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The below mentioned article provides an overview on Bacteriology of Water:- 1. Bacteriology of Water 2. Biological Hazards of Water Supplies 3. Bacteriological Indicators 4. Bacteriological Diagnosis 5. Presumptive Coliform Test 6. Differential Coliform Test 7. Interpretation of Total Count 8. Biological Examination 9. Bacteriological Examination of Sewage and Sewage Effluents 10. Isolation of Pathogens.
Contents:
- Bacteriology of Water
- Biological Hazards of Water Supplies
- Bacteriological Indicators
- Bacteriological Diagnosis
- Presumptive Coliform Test
- Differential Coliform Test
- Interpretation of Total Count
- Biological Examination of Water
- Bacteriological Examination of Sewage and Sewage Effluents
- Isolation of Pathogens from Water and Sewage
1. Bacteriology of Water:
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Soft water should be free from microorganisms and chemical substances and drinking water in particular should not only be safe but also pleasant to drink. Drinking water is liable to contamination with sewage and other excreted materials which may cause intestinal and systemic infections. Much of the ill-health in developing countries is attributable to lack of drinking water.
The hazards of water pollution are classified into two broad groups:
(1) Biological, and
(2) Chemical.
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Chemical pollutants of diverse nature (e.g. detergents, solvents, cyanides, heavy metals, minerals, sulfides, ammonia, toxic compounds) derived from industrial and agricultural wastes are increasingly finding their way in the public water supplies.
2. Biological Hazards of Water Supplies:
Faecal pollution of water supplies may lead to introduction of a variety of intestinal pathogens that comprise water-borne diseases. The object of microbiological study of water supplies is to detect whether water has been polluted by pathogenic organisms such as shigella, salmonella, cholera vibrios, etc.
Though it would be ideal to demonstrate the possible pathogenic organism in water, but, if present, they are usually few and for outnumbered by non-pathogenic organisms.
As a result their detection is not only technically difficult but also costly in time and money. Therefore we rely on tests that detect indicators of human/animal pollution in water, the presence of commensal bacteria of intestinal origin. The organisms most commonly used as indicators of pollution are Esch. coli and coliform group.
3. Bacteriological Indicators:
These are based on organisms indicative of pollution of water by human/animals faeces such as:
(1) Esch. coli and coliform group as whole.
(2) Faecal streptococci (Str. faecalis), and
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(3) Clostridium perfringens.
While evaluation faecal pollution of water supplies, one has to keep in view the bacteriological flora in water as natural almost always contains a few harmless organisms. 
1. Coliform:
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Lactose fermenting coliforms:
Esch. coli, Klebsiella, Enterobacter. Coliform groups include both faecal and non-faecal organisms. Esch. coli is the typical example; Klebsiella sp is an example of non-faecal groups. All coliforms are assumed to be of faecal origin unless a non- faecal origin can be proved.
For the following reasons, coliforms are chosen as indicators of faecal pollution:
(i) Commensals:
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Esch. coli are present in great abundance in intestine of man—about 200- 400 billion of these organisms are excreted by a man per day and their presence in water is an indication of faecal contamination.
(ii) Detection:
Esch. coli and other coliforms can easily be detected in cultures, even one bacterium in 100 ml water.
(iii) Survival:
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Esch. coli can survive longer for days or weeks, while the pathogens tend to die out more rapidly.
(iv) Resistance:
Coliform offers greater resistance to purification process (e.g. chlorination) than pathogens. Thus when Esch. coli are present in sample of water, the assumption is the probable presence of intestinal pathogen in the water supply.
2. Faecal Streptococcus ( str. faecalis):
They are regularly found in faeces though their number is much less than Esch. coli. Their presence in water are not regarded as confirmatory evidence of recent faecal contamination of water in doubtful cases.
3. CI. Perfringens:
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They also occurs in faeces regularly. They are also excreted in much smaller number than Esch. coli. Presence of spores indicate faecal pollution.
4. Bacteriological Diagnosis:
I. Collection of Samples:
Samples are to be collected in sterilised glass bottles of 230 ml with ground glass stoppers protected in kraft paper:
1. Sampling from Tap Pump Outlet:
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Before collecting sample the mouth of the tap or hand pump should be flamed with a blow lamp or spirit lamp. A tap is turned at maximum rate and the water flows for 5 minutes, bottles are then filled with water.
2. Samples of Water from Reservoir (stream, rivers, lakes and tanks):
The stopper is removed carefully by one hand and with another hand the bottle is held at the base and submerged in a depth of about 20 cm with the mouth upwards. If there is a current, the bottle should be faced to the current.
3. Sampling from a Dug Well:
A stone of suitable size is attached to the sampling bottle with a string. Then a clean cord of suitable length is tied with the bottle and lowered to the required depth and filled with water. The bottle should not touch the sides of wall any time, water-filled bottle is then quickly raised to the surface and stoppered.
Transport:
The sample should be examined within one hour and up to 3 hours; where delays for more than 3 hours are expected the bottles should be kept on ice or in an icebox.
Neutralisation of Chlorine:
When a sample is collected from a chlorinated water supply the chlorine should be neutralized immediately by sodium thiosulphate.
II. Methods of Analysis:
Standard tests usually employed in water bacteriology are:
(a) Presumptive coliform test and differentiate or confirmatory tests for Esch. coli, Str. faecalis.
(b) Detection of Str. faecalis and CI. perfringens.
(c) Colony count-enumeration of viable bacteria (also known as plate count).
5. Presumptive Coliform Test:
Two methods are in use for estimation of probable number of coliform bacilli in water, multiple tube method and membrane filter (MF) method. The multiple tube method is widely used.
