Salmonella Bacteriological Analytical Manual (BAM)

Salmonella
Bacteriological Analytical Manual (BAM)

Chapter 5
Introduction :
Several changes are being introduced in BAM 8th edition, which are as follows:
❏ Rappaport Vassiliadis (RV) medium : Now RV medium is used for both high and low microbial
load products. In previous edition it was recommended for analysis of shrimps only. RV replaces
Selenite Cystine medium for analysis of all foods except gaur gum.
❏ Lysine Iron Agar (LIA) : There is a reduction in incubation period of LIA slants. LIA slants
examined, and it gave all the distinctive results within 24 hrs of incubation. No significant changes
observed when incubated further 24 hrs. So, In this edition LIA slants are incubated for 24 hrs as
like TSI slants.
❏ Procedure for surface disinfection of egg shells: Previously, eggs surfaces were disinfected by
soaking them in 0.1% mercuric chloride solution for 1 hr followed by 70% alcohol for 30 min. Now
they are disinfected with 3:1 solution consisting 3 parts of 70% alcohol and 1 part of potassium
iodide solution for 10 sec. As hgcl2 classified as hazardous waste.
❏ Method for Gaur gum : When gaur gum pre-enriched with 1:9 (sample:broth) ratio,
becomes viscous and non pipettable. In this edition, addition of enzyme cellulose to the
pre-enrichment medium results in pipettable mixture.
❏ Picking colonies : In the absence of typical or suspected colonies on selective agar
medium, pick up the colonies which are atypical toTSI and LIA. This recommendation is
based on the fact that up to 4% of all Salmonella cultures isolated by FDA analysts from
certain foods, especially seafoods, during the past several years have been atypical.
Media and Reagents :
1. Lactose Broth.
2. Trypticase soya broth.
3. Tryptone Broth.
4. Selenite Cystine.
5. RV medium.
6. Tetrathionate broth (TTB)
7. Xylose Lysine Deoxycholate Agar (XLD)
8. Hektoen Enteric Agar (HEA)
9. Bismuth Sulfite Agar (BSA)
10. Triple Sugar Iron Agar (TSI)
11. MR VP medium
12. Simmons Citrate.
13. Urea agar.
14. Dey-Engley medium
15. Kovacs reagent.
16. VP medium test reagent.
17. Methyl red test reagent.
18. Salmonella Antiserum O, H and Vi.

Procedure :
For dried egg yolks, liquid milk, prepared powdered mixes and infant feedings containing egg yolk:
If frozen thaw at 45℃ for 15 min in water bath or within 18 hrs at 2℃-5℃
Take 25 g of sample
Non powdered
Powdered
Add 225 ml of lactose broth add 15 ml of lactose

broth stir it
Add 3 additional
portions 10,10,190ml (225)
Adjust pH to 6.8±0.2 if necessary

❏ Eggs:
Shell eggs Liquid whole eggs

Hard boiled eggs
Disinfect eggshells Take 375ml of composite sample Pulverize and weigh

25g of egg
Crack 20 eggs and mix keep at RT for 96±2 hrs Add 225
ml of TSB
Add 2L of trypticase soya broth Add 3375 ml of TSB supplemented with mix well
Ferrous sulfate
❏ Nonfat dry milk: Instant, non instant and whole dry milk :
Take 25 g of sample in 225 ml of Brilliant Green water
Keep it undisturbed for 60±2 min
Incubate at 35℃±2 hrs
Note: For non instant and dry whole milk 25g of analytical units may not be composited.
❏ Casein, Soya flour, and fresh frozen dried products, Vegetables :
● For frozen products thaw them before analysis.
● In case of vegetables use Universal pre enrichment Broth.
Take 25 g of sample in 225 ml of Lactose broth
Incubate at 35℃ for 24±2 hrs

❏ For Dried yeast, Frosting and Topping mixes:
Take 25 g of sample in 225 ml of
Trypticase Soy broth and nutrient broth resp.
Incubate at 35℃ for 24±2 hrs

❏ Spices
● Pepper, celery seed and flakes, chilli powder, sesame seed, vegetable flakes: Pre- enrich the
samples in Trypticase Soy Broth.
● Onion flakes, onion powder and garlic flakes : Pre-enrich sample in Trypticase Soy Broth with
added K2SO3 before autoclaving .
● Allspice, cinnamon, cloves and oregano: Dilute them beyond their toxic level. Allspice, cinnamon
and oregano dilute to 1:100 sample:broth ratio and for cloves at 1:1000 ratio.
❏ Candy and candy coatings including chocolate : Pre- enrichment in reconstituted nonfat dry
milk with added 1% Brilliant green dye.
❏ Coconut : Pre-enrichment in Lactose broth . Surfactants like Tergitol anionic 7 and Triton X-100
use to minimum quantity needed to initiate foaming..
❏ Food dyes and food coloring substances: For pH above 6 , pre-enrichment in Lactose Broth.
For pH below 6, pre-enrichment in Tetrathionate broth mix well.
❏ Gelatin : Pre-enrichment in lactose broth with added 5% aqueous papain solution.
❏ Meat, meat substitutes, meat by-products, animal substances, meals : pre-enrichment in
Lactose Broth.
❏ Mangoes: Pre-enrichment in Buffered Peptone Water.
❏ Tomatoes : Pre-enrichment in Universal pre-enrichment broth.
❏ Animal Food: Pre-enrichment in Buffered Peptone Water,
❏ Environmental testing : Sample environmental surfaces with swabs place in whirl-pak bag
containing Dey-Engley Broth enough to cover the swab. After taking swabs, pre-enrichment in
Lactose broth.
Isolation of Salmonella:
❏ After pre-enrichment:
For gaur gum For other food samples
1ml to 10 ml selenite Cystine and 1 ml to 0.1ml to 10 ml RV medium and 1 ml
10 ml TTB to 10ml TTB
SC and TTB incubated at High microbial load low microbial

