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Isolation and Identification of Two Bacterial Unknowns
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Isolation of individual cells from mixed culture:

  

Materials Required:

 

  1. Mixed culture of organism.
  2. Inoculation loop
  3. Nutrient agar plate.
  4. Bunsen burner
  5. Incubator

 

 

Procedure

 

  1.  Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot.
  2. Take the test tube containing the nutrient broth with mixed culture of organism that has been kept for 24 - 48 hours.
  3. Using aseptic technique loopful of organism from the broth.
  4. Quadrant streak the culture on the nutrient agar surface.
  5. Incubate the plates at 37 degree celcius for 24 hours. 
  6.  After incubation the isolated colonies are streaked aseptically on to nutrient agar slant for further studies

 

Indole test:

 

 

Materials Required:-


  1. Culture:24-48 hour bacterial culture.
  2. Media: Peptone water

 

Procedure:

 

  1. Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot.
  2. Take the test tube containing the nutrient broth culture of organism that has been kept for 24 - 48 hours.
  3. Remove the cap of the nutrient agar slant.
  4. Using aseptic technique organism from the nutrient agar slant.
  5. Take the test tube labeled indole.
  6. Inoculate the broth with the inoculation loop containing the inoculum from the nutrient agar slant.
  7. Incubate the tube for 24 to 48 hours at 37°C.
  8. After incubation add 5 drops of  Kovac’s reagent .
  9. Shake gently for several minutes.
  10. Red ring formation within 15 to 20 minutes is a positive result. No red ring formation after 15 to 20 minutes is a negative result.

 

Methyl red test:

  

Material Required:

 

                         Medium:  MR-VP Broth

 

Procedure:

 

  1. Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot. From the rack, 
  2. Take the test tube containing the nutrient broth culture of organism that has been kept for 24 - 48 hours.
  3. Remove the cap of the nutrient agar slant.
  4. Using aseptic technique organism from the nutrient agar slant.
  5. Take the test tube labeled MR
  6. Inoculate the broth with the inoculation loop containing the inoculum from the nutrient agar slant.
  7. Incubate the tube for 24 to 48 hours at 37°C. 
  8. After incubation add5 drops of  Methyl red reagent. 
  9. Shake gently for several minutes. 
  10. Red color  formation within 15 to 20 minutes is a positive result. No red color formation after 15 to 20 minutes is a negative result.

 

Voges prausker test:

 

                         http://vlab.amrita.edu/?sub=3&brch=76&sim=215&cnt=2

 

 

Citrate test:
 

                         Medium:  Citrate medium

 

Procedure:

 

  1. Sterilize the loop in the blue flame of the Bunsen burner till red hot and then allowed to cool. 
  2. Take out an isolated colony of the organism from the  nutrient agar tube with the cooled loop aseptically.
  3. Again flame the neck of the tube and replace the tube in the test tube rack.
  4. Take a sterile citrate slant tube remove the cap and flame the neck of the tube. 
  5. Inoculate the entire surface of the citrate slant (slope) with the provided growth from the nutrient gar slant culture using the inoculating loop (do not stab the butt). The slant of the medium is inoculated by streaking the surface of the agar in a zigzag manner.
  6. Again flame the neck of the citrate tube and place it in the test tube rack 
  7. Tighten the cap and incubate at 37°C for 24-48 hours.
  8. Obtain the tubes from the incubator and observe the colour change.
  9. Blue colour formation indicates a positive test and green color indicates the negative test.

                                       

 TSI:

 

                      http://vlab.amrita.edu/?sub=3&brch=76&sim=216&cnt=2

 

Urease:

 

                       http://vlab.amrita.edu/?sub=3&brch=76&sim=214&cnt=2

 

SIM agar:

 

                       http://vlab.amrita.edu/?sub=3&brch=73&sim=697&cnt=2

 

Gelatin Hydrolysis

 

Procedure

 

  1. Sterilize the needle in the blue flame of the Bunsen burner till red hot and then allowed to cool. 
  2. Stab  inoculate one nutrient gelatin tube with the organism. 
  3. Incubate at 37oC .The reason is gelatin melts at 28º C.   
  4. After incubation gently place the two tubes in the refrigerator for 15 minutes.   
  5. After 15 minutes, if the gelatin remains LIQUID, the result is POSITIVE
  6. If the gelatin is SOLID, the result is negative.  

 

Nitrate Reduction test.

 

                        Media:  Nitrate broth

 

Procedure:

 

  1.  Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot.
  2. Take the test tube containing the nutrient broth culture of organism that has been kept for 24 - 48 hours.
  3. Open the lid of the nutrient agar slant.
  4. Using aseptic technique organism from the nutrient agar slant.
  5. Take the test tube labeled nitrate broth
  6. Inoculate the broth with the inoculation loop containing the inoculum from the nutrient agar slant. 
  7. Incubate the tube for 24 to 48 hours at 37°C.
  8. After incubation add 5 drops of  Reagent A and 5 drops of reagent B.
  9. A red or pink colour,after the addition of reagents indicates  which should develop nitrate reduction. No red color formation after 15 to 20 minutes is a negative result.

 

Catalase test:

 

http://vlab.amrita.edu/?sub=3&brch=73&sim=703&cnt=2

 

Oxidase test:

 

  1. Scrape some fresh growth from the culture slant with a disposable loop or stick and rub  on the oxidase disc 
  2. Examine for blue colour within 10 seconds which indicates a positive test.
  3. Negative result:  no blue colour

 

Inoculation of culture into different agar plate.

 

Materials Required:

 

  1. Bunsen Burner
  2. Inoculating loop
  3. Bacterial culture in nutrient agar slant

 

Starch Hydrolysis
 

 

Procedure

 

  1.  Using aseptic technique take organism from the nutrient agar slant.
  2. The culture is the streaked on the agar surface.
  3. The plates are incubated at 37 degree celcius for 24 hours.
  4. After incubation few drops of iodine is added in the medium.
  5. The absence of blue color in the medium indicates the starch hydrolysis of the organism. The presence of blue color is a negative test.

 

Lipid Hydrolysis
 

 

Procedure

 

  1. Using aseptic technique take organism from the nutrient agar slant.
  2. The culture is the streaked on the agar surface.
  3. The plates are incubated at 37 degree celcius for 24 hours.
  4. After incubation presence of opaque zones around the colonies indicates the hydrolysis of lipid.Absence of opaque zones is a negative test for lipid hydrolysis.


 

 

Growth on selective and Differential Media

 

Materials Required

 

  1. Mannitol salt agar
  2. MacConkey’s agar
  3. Eosin Methylene Blue Agar
  4. Blood Agar

 

Procedure

 

  1. Using aseptic technique take organism from the nutrient agar slant.
  2. The culture is the streaked on the agar surface.
  3. The plates are incubated at 37 degree celcius for 24 hours 
  4. Observe the result.

 

  

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