1. APPARATUS
1.1. Autoclave
1.2. BOD - Incubator
1.3. Bacteriological Incubator
1.4. pH – meter
1.5. Hot air Oven
1.6. Laminar Air flow
1.7. Analytical balance
1.8. Spirit Lamp / Bunsen Burner
1.9. pipette
1.10. Petri dishes
1.11. Test tubes
1.12. Media bottles
2. REAGENTS AND MEDIAS
2.1 Fluid Lactose Medium
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Beef Extract 3.0 g
Pancreatic Digest of Gelatin 5.0 g
Lactose 5.0 g
Water 1000 ml
Final pH after Sterilization 6.9 ± 0.2
Mix, and dispense as desired and sterilize in an autoclave at 121–1240C for about 30 min. Cool as quickly as possible after sterilization.
If required add surface active agents or inactvators of antimicbial agents.
0.1 to 1.0 % w/v Polysorbate 20 or Polysorbate 80 may be added. Sterilize by heating in an autoclave at 121–124OC for about 30 min.
2.2 Soyabean Casein Digest Medium
2.3 Prepare media as per the current version of Respective GTP.
2.1.1. If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Purified Water 1000 ml
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after Sterilization between 7.1 to 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at about 121–124 0C for about 30 min.
2.4 Fluid Tetrathionate Medium
2.5 Prepare media as per the current version of Respective GTP..
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic Digest of Casein 2.5 g
Peptic Digest of Animal Tissue 2.5 g
Bile Salts 1.0 g
Calcium Carbonate 10.0 g
Sodium Thiosulphate 30.0 g
Water 1000 ml
Heat the solution of solids to boiling. On the day of use, add a solution prepared by dissolving 5 g of Potassium Iodide and 6 g of Iodine in 20 ml of water. Then add 10 ml of a solution of brilliant green (1 in 1000) and mix. Do not heat the medium after adding the brilliant green solution.
2.6 Brilliant Green Agar
2.7 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Yeast Extract 3.0 g
Peptic digest of animal tissue 5.0 g
Lactose 10.0 g
Sucrose 10.0 g
Sodium Chloride 5.0 g
Brilliant Green 12.5 g
Phenol red 20.0 g
Agar 12.0 g
Water 1000 ml
Sterilize by maintaining at 121-124 OC for 30 min.
2.8 Xylose – Lysine - Deoxycholate Agar
2.9 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Xylose 3.5 g
L-Lysine 5.0 g
Lactose 10.0 g
Sucrose 7.5 g
Sodium chloride 5.0 g
Yeast Extract 3.0 g
Phenol red 80 mg
Agar 13.5 g
Sodium Deoxycholate 2.5 g
Sodium Thiosulphate 6.8 g
Ferric Ammonium Citrate 800 mg
Water 1000 ml
Final pH: 7.4 ± 0.2
Heat the mixture of solids and water, with swirling, just to the boiling point. Do not over heat or Sterilize. Transfer at once to a water bath maintained at about 50°C, and pour into plates as soon as the medium cooled.
2.10 Deoxycholate citrate agar
2.11 repare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Beef extract 10.0 g
Meat peptone 10.0 g
Lactose monohydrate 10.0 g
Sodium citrate 20.0 g
Ferric citrate 1.0 g
Sodium deoxycholate 5.0 g
Agar 13.5 g
Neutral red 20.0 mg
Purified water 1000 ml
Final pH 7.3 ± 0.2
Heat gently to boiling for 1 min, cool to 500C and pour into Petri dishes. Do not heat in Autoclave.
2.12 Triple Sugar Iron Agar
2.13 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic digest of casein 10.0 g
Peptic digest of animal tissue 10.0 g
Lactose 10.0 g
Sucrose 10.0 g
Dextrose 1.0 g
Ferrous ammonium Sulphate 0.2 g
Sodium Chloride 5.0 g
Sodium Thiosulphate 200 mg
Agar 13.0 g
Phenol Red 25 mg
Water 1000 ml
PH after Sterilization 7.3 ± 0.2
Sterilize the medium in an autoclave at 121–1240C for about 30 min.
Incubate all the above media for 48 h at 32.5OC ? 2.50C before use. Discard any contaminated media.
3. PROCEDURE
3.1. PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
3.1.1. Water - Soluble Products :
3.1.1.1. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Soyabean casein digest medium 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
3.1.1.2. If necessary, adjust the pH to about 7.0 ± 0.2.
3.2. Enrichment
3.2.1. 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 Soyabean casein digest medium to make 100 ml and incubate at 300C to 350C for 18 to 24 hours.
3.2.2. Examine the tubes for growth and if growth is present, mix by gentle shaking.
3.2.3. Transfer 1 ml of the enrichment culture to 10 ml Fluid Tetrathionate medium, mix and incubate at 410C to 430C for 18 - 24 hours.
3.3. Primary Test
3.3.1. After incubation streak, by means of sterilized wire loop, portions from both the media on the surfaces of any two of the following media.
3.3.1.1. Brilliant Green Agar Medium contained in petridishes
3.3.1.2. Xylose-Lysine-Desoxycholate Agar Medium contained in petri dishes
3.3.1.3. Deoxycholate citrate agar Medium contained in petridishes
3.3.2. Cover and invert the petridishes and incubate at 300C to 350C for 18 to 72 hours.
3.3.3. After incubation, examine the colonies.
3.3.4. 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.
3.3.5. If colonies matching the description in Table given below are found, proceed with further identification.
Morphological Characteristics of Salmonella speciesOn Selective Agar Medium
TABLE - I
Medium Description of Colony
Brilliant Green Agar Small, Transparent and colorless or pink or opaque - white colonies often surrounded by a pink or red zone
Desoxycholate Citrate Agar Well developed , Colorless colonies
Xylose, Lysine, Desoxycholate Agar Well-developed, red with or without black centers
3.4. Secondary Test (Confirmatory test)
3.5. Observe the Grams reaction of the suspected colonies as per Respective GTP.
3.5.1.
3.5.2. 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.
3.5.3. Incubate the Triple Sugar Iron slants at 32.50C ? 2.50C for 24-38 hours.
3.5.4. 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.
3.6. Note :
3.6.1. 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 (NOTCH 6017 or equivalent) prepared from a 24 h culture in Nutrient broth, for the inoculation of tubes.
3.6.2. The test is invalid if the results do not indicate that the control contains salmonellae.
autoclave, bod - incubator, bacteriological incubator, general testing procedure
