MICROBIOLOGY biochemical test detailed.pptx

microbiology biochemical test with procedure and preparation methods

1. MICROBIOLOGY Biochemical Test in Microbiology Submitted by Abhijit padhi

2. Biochemical test Biochemical tests are laboratory procedures that use specific chemical reactions to identify and characterize microorganisms, such as bacteria. These tests are often used to identify the presence of specific enzymes or metabolic pathways in a microorganism, which can help to distinguish it from other microorganisms. Some common biochemical tests used in bacteriology include: Oxidase test:  This test is used to detect the presence of the enzyme cytochrome oxidase, which is involved in the metabolism of oxygen. Catalase test:  This test is used to detect the presence of the enzyme catalase, which helps to break down hydrogen peroxide. Indole test:  This test is used to detect the presence of the enzyme tryptophanase, which breaks down the amino acid tryptophan. Nitrate reduction test:  This test is used to detect the ability of a microorganism to reduce nitrate to nitrite. Gelatinase test:  This test is used to detect the ability of a microorganism to produce the enzyme gelatinase, which breaks down gelatin.

3. MacConkey Agar Composition Peptone 20gm/1ltr. Lactose monohydrate  10gm /1ltr. Bile salts 1.5gm /1ltr. Sodium chloride 5gm /1ltr. Neutral red 0.03gm /1ltr. Crystal Violet 0.001gm /1ltr. Agar 13.5gm /1ltr. ph should be 7.1 ±0.2 MacConkey agar is used for the isolation of gram-negative enteric bacteria. Escherichia coli red/pink non-mucoid Aerobacter aerogenes pink mucoid Enterococcus  species red minute, round Staphylococcus  species pale pink opaque Pseudomonas aeruginosa green-brown fluorescent growth

4. Procedure Suspend 49.53 grams of dehydrated medium in 1000 ml of distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.  Cool to 45°C -50°C. Mix well before pouring into sterile Petri plates.

5. MSA media (Mannitol-Salt agar) Composition Peptone 10gm/1ltr. Nacl 75gm/1ltr. D-mannitol 10gm/1ltr. Yeast extract 1gm/1ltr. Phenol red 0.025gm/1ltr. Agar 15gm/1ltr. ph should be 7.2±0.2 Organisms Results Staphylococcus aureus Yellow colonies surrounded by the yellow zone Staphylococcus epidermidis Pink or Red colonies Micrococci Red colonies Escherichia coli No growth

6. Procedure Suspend 111 grams of Mannitol Salt Agar in 1000 ml of distilled water. Boil to dissolve the medium completely. Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes. If desired, sterile Egg Yolk Emulsion (E7899) can be added to a final concentration of 5% v/v after autoclaving. Pour cooled Mannitol Salt Agar into sterile petri dishes and allow to cool to room temperature.

7. EMB (Eosin-methylene blue) Composition Peptic digest of animal tissue 10gm /1ltr. Dipotassium phosphate 2gm /1ltr. Lactose 5gm /1ltr. Sucrose 5gm /1ltr. Eosin – Y 0.400gm /1ltr. Methylene blue 0.065gm /1ltr. Agar 13.5gm /1ltr. ph should be 7.2±0.2 Organisms Growth Escherichia coli Blue-black bull’s  eye; may have a  green metallic sheen Pseudomonas aeruginosa Colorless Enterobacter aerogenes Good growth; pink, without sheen Klebsiella pneumoniae Pink, mucoid colonies Proteus mirabilis Luxuriant growth; colorless colonies Salmonella  Typhimurium Luxuriant growth; colorless colonies

8. Proedure Suspend 35.96 grams in 1000 ml distilled water. Mix until the suspension is uniform. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.  AVOID OVERHEATING. Cool to 45-50°C and shake the medium in order to oxidize the methylene blue (i.e. to restore its blue color) and to suspend the flocculent precipitate. Pour into sterile Petri plates. Allow plates to warm to room temperature. The agar surface should be dry before inoculating. Inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface and streak for isolation with a sterile loop. Incubate plates aerobically at 35-37°C for 18-24 hours and protect from light. Examine plates for colonial morphology. If negative after 24 hours, reincubate an additional 24 hours.

9. Catalase Test slide method use a loop or sterile wooden stick to transfer a small amount of colony growth to the surface of a clean, dry slide. Place a drop of 3% H 2 O 2 in the glass slide. Observe the evolution of oxygen bubbles.

