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8. Isolation and identification of salmonella from seafoods
8. Isolation and identification of salmonella from seafoods
Salmonella are Gram-negative motile rods which are catalase-positive, oxidase-negative and facultatively anaerobic bacteria of Enterobacteriaceae family with over 2000 serovars. Salmonella cause infective type of food poisoning (salmonellosis) in humans upon consumption of contaminated water and food. The most frequent sources of Salmonella food poisoning are poultry, meat, milk, cream and egg. Salmonellosis develops within a short incubation period of 24 hours or less, causing diarrohoea, vomitting, abdominal pain, fever and acute cholera like diseases. Such disease, usually subside in 2-4 days, but, a few develop into typhoidal or septicaemic-type fever. Salmonella especially S. Typhimurium occurs in seafoods. Salmonella should be absent in fish and fishery products.
Materials required
- Lactose Broth (LB)
- Selenite Cystine Broth (SCB)
- Tetrathionate Broth (TTB)
- Bismuth Sulphite Agar (BSA)
- Xylose Lysine Deoxycholate (XLD) Agar
- Brilliant Green Agar (BGA)
- Salmonella-Shigella Agar (SSA)
- Triple Sugar Iron (TSI) Agar
- Lysine Iron Agar (LIA)
- Urea Broth
- Lysine Decarboxylase Broth
- Tryptone Broth
- KCN Broth
- Glucose Phosphate Broth
- Simmons Citrate Agar
- Trypticase Soya Broth
- Brain Heart Infusion (BHI) Broth
- Bromocresol Purple Sugar (Lactose, Sucrose, Dulcitol) Broth
- Malonate Broth
- ONPG (O-Nitrophenyl b-D-Galactopyranoside) compound
Procedure
Isolation of salmonella which are generally encountered in very low numbers in foods involves subjecting the sample to pre-enrichment, selective enrichment, plating on selective agar media and confirming the typical colonies appearing on agar plates by biochemical tests.
About 25 g of seafood sample is homogenized with 225 ml of lactose broth (LB) and incubated at 37oC for 24 hours for pre-enrichment. One ml each of pre-enriched sample is transferred to 10 ml of Selenite cystine broth (SCB) and 10 ml of Tetrathionate broth (TTB) and incubated at 37oC for 24 hours for enrichment. A loopful of enriched sample is streaked onto bismuth sulphite agar (BSA) plates or Xylose-Lysine Deoxycholate (XLD) agar(XLD) plates or brilliant green agar (BGA) plates and incubated at 37oC for 24 to 48 hours. After incubation, typical colonies characteristic of Salmonella developing on different selective agar plates are picked and subjected to biochemical tests for confirmation.
Colony morphology on selective agar plates:
On BSA plates: They appear as brown grey to black, sometimes with metallic sheen. Surrounding medium is usually brown at first, turning black with increasing incubation time. Some strains produce green colonies with little or no darkening of surrounding medium. Super heating and lack of proper agitation results in the reduction of bismuth sulphite indicator to sulphide and caramelization of dextrose. Since Salmonella reduces bismuth sulphite to sulphide, they appear as black colonies on BSA plates.
On XLD agar plates: They appear as pink or red colonies with or without black center.
Reaction of Salmonella to biochemical tests
Tests |
Results |
|
TSI Urease |
A/Alk with / without H2S production |
Agglutination test
Tests |
Results |
|
Polyvalent Flagellar test (H-antigens) |
+ + |
Media composition
Lactose Broth (LB)
Beef Extract : 3.0 g Peptone : 5.0 g Lactose : 5.0 g Distilled water : 1000.0 ml pH : 6.9 ± 0.1 |
Sterilize at 121oC for 15 minutes.
Selenite Cystine Broth (SCB)
Peptone : 5.0 g Lactose : 4.0 g Sodium biselenite : 4.0 g Disodium hydrogen phosphate : 10.0 g L-Cystine : 0.01 g Distilled water : 1000.0 ml pH : 7.0 ± 0.2 |
Suspend the ingredients in sterile distilled water, heat to dissolve, distribute appropriate quantities in sterile test tubes and sterilize in a boiling water bath or free flowing steam for 10 minutes. The prepared medium should be of pale straw colour. Autoclaving or overheating to be avoided as it allows precipitation of selenium.
Tetrathionate Broth (TTB)
Peptone : 18.0 g Calcium carbonate : 25.0 g Sodium thiosulphate : 38.0 g Yeast extract : 2.0 g Sodium chloride : 5.0 g D-Mannitol : 2.5 g Dextrose : 0.5 g Sodium deoxycholate : 0.5 g Brilliant green : 0.01 g Distilled water : 1000.0 ml pH : 7 ± 0.2 |
Suspend the ingredients in sterile distilled water, heat to boil to dissolve, cool to 450C, add 40 ml of iodine solution (8 g potassium iodide and 5 g iodine per 40 ml distilled water), mix and dispense in appropriate quantities in sterile test tubes. The iodine solution should be mixed only on the day the medium is to be used. Do not autoclave.
Bismuth Sulphite Agar (BSA)
Beef extract : 5.0 g Peptone : 10.0 g Dextrose : 5.0 g Disodium hydrogen phosphate : 4.0 g Ferrous sulphate : 0.3 g Bismuth sulphite : 8.0 g Brilliant green : 0.0025 g Agar : 20.0 g Distilled water : 1000.0 ml pH : 7.7 ± 0.2 |
Heat to dissolve the medium in sterile distilled water, cool to about 50oC and pour in to sterile Petri plates. Do not autoclave.
Xylose-Lysine Deoxycholate (XLD) Agar
Xylose : 3.5 g L-Lysine : 5.0 g Lactose : 7.5 g Sucrose : 7.5 g Yeast extract : 3.0 g Sodium chloride : 5.0 g Sodium deoxycholate : 2.5 g Sodium thiosulphate : 6.8 g Ferric Ammonium citrate : 0.8 g Phenol red : 0.08 g Agar : 15.0 g Distilled water : 1000.0 ml pH : 7.4 ± 0.2 |
Heat to dissolve the medium in sterile distilled water. Do not autoclave.
Brilliant green agar:
Peptone : 10.0 g Yeast extract : 3.0 g Sodium chloride : 5.0 g Lactose : 10.0 g Sucrose : 10.0 g Phenol red 90.2% aqueous solution) :40.0 ml Brilliant green (1% aueos solution): 12.5 ml Agar : 15.0 g Distilled water : 948 ml pH : 6.9 |
Dissolve solid ingrediemts by boiling, then add solutions, dispense in 100 ml amounts to flasks and sterilize at 1210C for 15 minutes.
Last modified: Thursday, 23 December 2010, 6:58 AM