EXPERIMENT NO -7

Aim Cultivation of anaerobic bacteria in an anaerobic jar.

RequirementAnaerobic jar, candle, petroleum jelly, soil sample, distilled water tube,
inoculating needle incubator, nutrient medium, autoclave etc.
PrincipleThe 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.
Procedure1.

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 tubes
10. 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 hours
15.
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

1
2
3
4

5 min

7min

10 min

1:80
1:90
1:100
1:110

for disinfectant
Sr

Disinfectant
dilution

Presence of growth in the nutrient broth ( time in

minutes)

2.5 min
1
2
3
4

5 min

7min

10 min

1:400
1:450
1:500
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=
circled

reciprocal of the chemical dilution

________________________________
Reciprocal of the phenol dilution
circled.

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