Organisms indicating sewage pollution: EXAMINATION OF WATER Organisms indicating sewage pollution: @ E. coli type I: Found in intestines of Man and animals. It is indole positive & produces gas at 44 °C (Eijkman test) @ E. coli type II: When detected in drinking water it will not suggest sewage pollution. It is indole negative & Eijkman test negative

Other organisms contaminating water: @ Klebsiella pneumoniae @ Enterobacter @ Citrobacter @ Enterococcus faecalis @ Cl. perfringes @ Salmonella @ Shigella

Significance of Coliforms: @ Coliforms are derived from intestines of animals & birds. @ They are found in small numbers in pure water supplies. @ Hence their number should be very high to indicate sewage pollution.

Diseases caused by sewage pollution @ Enteric fever @ Dysentery, @ Food-poisoning @ Cholera @ Jaundice (Hepatitis) @ Gastroenteritis in children, @ Poliomyelitis @ Aseptic meningitis, @ Intestinal worms.

Collection of water specimens @ Use 6-oz bottles ( 230 ml ) bottles @ Mouth of tap is flamed. Water is run for 5 min @ From streams, rivers & lakes, bottle is immersed with base down & turned against the current; or horizontally if current is not available.

@ Bottles should reach lab. within 3 hrs. Otherwise keep in ice. @ Neutralize chlorinated water by adding sodium thiosulphate to avoid killing of bacteria in water by Chlorine. @ Before testing, bottle must be shaken vigorously.

Methods of Water Examination Water is examined by the following methods to check if it not affected by sewage pollution: @ Presumptive coliform count @ Differential coliforms test @ Viable plate count @ Membrane filtration

Presumptive coliform count: @ Use the multiple tube technique to count number of coliforms in water @ Use double strength Mc Conkey broth in bottles with Durham’s tubes for gas. @ Acid is detected by an indicator @ Acid & gas indicate growth of coliforms @ Count is read from Mc Crady's tables.

(1) For Treated Water Samples Mac Crady Probability Tables for estimating the MPN of Faecal Coliform Bacteria (1) For Treated Water Samples

(2) For Untreated Water Samples

(3) For Untreated Water Samples

Differential Coliforms test @ A differential test for E. coli type I. @ The test is Eijkman test. Use single strength Mc Conkey broth with Durham’s tubes for gas at 44 °C @ Confirm result by a positive indole test @ To suppress growth of Cl. perfringes, use brilliant green broth .

Viable plate count: @ Prepare 2 sets of serial dilutions of water in tubes using Ringer’s solution. @ Pour water from each dilution in plates containing melted yeast extract agar. @ Incubate one set at 37°C, 2nd set at 22 °C (to detect saprophytes in soil) @ Count colonies, multiply the dilution

Membrane filtration: @ Water sample is filtered through a membrane filter, using a vacuum pump. @ The membrane with organisms on it is cultured on a pad of selective lactose broth medium. @ Incubate, colonies are counted, to give the presumptive number of E.coli in water

Significance of Bacteria detected: @ E.coli: Indicates recent fecal pollution @ Other coliforms: Derived from soil & vegetation, hence no faecal pollution @ Cl. perfringes: Its spores need a long time to germinate, hence indicates remote fecal pollution

Bacteriological standards: Class No. coliforms No. E. coli per 100 ml per100 ml 1- Excellent 0 0 2- Satisfactory 1-3 0 3- Suspicious 4-10 0 4- Unsatisfactory more than 10 0.1 or more

Examination of Milk Count of Viable Bacteria: @ Milk is serially diluted, count viable bacteria by the melted agar method as for water. Incubate at 30°C for 3 d. 2. Methlylene blue reduction test: @ In presence of bacteria, M.B. is reduced to colorless. Blue colour means no contamination of milk

3. Count of coliform bacilli: @ Use the multiple tube technique as done for water examination. 4. Detection of Brucella: @ Milk ring test: Add a suspension of Brucella stained with haematoxylin dye to the milk. A blue ring means Brucella @ Whey agglutination test: Add cheese making rennet to skimmed milk to have a clear whey. Add Brucella antigen to the whey to get agglutination.

5. Phosphatase test: @ Test for effective pasteurization. @ To detect phosphatase enzyme in milk. @ If the enzyme is absent, milk is pasteurized @ Add disodium nitrophenyl phosphate to milk. This reacts with phosphatase enzyme to give nitrophenol (yellow colour). Yellow colour indicates unpasteurized milk.

6. Turbidity test: @ This is a test for sterilized milk. @ Add ammonium sulphate, filter, & boil filtrate for 5 min. Look for turbidity. @ Turbidity is due to precipitated coagulable proteins. Milk is not sterile @ No turbidity: Proteins are precipitated by ammonium sulphate. Milk is sterile.