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After completing this practical you will be able to:
1- Know various methods used for testing potable water.
2- Test the quality of the potable water for drinking.
Drinking water is acceptable and fit for drinking when it is clear, colourless, odourless and without disagreeable taste. Microscopically it should be free from pathogenic organisms.
Natural sources of water usually contain some saprophytic bacteria, such as Pseudomonas, Serratia, Flavobacterium, Chromobacterium, Acinetobacter, Alcaligenes, etc. These saprophytes are harmless.Water gets contaminated by pathogens which are introduced into water by sewage pollution. A wide varieties of diseases are transmitted by contaminated water. There are some indicator organisms, whose presence indicates the contamination of water with fecal matter. These are:
2 Fecal (thermotolerant) coliforms.
3 Faecal Escherichia coli.
4 Faecal Streptococci, and
5 Clostridium perfringens.
Coliforms are defined as members of the family Enterobacteriaeceae which grow in the presence of bile salts and produce acid and gas from lactose within 48 hours at 37°C. In order to include anaerogenic bacteria and those which are nonlactose fermenters, it has been modified as the members of the Enterobacteriaeceae capable of growing galactosidase at 37°C that normally posses.The total coliform count is widely regarded as the most reliable indicator of potable water quality. However, the presence of coliforms not necessarily indicate faecal or sewage contamination, because, these organisms are present in large quantities in the environment.
Faecal or thermotolerant coliforms
These satisfy the criteria for coliforms but have the additional property of the ability to grow at a higher temperature 44°C.
Faecal Escherichia coli
These are defined as thermotolerant coliforms which ferment lactose (or) mannitol at 44°C with the production of acid and gas within 24 hours and also form indole from tryptophan at 44°C. The presence of E. coli is considered as the contamination of water with feces of human or animal origin.
These are catalase negative, Gram positive cocci present in the intestinal tract of man and animals. These organisms have the Lancefield group D antigen, hydrolyse aesculin and can grow at 45°C, in the presence of azide and 40% bile. Such organisms which can survive 60°C for 30 min and can grow at 10°C at pH 9.6 and in 6.5% of sodium chloride (NaCl) belong to the genus Enterococcus (Enterococcus faecalis and E. faecium) .
The presence of this organism in water in the absence of other indicators of contamination of water implies remote or intermittent fecal pollution. Viruses in water are destroyed by chlorination. When the concentration of free residual chlorine is at least 0.5 mg per litre, for a minimum contact period of 30 minutes at pH below 8 and a turbidity of 1 nephelometric turbidity unit or less, protozoa such as Entamoeba histolytica, Giardia species and Balantidium coli may be present in the drinking water. Coliforms are not the reliable indicator of protozoal contamination.
Collection of water samples
Water is collected in heat sterilised bottles containing a sufficient volume of sodium thiosulphate to neutralise the bactericidal effect of any chlorine or chloramine in the water. For collection from tap, water is allowed to run to waste for 2–3 min. before collecting it into the bottle. When collecting water samples from lakes or streams the bottle is opened at a depth of about 30 cm with its mouth facing the current. At least 100 ml of water to be tested is collected in each bottle.
After collecting the water in the bottle, the bottle is stoppered, and sent to the laboratory as quickly as possible within 6 hours keeping it in a cool container and protecting it from light.
The following tests can be done for bacteriological analysis of water:
1 Plate count.
2 Detection of coliform bacteria and E. coli.
a Presumptive coliform count: multiple tube technique.
b Differential coliform test.
c Membrane filtration method.
3 Detection of faecal streptococci.
4 Examination of Cl. perfringens, and
5 Test for pathogenic bacteria.
This consists of inoculating the nutrient agar with water to be tested and incubating the agar aerobically in parallel at 37°C for 1–2 days and at 22°C for 3 days. After incubation number of the colonies formed in the agar are counted. Those which grow at 37°C are associated with organic material of human or animal origin and those growing at a lower temperature are mainly saprophytes that normally inhabit water, soil, and vegetables. The agar count at 22°C gives an indication of the amount of decomposing organic matter in the water available for bacterial nutrition. Though these are non-pathogenic the greater the amount of organic matter present, the more likely is the water to be contaminated with parasitic and potentially pathogenic organisms.
The agar count at 37°C is a more important index of dangerous pollution.
Detection of coliform bacteria and E.coli
Presumptive coliform test – Multiple tube
The test is called presumptive, because of the presumption that the reactions are due to coliforms organisms. The count is made by adding varying quantities of water (0.1 ml–50 ml) to bile salt lactose peptone water with an indicator for acidity. Acid and gas formation indicate the growth of coliform bacilli.
