Detection of Salmonella Species

 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. Selenite - Cystine Medium
2. Tetrathionate Brilliant Green Bile Broth Medium
3. Brilliant Green Agar
4. Xylose – Lysine - Desoxycholate Agar
5. Bismuth Sulphite Agar
6. Deoxycholate citrate agar
7. Triple Sugar Iron Agar
8. Prepare media (2.1 – 2.7 )as per the current version of Respective GTP.

1. Incubate all the above media for 48 h at 32.5^OC ± 2.5⁰C before use.  Discard any contaminated media.?

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1. PROCEDURE    
   1. Pre-Treatment Of The Preparation Being Examined
      1. Water - Soluble Products :
      2. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 ml with the same medium
      3. If necessary, adjust the pH to about 7.0 ± 0.2.
2.  Enrichment
   1. Take 10 ml or 10 g, the quantity corresponding to 1.0 g or 1.0ml (as per the specification) of the pretreated sample in a suitable vessel, add a volume of Fluid Lactose medium to make 100 ml and incubate at 32..5⁰C ± 2.5⁰C for 24 to 48 hours.
   2. Examine the tubes for growth and if growth is present, mix by gentle shaking.
   3. Pipette 1 ml portions into tubes containing, respectively, 10 ml Tetrathionate brilliant green bile broth medium and 10 ml  Selenite-Cysteine medium, mix and incubate at 32.5⁰C ± 2.5⁰C for 12 - 24 h.
   4. After incubation streak, by means of sterilized wire loop, portions from both the media on the surfaces of any two of the following media.
      1.   Brilliant Green Agar Medium
      2. Xylose-Lysine-Desoxycholate Agar Medium
      3.   Bismuth Sulphite Agar Medium
      4.   Deoxycholate citrate agar Medium
   5. Cover and invert the petridishes and incubate at 32.5⁰C ± 2.5⁰C for 24 to 48 hours.
   6. After incubation, examine the colonies.
   7. If none of the colonies conforming to the description given in table are obtained, then the sample passes the test for absence of genus Salmonellae.
   8. If colonies matching the description in Table given below are found, proceed with further identification.

Morphological Characteristics of  Salmonella species

On Selective Agar Medium                                                                                                            

TABLE - I

1.        Observe the Grams reaction of the suspected colonies as per  Respective GTP.
2. If colonies of gram negative rods conforming to the description given in table are obtained proceed with further identification by transferring representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple sugar-iron medium by first streaking the surface of the slant and then stabbing the wire, well beneath the surface.
3. Incubate the Triple Sugar Iron slants at 32.5⁰C ± 2.5⁰C  for 24-38 hours.
4. If examination discloses no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from Hydrogen Sulphide production), the specimen meets the requirements of the test  for the absence of the genus salmonellae.
5. Note :
   1. Carry out control test by repeating the primary and secondary tests using 1 ml of the -enrichment culture and a volume of broth containing 10 to 50 Salmonella abony  prepared from a 24 h culture in Nutrient broth, for the inoculation of tubes.
   2. The test is invalid if the results do not indicate that the control contains salmonellae.

Autoclave, general testing procedure, laminar air flow, bod - incubator