EXPERIMENT NO -7

Aim – Cultivation of anaerobic bacteria in an anaerobic jar.

Requirement-

Anaerobic jar, candle, petroleum jelly, soil sample, distilled water tube, inoculating needle incubator, nutrient medium, autoclave etc.

Principle-

The appropriate method for the cultivation of anaerobes should be chosen with consideration of the sensitivity of the given organism to oxygen concentration and/or redox value within the media or the surrounding atmosphere. For the cultivation on of bacteria sensitive to even trace amount of oxygen (e.g. methanogens sulphate reducing bacteria) the best technique is the use of an anaerobic system filled with an appropriate gas and the only contact with the outside world is through a channel chamber that that removes oxygen wherematerials are introduced into the system.

Microbes that are not sensitive to trace amount of oxygen and are able to survive temporarily high oxygen concentration by forming endospores can be cultivated in anaerobic jars or in semisolid media, containing special reductive again however colonies formed inside ”anaerobic” agar deep tubes are difficult to examine and isolate this problem can be solved by using marino-plates. Combination of petri-dish and an “anaerobic” agar.

Procedure-

1. Prepared the nutrient agar plate/bismuth sulphite agar plate.

2. Prepared a dilution of soil sample aseptically.

3. The diluted sample of soilwere streaked on the surface of nutrient

agar bismuth sulphite agar aseptically.

4. Put the inoculated petri-disheswith their surface down into the anaerobic jar.

5. Burn the candle inside the jar to utilized the complete, oxygen in the jar. So it creates the

anaerobic condition in the jar.

6.Sealed the mouth of the jar with petroleum jelly.

7. The jar was kept in the incubator for 1 day to 1 weak.

Observation

Result- The cultivation of anaerobic bacteria was seen in the plateby using anaerobic jar.

Aim: To determine the phenol coefficient of given disinfectant.

Requirements : Test tube, pipette, phenol concentration( 1:80, 1:90, 1:100 and 1:110), test disinfectant (1:400, 1:450, 1:500 and 1:550) beaker, measuring cylinder, micropipette, D/W bacterial culture ( Salmonella typhi) and nutrient broth.

Principle:

Disinfectants are chemical agents (usually liquid) that used on the surface of non-living (inanimate) object/material to lower the level of microbes on that surface. The chemical used to wipe down your laboratory work area probably contain the strong disinfectant. Many house hold cleaning agents such as ammonia and bleach are also disinfectants. Most household disinfectants are strong oxidizing agent.

When considering the relative effectiveness of different disinfectants against bacteria, some yardstick of comparison is necessary. The official method to measure the effectiveness of a disinfectant is to determine its phenol coefficient, where the effectiveness of various phenolic based chemicals, is compared to phenol under standardized experimental conditions.

Phenol and its derivatives, called phenolics, kill bacteria by damaging the plasma membrane, inactivating enzymes and denaturing protein.

The phenol coefficient is a comparative test using a standard organism such as Salmonella typhi or Staphylococcus aureus. The experiment deals with comparing the effectiveness of disinfectant and find out the phenol coefficient.

Procedure:

1. Prepare the nutrient broth as per the standard protocol.2. Transfer 5 ml of nutrient broth in each test tube and sterilized the test tube in

autoclave at 15 lbs pressure for 15-20 mins at 121C.3. Prepare the dilution of phenol like 1:80, 1:90, 1:100 and 1:110 in four different beaker

or volumetric flask and label properly.4. In same manner , prepare the dilution of disinfectant like 1:400, 1:450, 1:500 and

1:550 in four different beaker or volumetric flask and label properly.5. Now, take 5ml of each phenol dilution 1:80, 1:90, 1:100 and 1:110 in four test tube

and labelled them.6. Also take 5ml of each disinfectant dilution1:400, 1:450, 1:500 and 1:550 in four test

tube and labelled them.7. These tube are the main tubes.

8. After sterilization of nutrient broth, cool it( 32 test tubes of nutrient broth).9. Divide in two set each set should contain 16 tubes10. Set of phenol dilution contains 16 test tubes with label as

1:80 for 2.5 minutes, 5 minutes, 7 minutes and 10 minutes.1:90 for 2.5 minutes, 5 minutes, 7 minutes and 10 minutes.1:100 for 2.5 minutes, 5 minutes, 7 minutes and 10 minutes.1:110 for 2.5 minutes, 5 minutes, 7 minutes and 10 minutes.

11. Repeat the same procedure for the disinfectant dilutions.12. Now all the tube are inoculated with 0.2 ml of test organism Salmonella typhi or Staphylococcus aureus.13. After the respective incubation at 17 C or room temperature, then the tube

contain of each dilution ( phenol and disinfectant) were transfer to nutrient both with aseptic condition .

14. All the tubes are incubated at 37 C for 24 to 48 hours15. Observe the growth by examine the turbidity.

Observation table for phenol

Sr Phenol dilution Presence of growth in the nutrient broth ( time in minutes)

2.5 min 5 min 7min 10 min1 1:802 1:903 1:1004 1:110

for disinfectant

Sr Disinfectant dilution

Presence of growth in the nutrient broth ( time in minutes)

2.5 min 5 min 7min 10 min1 1:4002 1:4503 1:5004 1:550

Circle the dilution of the test substance that killed the bacteria in 10 mins but in in 5 mins

CALCULATION

Phenol coefficient of the substance= reciprocal of the chemical dilution circled

________________________________

Reciprocal of the phenol dilution circled.

A phenol coeffieient greater than 1 suggest that the test chemical is more effective than phenol.