The following points highlight the top four tests for the presence of coliforms in water. The tests are: 1. Sanitary Tests for Coliforms 2. The Most Probable Number of Coliforms 3. The Defined Substrate Test 4.IMViC Tests.
Contents
Test # 1. Sanitary Tests for Coliforms:
The original test for the presence of coliform in water is done by standard multiple tube fermentation technique.
This method involves the three routine standard tests:
(a) The presumptive test,
(b) The confirmed test, and
(c) The complete test (Fig. 31.3).
(i) Presumptive Test:
A series of fermentation tubes each containing lactose broth or lauryl tryptose broth of known concentration, are inoculated with known amount of water. These tubes are incubated for 24 to 48 hours at 35°C (Fig. 31.3A).
Generally, five fermentation tubes containing single or double strength broth are inoculated with 10 ml water, 5 tubes with 1 ml water and 5 with 0.1 ml water. At the end of 24 hours of incubation, the tubes indicate that the coliforms are absent. These tubes are incubated for an additional 24 hour to be sure for the absence of coliforms (i.e. gas production).
Chambers (1950) showed that 40 to 390 million per ml coliforms were required to produce visible gas in fermentation broth. The average number was 170 million per ml. However, in most of the cases 75 million coliforms per ml were required to produce the gas. This difference may be due to the ratio of coliforms to non-coliforms. The non-coliforms, if in high number, reduce the gas formation.
(ii) Confirmed Test:
If a positive test of gas production is obtained, it does not mean that coliforms are present. The other organisms too also give false positive presumptive test because they are also capable of fermenting lactose with formation of acid and gas. The positive presumptive test is resulted due to synergism i.e. joint action of two microorganisms on a carbohydrate with production of gas which is not formed if both are grown separately.
In addition, if yeasts, species of Clostridium and some other microorganisms are present, gas is also produced. Therefore, a confirmed test is performed for the presence of coliforms. All fermentation tubes showing gas within 48 hours at 35°C are used for confirmed test.
It is of two types as described below:
The positive presumptive fermentation tube is gently shaken. A drop of its culture is transferred to brilliant green lactose bile broth fermentation tube (Fig. 31.3B). The tubes are incubated for 48 hours at 35°C. The appearance of gas within this period indicates for positive confirmed test. The dye (brilliant green) inhibits the Gram-positive bacteria and synergistic reactions of Gram- positive and Gram-negative bacteria for a common food base.
The second confirmed test is done by eosine methylene blue (EMB) agar or endo agar method. In eosine methylene blue agar method, a definite amount of two stains (eosin and methylene blue) is added to a melted lactose agar.
The medium is poured into Petri dishes. Over the surface of EMB agar medium, a loopful culture from each positive fermentation tube is streaked. Plates are incubated at 35 °C for 24 hours keeping them in inverted position.
There develops three types of colonies:
(a) Typical colonies (nucleated, with or without metallic sheen),
(b) Atypical colonies (opaque, non- nucleated mucoid after 24 hours of incubation, pink), and
(c) Negative colonies (all other types).
The development of typical colonies shows that the confirmed test is positive.
The endo agar medium is prepared by adding basic fuchsin (previously decolourised with sodium sulfite) to a melted lactose agar base. Medium is poured into Petri dishes. A loopful culture from each fermentation tube is streaked over the surface of medium. The plates are incubated for 24 hours at 35°C. Different types of colonies appear after 24 h. After lactose fermentation, acetaldehyde is produced which is trapped in endo agar.
Acetaldehyde reacts with sulfite to form an additional compound. This results in release of the basic fuchsin from the combination into the agar. Consequently,, agar turns into a deep red colour. The metallic gold like sheen appearing on the surface of typical colonies is due to the precipitation of liberated stain. The stain which is restored appears purple in colour.
(iii) Completed Test:
In the last the completed test is performed to ascertain about the presence of coliforms in water.
The purpose of the completed test is to determine whether:
(a) The colonies growing on EMB or endo agar are again capable of fermenting lactose and forming acid and gas, and
(b) The organisms transferred to agar slants show the morphological appearance of coliform group.
Each colony that gives positive confirmed test is transferred to lactose fermentation tube and to nutrient agar slants (Fig. 31.3C).
