isolation and detection of salmonella in food
TRANSCRIPT
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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