it is very useful

1. Biochemical test

2. Spore staining

3. Spore staining
procedure
• Materials required
• 48-72 hrs old slant culture of
bacillus sp., malachite green
& Saffranin ,glass slide ,
inoculation loop , etc.

4. procedure
• The glass slide was cleaned well and a smear of given
organisms were made on the slide and dried and heat
fixed.
• Malachite green was applied and the slide was placed
on the hot water bath allowing the smear preparation
to retain for 2-3 minutes.
• Care was taken to see that the stain does not get
evaporated.
• The slide was cooled and then washed with running
water.
• Saffranin was applied and kept for 30 sec.
• Again the slide was washed in water and examined
under oil immersion.

5. Gram staining

6. Gram staining
procedure
• Materials required
• slides, inoculation loop , burner, wash
bottle, microscopic lens , cleaning paper
, culture ,crystal violet ,ethanol ( de
colorizing agent) ,saffranin , etc.

7. procedure
• The glass slide was cleaned.
• A smear of the given culture was made on the glass slide using
sterile techniques.
• The smear was air dried or heat fixed.
• Gram crystal violet was flooded over the smear and left for 60 sec.
• Then it was washed with water .
• Gram iodine was applied as a mordant for 30 sec.
• The slide was again washed with water.
• Decolorizing agent was added drop wise till the primary stain stops
draining from the smear and washed with water.
• Saffranin was added as a counter strain for 60 sec.
• Again the slide was washed with water and then dried with blotted
sheet and examined under the microscope.

8. Methyl red

9. Methyl red
procedure
• Materials required
• Test tube, conical flask, MR-VP broth,
methyl red , cotton, inoculation loop ,etc.
• MR-VP BROTH:
• Peptone-7.0g
• Dextrose-15.0g
• Potassium phosphate-5.0g
• Distilled water-1000ml
• PH-6.9

10. procedure
• Methyl red
• Dissolve 0.1g of methyl red in 300ml of 95% ethyl
alcohol .dilute to 500ml with distilled water.
• Procedure
• 5ml of MR-VP broth is dispersed into the test tubes
and sterilized at 151b pressure.
• The tubes were inoculated with loop inoculation .
• Inoculated tubes were incubated for 24-38 hrs at 37c.
• One un inoculated tube will serve as a control .
• After incubation methyl red solution was added
and observed the results.

11. Citrate utilization

12. Citrate utilization
procedure
• Materials required
• test tube, conical flask , inoculation loop, culture, simmon
citrate agar etc.,
• Simmon citrate agar:
• Ammonium di hydrogen phosphate-1.0g
• Di potassium phosphate-1.0g
• Sodium chloride-5.0g
• Sodium citrate-2.0g
• Magnesium sulphate-0.2g
• Agar-15.0g
• Bro mothymol blue- 0.08g
• PH-6.9

13. procedure
• 5ml of simmon citrate agar was dispersed in test
tube and sterilized.
• After sterilization ,slant was prepared along with
stab.
• Inoculate the given culture by means of slant &
stab inoculation .
• One un-inoculated tube will serve as a control.
• All the tubes are incubated at 37c for 24 hrs.
• After incubation observe the results.

14. Catalase test

15. Catalase test
procedure
• Materials required
• Trypticase soy agar, culture, conical flask , petri plate ,
inoculation loop,3% hydrogen peroxide, cotton ,marker
, etc.,
• Trypti case soy agar
• Trypticase-15.0gm
• Phytane-5.0gm
• Sodium chloride-5.0gm
• Agar-15.0gm
• Distilled water-1000ml
• PH-7.3

16. procedure
• Prepare trypticase soy agar plates.
• inoculate the given culture by means of simple
streaking .
• Incubate all the plates at 37c for 24 hrs .
• After incubation 3% hydrogen peroxide were
added to the plates.
• Observed the results.

17. Oxidase test

18. Oxidase test
procedure
• Materials required
• Trypticase soy agar plates, culture, p-amino
dimethyl aniline oxalate, inoculation loop etc.,
• Trypticase soy agar;
• Tryp ticase- 15.0gm
• Phytane – 5.0gm
• Sodium chloride – 5.0 gm
• Agar – 15.0gm
• Distilled water – 1000ml
• PH – 7.3