load
35℃ for 24±2 hrs RV at 42℃ for 24±2 hrs RV at 42℃ for
24±2 hrs
TTB at 43℃ for 24±2 hrs TTB at 35℃

for 24±2 hrs
Streak loopful of above on XLD, HEA and BSA solid media

❏ Typical Colonies on :
● XLD Agar : Pink colonies with or without black centers.
● HEA : Blue green to blue colonies with or without black centers..
● BSA : Brown, gray or black colonies sometimes they have metallic sheen.
Select typical, suspected or atypical in case absence of typical colonies on solid media , test them for
TSI agar slant and LIA slants.
Biochemical tests :
Biochemical test Reaction Interpretation for
Salmonella
TSI Agar slant 24 hrs at 35℃ Pink slant yellow butt Positive
LIA slant 24 hrs at 35℃ Purple slant purple butt Positive
H2S (TSI and LIA) 24 hrs at Blackening Positive

35℃
Urease 24 hrs at 35℃ Change in color to purple-red Negative
Lysine Decarboxylase broth 48 Change in color to yellow Negative

hrs at 35℃
Phenol red Dulcitol broth 48 hrs Acid/ Gas production Positive

at 35℃
Indole test 24 hrs at 35℃ Addition of Kovac’s reagent Negative

pink-red color ring forms
Phenol red lactose broth 48 hrs Acid/Gas production Negative

at 35℃
Biochemical tests
Biochemical tests Reaction Interpretation for
Salmonella
MR test 48 hrs at 35℃ After addition of MR indicator Positive

media color changes to diffuse
red color
VP media 48 hrs at 35℃ After addition of VP reagents, Negative

media color changes to pink-red
Simmons Citrate 96 hrs at 35℃ Media color changes to blue Positive
Serological Somatic(O) test Agglutination Posiitive
Serological flagellar (H) test Agglutination Positive
Potassium cyanide broth test 48 Turbidity Negative

hrs at 35℃ (KCN)
Malonate broth test 48 hrs at Media color changes to Blue Negative

35℃
Note: For KCN and malonate test use Tryptone broth culture incubate at 35℃ for 24 hrs.

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Salmonella

Bacteriological Analytical Manual (BAM)

17. Methyl red test reagent.

18. Salmonella Antiserum O, H and Vi.

Add 225 ml of lactose broth add 15 ml of lactose

Adjust pH to 6.8±0.2 if necessary

Shell eggs Liquid whole eggs

Disinfect eggshells Take 375ml of composite sample Pulverize and weigh

Take 25 g of sample in 225 ml of Brilliant Green water

Keep it undisturbed for 60±2 min

Incubate at 35℃±2 hrs

Take 25 g of sample in 225 ml of Lactose broth

Incubate at 35℃ for 24±2 hrs

Take 25 g of sample in 225 ml of

Trypticase Soy broth and nutrient broth resp.

Incubate at 35℃ for 24±2 hrs

For gaur gum For other food samples

1ml to 10 ml selenite Cystine and 1 ml to 0.1ml to 10 ml RV medium and 1 ml

10 ml TTB to 10ml TTB

SC and TTB incubated at High microbial load low microbial

TTB at 43℃ for 24±2 hrs TTB at 35℃

Streak loopful of above on XLD, HEA and BSA solid media

LIA slant 24 hrs at 35℃ Purple slant purple butt Positive

H2S (TSI and LIA) 24 hrs at Blackening Positive

Urease 24 hrs at 35℃ Change in color to purple-red Negative

Lysine Decarboxylase broth 48 Change in color to yellow Negative

Phenol red Dulcitol broth 48 hrs Acid/ Gas production Positive

Indole test 24 hrs at 35℃ Addition of Kovac’s reagent Negative

Phenol red lactose broth 48 hrs Acid/Gas production Negative

MR test 48 hrs at 35℃ After addition of MR indicator Positive

VP media 48 hrs at 35℃ After addition of VP reagents, Negative

Simmons Citrate 96 hrs at 35℃ Media color changes to blue Positive

Serological Somatic(O) test Agglutination Posiitive

Serological flagellar (H) test Agglutination Positive

Potassium cyanide broth test 48 Turbidity Negative

Malonate broth test 48 hrs at Media color changes to Blue Negative

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