Introduction to Lab Ex. 20: Enumeration of Bacteria -

Most Probable Number method

Membrane Filter method

Water Quality

• Microbes are filtered from water that percolates into groundwater.

• Some pathogens are transmitted to human in drinking and recreational water.

• Resistant chemicals may be concentrated in the aquatic food chain.

• Mercury is metabolized by certain bacteria into a soluble compound, concentrated in animals

Waterborne Diseases

Table 27.2

Most Probable Number & Membrane Filter methods

These are methods used to enumerate the numbers of bacteria in water samples.

The Most Probable Number method is used to check potability (if water is safe enough to be drinking water) of water.

The MPN method looks for the presence of potential pathogenic bacteria that may be in the water due to fecal contamination of the water supply.

Water supplies are generally derived from ground sources and have to be checked for safety levels of bacterial contamination.

MPN method enumerates the enteric bacteria called coliforms, specifically fecal coliforms (E. coli)

Coliforms are Gram negative bacilli that have the ability to ferment lactose with the production of acid and gas.

Fecal coliforms are those coliforms that are normally found in the feces of warm blooded animals (including humans)

MPN method thus enumerates the fecal coliforms in water samples.

E. coli is thus used as an indicator organism.

MPN test includes 3 levels of testing: Presumptive, Confirmed, Completed.

The presumptive test looks for presence of fecal coliforms in the water sample by inoculating lactose broths with the water sample.

Those tubes that show presence of acid and gas are scored + and those with no acid/gas as -. Three sets of lactose broths are inoculated with varying dilutions of the sample:

first set of 3 or 5 tubes inoculated with 10ml of sample; second set of tubes inoculated with 1ml of sample; third set of tubes inoculated with 0.1ml of sample.

The combination of positives in the 3 sets is used to figure out the MPN /100ml of water using the table provided.

The tubes that show positive in the presumptive test should be confirmed to contain E.coli.

This done in the confirmed test using the selective/differential medium EMB (that uniquely highlights E.coli growth on it).

The completed test is done only where legal issues are involved wherein the bacterial culture is then identified by a full complement of tests including gram stain to show that it is indeed E.coli

 Water double strength single strength

single strength sample

10 ml sample 1.0 ml sample 0.1 ml sample

 *Normally 5 Durham tubes are inoculated but this exercise is modified to three tubes in

the interest of economy.

Bacteriological analysis of water: Most Probable Number (MPN) technique

coliform: acid and gas from lactose <24 hours/370Cindicator organism: E. coli

Table 1.Table of Most Probable Numbers (MPN) Per 100 ML of Sample using Three Tubes of Each Dilution

Number of positive Number of positive tubes in dilutions tubes in dilutions MPN per MPN per 10 ml 1 ml 0.1 ml 100 ml 10 ml 1 ml 0.1 ml 100 ml 0 0 0 2 0 0 9.1 0 1 0 3 2 0 1 14 0 0 2 6 2 0 2 20 0 0 3 9 2 0 3 26 0 1 0 3 2 1 0 15 0 1 1 6.1 2 1 1 20 0 1 2 9.2 2 1 2 27 0 1 3 12 2 1 3 34 0 2 0 6.2 2 2 0 21 0 2 1 9.3 2 2 1 28 0 2 2 12 2 2 2 35 0 2 3 16 2 2 3 42 0 3 0 9.4 2 3 0 29 0 3 1 13 2 3 1 36 0 3 2 16 2 3 2 44 0 3 3 19 2 3 3 53 1 0 0 3.6 3 0 0 23 1 0 1 7.2 3 0 1 39 1 0 2 11 3 0 2 64 1 0 3 15 3 0 3 95 1 1 0 7.3 3 1 0 43 1 1 1 11 3 1 1 75 1 1 2 15 3 1 2 120 1 1 3 19 3 1 3 160 1 2 0 11 3 2 0 93 1 2 1 15 3 2 1 150 1 2 2 20 3 2 2 210 1 2 3 24 3 2 3 290 1 3 0 16 3 3 0 240 1 3 1 20 3 3 1 460 1 3 2 24 3 3 2 1100 1 3 3 29

Multiple-Tube Method

Figure 6.18a

Multiple-Tube Method

Figure 6.18b

Bacteriological analysis of water: Membrane filter technique