The tubes are incubated at 35°C for 48 hours. Production of gas in fermentation tubes and, demonstration of Gram-negative, non spore forming rods on the agar slants constitute a positive completed test for coliforms. The absence of gas and the rod production confirms for negative test of coliforms.
Test # 2. The Most Probable Number of (MPN) of Coliforms:
For the first time Hoskins (1934) computed the MPN to evaluate coli-aerogenes test by fermentation tube method. Table 31.3 is based on the general formula of Hoskins (1934) for calculating the numbers of coliform microorganisms present in 100 ml of water.
The figures are based on the use of five tubes as described in presumptive test. By referring to a MPN table (Table 31.3.), a statistical range of the coliform numbers is determined by observing the number of broth tubes producing gas. Confirmed and positive tests of Fig. 31.3B are used to calculate the MPN.
The Membrane Filter Technique:
Goetz and Tsuneishi (1951) described a new method for enumeration of coliform microorganisms in water to which they named as millipore filter technique. However, it is often referred to as membrane filter technique. This technique has been accepted as the standard method for the microbiological examination of sewage, water, etc.
The filtering apparatus consists of a glass or stainless steel funnel, and a flask. The funnel of stainless steel is clamped to a base containing a molecular filter. The stem of base is inserted into a fitter flask through a rubber stopper (Fig. 31.4 B).
A sterile membrane filter (0.45 µm) disk is placed in the sterilized holding apparatus (A). A volume of water is passed through filter disk. Bacteria present in water sample are retained on filter disk. The sides of funnel and membrane are rinsed with sterile distilled water.
Thereafter, the membrane filter disc is aseptically removed by a sterile forceps and placed on absorbent disk saturated with culture medium and contained in Petri dish (C). The medium passes through the pores of membrane and nourishes the bacteria present on it (D). After proper incubation at 35°C for 24 h each bacterium multiplies to form a visible colony on membrane (D). The colonies are easily counted. This method has both advantages and disadvantages.
Advantages to Membrane Filter Technique:
Following are some of the advantages of membrane filter technique:
(a) It permits the small numbers of bacteria from large quantities of water. Therefore, it increases the accuracy and reliability of counting bacterial colonies.
(b) It does not allow to spread the combination of any number of bacteria from a few to 5,000 at a time. Therefore, there is no need of making dilutions of water.
(c) It permits the separation of bacteria from their nutrients at any time.
(d) It allows the direct counting of microorganisms instead of counting most probable number.
(e) It is time saving method, permitting faster differentiation of bacteria and giving a permanent record if filter disks are preserved.
(f) This method is very useful in emergency.
Disadvantages to Membrane Filter Technique:
This technique has the following disadvantages:
(a) In turbid waters containing algal growth and other materials the pores of membrane filter are clogged. Therefore, the filter prevents the testing of sufficient sample and fails to give accuracy of coliforms.
(b) High populations of non-coliforms and other bacteria cause overgrowth. Therefore, these cannot be counted accurately.
(c) Metals and phenols can be absorbed to membrane filter and, therefore, inhibit growth of bacteria retained on membrane filter.
Test # 3. The Defined Substrate Test:
The defined substrate test is used for both coliforms and E. colt in a single 100 ml water sample.
A specialised medium containing 0-nitrophenyl-β-D-gaIactopyranoside (ONPG) and 4- methylumbelliferyl-β-D-glucuronide (MUG) as the only nutrients are added with a water sample of 100 ml. The incubation bottles containing water plus both nutrients along with control are incubated at 35°C for 24 hours.
If coliforms are present the medium will turn yellow within 24 hours due to break down of ONPG. When E. coli is present, the MUG is changed to yield a fluorescent product.
The fluorescence is observed under long-wavelength UV light for the presence of E. coli. If the colour does not develop and give negative report for the presence of coliform, the water is suitable for drinking. If coliforms are present, E. coli or faecal coliforms must be detected in water.
Test # 4. IMViC Tests:
E. coli and Aerobacter aerogenes are the most important bacteria of coliform group, the former is commonly known as faecal contaminant and the later as non-faecal contaminant. Morphologically both are similar and cannot be distinguished. Therefore, biochemical tests are being used to differentiate each of them.
The tests differentiating them are collectively known as IMViC tests (or reactions). The abbreviation IMViC is prepared by using first letter of four different tests viz., indole, methyl red, Voges-Proskauer, and citrate tests. The reactions of E. coli and A. aerogenes are shown in Table 31.4.