10. Urease test Composition Peptone 1 gm/L Dextrose 1gm/L NaCl 5gm/L K2HP O4 2gm/L Phenol red few drops Agar 15gm/L Urea 20gm/L ph should be 6.8 ±0 .2 Positive Reaction:  Development of an intense magenta to bright pink color in 15 min to 24 h. Examples:   Proteus   spp ,  Cryptococcus   spp ,  Corynebacterium   spp ,  Helicobacter pylori ,  Yersinia   spp ,  Brucella   spp , etc. Negative Reaction:   No color change. Examples:   Escherichia, Shigella, Salmonella , etc.

11. procedure Take 20 ml dil. Water in a clean and dry culture bottle. Then weight all composition for 20 ml (peptone 0.02 gm, dextrose 0.02 gm, NaCl 0.1 gm K 2 HPO 4 0.04 gm, phenol red 4 drops, agar 0.30 gm, urea 0.40 gm) and add in the bottle one by one. After that, maintain the pH and transfer 10-10 ml media in two clean and dry tests then add 0.15 gm agar/test tube and cover the test tube tightly with 4 layer paper. After that, autoclave the media for 45 minutes. Then add 0.20 gm urea/ test tube under the laminar air flow and vertex it for a few seconds then prepare a slant in the test tube for streaking. After solidifying the agar well, a cool streak isolation bacteria ( E . coli ) in test tube slant media. Incubate it 48 hours. After incubation, observe the result.

12. Citrate test Composition Sodium chloride 5gm/L Sodium citrate 2gm/L Ammonium dihydrogen phosphate 1gm/L Dipotassium phosphate 1gm/L Magnesium sulphate 0.2gm/L Bromothymol blue 0.08gm/L Agar 15gm/L ph should be 6.9 ±0.2 Positive Reaction:  Growth with color change from green to intense blue along the slant. Examples:   Salmonella, Edwardsiella , Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia , etc. Negative Reaction:  No growth and No color change; Slant remains green. Examples:   Escherichia, Shigella, Morganella , Yersinia  etc.

13. procedure Take 20 ml dil. Water in a clean and dry culture bottle. Weight all composition (sodium chloride 0.10 gm, sodium citrate 0.04 gm, ammonium dihydrogen phosphate 0.02 gm, dipotassium phosphate 0.02, magnesium sulphate 0.004 gm, bromothymol few drop) and add it in the dil. Water. After that, maintain the pH and transfer 10-10 ml media in two clean and dry test tubes then add 0.15 gm agar/ test tube and cover the test tube tightly from 4 layer paper. After solidifying the media, cool streak the isolation bacteria ( E. Coli ) in the test tube slant media. Incubate it for 24 hours and observe the result.

14. Nitrate Reduction Test Composition Peptone 5gm/ ltr . Meat extract 3gm/ ltr . Potassium nitrate 1gm/ ltr . ph should be 7.0 ± 0.2 Preparation of 0.8% sulfanilic acid (Regent A) Sunfanilic acid  0.8 grams Distilled water  70 mL Glacial acetic acid  30 mL Preparation of 0.5% -naphthol (Reagent B) -naphthol  (N, N-dimethyl- α- napthylamine ) 0.5 grams Distilled water  70 mL Glacial acetic acid  30 mL

15. Procedure Determination of nitrate reduction to nitrite is a two step process. First, the reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B, then if necessary, the reduction of nitrate beyond nitrite is determined by the addition of Nitrate Reagent C (zinc dust). Inoculate the nitrate broths with bacterial suspension. Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours. After incubation look for N2 gas first before adding reagents. Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B. Observe for the reaction (color development) within a minute or less. If no color develops add zinc powder. Observe for at least 3 minutes for a red color to develop after addition of zinc.

16. Lipase Test Method Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr Agar 15gm/ ltr . Egg yolk 40ml/ ltr . ph should be 7.0 ±0.3 Positive Control :  Staphylococcus aureus  ATCC 12600 Negative Control :  Clostridium difficile  ATCC 9689/  Clostridium perfringens  ATCC 12924

17. Procedure take 25 ml dil. Water in a clean and dry culture bottle. All composition (peptone0.125 gm, yeast 0.075 gm, sodium chloride 0.125 gm) are added to the dill. Water. Then maintain the pH and add the 0.375 gm agar. After that autoclave the media for 45 min. during the autoclave, prepare the petri plate to give the UV for pouring the media. After autoclave and before pouring the media add 1 ml egg yolk in the shake and pour it under the laminar air flow 10 min. UV of media. After solidifying the agar and drying media, take a sterile loop full of isolated bacteria and streak it as a straight line on the plate. Incubate anaerobically in a gas pack jar immediately after streaking and transfer into the incubator maintained at 35-37°C for 24 to 48 hours for anaerobes and for aerobes incubate the plate at 35-37°C for 24 to 48 hours. Positive test – A positive lipase test is noted by the appearance of an iridescent sheen. Immediately around colonies that can be seen when the plate is held at an angle to a light source. Negative test – A negative lipase test is indicated by the absence of an iridescent sheen.

