EXPERIMENT NO. AIM Determination of total coli form & fecal contamination in drinking water. Requirment 1.

1. Single & double strength lactose broth 2. Water sample 3. Sterile test tube 4. Distilled water PrincipleColiform bacteria are a commonly used bacterial indicator of sanitary quality of foods and water. They are defined as rod-shaped Gram-negative non-spore forming bacteria which can ferment lactose with the production of acid and gas when incubated at 3537C. Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the feces of warm-blooded animals. While coli form themselves are not normally causes of serious illness, they are easy to culture and their presence is used to indicate that other pathogenic organisms of fecal origin may be present. Such pathogens include bacteria, viruses, or protozoa and many multicellular parasites. Fecal coli form bacteria are no disease causing organisms which are found in the intestinal tract of all warm-blooded animals. Each discharge of body wastes contains large amounts of these organisms. The presence of fecal coliform bacteria in a stream or lake indicates the presence of human or animal wastes. The number of fecal coliform bacteria present is a good indicator of the amount of pollution present in the water. Most waterborne disease-causing organisms originate in human or animal bodies and are discharged as part of body wastes. Due to the relatively small numbers of disease causing organisms, it is very difficult to isolate and identify specific disease causing bacteria. Since fecal coliform bacteria originate in the same location, they are used as an indicator of possible disease hazards in a body of water. The presence of very few fecal coliform bacteria would indicate that a water source probably contains no disease producing organisms, while the presence of large numbers of fecal coliform bacteria would indicate a very high probability that the water source could contain disease producing organisms. For this reason, regulatory agencies with responsibility for protection of public health have established water quality standards which include maximum levels of fecal coliform bacteria. PROCEDURE 1. Single and double lactose broth fermentation were prepared and autoclaved. 2. Six test tube of single strand lactose broth (each containing 10ml)were prepared & test tube no. were labeled from 1-6

3. 3 test tube of double strength lactose fermentation broth (each containing 10ml) were prepared &test tube no. were labeled from 7-9 4. 0.1ml of water sample added into test tube no. 1-3 and 1ml of water sample is added into test tube 4,5,6 5. 10ml water sample was added in test tube no. 7, 8, 9 .these test tube were incubated at 37c for 24 hour. After incubation period test tube were observed. Result Precaution 1. All glass wares should be sterile. 2. Petri dishes should be properly sealed before incubation.

Experiment No.Objective: Testing of drinking water by MPN ( Most Probable Number ) test. Requirements: single and double strength lactose fermentation broth, water sample, 10ml and 1mL pipette, sterile test tubes, distilled water, etc. Principle:- Coliform group is a group of short rod-shaped bacteria, which normally parasitize in animal intestines; numerous coliform groups exist in feces. Although most of these bacteria wont cause disease, it can serve as an index whether the water is polluted by feces. From a scientific view, the coliform group consists no-spore bacillus that could decompose lactose and produce gas; they are aerobic and facultative, and shows as gram negative under microscope observation. Since coliform is unable to reproduce directly in the water, whereas the feces of warm-blooded animals generally contain this kind of bacteria, if we can detect large numbers of coliform from water, it means the water is polluted by human or animals excreta within a short period of time. This is because coliform and other pathogenic bacteria come from warm-blooded animals, which the survival time of coliform is longer than pathogenic bacteria. If we cant detect coliform group from water, chances are less for the water containing other pathogenic bacteria. Therefore, coliform group is a common biological index to estimate water quality. Total

coliforms can be detected and enumerated in the multiple-tube technique . The most accurate number of coliform bacteriais obtained by testing a large sample of water. In this method, coliforms are detected in two stages. In the presumptive test, dilutions from a water sample are added to lactose fermentation tubes. The lactose broth can be made selective for gram negative bacteria by adding lauryl sulfate or brilliant green and bile. Fermentation of lactose to gas is a positive reaction. Samples from the positive presumptive tube at the high. Most probable number (MPN) testing uses statistical tables to provide quantitative microbiological data by completing multiple presence/absence tests. In this method, multiple vials or wells are filledwith the sample water and media. The vials or plates are incubated for 24-48 hours, and each vial or well is assessed for color change (for total coliform/fecal coliform) or UV-fluorescence (for E. coli).The number of positive and negative vials or wells is compared to a table and a numerical contaminationvalue (in MPN/100 mL) is assigned. The number of vials or wells determines the range of the test for example, five tubes can have a results range of 0-84 MPN/100 mL. Some commercial tests have significantly higher ranges, up to 2,419 MPN/100 mL..

