How to Detect the Presence of Bacteria in Water? | Water Engineering

For detecting the presence of bacteria of coliform group and measuring their concentration in water the following methods are adopted:

I. Total Count or Agar Plate Count Test:

In this lest, bacteria are cultivated on specially prepared medium of agar for different dilutions of sample of water with sterilised/distilled water. The dilutions commonly used are 1/10, 1/100 etc. The diluted sample is placed in an incubator for 24 hours at 37°C (human body temperature) or for 48 hours at 20°C.

These represent the so called hot counts and cold counts respectively. The bacterial colonies which are formed are then counted and the results are computed per 100 ml. For drinking water the total count should not exceed 1 per 100 ml.

II. E-Coli Test:

This test is divided into the following three parts:

(a) Presumptive test

(b) Confirmed test

(c) Completed test

(a) Presumptive-Test:

The presumptive test is based on the ability of coliform group to ferment the lactose broth and producing gas. The confirmed test consists of growing cultures of coliform bacteria on media which suppress the growth of other organisms. The completed test is based on the ability of the culture grown in the confirmed test to again ferment the lactose broth.

In this test the following procedure is adopted:

(1) Definite amounts of diluted samples of water are taken in multiples of ten, such as 0.1 ml, 1.0 ml, 10 ml, etc.

(2) The water sample is placed in standard fermentation tubes containing lactose broth.

(3) The tube is placed in an incubator and kept there at a temperature of 37°C for a period of 48 hours.

(4) If gas is seen in the tube after this period is over, it indicates the presence of E-coli group and the result of test is treated as positive. If no gas is seen, it indicates the absence of E-coli group and the result of test is treated as negative.

(5) A negative result of presumptive test indicates that water is fit for drinking.

(b) Confirmed Test:

The confirmed test is required because the production of gas in the lactose broth during the presumptive test does not necessarily indicate the presence of bacteria of the coliform group, as there may be other bacteria present which also fermant lactose.

The confirmed test is carried out in one of the following ways:

(1) A small portion of the incubated material from the fermentation tubes showing gas in the presumptive test is carefully transferred to another fermentation tube containing brilliant green lactose bile broth. This tube is placed in an incubator and kept there at a temperature of 37°C for a period of 48 hours.

If gas is seen in the tube the presence of E-coli group is confirmed and the completed test becomes essential. The brilliant green lactose bile broth is used in this test because it inhibits the growth of bacteria other than the bacteria of coliform group.

(2) A small portion of the incubated material from the fermentation tubes showing gas in the presumptive test is marked as streaks on the plates containing Endo or Eosin-methylene-blue agar. The plates are kept in an incubator at a temperature of 37°C for a period of 24 hours. If coloines of bacteria are seen after this period, it indicates the presence of E-coli group and the completed test becomes essential.

(c) Completed Test:

This test is carried out by introducing or inoculating bacterial coloines or cultures grown in the confirmed test into lactose broth fermentation tubes and agar tubes. The tubes are placed in an incubator and kept there at a temperature of 37°C for a period of 24 to 48 hours.

If gas is seen in the tubes after this period, it indicates the presence of E-coli group and the result of the test is treated as positive and further detailed tests are carried out to detect the particular type of bacteria present in water. Again the absence of gas indicates negative result and water is considered to be safe for drinking.

III. Membrane Filter Technique:

It is the simplest and the most recent method used for detecting the presence of bacteria of coliform group and measuring their concentration in water. In this method the water sample is filtered through sterile membrane having porosity 80% and microscopic pores (pore size 5 to 10 mμ.). The bacteria present in water are retained on the membrane.

The membrane with the bacteria so isolated is then put in contact with a suitable nutrient (M-Endo’s medium) which inhibits the’ growth of bacteria other than the bacteria of coliform group. It is then placed in an incubator and kept there at a temperature of 37°C for a period of 20 hours. The bacteria of coliform group if present in water are developed into visible colonies which can be counted with the help of microscope.

