Identification of Cultures

1.    APPARATUS

1. Autoclave
2. BOD - Incubator
3. Bacteriological Incubator
4. pH – meter
5. Hot air Oven
6. Laminar Air flow
7. Analytical Balance
8. Spirit Lamp / Bunsen Burner
9. Micropipette
10. Petri dishes
11. Test tubes
12. Media bottles   

2.   REAGENTS   AND  MEDIAS

1. Prepare media as per the current version of Respective GTP.
   1. Pseudomonas agar for fluorescein
   2. Pseudomonas agar for pyocyanin
   3. Potato dextrose Agar Slants
   4. Vogel-Johnson agar
   5. 3% hydrogen peroxide solution
   6. Eosin methylene blue Agar
   7. Kovac’s Indole reagent
   8. Cetrmide Agar
   9. MacConkey’s agar
   10. Triple Sugar Iron Agar
   11. Nitrate Broth
   12. α-napthylamine solution
   13. Sulphanilic Acid
   14.  Gram’s Iodine Solution
   15. Sabouraud Chlorampenical Agar
   16. Oxidase disc
   17. Cotton Blue solution/ Lacto phenol blue
   18. Phenol solution (1 in 100)
   19. Ether
   20. MacConkey’s broth
   21. Bismuth Sulphite Agar Medium
   22. Gram’s Crystal violet solution (1%).
   23. Gram’s  Decoluoriser / 70% Alcohol.
   24. Gram’s Safrranine solution
   25. Nutrient broth
   26. Fluid Tetrathionate medium
   27. Peptonised milk (15%)
   28. 1 N Ferric chloride solution
   29. Tryptone Soy agar
   30. Saline solution (0.9%)

3.  Procedure

Identification Of Stock Cultures Before Fresh Transfer

3.1.   Test For Staphylococcus aureus :

Streak for isolation onto Vogel - Johnson  agar and incubate at 32.5°C±2.5° c for  24 hrs. reincubates for an additional 24hrs. A presumptive positive occurs as black colonies with yellow zone. Confirm by conducting a gram stain and coagulase test. If the gram stain shows gram positive cocci, and if the coagulase test is positive then the culture confirm as S. aureus                    

3.2. Test For Candida albicans .   

1. Streak for isolation on Sabouraud Chlorampenical Agar and incubate for 48 hrs at  20° c –25 ⁰ c.A presumptive positive occurs as white smooth /  mucoid colonies, circular or Hemispherical with a slight mycelial fringe. Confirm by conducting a gram stain, If gram stain shows gram negative yeast then the culture is considered as

C. albicans.

3.3. Test For Pseudomonas aeruginosa

Oxidase and Pigment Tests:With the aid of an inoculating loop, streak the enriched culture on Cetrimide agar medium and incubate for 24 hours at 32.5 ± 2.5°C, greenish colonies will be seen. Streak the isolated colonies on the agar surfaces of Pseudomonas agar medium for detection of Fluorescin and pseudomonas agar medium for detection of Pyocyanin.Cover and invert the inoculated media, and incubate at 32.5 ±2.5^OC for not less than 3 days.Examine the streaked surface for florescin under ultraviolet light.

3.3.1.      Confirm colonial growth by gram staining and oxidase test.

3.3.2.      For Oxidase test, place or transfer colonies on oxidase disks. if there is development of purple colour within one minute and gram staining shows gram negative rods, the culture confirms positive for pseudomonas aeruginosa.

3.4.       Test For Escherichia coli

3.4.1.      With the help of an inoculating loop transfer the culture to 100 ml Nutrient broth and incubate at 32.5±2.5°C for 24 hr.

3.4.2.      Pipette 1 ml of the enrichment culture into a tube containing 5 ml of MacConkey’s broth and incubate at 32.5±2.5°C for 48 hr. Upon incubation there should be production of acid and gas.

3.4.3.      By means of an inoculating loop, streak a portion from the MacConkey’s broth tube on the surface of MacConkey agar medium. Also transfer 0.1 ml from above MacConkey’s broth tube to a tube containing 5 ml of Peptone water. Carry out incubation of MacConkey agar plate.

3.4.4.       Upon incubation, on MacConkey agar there should be growth of red, generally non-mucoid colonies, which may have surrounding reddish zone of bile precipitation.

3.4.5.      Carry out Gram staining by using the colonial growth obtained on MacConkey agar. Gram staining should show presence of Gram-negative rods ( Cocco-bacilli).

3.4.6.      Transfer the colonial growth from MacConkey agar, by means of an inoculating loop, to the surface of Levine Eosin-Methylene blue agar medium, plated on petridishes and incubate at 32.5±2.5 °C for 48 h and also inoculated into Peptone water and incubate at 43 – 45°C for 24 hrs.

3.4.7.      Upon incubation, if the colonies exhibit both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light

3.4.8.      Carry out detection of Indole production by adding 0.5 ml of Kovac’s reagent to the above peptone water tube, shake well and allow to stand for one minute. There should be development of a red colour in the reagent layer indicating the presence of indole, the culture confirms to be Escherichia coli..

3.5.          Test For Salmonella speciess.

3.5.1.      With the help of an inoculating loop transfer the culture to 100 ml Nutrient broth and incubate at 35±2°C for 24 hrs.

3.5.2.      Pipette 1 ml portion of this enrichment culture and transfer into a tube containing 10 ml Fluid Tetrathionate medium, mix and incubate at 32.5±2.5°C for 12 - 24 hrs.

3.5.3.      After incubation streak, by means of sterilized wire loop, portion from the above Fluid Tetrathionate medium on the surfaces of Bismuth Sulphite Agar Medium .

3.5.4.      Cover and invert the petridishes and incubate at 32.5±2.5^OC for 24 hrs.

3.5.5.      After incubation, examine the colonies.

3.5.6.      If the colonies conforming to the description given in table are obtained, then the culture confirms to be Salmonella sps.

3.5.7.      Carry out Gram staining by using the colonial growth obtained as above. Gram staining should show the presence of Gram-negative rods. Proceed with further identification by transferring representative colonies individually, by means of an inoculating wire, to a Triple sugar-iron medium by first streaking the surface of the slant and then stabbing the wire, well beneath the surface.  Incubate at 32.5±2.5°C for 24-38 hrs.

3.5.8.      Upon incubation, tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from Hydrogen Sulphide production), confirm the culture to be Salmonella species.

TABLE - I

3.6.         Test For Aspergillus niger

3.6.1.      Carry out mounting of the culture using Lacto phenol / Cotton Blue stain.

Macroscopic observation : Mycelium with Conidiophores bearing black coloured conidia.

Microscopic observation: Conidiophores showing foot cell, sterigma and round shaped, blue coloured conidia should be present.

3.7.         Test For Bacillus subtilis

3.7.1.      Isolate the refrigerated stock culture on TSA plate and incubate for 30⁰ c -35 ⁰ c for good growth.

3.7.2.      Carry out following tests, which should reveal results as follows:

 Microscopic observation:

• Upon Gram staining Gram positive rods should be observed.

•  Presence of spores should be detected by using spore staining technique current version of Respective GTP.   

1. Nitrate reduction     :  The culture should give a positive test.
2. Catalase test             :  The culture should give a positive test.

Cultures, analytical balanc, micropipette, general testing procedure