Differential coliform test
This is called Eijkman test. This test is usually employed to find out whether the coliform bacilli detected in the presumptive test are E. coli or not. After the usual presumptive test, subcultures are made from all the bottles showing acid and gas to fresh tubes of single strength MacConkey medium already warmed to 37°C. They are incubated at 44°C for 24 hours. Those showing gas in Durham’s tubes contain E. coli. From the number of positive tubes obtained, results are read off the probability tables. Further confirmation of the presence of E. coli is done by testing for indole production and citrate utilization tests.
Membrane filtration method
A measured volume of water is filtered through a Millipore filter. All the bacteria present are retained on its surface. This is then placed on suitable media and incubated at the appropriate temperatures. Colonies on the surface of the membrane are counted. After 18 hours of incubation the presumptive coliform counts and detection of E. coli can be directly made.
Detection of faecal streptococci
Subcultures are made into tubes containing 5 ml of glucose azide broth from the positive bottles in the above test. Streptococcus faecalis if present produces acid in the medium within 18 hours at 45°C.
Examination for Cl. perfringens
Water is incubated in litmus milk medium at 37°C for 5 days, if positive, stormy fermentation occurs.
Examination for pathognic bactrial cells test
For other special pathogens like Salmonella, Vibrio, etc. corresponding special media are used.
In this chapter Presumptive coliform test and differential coliform test will be discussed.
Bacteriological incubator, autoclave and water bath.
MacConkey broth, brilliant green bile broth Preparation of MacConkey broth: This is prepared by mixing pancreatic digest of gelatin, 20 grams; lactose, 10 grams; bile salt, 5 grams; bromocresol purple, 0.01 grams and final pH (at 25°C) is adjusted at 7.3 ± 0.2.
Preparation of double strength medium: It is prepared by suspending 35 grams in 500 ml of distilled water and heated to dissolve the medium completely. 50 ml and 10 ml of the media are dispensed into screw capped bottles with inverted Durham’s tubes and are sterilized by autoclaving at 15 lbs (121°C for 15 minutes).
Preparation of single strength medium: It is prepared by suspending 3.5grams in 100 ml of distilled water. 5 ml quantities of the medium are dispensed in screw capped bottles with inverted Durham’s tubes and are sterilized by autoclaving at 15 lbs (121°C for 15 minutes).
Preparation of brilliant green bile broth (BGBB): This is prepared by mixing peptic digest of animal tissue, 10 grams; lactose, 10 grams; ox gall, 20 grams and brilliant green, 0.0133 gm and adjusting the final pH (at 25°C) to 7.2 ± 0.2. 4 grams of the medium is suspended in 100 ml distilled water and mixed well. The media is distributed in 5ml volumes in test tubes with inverted Durham’s tubes and sterilized by autoclaving at 15 1bs pressure (121°C) for 15 minutes.
Water specimen to be tested.
1- Collect 500 ml of water in a sterile bottle.
2- Inoculate water sample immediately into the MacConkey broth medium.
3- Inoculate 50 ml of water into 50 ml double strength media (1 bottle)
4- Inoculate 10 ml of water into 10 ml double strength medium (5 bottles).
5- Inoculate 1 ml of water into 5ml single strength medium (5 bottles).
6- Incubate all the bottles at 37°C for 48 hours.
7- Check for acid and gas after 24 hours and 48 hours.
8 -Any positives are inoculated into brilliant green bile broth and peptone water.
9 -Incubate at 44°C in a water bath overnight.
Sterility of media and strength of media should be properly checked.
Observe for the presence of gas and production of indole. Presence of gas in brilliant green bile broth and indole production at 44°C is indicative of the presence of E. coli. For interpretation refer MacGrady’s table.
RESULTS AND INTERPRETATION
Presumptive coliform count The no. of bottles showing acid and gas is counted and compared with the MacGrady’s table. Result is expressed as- coliforms most probable no (MPN)/
100ml. e.g. for Presumptive coliform count No. of tubes giving positive reaction 50 ml 10 ml 1 ml * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *1 * * * *2 * * * 2
Result: Coliforms most probable no 10/100 ml.
Differential coliform count
The no of tubes showing positive results i.e. both gas productions in BGBB and Indole test at 44°C is compared with MacGrady’s table. Result is expressed a Escherichia coli Most probable No (MPN) / 100 ml. Eg. for Differential coliform counts: No. of tubes giving positive reaction.
50 ml * 10 ml * *1 ml
1 * * * * * 1 * * * * *1
Results: Escherichia coli MPN—— 5 /100 ml
Grades of the quality of drinking water supplied as determined by results of periodic Escherichia coli and coliform counts is listed in the table 84-1.
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وجعله في ميزان حسناتكم
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