18. Proteus test Composition Peptone 5gm/ ltr . Dextrose 1gm/ ltr . Yeast extract 3gm/ ltr . Agar 15gm/ ltr . Milk powder 20gm/ ltr . ph should be 6.8 ± 0.2 Positive should be create zone around bacteria

19. Procedure take 25 ml of dill water in a clean and dry culture bottle. Weigh all composition (peptone 0.125 gm, dextrose 0.025 gm, yeast exract 0.075 gm, milk powder 0.50 gm) and add I dilute water. After that, maintain the pH and add 0.375 gm agar and autoclave the media for 45 min. During the autoclave, prepare the petri plate for pouring the media, so give the UV of the petri plate for 10 min. After autoclave, pour the media in the ready plate then leave the media in UV for 15 min. after that dry the media for 15 min. After that, strip the isolated bacteria with the help of a sterile wire loop ( E. Coli ) on agar media to a straight line. Incubate it for 24 hours and observe the result.

20. Hydrogen sulphate (H2S) Test Composition Yeast extract 3gm/ ltr . Peptone 30gm/ ltr . Ammonium ferrous sulphate 0.2gm/ ltr . Sodium thiosulphate 0.025gm/ ltr . Agar 3gm/ ltr . ph should be 7.3 ± 0.2 H 2 S Test Positive Bacteria Proteus spp., Citrobacter spp., Salmonella spp., Staphylococcus saprophyticus, Campylobacter   spp ., etc.  H 2 S Test Negative Bacteria Klebsiella pneumoniae, Shigella spp., Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Vibrio cholerae, Yersinia pestis , etc. 

21. Procedure An iron compound and a sulphur compound are included in the test medium to test for the production of hydrogen sulphide gas. Hydrogen sulphate is production if the sulphur compound is reduced by the bacterial strain. This test thus determines whether the microbe reduces sulphur- containing compound to sulphide during the process of metabolism. H 2 S is produced by certain bacteria through reduction of sulphur containing amino acids like cystine, methionine or through the reduction of inorganic sulphur compound such as thiosulphates, sulphate or sulphites during protein degradation or when anaerobic respiration shuttles the electrons to sulphur instead of to oxygen. In either case H 2 S is produced (hydrogen sulphide gas) which reacts with the iron compound to from the back precipitate of ferric sulphide. The lack color acts as an indicator for the presence of hydrogen sulphide. The detection of hydrogen sulphide (H 2 S) gas produced by an organism is used mainly to assist in the identification of that particular organism.

22. Starch hydrolysis Composition Yeast extract 3gm/ ltr . Starch 5gm/ ltr . Agar 15gm/ ltr . ph should be 6.8 ±0.2 Starch hydrolyzing (amylase producing) bacteria:  S. bovis , Bacillus subtilis, B. cereus, B. megaterium, Clostridium perfringens,  etc. Starch non- hydrolyzing (amylase non-producing) bacteria:   Corynebacterium diphtheria, Clostridium difficile, C. botulinum , bile-esculin positive viridans   Streptococci  except  S. bovis , E. coli, S. aureus, Pseudomonas aeruginosa, P. putida , etc. 

23. Procedure Take a clean and washed culture bottle and pour 25 ml distilled water in the bottle. Weigh these reagets : 0.07 gm of beef extract, 0.25 gm of starch and 0.37 gm of agar added in the culture bottle. Maintain the pH at 6.8 ± 0.2, diluted HCL or NaOH to maintain pH. Pour the media into test tubes and cover it with 4 layers paper and a rubber band. Autoclave the media at 121°C for 15 minutes at 15 PSI pressure. After autoclave, let the media cool down for some time. During the autoclave, prepare the petri plate for pouring the media, so give the UV of the petri plate for 10 minutes. After the autoclave, pour the media in the ready plate. Then leave the media in UV for 15 minutes, after drying the media for 15 minutes. After that strip, the isolated bacteria with the help of a sterile wire loop on agar media to a straight line. Incubate it for 24 hours and observe the result. Following incubation, add a few drops of iodine solution directly over the colonies and observe for the formation of a clear halo around the colonies.

