1. Microbiology of Water
Dr.GulveR.M

2. Water is very essential factor needed by man (used
Water is very essential factor needed by man (used for cooking, drinking, etc.)
-Open and widely accessible water, making it susceptible to contamination by chemicals and bacterial pathogens
-Once water get contaminated, it would be harmful for human consumption.

3. Detection of Microorganisms in water
Indicator and index microorganisms are
1) Coliform
2) Faecal coliforms
3) E. Coli
Detection techniques of these microorganisms are
1) Multiple tube fermentation or MPN method
2)Membrane filtration method

4. Water Borne Diseases
1)Dysentery - Shigella/Salmonella bacteria - gastro-intestinal food/water
2)Typhoid - Salmonella typhi bacteria - gastro-intestinal water/food borne. Salmonellosis - due to many Salmonella species. Water/food/direct contact borne.
3)Vibrio illness caused by the bacteria of Vibrio vulnificus, Vibrio alginolyticus and Vibrio parahaemolyticus commonly found in seafood and recreational water.
4) Cholera - Vibrio cholerae bacteria - gastro-intestinal often waterborne
5) Chronic granulomatous disease - caused by the Mycobacterium marinum infection and localized in skin, frequently occurred with aquarium keepers.
6)Diarrheal disease due to E. coli.

5. Hepatitis A: inflammation and necrosis of liver
Viral Sources of Waterborne Disease
Hepatitis A: inflammation and necrosis of liver
Norwalk-type virus: acute gastroenteritis
Rotaviruses: acute gastroenteritis, especially in children
Enteroviruses: many types affect intestines and upper respiratory tract
Reoviruses: infects intestines and upper respiratory tract

6. Coliforms
Coliforms- refers to the various genera of the family Enterobacteriaceae which are lactose fermentors and are commonly found contaminants in water.
Organisms that are under the genus Escherichia, Enterobacter, Klebsiella, Serratia, Citrobacter.
Collectively, this group of Gram-negative bacilli are referred to as "coliforms" because they share similar morphological and biochemical characteristics.
Most of these organisms are members of the normal flora of humans and/or animals and are considered opportunistic pathogens

7. E. coli are found in intestine, their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. Indicator organisms indicate that water received contamination of intestinal origin.
E. coli are Gram negative bacterium that is commonly found in the lower intestine of warm-blooded animals,
while other coliforms( Enterobacter, Klebsiella) can be found on plants and in soil.

8. Indicators of fecal pollution:
Coliforms organisms are called indicators of fecal pollution. Water may contain many types relatively harmless saprophytes whose normal habitats are soil, water and air. Water may be contaminated by domestic sewage which may contain human pathogens such agents of typhoid or paratyphoid fevers , dysentery, hepatitis and cholera. The causative agent of these disease are considered to be of fecal origin and are primary importance in water examination practically isolation of pathogenic organisms is very difficult job and also time consuming. Therefore some alternative must be there in order to show the presence of this pathogen thus E.coli or coliform organisms are considered as ‘Index of water pollution’ because of

9. Definition of coliform group: The coliform group of bacteria includes all the aerobic, and faculatatively anaerobic, Gram negative non spore forming bacilli that produce acid and gas from the fermentation of lactose.The classical species of this group are Escherichia coli and Enterobacter aerogenes. The relationship of these organisms to others of the enteric group Salmonella, Shigella, Prtotens, Pseudomonas all of these are gram negative, non spore forming bacilli. E.coli and Enterobacter aerogenes are closely related with each other on the basis of morphology and cultural characteristics. But some important differences in between these two organisms are as fallows. E.coli is a normal inhabitant of the intestinal tract of man and animals means always found in intestine but E. arongenes most frequently found in soil, on grains and plants but may occur in the feces of man and other animals. The Escherichia coli being strictly intestinal, their presence in water ensures fecal pollution. E. coli can be easily isolated from water using selective media like MacConkeys agar. Hence, the E. coli is the most perfect index of water pollution. Escherichia coli can be differentiated from the Enterobacter aerogenes on the basis of IMViC tests and Eijkman test. These biochemical tests differentiated these two microorganisms from each other.

10. Examination of Water Multiple Tube Fermentation Technique or MTFT ( Qualitative
Several selective and differential media use in the process of examining water for coliform organisms. The examination involves three successive process or steps.
i) The presumptive test ii) The confirmed test and iii)The completed test
Presumptive test:-In this test consists of inoculating lactose bile broth tubes or macConkey’s broth tubes with appropriate amount of the water samples. These inoculated tubes are incubated at 370Cfor 24 hrs. After incubation tubes are observed for gas are called presumptive positive tubes.The medium used is selective medium for coliform organisms. Coliform organisms are able to ferment lactose and from acid and gas. All fermentation tube showing acid and gas within 48 hrs. at 370Cshall be utilized in the confirmed test.

11. Multiple Tube Fermentation Technique to determine Most Probable number
uses a specified number of test tubes to statistically predict the number of organisms present (based on the expected population of organisms in the sample)
tubes may also contain an inverted inner vial (Durham tube) for gas collection
ideal for wastewater samples and non-potable samples, because the analyst can accommodate highly turbid samples by diluting prior to analysis

12. Bacteriological analysis of water:
Most Probable Number (MPN) technique
coliform: acid and gas from lactose <24 hours/370C
indicator organism: E. coli
Water double strength single strength single strength
sample
10 ml sample ml sample ml sample
*Normally 5 Durham tubes are inoculated but this exercise is modified to three tubes in the interest of economy.

14. Confirmatory Test
BGLB tubes (10ml ) with Durham tubes were prepared and sterilized.
Loopful of suspension from the positive presumptive tubes was inoculated and incubated at 35°C for 48hours
EMB plate is inoculated and incubated at 35C
Loopful of suspension was inoculated and incubated at 44°C for 24 hours.
Sometimes an IMViC reaction is inoculated
BGBL : Turbidity and gas production is observed.
EMB: Green metallic sheen growth is observed on the plate.
IMViC reaction : ++--
Those positive for the confirmatory test were subjected to the completed test.

16. Confirmatory Test
E. coli E. aerogenes