INTRODUCTION:
Salmonella is a Gram-negative, motile bacterium. It was named after Daniel Elmer Salmon, an American veterinary pathologist, although it was his partner and contemporary Theobald Smith (better known for his work on anaphylaxis)who first discovered the bacterium in 1885 from pigs. Salmonella is a genus of rod-shaped Gram-negative enterobacteria that auses typhoid fever, paratyphoid fever, and food borne illness. Salmonella species are motile and produce hydrogen sulfide.
PREPARATION OF MEDIA:
Sodium hydroxide 1N: Dissolve 4.0 g of sodium hydroxide in water to make 100 mL.
Hydrochloric Acid 1N: Dissolve 8.5 ml of Hydrochloric Acid in water to make 100 mL.
Soyabean-Casein Digest Broth Medium/ Xylose-Lysine-Desoxycholate Agar Medium (XLD): Reconstitute dehydrated media as directed by the manufacturer and sterilize in an autoclave at 121°C for 20 minutes.
Rappaport Vassiliadis Salmonella Enrichment Broth: Reconstitute dehydrated media as directed by the manufacturer and sterilize in an autoclave at 115°C for 20 minutes.
PROCEDURE:
Sample Preparation and Pre enrichment:
Aseptically add 10g of specimen if it’s a solid or 10ml accurately measured, if the specimen is a liquid to make 100ml of Soyabean-Casein Digest broth Medium unless otherwise described in the specific STP.
Negative Control: Take 100 mL of sterile Soyabean-Casein Digest Medium without any inoculation.
Positive Control: Add 1mL Salmonella typhimurium culture suspension in to 100ml Soyabean-Casein Digest broth Medium.
Note: Incase Salmonella typhi is specified in the individual specification instead of Salmonella typhimurium use the same for positive culture.
Incubate the sample, negative control and positive control 30-35°C for 18 - 24hours.
Examine the medium for growth.
Interpretation:
Negative control flasks should not show any growth.
Positive control growth should be present.
If growth is present (turbidity observed) in the sample flask, or the media difficult to observe the growth with sample (Likely some powders and capsules can alter the color and the clearness of the media) mix by gentle shaking and proceed to test for Salmonella species. If there is no growth in the sample flask, that indicates, sample meets the requirement for absence of Salmonella species.
Note: Incase of water sample add 10 ml sample in 100 ml Soyabean-Casein Digest broth Medium for test and negative control with out adding anything and positive control by adding Salmonella typhimurium culture in 10 ml media.
Selective Enrichment and Conformation test :
Pipette each 0.1mL sample portions from Soyabean-Casein Digest broth Medium to tubes containing 10 ml of Rappaport Vassiliadis salmonella Enrichment Broth mix and incubate at 30-35°C for 18 to 24 hours.
Maintain a negative control by the omitting the sample and standard culture in respective tubes.
Maintain a positive control by inoculating the standard cultures of Salmonella typhimurium in the respective tube.
Note: Incase Salmonella typhi is specified in the individual specification instead of Salmonella typhi use Salmonella typhimurium for positive culture.
Streak portions of sample, on the surface of Xylose-Lysine- Desoxycholate Agar, by taking a loop full of Rappaport Vassiliadis salmonella Enrichment Broth.
Maintain a negative control by the omitting the sample and standard culture in respective plates.
Maintain a positive control by streaking the standard cultures of Salmonella typhimurium in the respective plates.
Cover and invert the dishes and incubate at 30 to 35°C for 18 to 48 hours.
Examine the plates.
Interpretation:
Negative plates of Xylose-Lysine-Desoxycholate Agar should not show any colonies.
Positive plates should show characteristic growth.
If colonies on the sample plates of Xylose-Lysine-Desoxycholate Agar do not match to the description given in the Table-1, then the sample meets the requirements for the absence of Salmonella species. If require perform identification test.
Salmonella, general testing procedure