24. Methyl red (MR) Composition Yeast extract 7gm/ ltr . Dextrose 5gm/ ltr . Dipotassium phosphate 5gm/ ltr . ph should be 6.9 ± 0.2 MR positive:    Escherichia coli  (ATCC25922) MR negative:   Enterobacter aerogenes  (ATCC13048)

25. Procedure Inoculate MRVP broth with a pure culture of the organism. Incubate at 35°-37°C for a minimum of 48 hours in ambient air. Add 5 or 6 drops of methyl red reagent per 5 mL of broth. Observe for the color change in the broth medium.

26. Voges-Proskauer (VP) Composition Yeast extract 7gm/ ltr . Dextrose 5gm/ ltr . Dipotassium phosphate 5gm/ ltr . ph should be 6.9 ± 0.2 VP Positive Bacteria VP Negative Bacteria Klebsiella spp., Enterobacter spp., Viridans Streptococci (except S. mitis, and S. vestibularis) Proteus mirabilis, Hafnia spp., Serratia spp., Staphylococcus aureus, Listeria spp., V. cholerae  Escherichia spp., Proteus vulgaris, Citrobacter freundii , Morganella morganii, Shigella spp., Yersinia spp., V. parahaemolyticus  5% Alpha-naphthol Solution ( Barritt’s Reagent A) and 40% KOH or NaOH solution ( Barritt’s Reagent B) are required.  Preparation of Barritt’s Reagent A Dissolve 5 grams of α-naphthol reagent in 100 mL of 95% ethanol. The reagent can be stored for up to 3 weeks in a dark place at 4 to 8°C.   Preparation of Barritt’s Reagent B Dissolve 40 grams of KOH pellet in 100 mL of sterile distilled water. The reagent can be stored for up to 3 weeks at 4 to 8°C.

27. Procedure Using a sterile inoculating loop, pick up well-isolated colonies of sample bacteria from 18 to 24 hours old culture and inoculate the broth. Incubate the tubes aerobically for 18 to 24 hours at 35±2°C. Following incubation, transfer 2 mL of broth to a clean (sterile if possible) test tube.  Add 6 drops of Reagent A (5% α-naphthol solution) and mix properly by shaking. Add 2 drops of Reagent B (40% KOH solution) and mix properly by shaking.  Observe for the formation of red-pink color at the surface of the medium within 30 minutes. Continuously shake the tube vigorously during the 30-minute waiting period.   If no color is developed (negative reaction) re-incubate the remaining broth for additional 24 hours and test again.

28. Indole test Composition Tryptophan 10gm/ ltr . Sodium chloride 5gm/ ltr . Indole Kovacs Reagent: p-Dimethylaminobenzaldehyde 50.0 gm Hydrochloric Acid, 37% 250.0 ml Amyl Alcohol 750.0 ml Positive:  A positive reaction is denoted by the appearance of a blue to blue-green color change on the bacterial smear within 2-3 minutes. Negative:  Negative reactions remain colorless or light pink. Note: Positive reaction is Red-violet in the case of Providencia alcalifaciens .

29. Procedure Take sterilized test tubes containing 4 ml of tryptophan broth. inoculate the tube aseptically by taking the growth from 18-24 hrs. ’culture. Add 0.5 ml of Kovac’s reagent to the broth culture. Observe the presence or absence of a ring. Positive:  Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding the reagent. Examples:   Aeromonas hydrophila ,  Aeromonas punctata ,  Bacillus alvei , Edwardsiella  sp.,  Escherichia coli ,  Flavobacterium  sp.,  Haemophilus influenzae ,  Klebsiella oxytoca ,  Proteus  sp. (not  P. mirabilis  and  P. penneri ),  Plesiomonas shigelloides , Pasteurella multocida ,  Pasteurella pneumotropica ,  Enterococcus faecalis , and  Vibrio  sp. Negative:  No color change even after the addition of appropriate reagent. Examples:  Actinobacillus  spp.,  Aeromonas salmonicida ,  Alcaligenes  sp., most  Bacillus  sp.,  Bordetella  sp.,  Enterobacter  sp.,  Lactobacillus  spp., most  Haemophilus  sp., most  Klebsiella  sp.,  Neisseria  sp.,  Pasteurella haemolytica ,  Pasteurella ureae ,  Proteus mirabilis ,  P. penneri ,  Pseudomonas   sp ., Salmonella  sp.,  Serratia  sp.,  Yersinia  sp.

