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SOP for

BACTERIAL COUNT BY MPN METHOD

Aim: To determine bacterial count in the given sample powder using MPN method. Background: Bacterial count using MPN method involves serial dilution of the given sample in sterile bacterial growth media. As one serially dilutes the culture suspension the bacterial number decreases with each dilution. At one point the number of microbes in the tube falls down to such a critical level that no transfer is achieved in subsequent serial dilution. Thus subsequent tubes in he series do not receive any microbe and hence continue to remain sterile. The number of tubes receiving microbial culture (and showing growth after incubation) depends on the number of microbes present in the original sample. Higher the number in original sample higher is the dilution of the last tube that shows growth. One can then estimate the number of microbe in the original sample from the dilution factor of last tube that shows growth. Scope: The assay can be used to take microbial count of both liquid and solid (powder) samples. Media, diluents buffer, temp etc could be modified depending on the specific needs of the microorganism. Requirements:

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Glassware: 100 ml flasks, 20 ml testube, Exact number of tubes will depend on the dilutions planned for the assay. Chemicals: Growth media: Tryptone soya broth Diluent: pH 7.2 0.01 M tris (hydroxymethyl)-amino-methane (Tris) or 0.01 M or 0.02 M sodium Phosphate buffer with 0.01% tween 80.

Other: 1ml sterile tips, 1000 microliter Micropipette, Clean Laminar airflow unit. Incubator, Orbital shaker, Sterile spatula, Weighing balance Vortex mixer

Procedure: Note: All transfers are to be made aseptically in a laminar airflow unit. All glassware, tips etc used must be sterile and free of any residual microbicidal chemicals, e.g. detergents. 1. Weigh 10 gram of powder to be analyzed and aseptically mix it in 90 ml of sterile diluent buffer. 2. Keep the flask on orbital shaker for at least 1 hr for continuous shaking at 100 rpm to ensure complete dispersion of microbes into the diluent. 3. Transfer 1 ml aliquot of this suspension aseptically to another flask containing 99 mL of sterile diluent buffer and shake vigorously for complete dispersal of the microbial suspension. 4. Transfer 1 ml aliquot from this flask in 9 ml sterile TSA broth taken in 20 25 ml capacity testube. Vortex the tube vigorously for 60 sec and transfer one ml of this suspension in a fresh tube containing 9 ml of sterile TSA broth. Continue serial dilution of the microbial suspension in the broth till desired level. If the expected count is in the range of 10 10 then one should do serial dilution atleast up to 10 15 ____________________________________________________________________ _
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5. The assay should d be done in triplicates to get statistically correct readings. 6. Incubate tubes in incubator for five days and record readings on fifth day. Observe the last tube that shows growth in each set. Interpretation After incubation, the pattern of positive and negative tubes is noted, and a standardized MPN Table (shown below) is consulted to determine the most probable number of organisms (causing the positive results) per unit volume (per gram) of the original sample. In order to estimate the number of organisms per ml of the sample, locate the three sets of tubes that show dilution of the organisms to extinction. Compare the results with the readings given in MPN chart. Determine the average number of microbes from the chart.

MPN chart Second First set set 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0 2 0 2 0 2 0 2 0 3 0 3 0 3 0 3 1 0 1 0 1 0

Third set 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2

Microbes present 0.03 0.03 0.06 0.09 0.03 0.061 0.092 0.12 0.062 0.093 0.12 0.16 0.094 0.13 0.16 0.19 0.036 0.072 0.11

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1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

0 1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3

3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

0.15 0.073 0.11 0.15 0.19 0.11 0.15 0.2 0.24 0.16 0.2 0.24 0.29 0.091 0.14 0.2 0.26 0.15 0.2 0.27 0.34 0.21 0.28 0.35 0.42 0.29 0.36 0.44 0.53 0.23 0.39 0.64 0.95 0.43 0.75 1.2 1.6 0.93 1.5 2.1 2.9 2.4 4.6 11 24

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The number of microbes so estimated are then multiplied with the dilution factor to give final reading per gram of powder.

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