Multiple Tube Method:
By this test the most probable number (MPN) of coliform organisms are detected in 100 ml water.
Media:
Double strength and single strength modified MacConkey fluid medium containing bromocresol blue sterilised in bottles tubes containing Durham tube of indication of gas production.
Procedure:
Measured amounts of sample of water are added by sterile graduated pipettes as:
(i) 50 ml of water to 10 ml double strength medium.
(ii) 10 ml water to 10 ml double strength medium.
(iii) Five 1 ml quantities to each 5 ml double strength medium.
(iv) 0.1 ml quantities of water to each single-strength medium.
The bottle/tubes are incubated at 37°C for 48 hours. An estimated coliform count is made from the tubes showing acid and gas production by the help of a Statistical table. The reaction may occasionally be due to combination of organisms or due to other organisms.
6. Differential Coliform Test:
Eijkman test is usually done to confirm whether coliform bacilli detected in the presumptive test are Esch. coli or not. Esch. coli produces gas when grown in bile salt lactose peptone water at 44°C but a typical coliform bacilli tube is sub-cultured in two tubes of single strength — MacConkey’s medium with inverted Durham tube at 44°C in water bath for 24 hours. Those yielding gas in Durham’s tube contain Esh. coli.
Confirmation of Esch. coli may be made by test for indole production as shown in the following table :
Sometimes spore bearing bacilli may give false positive reactions in the presumptive test, hence the presumptive positive tubes should preferably be placed on solid medium to confirm the presence of coliform.
Membrane Filter (MF) Method:
The membrane filter technique is used in some countries for the presence of coliform as standard test. A measured volume of water is filtered through a membrane specially made of cellulose ester. Bacteria are retained on the surface of membrane.
The membrane is inoculated (face up-wards) in suitable medium for 15-20 hours and the number of colonies are counted directly. An advantage in that the bacterial colonies can be counted in 20 hours but it is not suitable for turbid water.
B. Detection of Faecal Streptococci and CI. Welchii:
Their presence in water provides useful confirmatory evidence of their pollution of water in doubtful cases. Str .faecalis. Subcultures from presumptive positive bacteria bottles in coliform test are made into tubes containing 5 cc of glucose azide broth and incubated at 45°C.
Production of acid in the medium indicates presence of Str. faecalis.
CI. welchii:
The water in question is inoculated in litmus milk medium and incubated anaerobically at 37°C for 5 days and looked for stormy fermentation.
C. Colony Count:
One ml. test sample of water is placed in petri dish (10 cm diameter) and then 10 ml. melted yeast agar (45°C-50°C) is poured on the water, mixed thoroughly and allowed to solidify. One more plate is prepared. One plate is incubated at 22°C and other at 37°C for about 18-24 hours. After incubation the colonies are counted using hand lens. Each colony represents one viable bacterium in the original specimen.
Colony count provides an estimate of total numbers of viable bacteria present in 1 ml specimen. Sample may be diluted when it is tubed and total count is made by multiplying the number of colonies with the dilution factor.
7. Interpretation of Total Count:
(i) Growth at 22°C indicates the amount of decomposing organic matter present in water. Organisms growing at 22°C are usually non-pathogenic and however, in the presence of excess organic matter the sample is likely to be contaminated by parasites and potentially pathogenic organisms.
(ii) Growth at 37°C is more important index of contamination of water sample. A high count of bacteria at 37°C is undesirable for water consumption purpose. It also causes spoilage of food and drink at home and also in factory.
8. Biological Examination of Water:
Sometimes water may contain microscopic organisms such as algae, fungi, yeast, protozoan, minute worms, etc. which are collectively known as plankton. These plankton organisms are index of pollution and produce objectionable taste in water.
Interpretation of results: It is not possible to lay down a rigid bacteriological standard to which all drinking water supplies should conform. As already mentioned, bacterial flora of water differs widely to different sources such as well, river, lake or reservoir.
On the basis of repeated tests of water a standard has been suggested by pioneer workers in the field into Class 1, Class 2, Class 3, and Class 4 types — based on presumptive coliform count per 100 ml sample shown in below table.
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Throughout the year, 50% of samples should fall into Class 1, 80% should not fall below Class 2 and the remainder should not fall below Class 3.
(ii) With deep tube well or other pure waters that are normally into Class 1, sudden deterioration in standard even to Class 2 is to be considered as significant.
9. Bacteriological Examination of Sewage and Sewage Effluents:
Method is same as for water:
1. Viable Bacteria:
It is estimated by plating and counting colonies. A small amount of specimen of water is used for testing coliform bacilli. Amount of water to be tested depends on the likely extent of pollution of the effluent. Bacterial count in sewage water varies between 1 million in 100 million per ml crude sewage.
2. Enteric Pathogens:
Sewage water for membrane filter technique is employed during isolation of salmonellae from communal sewage.
10. Isolation of Pathogens from Water and Sewage:
Sometimes water may have to be examined for presence of specific pathogens (e.g. Salmonella, Shigella, Vibrio cholera, etc.).
1. Concentration:
Concentration of the organisms in the sample yields better result. Sample may be concentrated by membrane filter technique. Pathogenic bacteria retained on the surface of membrane are transferred into a suitable differential medium and incubated.
2. Dilution Method:
As the number of pathogenic bacteria present may be scanty, larger volume of multiple samples may have to be examined for isolating the organisms. A volume of 10 ml of enrichment and selective media (selenite broth for salmonella, alkaline peptone water for Vibrio cholera), is mixed with nine times its volume of water and incubated for 24 hours and then sub-cultured in suitable media.
Isolated organisms are identified by biochemical test and serotyping.