The advantages of this method are:

(i) The test is simple and it can be readily performed in the field;

(ii) The procedure is more precise;

(iii) It requires much less time than the presumptive and the confirmed tests; and

(iv) Even the densities of bacteria of coliform group less than 1 per 100 ml can be detected.

E-Coli Index (or Coliform Index) and Most Probable Number (MPN) Index:

E-coli index (or coliform index) is a measure of the concentration of coliform bacteria or E-coli in water. For a single water sample it is defined as the reciprocal of the smallest quantity of the water sample in ml, which would give a positive E-coli test. For example, if three dilutions of water sample, 1:1, 1:10, and 1:100, are tested and a positive result is found in both the higher dilutions, the smallest quantity of the water sample that will give a positive result is 1/100 ml.

Thus by definition, the E-coli index for the water sample is 100. However, if in any case a negative result is obtained in a dilution between two positive results, then the positive observation at the higher dilution is interchanged in the record with its next lowest negative result and the index is based on the changed data.

Further a more accurate value of E-coli index would be obtained if it is computed from a series of tests, for which the following procedure is adopted:

(i) Record in decreasing order of magnitude the quantities of water sample tested.

(ii) Record the total number of tests carried out and the number of positive results obtained for each quantity of water sample tested.

(iii) Record the ratio of the number of positive results obtained to the total number of tests carried out in each case.

(iv) Record the difference between successive ratios.

(v) Find the product of each difference and the reciprocal of the larger quantity of water sample.

(vi) The sum of these products gives the E-coli index.

The following example is given to illustrate the above noted procedure for computation of E-coli index:

Example:

For determining E-coli index tests were carried out on water samples collected daily for a month at a particular filter plant and the results obtained are as indicated in the first three columns of the following table. Column (1) indicates the quantities of water sample tested; column (2) indicates the total number of tests carried out; and column (3) indicates the number of positive results obtained in these tests. The remaining three columns in the same table indicates the various computations.

Column (4) indicates the ratios of the number of positive results obtained to the total number of tests carried out, i.e., column (3) ÷ column (2) column (5) indicates the difference between successive ratios; and column (6) indicates the product of the difference in ratios and the reciprocal of the larger quantity of water sample in column (1). The sum of column (6) gives the value of E-coli index which in this case is obtained as 12.563.

The reciprocal method of determining E-coli index is not altogether satisfactory because of anomalies that may arise in its use. In the reciprocal method, observations in column (3) must fall on the probability curve to give consistent values in column (6). For example, if in line 4 column (3), the figure were 2, the value in column (6) would be 6.7 which is contrary to the fact that for a greater number of positive results we should have a higher index.

The error results from the fact that a value of 2 in column (3) falls off the probability curve. Thus on account of the anomalies in this method another method called ‘most probable number’ method is used in which the concentration of coliform bacteria or E-coli in water is indicated in terms of Most Probable Number (MPN) index, or (Most Probable Number (MPN) of E-coli) which is defined below.

Most Probable Number (MPN) index or (Most Probable Number (MPN) of E-coli is defined as “that bacterial density, which if it had been actually present in the water sample under examination, would more frequently than any other have given the observed analytical results”.

In other words Most Probable Number (MPN) index may be defined as that bacterial density which is most likely to be present in water. For determining MPN index bacteriological tests (confirmed or completed) are carried out by taking standard quantities of water sample (or standard samples).

The standard samples in these tests may consist of five 10 ml (or 100 ml) quantities of water sample, or a combination of five 10 ml, five 1.0 ml and five 0.1 ml quantities of water sample. The other combinations of the quantities of water sample which may also be used in the standard samples in these tests are five 100 ml, five 10 ml and five 1.0 ml quantities of water sample, or five 1.0 ml, five 0.1 ml and five 0.01 ml quantities of water sample. The number of positive results of these tests is recorded and corresponding to the positive results of the tests the MPN index which gives the values of MPN index per 100 ml.

The concept of MPN index is based on the laws of probabilities and statistics and hence it provides a more rational approach for indicating the concentration of coliform bacteria or E-coli in water.