Isolation and enumeration of salmonellae from foods

Isolation and enumeration of salmonellae from foods

Salmonella is a Gram-negative rod-shaped, motile nonspore forming bacterium. There is a widespread occurrence in animals, especially in poultry and swine. Environmental sources of the organism include water, soil, insects, animal feces, raw meats, raw poultry, and raw seafood, etc. S. typhi and the S .paratyphi are normally septicemic and produce typhoid or typhoid-like fever in humans. Other forms of salmonellosis generally produce gastroenteritic symptoms. After ingestion through food/water penetration and passage of salmonellae from gut lumen into epithelium of small intestine where inflammation occurs; there is evidence that the organism may produce an enterotoxin. A wide variety of foods such as raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog legs, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, chocolate etc has been reported to be associated with infection caused by salmonellae. It is estimated that from 2 to 4 million cases of salmonellosis occur in the U.S. annually.

Procedure for detection and enumeration of Salmonella.

The detection of salmonellae in food samples involve several steps such as primary enrichment, selective enrichment and biochemical as well as serological characterizations

Primary enrichment: Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth and blend (sample to media ratio should be 1: 9). Aseptically transfer homogenized mixture to sterile, wide-mouth, screw-cap jar or other appropriate container and incubate at 37^oC for 24 hrs. For enumeration of salmonellae primary enrichment should be done as 3-tube decimal dilution method. For this 10 ml of the homogenate should be aseptically transferred into the first set of three test tubes containing 10ml double strength lactose broth. The other two sets of test tubes containing 10 ml single strength lactose should be inoculated with 1 ml and 0.1 ml homogenate respectively. All the tubes should be incubated at 37^oC for 24 hrs.

Selective enrichment: selective enrichment should be done either in selenite cystine broth (SCB) or tetrathionate broth (TTB). For this 1 ml culture from tubes showing growth (turbidity) after primary enrichment should be aseptically transferred into 10 ml SCB or TTB. Incubate at 37^oC for 24 to 48 hrs.

Selective plating: One loopful culture from selective enrichment media should be streaked on to any of the following selective agar media and incubate at 37^oC for 24 to 48 hrs.

Xylose Lysine Desoxycholate (XLD) agar

Bismuth Sulphite Agar (BSA)

Hektoen enteric Agar (HEA)

After incubation typical salmonella colonies (i.e red colonies with black center on XLDA, lustrous black with silvery white metallic sheen on BSA and blue-green colonies with black center on HEA) should be picked and subjected to biochemical tests.

Biochemical tests for Salmonellae.

Test	Reaction
Fermentation of lactose	No acid, no gas
Fermentation of sucrose	No acid, no gas
Fermentation of salicin	No acid, no gas
Fermentation of dulcitol	Acid and gas
Indole production	Negative
Urease test	Negative
Citrate utilization	Positive
O/F test	Fermentative
Reaction on Triple Sugar Iron agar (TSIA)	Alkaline slant (red) and Acid 9yellow) but (K/A), H2S and gas +/-
Reaction on Lysine iron gar (LIA)	Alkaline purple) slant and but (K/A), H2S and gas +/-
Polyvalent somatic (O) test	Agglutinative
Polyvalent Flgellar (H) test	Agglutinative

Cultures showing typical reactions match with that of salmonellae in the biochemical test should be referred for their MPN reading in selective enrichment step and the MPN index per gram or 100 ml of salmonellae should be find out from the standard table.

Isolation and enumeration of shigellae from foods

Shigellae are Gram-negative, facultatively anaerobic, nonmotile, non-sporeforming rods in the family Enterobacteriaceae. The illness caused by Shigella (shigellosis / bacillary dysentery) accounts for less than 10% of the reported outbreaks of foodborne illness. Shigellosis, although commonly regarded as waterborne, is also a foodborne disease restricted primarily to higher primates, including humans. It is usually spread among humans by food handlers with poor personal hygiene. The organism is frequently found in water polluted with human feces. The disease is caused when virulent Shigella organisms attach to, and penetrate, epithelial cells of the intestinal mucosa. After invasion, they multiply intracellularly, and spread to contiguous epitheleal cells resulting in tissue destruction. Some strains produce enterotoxin and Shiga toxin (very much like the verotoxin of E. coli O157:H7. Salads, raw vegetables, milk and dairy products, and poultry are reported to be associated with infection caused by shigellae. Contamination of these foods is usually through the fecal-oral route. Fecally contaminated water and unsanitary handling by food handlers are the most common causes of contamination. The genus Shigella consists of four species: S. dysenteriae (subgroup A), S. flexneri (subgroup B), S. boydii (subgroup C), and S. sonnei (subgroup D). Shigella organisms may be very difficult to distinguish biochemically from Escherichia coli.

Enrichment: Aseptically weigh 25 g sample into 225 ml Shigella broth to which novobiocin (0.5 ?g/ml) has been added. Incubate at 44?C water bath for 24 h. For enumeration of shigellae, primary enrichment should be done in 3-tube decimal dilution method using Shigella broth (supplemented with novobiocin) as described above.

Platting : One loopful culture from selective enrichment media should be streaked on to MacConkey agar and incubate at 37^oC for 24 to 48 hrs. After incubation typical colonies (Shigella colonies are slightly pink and translucent, with or without rough edges on MacConkey agar ) should be picked and subjected to further biochemical and serological characterizations.

Biochemical tests for Shigellae.

Test	Reaction
Fermentation of lactose	No acid, no gas
Fermentation of sucrose	No acid, no gas
Fermentation of salicin	No acid, no gas
Citrate utilization	Negative
H2S production	Negative
Urease	Negative
Methyl red	positive