ISOLATION AND DETECTION OF SALMONELLA IN FOOD SALMONELLA Enterobacteriaceae Major food borne pathogenic bacterium Non-sporeforming Gram-negative Facultative anaerobe Motile by peritrichous flagella Chemoorganotroph Causes: typhoid fever; salmonellosis ISOLATION AND DETECTION INVOLVES: 1. non-selective pre enrichment 2. selective enrichment 3. selection and differentiation 4. presumptive and confirmatory testing PROCEDURES AND PRINCIPLES A. PRE-ENRICHMENT AND ENRICHMENT Allows recovery of injured cells Increases the number of Salmonella in proportion Dilutes toxic substances in the food which may hinder the growth of microorganisms MEDIA USED: 1. Lactose Broth (LB)- preenrichment - allows optimal growth and multiplication of bacteria * Salmonella does not ferment the lactose. it is the accompanying bacteria in the food that ferments the lactose. * Salmonella utilizes the other metabolites produced by the lactosefermenting organisms 2. Selenite cystine broth (SCB)- selective enrichment -inhibits the growth of coliform bacteria and enterococci (Salmonella, Shigella, Proteus, Pseudomonas are slightly inhibited) in the first 6-12 hrs of incubation effect declines after this period Inhibitory 3. Tetrathionate broth (TB)- selective - enrichment tetrathionate and excess thiosulfate suppress coliforms and other accompanying bacteria - tetrathionate reducing bacteria can multiply more or less normally in this medium * acidic tetrathionate decomposition products are formed which are neutralized by calcium carbonate. * bile salts largely inhibit all microorganisms which do not normally live in the intestine. * the addition of brilliant green dye suppresses all the Gram-positive microbial flora and majority of Gram-negative rods. * the resulting culture medium has a very strong inhibitory action *it sometimes better, therefore to omit the brilliant green in order to obtain satisfactory yields of Salmonella 1: Selenite cysteine broth (sterile) 2: Selenite cysteine broth incubated with Salmonella 3: Tetrathionate broth (sterile) 4: Tetrathionate broth with Salmonella 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 Shigella A. brilliant green agar dye: suppresses all the Gram-positve microbial flora and majority of Gram-negative rods B. Phenol red: ph indicator Yellow: acid production in the fermentation of lactose and/or sucrose in the medium Deep red: alkaline condition SALMONELLA ON BGA - (+) colorless, pink to fuschia, transluscent to opaque with surrounding pink to red medium; some Salmonella appear as transparent green colonies if surrounded by organisms fermenting lactose and/or sucrose 2. Xylose lysine desoxycholate agar (XLDA) Phenol red: pH indicator * degradation of xylose, lactose, and sucrose to organic 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 to cadaverine can be recognized by the appearance of purple coloration around the colonies due to an increase in pH -(+) pink colonies with/without black centers; large, many Salmonella colonies may have large glossy black centers or may appear as almost completely black colonies 3. Bismuth sulfite agar (BSA) Brilliant green dye and bismuth sulfite Selective agents that largely inhibits accompanying microflora Sulfur compounds substrate for H2S production Provide • • Freshly prepared medium is strongly INHIBITORY; thus, especially suitable for heavily contaminated samples Colonies of H2S-positive Salmonella exhibit blackening due to formation of Fe2S3 • Reduction of bismuth ions to metallic bismuth produces a metallic brown/black luster (usually only appears after 48hrs of incubation; after 4 days at 4ºC, the inhibitory action of the medium is not as strong and it should then be used for less heavily contaminated specimens (+) brown, gray or black colonies, sometimes with metallic sheen; surrounding medium is usually brown at first, turning black with increasing incubation time PRESUMPTIVE & CONFIRMATORY TESTING Media used: Triple Sugar Iron Sugar (TSI) Rapid Urea Broth Lysine Decarboxylase Broth (LD Broth) 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 hydrogen sulfide by several species of bacteria; H2S reacts with an iron salt to give black Fe2S3 • • • Formation of H2S causes blackening of the medium especially on the butt area of the slant TSI may also show gas production (formation of bubbles) Salmonella and Proteus use the protein at the aerobic surface of the slant as Carbon source, increasing the pH; thus, turning the slant red (+) red (alkaline) slants and yellow (acidic) butts, with or without blackening of agar Sugar/s fermented None Butt Red Slant Red Mcgs P. aeruginosa Salmonella, Shigella E. coli, Klebsiella Glucose Lactose or sucrose Yellow Yellow Red Yellow 1. Rapid Urea Broth -differentiation medium for detecting Gram (-) bacteria which metabolize urea Phenol Red: pH indicator • • • • Only supports growth of microorganisms such as Proteus which utilize urea as their sole carbon source Microorganisms as said above produce urease making them metabolize urea to carbon dioxide and ammonia When medium becomes alkaline, pH indicator changes its color to purple red and medium becomes turbid which is a result of microbial growth (+) purple red broth (Salmonella) is urease negative 3. Lysine Decarboxylase Broth (LD Broth) -used to differentiate Enterobacteriaceae especially Salmonella and Arizona From other microorganisms, based on their ability to decarboxylate lysine with the use of lysine decarboxylase (LDC) Components: gelatin peptone & yeast extract: provide growth nutrients Dextrose: fermentable sugar Bromcresol purple: pH indicator (yellowacid production from fermentation of dextrose; purple red- L-lysine is decarboxylated to cadaverine, increasing the pH) • • As decarboxylation only occurs in an acidic medium (pH below 6.0), the culture medium must first be acidified by glucose fermentation; thus this medium can only be used for the differentiation of glucose fermenting cultures LDC-negative, glucose-fermenting microorganisms cause the medium to become yellow (+) broth retains purple color (Salmonella, with the exception of S. typhi, gives a positive result) 3. TRYPTOPHANE/TRYPTONE BROTH (WITH KOVAC’S REAGENT) tests the ability of a microorganism to produce the enzyme tryptophanase tryptophanase can hydrolyze the tryptophan in the medium, producing indole COMPONENTS: Peptone from casein (tryptone): contains high proportion of tryptophan Kovac’s reagent: detects the presence of indole, turning the interface of the medium deep red (+) deep red color at the interface of the medium (Salmonella and Arizona are tryptophanase negative) ----END. Thank you! Prepared by Team Lara
Report "Isolation and Detection of Salmonella in Food"