The test for Identification of Salmonella species is provided to determine compliance with the requirements given in individual monograph / specifications.
PRINCIPLE
Identification of Salmonella species which is pathogenic bacteria causes disease in human body. It can be identified by using specific, differential media which only supports growth of Salmonella.
REAGENTS AND MEDIA
1. Selenite F Broth
Peptone 5.0 g
Lactose 4.0 g
Disodium Hydrogen Orthophosphate 10.0g
Sodium Hydrogen Selenite 4.0 g
Water 1000 mL
Dissolve, distribute in sterile containers and sterilize by maintaining at 100 OC for 30 min.
2. Fluid Lactose Medium
Beef Extract 3.0 g
Pancreatic Digest of Gelatin 5.0 g
Lactose 5.0 g
Water 1000 mL
Cool as quickly as possible after sterilization. pH after sterilization 6.9 ± 0.2
3. Fluid Selenite - Cystine Medium
Pancreatic Digest of Casein 5.0 g
Lactose 4.0 g
Sodium Phosphate 10.0 g
Sodium Acid Selenite 4.0 g
L - Cystine 10.0 g
Water 1000 mL
Final pH 7.9 ± 0.2. Mix and heat to effect solution. Heat in flowing steam for 15 minutes.
Do not Sterilize.
4. Fluid Tetrathionate Medium
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.
5. Tetrathionate Broth
Beef Extract 0.9 g
Peptone 4.5 g
yeast Extract 1.8 g
Sodium Chloride 4.5 g
Calcium Carbonate 25.0 g
Sodium Thiosulphate 40.7 g
Water 1000 mL
Mix, bring to the boil, cool to below 45 OC , and add a solution of 6 g of Iodine and 5 g of Potassium Iodide in 20 mL of water. Mix, and distribute in sterile containers.
Do not heat after Iodine has been added. The medium with Iodine should be used on the day it is prepared. The base medium without Iodine may be stored indefinitely after sterilization.
6. Nutrient Broth
Beef Extract 10.0 g
Peptone 10.0 g
Sodium Chloride 5.0 g
Water 1000 mL
Dissolve with the aid of heat. Adjust to pH 8.0 to 8.4 with 5 M Sodium Hydroxide and boil for 10 min. Filter, adjust to pH 7.2 to 7.4, and sterilize by maintaining at 115 OC for 30 min.
7. Brilliant Green Agar
Peptone 10.0 g
Yeast Extract 3.0 g
Lactose 10.0 g
Sucrose 10.0 g
Sodium Chloride 5.0 g
Brilliant Green 12.5 g
Agar 12.0 g
Water 1000 mL
Mix, allow to stand for 15 min. sterilize by maintaining at 115 OC for 30 min. and mix before pouring.
8. Desoxycholate Citrate Agar
Beef Extract 5.0 g
Peptone 5.0 g
Lactose 10.0 g
Trisodium Citrate 8.5 g
Sodium Thiosulphate 5.4 g
Ferric(III) Citrate 1.0 g
Sodium Desoxycholate 5.0 g
Neutral Red 0.02 g
Agar 12.0 g
Water 1000 mL
Mix and allow to stand for 15 min. with continuous stirring, bring gently to the boil and maintain a boiling point until solution is complete. Cool to 80 OC , mix, pour and cool rapidly.
Care should be taken not to overheat Desoxycholate citrate agar during preparation. It should not be re-melted and the surface of the plates should be dried before use.
9. Bismuth Sulphite Agar
1.
Beef Extract 6.0 g
Peptone 10.0 g
Agar 24.0 g
Ferric(III) Citrate 0.4 g
Brilliant Green 10.0 mg
Water 1000 mL
Dissolve with the aid of heat and sterilize by maintaining at 115 OC for 30 min.
2. Ammonium bismuth Citrate 3.0 g
Anhydrous Sodium Hydrogen Orthophosphate 5.0 g
D-Glucose Monohydrate 5.0 g
Water 100 mL
Mix , heat to boiling, cool to room temperature, add 1 volume of solution (2) to 10 volumes of solution (1) previously melted and cooled to a temperature of 55 OC and pour.
Bismuth sulphite agar should be stored at 2 - 8 OC for 5 days before use.
10. Triple Sugar Iron Agar
Beef Extract 3.0 g
Yeast Extract 3.0 g
Peptone 20.0 g
Lactose 10.0 g
Sucrose 10.0 g
D-Glucose Monohydrate 1.0 g
Iron (III) Sulphate 0.2 g
Sodium Chloride 5.0 g
Sodium Thiosulphate 0.3 g
Phenol Red 24.0 mg
Agar 13.0 g
Water 1000 mL
Mix, allow to stand for 15 min. bring to and maintain at boiling point until solution is complete, mix, distribute in tubes and sterilize by maintaining at 115 OC for 30 min. Allow to set in a sloped form with a butt about 2.5 cm long.
11. Urea Broth
Potassium Dihydrogen Orthophosphate 9.1 g
Anhydrous Disodium Hydrogen Orthophosphate 9.5 g
Urea 20.0 g
Yeast Extract 0.1 g
Phenol Red 0.01 g
Water 1000 mL
Mix, sterilize by filtration and distribute aseptically in sterile containers.
12. Xylose-Lysine-Desoxycholate Agar Medium
Xylose 3.5 g
Lysine 5.0 g
Lactose 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 18.0 mg
Agar 13.5 g
Sodium Desoxycholate 2.5 g
Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 800.0 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 overheat or sterilize. Transfer at once to water batch maintained at about 50 OC and pour into plates as soon as the medium has cooled.
