Number of Micro Organisms Present of Water

Determine the total number of micro-organisms present, water is filtered at 0.45 microns, and membranes are placed on MacConkey agar No. 3 and Pseudomonad CFC agar. Following incubation, at 22°Cover 3 nights (Pseudomonads) and 30°C over 3 nights (Coliforms), the number of colonies is counted.

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3. APPARATUS

3.1 Incubator at 22 ± 2°C

3.2 Incubator at 30-35°C

3.3 Autoclave

3.4 Sterile Petri dishes (90mm diameter)

3.5 100ml sterile bottles and caps

3.6 2000ml flask

3.7 Balance

3.8 Water bath 45-50°C

3.9 Laminar flow cabinet

3.10 Refrigerator 4 +/- 2°C

3.11 Sterile filter holder

3.12 Sterile membrane filters of maximum 0.45 um pore size

3.13 Vacuum flask

3.14 Vacuum pump

4. Reagents for Determine Microbially 

4.1 MacConkey No. 3 Agar, Oxoid CM0115 or equivalent. Prepare and sterilize according to manufacturers’ instructions.

4.2 Pseudomonad Agar Base, Oxoid CM0559 or equivalent. Prepare and sterilize according to manufacturers’ instructions. Store sterilized base agar at 2-8°C until required.

4.3 CFC Selective Agar Supplement, Oxoid SR0103 or equivalent. Rehydrate 1 vial as directed by the supplier and add 500ml of Pseudomonad agar base cooled to 50°C. Mix well and pour into sterile Petri dishes. If necessary, the prepared plates may be stored at 2-8°C for up to 4 weeks. Dry the surface of the plate in a laminar flow cabinet before use.

5. Procedure

5.1 Pseudomonad count

Filter 100 ml through a 0.45 m sterile membrane filter. Aseptically remove the filter from the filtration holder and place it on the pseudomonad CFC agar plate. Incubate the plate, without inverting at 22°C ± 2°C over 3 nights.

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

If no colonies are recovered record the results as 0 cfu/100ml.

If the number of colonies is between 1 and 300 record the results as cfu/100ml of a sample.

If the average number of colonies is more than 300, record the result as >300/100 cfu/ml.

5.2 MacConkey Agar No. 3

Filter 100 ml through a 0.45 m sterile membrane filter.

Aseptically remove the filter from the filtration holder and place it on the MacConkey No. 3 agar plate. Incubate the plate, without inverting at 30-35°C over 3 nights.

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

If no colonies are recovered record the results as 0 cfu/100ml.

If the number of colonies is between 1 and 300 record the results as cfu/100ml of a sample.

If the average number of colonies is more than 300, record the result as >300 cfu/100ml.

>Moisture content analysis, the Water content of the soil, and Volatile matter

>Determination of hardness of water and Conductivity of water

>Sampling methods Microbiological testing