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Biochemical test

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Biochemical test

  1. 1. Biochemical test
  2. 2. Spore staining
  3. 3. Spore staining procedure • Materials required • 48-72 hrs old slant culture of bacillus sp., malachite green & Saffranin ,glass slide , inoculation loop , etc.
  4. 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. 5. Gram staining
  6. 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. 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. 8. Methyl red
  9. 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. 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. 11. Citrate utilization
  12. 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. 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. 14. Catalase test
  15. 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. 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. 17. Oxidase test
  18. 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

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