Incubate all the above media for 24 h at 37 OC before use. Discard any contaminated media.
Instead of preparing media, one can also use dehydrated media of Himedia/Difco and rehydrate the required quantity as per instructions on the bottle label, dispense in required quantities and sterilize at 15 lbs and 121 OC for 15 min
TABLE - I
PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
Water Soluble Products :
1. Dissolve or dilute 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in sterile 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.
2. If necessary, adjust the pH to about 7.0
Non-Fatty Products in Water :
1. Suspend 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in sterile Lactose medium shown not to have anti-microbial activity under the medium broth or another suitable
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3 . A suitable surface-active agent such as 0.1 % w/v of Polysorbate 80 may be added to assist the suspension of poorly wettable substances.
4. If necessary, adjust the pH of the suspension of about 7.0.
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80.
2. If necessary, heat to not more than 40 OC.
3. Mix carefully while maintaining the temperature in a water bath or in an oven.
4. Add 85 mL of sterile Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not more than 40 OC if necessary.
5. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min.
6. If necessary, adjust the pH of the emulsion to about 7.0.
For a fluid specimen in Aerosol form :
1. Chill the container in an Alcohol-dry ice mixture for approx. 1 h., cut open the container, allow it to reach room temperature, permit the propellant to escape , or warm to drive off the propellant if feasible and transfer the quantity of test material required of the procedures as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residue.
PROCEDURE
As per BP
A. Enrichment
Incubate the solution, suspension or emulsion obtained by appropriately pre-treating the preparation being examined as described above at 35 - 37 OC for 5 - 24 h, as appropriate for enrichment.
B. Primary Test
1. Transfer 10 mL of the enrichment culture to 100 mL of Tetrathionate bile brilliant green broth and incubate at 42 - 43 OC for 18 - 24 h.
2. Subculture on at least two of the following three agar media : Desoxycholate citrate agar; Xylose, Lysine, Desoxycholate agar and Brilliant Green Agar.
3. Incubate at 35 - 37 OC for 24 - 48 h.
4. If any colonies conforming to the description in Table II are produced, carry out the secondary test.
C. Secondary Test
1. Subculture any colonies showing characteristics given in Table I on Triple sugar iron agar using surface and deep inoculation. (This can be done by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle and incubating at 35 - 37 OC for 18 - 24 h.)
2. The presence of Salmonella is provisionally confirmed if, in the deep culture but not in the surface culture, there is a change of color from red to yellow and usually a formation of gas, with or without production of Hydrogen sulfide in the agar.
3. Precise confirmation may be carried out by appropriate biochemical and serological tests.
4. The preparation being examined passes the test if, in the primary test, cultures of the type described do not appear or if, in the secondary test, the confirmatory biochemical and serological tests are negative.
As per IP’96
A. Enrichment
1. Place the prescribed quantity in a sterile screw-capped jar, add 100 mL (unless otherwise directed) of Nutrient broth, allow to stand for 1 h (4 h for Gelatin ), and shake again.
2. Loosen the cap and incubate at 35 - 37 OC for 24 h.
B. Primary Test
1. Add 1 mL of the enrichment culture to each of two tubes containing
a) 10 mL of Selenite F broth and
b) 10 mL of Tetrathionate broth and incubate at 36 - 38 OC for 48 h.
2. From each of these two cultures inoculate three plates containing a layer of
a) Brilliant Green Agar
b) Desoxycholate Agar
c) Bismuth Sulphite agar
3. Incubate the plates at 36 - 38 OC for 18 - 24 h.
4. If nay colonies conforming to the description on Table I are produced, carry out the secondary test.
C. Secondary Test
1. Subculture any colonies showing the characteristics given in Table I in Triple sugar iron agar, by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle and at the same time inoculate a tube of Urea broth.
2. Incubate at 36 - 38 OC for 18 - 24 h.
3. The formation of acid and gas in the stab culture (with or without concomitant blackening ) and the absence of acidity from the surface growth in the triple sugar iron agar, together with absence of a red color in the urea broth, indicates the presence of Salmonella.
4. If acid but no gas is produced in the stab culture the identity of the organisms should be confirmed by agglutination tests.
As per USP
A. Enrichment
1. To the specimen contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL and incubate at 37 OC for 48 h.
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 Fluid Tetrathionate medium and 10 mL Fluid Selenite-Cysteine medium, mix and incubate at 37 OC for 12 - 24 h.
B. Primary Test
1. After incubation streak, by means of sterilized wire loop, portions from both the media on the surfaces of.
i. Brilliant Green Agar Medium
ii. Xylose-Lysine-Desoxycholate Citrate Agar Medium
iii. Bismuth Sulphite Agar Medium contained in petridishes.
2. Cover and invert the petridishes and incubate at 37 OC for 24 h.
3. After incubation, examine the colonies.
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.
C. Secondary Test ( Conformatory test)
1. 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. Incubate at 37 OC for 24-38 h.
2. If examination discloses no evidence ou 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.
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 (ATCC 6017) prepared from a 24 h culture in Nutrient broth, for the inoculation of tubes (a.) and (b.)
2. The test is invalid if the results do not indicate that the control contains salmonellae.
Note : Use the method as per the Pharmacopoeial status ( grade ) of the material. In case of In-house specification, follow the method as per IP or as specified.
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