30. Carbohydrate utilization test 1. Dextrose test Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . Dextrose 10gm/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure Take 10 ml dil. Water in a bicker and weigh all the ingredients (peptones 0.05 gm, NaCl 0.05 gm, yeast 0.03 gm, dextrose 0.10 gm, phenol red few drop) add in dil. Water. After that, maintain the pH then transfer 5-5 ml media I two clean and dry tests and cover the test tube with 4 layer of paper. After that place the media in the autoclave for 45 min. After the autoclave, place the media in cool water to cool it down. after that take culture broth and sterile pipette ad tips from UV then inoculate 100 µl culture broth of salmonella and E. Coli in dextrose broth separately. Place the inoculated tube into the incubator at 35-37°C for 24 to 48 hours. Positive result – change the media color into yellow from orange. Negative result – media color is not changed.

31. 2. Sucrose test Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . sucrose 10gm/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure Take 10 ml dil. water in a bicker and weight the compositions add in dil. water. After that, maintain the pH then transfer 5-5 ml media in two clean and dry tests and cover the test tube with 4 layers of paper. After that place the media in the autoclave for 45 min. After the autoclave, place the media in cool water to cool down. after that take culture broth and sterile pipette and tips from UV then inoculate 100 µl culture broth of Salmonella and E. coli in sucrose broth separately. Place the inoculated tube into the 35-37 °C incubator. Positive result – media colour changes from orange. Negative result – media colour is not changed.

32. 3. Lactose test Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . lactose 10gm/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure Take 10 ml dil. water in a bicker and weight all the ingredients add I dil. water. After that, maintain the pH then transfer 5-5 ml media in two clean and dry tests and cover the test tube with 4 layers of paper. After that place the media I the autoclave for 45 min. After that autoclave, place the media I cool water to cool water to cool down after that take culture broth and sterile pipette and tips from UV then inoculate 100 µl culture broth of salmonella and E. coli in lactose broth separately. Place the inoculated tube into the 35-37° C incubator. Positive result – change the media colour into yellow from orange. Negative result – media colour is not change.

33. 4. Maltose test Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . maltose 10gm/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure Take 10 ml dil. water in bicker and weight all the ingredients add in dil. water. After that, maintain the pH then transfer 5-5 ml media in two clean and dry tests and cover the test tube with 4 layers of paper. After that place the media in the autoclave for 45 min. After the autoclave, place the media in cool water to cool down. After that take culture broth and sterile pipette and tips from UV then inoculate 100 µl culture broth of salmonella and E. coli in maltose broth separately. Place the inoculated tube into the 35-37°C incubator for 24-48 hours. Positive result – change the media colour into yellow from orange Negative result – media colour is not change.

34. 5. D- mannitol Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . D- mannitol 10gm/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure Take 10 ml dil. water in a bicker and weight all the ingredients add in dil. water. After that, maintain the pH then transfer 5-5 ml media in two clean and dry tests and cover the tests tube with 4 layers of paper. After that place the media in the autoclave for 45 min. After the autoclave, place the media in cool water to cool down after that take culture broth and sterile pipette and tips from UV then inoculate 100 µl culture broth of salmonella and E. coli in mannitol broth separately. Place the inoculated tube into the 35-37°C incubator. Positive result – change media colour into yellow from orange. Negative result – media colour is not changed.

35. 6. Sorbitol test Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . Sorbitol 10gm/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure Take 10 ml dil. water in bicker and weight all the ingredients add in dil. water. After that, maintain the pH then transfer 5-5 ml media in two clean and dry tests and cover the test tube with 4 layers of paper. After that place the media in the autoclave for 45 min. After the autoclave, place the media in cool water to cool down. After that take culture broth and sterile pipette and tips from UV then inoculate 100 µl culture broth of salmonella and E. coli in maltose broth separately. Place the inoculated tube into the 35-37°C incubator for 24-48 hours. Positive result – change the media colour into yellow from orange Negative result – media colour is not change.

36. 7. Glycerol test Composition Peptone 5gm/ ltr . Sodium chloride 5gm/ ltr . Yeast extract 3gm/ ltr . glycerol 10ml/ ltr . Phenol red few drops ph should be 7.0 ± 0.2 Media color should be pink, red or dark orange Procedure after the maintenance the pH then transfer 5-5 ml of media in 2 clean and dry test tubes and covers the test tube from 2 layers paper. After that place the media to autoclave. After autoclave, place the media I cool water to cool down. After that take culture growth ad sterlite pipette ad tips from UV then inoculate 100 ml broth culture salmonella and E. coli in glycerol broth separately. Place the test tube in the incubator at 35°C for 24-48 hours. Positive result – change the media colour into yellow from orange Negative result – media colour is not change.