Table 5.1: Standard MPN No. table

Procedure: 1. Single and double strength lactose fermentation broth were prepared and autoclaved. 2. Six test tubes of Single strength lactose fermentation broth ( each containing 10mL) were prepared. And test tubes no. were labeled from 1 to 6. 3. Three test tubes of double strength lactose fermentation broth ( each containing 10mL) were prepared. And test tubes no. were labeled from 7 to 9. 4. 0.1mL of water sample was added into test tubes no. 1, 2, & 3 and 1mL of water sample was added into test tubes no. 4, 5, & 6. 5. 10 mL of water sample was added in to test tubes no. 7, 8, & 9. 6. These test tubes were incubated at 37oC ad for 24 to 48 hours. 7. After incubation period, test tubes were observed.

Result: The MPN No is neglegible according to standard MPN Table.

Precautions: 1. Broth and test tubes should be sterile.

EXPERIMENT No. AIM - Separation of amino acid by using paper chromatography. Requirement Amino acid , filter paper, pencil, Principle the different molecules or ions in the mixture will interact differently with the stationary phase of the chromatograph, and get separated in the process. The different techniques of chromatography

use different substances as the stationary and mobile phases. Chromatography can either be analytical or preparative. Analytical chromatography is used for determining the relative proportions of the different components in the mixture, while the separation of the different components is what preparative chromatography is used for.. Procedure 1. Draw a line 1.5cm above the bottom. 2. Use capillary tube provide to make four spots one of each amino acid ( Alanine, Leucine, Lysine ,valnine) 3. Along the pencil line. 4. Dry each spots for 5minute. 5. Pour 50 ml amino acid developing solution (4:1,ratio mixture of butanol and glacial acid that has been saturated with water). 6. Make the sure paper does not touch the glass. 7. Allow the chromatogram to develop undisturbed for 60-75 minute. 8. Dry paper and spray ninhydrin then again dry and measure the length of spot on the paper. Result Precaution

EXPERIMENT NO.

AIM - To isolates antibiotic bacteria from the soil. Material Requirement

Principle . Soil is the major reservoir of microorganisms that produce antibiotics. Considering that soil is Densely packed with microorganisms, it is not a wonder that many bacterial and fungal species have evolved over the eons to develop ways of inhibiting their neighbors for the benefit of their own growth. An antibiotic made by a microbe can inhibit many other soil microbes. The bacterial genera Bacillus and Streptomycin along with the fungal genera Penicilium and Cephalosporium are commonly found in soil. The genus Streptomycin are the most prolific Antibiotic producers and, although bacteria, are a unique subgroup of bacteria called the Actinomycetes. Although soil has historically been used to find new antibiotic producers, at present many of the old antibiotics are now being manipulated in the lab and chemical modified to form new versions of older antibiotics.

In this experiment you will try to isolate an antibiotic producing bacterium or fungus from the soil. If you succeed at that you will then test that isolate to determine what organisms might be inhibited by the antibiotic that it makes. Because we want to isolate both bacterial and fungal antibiotic producers, two different kinds of media will be used. Glycerol yeast extract media is for isolation of Streptomycin, while Seaboard dextrose agar medium will be used for the isolation of the fungi. One-half of the groups will look for bacterial isolates and the one half will be looking for fungal isolates. Procedure 1. Bring a specimen of soil from any location. 2. Weigh out 1 gram of soil. 3. Obtain 4 plates of the appropriate agar medium(depending on whether your group is searching for bacteriaor fungi). Label these plates 1/103 through 1/106. 4. Set up a series of 1/10 dilutions: a. Place 6 test tubes into a rack and fill each with 9ml of 0.9% NaCl solution. b. Place the gram of soil into the first tube with saline and SHAKE WELL. c. Let the soil particles settle after shaking and then transfer 1ml of the solution intotube 2. This is your 1/10 dilution. d. Repeat the above step for tubes 2-6, changing 1 ml pipets between each transfer. Be sure TO SHAKE EACH TUBE WELL before transferring into the next tube. These tubes will be dilutions of 1/102, 1/103, 1/104, 1/105,and 1/106. 5. Place 0.1ml of the 1/103 dilution onto the top of the appropriate agar. Place the spreader into the alcohol solution and then stick it into the flame to catch it on fire. DO NOT HOLD THE SPREADER IN THE FLAME. When the fire is out, you have a sterile spreader. Place the spreader on the agar with the 0.1ml sample and spread the sample all over the agar medium. 6. Your plates will be made from the 1/103 through 1/106 dilutions. 7. Incubate the plates at 30C until the next lab session. If you do not have sufficient colony numbers, re-incubate the plates and carry on the exercise at next period after.

Result Precaution