Enumeration of Microbes

ENUMERATION OF MICROBES

From Sewage

Sewage water consists of about 99% water with suspended waste materials. Various types of microbes enter the sewage, some of them are of human origin including human pathogens which cause various water borne diseases . Thus the microbes present in sewage are coliforms including E.Coli, Coliforms,faecal Streptococci, Vibrio cholera, Entamoeba and Hepatitis. Other types of microbes come from industrial waste or other sources

Methods of Enumerations

There are the following methods for the enumeration of the microbes from sewage water.

1) Standard plate count

This test is designed to enumerate total viable population. A measured amount of sewage water is diluted and thus transferred to the petri plates. These are mixed thoroughly with molten medium and plates are then incubated after solidification.

The number of microbes are then counted. The result of this method depends on:

• Composition of medium should be a general purpose medium which allows the growth of most of the microbes

• Temperature of incubation should be accurate for the growth of the microbes

2) Specific method for specific microbes

There are selective methods for specific group of microbes in microbiological examination of sewage. The most important test is for coliform present in sewage .c Coliforms group is defined to include all aerobic and facultative anaerobic gram negative, rods which ferment lactose with the production of acid in gas within 48 hours at 35 degrees celsius. Most important members found in sewage polluted waters are E.coli,E freundic and Enterobacter aerogens.

Presence of coliforms is detected by multiple tube fermentation technique which includes three steps

• Presumptive test

• Confirmed test

• complete test

Enumeration of coliforms

The presumptive test procedure may be used in estimating the number of coliforms present in a given sample. This is done by transferring the small measured amount of sewage water from positive lactose broth to a petri plate containing EMB Which are pink and mucoid represent enterobacter and colonies growth showing green metallic shine. Colonies are counted and the total number of coliform in a given volume of sewage is expressed.

Detection of faecal Streptococci

The faecal Streptococci group are indication of fish pickle pollution of water because the general habitat of these organisms is the intestine of men. These are gram positive ,cocci and ferment glucose with production of acid. Only multiple tube fermentation tests done (include two steps) for the detection of these faecal Streptococci.

1. Presumptive test

In this test the medium used is sodium azide glucose broth. Tubes are inoculated with sewage and incubated at 37 degrees Celsius. Tubes showing positive result i.e turbidity (this medium is selective for gram positive bacteria because sodium azide inhibit gram negative bacteria).

2. Confirmed test

Medium used for this is Ethyl Violet Azide Glucose (EVAG)broth. This medium inhibit virtually all bacteria accept faecal Streptococci. Broth from positive presumptive test is transferred to EVAG .The presence of Streptococci is indicated by the firm formation of a purple button at the bottom of the tube or occasionally by a dense turbidity.

For enumeration ,diluted sample can be plated on EVAG agar and colonies counted.

Total number of colonies multiplied by dilution factor gives number of Streptococci in savage

Drawbacks

• Only small sample can be examined at one time

• Several days are required for incubation of successive cultures

• The test is largely qualitative ,only relatively rough estimated the number of microbes

• Expensive medium and equipments are needed

Standard membrane filter technique

Membrane filters can be used for enumeration of microbes in a given sample of sewage, only large amount of sample can be passed through a multi pores assembly and then filterate is removed with the help of sterilised forceps and is placed on a suitable medium .The medium percolates through the pores of the filter and the colonies are formed on the filterate. After incubation ,these colonies are counted and expressed . Membrane filter technique can be used for enumeration of different groups of microbes present in sewage. This can be done by passing small amount of sewage water through filter and keeping the filter in specific media

Advantages

• It is possible to filter rapidly on the spot and immediately after collection

• large sample of sewage or water can be tested

• it gives a direct count

• It permits the concentration of small number of organisms from large quantity of samples

• Different types of microbes example Coliforms, faecal coliform, faecal streptococci or indeed any all organisms that can grow on any particular condition can be a numerated.

• It gives a permanent record in the form of preserved filter discs.

Disadvantages

The sample heavely contaminated with algae or other material or sewage with large sediments cannot be analysed by this method as formation of clogging or filter prevent testing of sample.

Enumeration by most probable number

This is a method which does not give the exact number of coliforms in the given sample but it provides an index of pollution, that is a very good idea about the most probable number of coliforms

Procedure

A large number of tubes are taken each with lauryl tryptose broth (lactose broth ). The tubes are inoculated with different volume of samples . Example: five tube with 10 ml sample, 5 take with 1 ml sample and five with 0.1 ml sample. After incubation the number of positive and negative types is calculated from each set. A combination of the number of tubes i.e positive and negative tubes , provides us an index of pollution which is usually expressed as the most probable number coliforms per 100 ML of sample . This is the most common method used to test the portability of water . By this method , the number of organisms present is not absolute but is a statistically estimated.

Significance of enumeration

• In food microbiology presence of specific bacteria ,its growth rate in food should be analysed ,to control safety hazards the capacity of spoiling the food and to ensure correct product characteristics.

• Some human activities in the mines support the growth of sulfur oxidising bacteria that produce sulfuric acid . The high concentration of this acid drop the pH level of the water 2 below to 0.0 causes corrosive nature of the liquid which is very harmful so enumeration of these bacteria from these water may help.