Isolation and Detection of Salmonella in Food

8/4/2019 Isolation and Detection of Salmonella in Food

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ISOLATION AND DETECTION OFSALMONELLA IN FOOD


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SALMONELLA EnterobacteriaceaeMajor food borne pathogenic

bacterium

Non-sporeformingGram-negative

Facultative anaerobe

Motile by peritrichous flagellaChemoorganotroph

Causes: typhoid fever;salmonellosis


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ISOLATION AND DETECTION INVOLVES:

1. non-selective pre enrichment

2. selective enrichment

3. selection and differentiation

4. presumptive and confirmatory testing


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PROCEDURES AND PRINCIPLES


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A. PRE-ENRICHMENT AND ENRICHMENT

Allows recovery of injured cells

Increases the number ofSalmonella

inproportion

Dilutes toxic substances in the foodwhich may hinder the growth ofmicroorganisms


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MEDIA USED:

1. Lactose Broth (LB)- pre-enrichment

- allows optimal growth andmultiplication of bacteria

* Salmonella does not ferment thelactose. it is the accompanying

bacteria in the food that ferments thelactose.

* Salmonella utilizes the othermetabolites produced by the lactose-

fermenting organisms


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2. Selenite cystine broth (SCB)- selectiveenrichment

-inhibits the growth of coliform bacteria and

enterococci (Salmonella, Shigella, Proteus,Pseudomonas are slightly inhibited) in the first6-12 hrs of incubation

Inhibitory effect declines after this period


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3. Tetrathionate broth (TB)- selective

- enrichment tetrathionate and excess

thiosulfate suppress coliforms and otheraccompanying bacteria

- tetrathionate reducing bacteria canmultiply more or less normally in thismedium


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* acidic tetrathionate decomposition productsare formed which are neutralized by calciumcarbonate.

* bile salts largely inhibit all microorganismswhich do not normally live in the intestine.

* the addition of brilliant green dyesuppresses all the Gram-positive microbialflora and majority of Gram-negative rods.


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* the resulting culture medium has a verystrong inhibitory action

*it sometimes better, therefore to omit the

brilliant green in order to obtain satisfactoryyields of Salmonella


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1: Selenite cysteine broth (sterile)2: Selenite cysteine broth incubated with Salmonella3: Tetrathionate broth (sterile)

4: Tetrathionate broth with Salmonella


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B. SELECTION AND DIFFERENTIATION

Media used: 1. Brilliant Green Agar (BGA)selective culture

medium -for the isolation of Salmonella, with the

exception of S. typhosa and ShigellaA. brilliant green agar dye: suppresses all the

Gram-positve microbial flora and majority ofGram-negative rods

B. Phenol red: ph indicatorYellow: acid production in the fermentation of lactose

and/or sucrose in the medium

Deep red: alkaline condition


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SALMONELLA ON BGA

- (+) colorless, pink to fuschia, transluscent toopaque with surrounding pink to red medium;some Salmonella appear as transparent greencolonies if surrounded by organisms fermenting

lactose and/or sucrose


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2. Xylose lysine desoxycholate agar (XLDA)

Phenol red: pH indicator

* degradation of xylose, lactose, and sucrose toorganic acids causes phenol red to change its

color to yellow * production of H2S react to form a precipitate of

black Fe2S3 in the colonies

* bacteria with decarboxylate lysine tocadaverine can be recognized by theappearance of purple coloration around thecolonies due to an increase in pH


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-(+) pink colonieswith/without black centers;large, many Salmonella

colonies may have largeglossy black centers ormay appear as almostcompletely black colonies


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3. Bismuth sulfite agar (BSA)

Brilliant green dye and bismuth sulfite

Selective agents that largely inhibits accompanying

microflora

Sulfur compounds

Provide substrate for H2S production


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Freshly prepared medium is stronglyINHIBITORY; thus, especially suitable forheavily contaminated samples

Colonies of H2S-positive Salmonellaexhibitblackening due to formation of Fe2S3


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Reduction of bismuth ions to metallic bismuthproduces a metallic brown/black luster(usually only appears after 48hrs of

incubation; after 4 days at 4C, the inhibitoryaction of the medium is not as strong and itshould then be used for less heavily

contaminated specimens


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(+) brown, gray or black colonies, sometimeswith metallic sheen; surrounding medium isusually brown at first, turning black with increasingincubation time


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PRESUMPTIVE & CONFIRMATORY TESTING

Media used:

Triple Sugar Iron Sugar (TSI)

Rapid Urea Broth

Lysine Decarboxylase Broth (LD Broth)


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1. Triple Sugar Iron Agar (TSI)-tests ability of an organism to ferment

glucose, lactose, and sucrose, and to

produce H2S

Phenol Red: pH indicator

Thiosulfate: reduced to hydrogensulfide by several species of bacteria;H2S reacts with an iron salt to giveblack Fe

2S

3


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Formation of H2S causes blackening of themedium especially on the butt area of theslant

TSI may also show gas production(formation of bubbles)

Salmonellaand Proteususe the protein at

the aerobic surface of the slant as Carbonsource, increasing the pH; thus, turning theslant red


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(+) red (alkaline) slants and yellow(acidic) butts, with or without blackening ofagar


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Sugar/sfermented

Butt Slant Mcgs

None Red Red P.

aeruginosaGlucose Yellow Red Salmonella,

Shigella

Lactose orsucrose

Yellow Yellow E. coli,Klebsiella


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1. Rapid Urea Broth-differentiation medium for detecting

Gram (-) bacteria which metabolize urea

Phenol Red: pH indicator


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Only supports growth of microorganismssuch as Proteuswhich utilize urea as their

sole carbon source Microorganisms as said above produce

urease making them metabolize urea tocarbon dioxide and ammonia

When medium becomes alkaline, pHindicator changes its color to purple red andmedium becomes turbid which is a result of

microbial growth (+) purple red broth (Salmonella) is urease

negative


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3. Lysine Decarboxylase Broth (LD Broth)

-used to differentiate

Enterobacteriaceae

especially Salmonella

and ArizonaFrom other

microorganisms,

based on theirability to decarboxylate lysine with

the use of lysine decarboxylase (LDC)


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Components:

gelatin peptone & yeast extract:provide growth nutrients

Dextrose: fermentable sugar

Bromcresol purple: pH indicator (yellow-acid production from fermentation ofdextrose; purple red- L-lysine isdecarboxylated to cadaverine,increasing the pH)


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As decarboxylation only occurs in an acidicmedium (pH below 6.0), the culture mediummust first be acidified by glucose

fermentation; thus this medium can only beused for the differentiation of glucosefermenting cultures

LDC-negative, glucose-fermentingmicroorganisms cause the medium tobecome yellow


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(+) broth retains purple color (Salmonella,with the exception of S. typhi, gives a positiveresult)


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3. TRYPTOPHANE/TRYPTONE BROTH(WITH KOVACS REAGENT)

tests the ability of a

microorganism to produce

the enzyme tryptophanase

tryptophanase can

hydrolyze the tryptophan

in the medium, producing

indole


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COMPONENTS:

Peptone from casein (tryptone):contains high proportion of tryptophan

Kovacs reagent: detects the presence

of indole, turning the interface of themedium deep red


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(+) deep red color at the interface of themedium (Salmonellaand Arizona aretryptophanase negative)


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----END.Thank you!

Prepared by Team Lara

Date post:	07-Apr-2018
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Isolation and Detection of Salmonella in Food

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