EXERCISE 6: Isolation, Detection and Enumeration of Staphylococcus aureus in Food

General Characateristics:

 Gram Positive
 Cocci in Shape
 Non-motile
 Facultative anaerobe
 Catalase positive
 Mesophilic (opt. T = 30-37̊ C)
 pH range 4.0 – 9.8 (opt. pH 6-7)
 Coagulase positive
 Produces heat stable enterotoxin which cause food poisoning
 Belong to the pyogenic (pus-producing) group of bacteria
 Part of the normal microflora of the nose, throat and skin
Selective Enrichment for Detecting S. aureus
Selective Enrichment is recommended for detecting small numbers of S. aureus in food samples of
processed foods.
 Giolitti –Cantoni Medium (GCM)
 A selective enrichment medium that provides optimal condition for the growth of Staphylococcus but
inhibits accompanying microorganisms and contaminants
 Pyruvate glycine and high concentration of mannitol promotes the growth of Staphylococcus
 Lithium chloride inhibits the growth of gram-negative contaminants
 Potassium tellurite inhibits the growth of gram-positive contaminants
 Results: black coloration of the culture medium due to the reduction of tellurite to tellurium indicates the
growth of Staphylococci
Enumeration of S.aureus
 Baird-Parker Agar (BPA)
 Specified for the surface plate count of coagulase-positive Staphylococci in foods
 Pyruvate and glycine selectively stimulates growth of Staphylococci particularly stressed cells
 Lithium chloride and tellurite inhibit the growth of contaminant microorganism and accompanying
microflora
Results:
 Staphylococcus aureus colonies – black, shiny, convex, 1.5 mm in diameter with narrow, white edge
surrounded by a clear zone, 2-5mm wide.
 Opaque rings within the clear zone are present after 48 hours of incubation
 Clearing of egg yolk is due to the production of fatty acids salts by lipolysis
 Blackening is due to the reduction of tellurite to tellurium
Confirmation of S. aureus

Confirmation of the identity of colonies in selective plating media is essential when observed reactions in plates
are insufficient

 Coagulase Test
 Recommended for the determination of coagulase production by Staphylococci
 A criterion potential pathogenicity of Staphylococci
 Coagulase is an enzyme capable of clotting plasma
 Staphylococci strains producing coagulase were usually pathogenic
 Coagulase positive Staphylococci will produce macroscopic clumping or solid clot
 Clotting is first evident when the plasma becomes viscous and flows sluggishly
 The clot may become so hard that the plasma fails to move at all even when the tube is tilted upside
down
 Most coagulase positive organisms clot the plasma within one hour
 Any degree of clotting, no matter how slight, is considered positive

 EDTA (Ethylene Diamine Tetraacetic acid)

 An anti-coagulant that chelates Ca2+ that is necessary for normal plasma clot formation where the
coagulase activity will only contribute to clot formation
 Brain heart Infusion Broth (BHIB)
 Is a general purpose liquid medium used in the cultivation of fastidious and non-fastidious organism
including aerobic and anaerobic bacteria
 A non-selective enrichment medium which provide S. aureus the essential energy and also for the
enhancement of coagulase enzyme
 Growth in the tubes is indicated by the presence of turbidity compared to an uninoculated control.
 Contains 6.5 % NaCl
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 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)- pre-enrichment
 - 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 lactose-fermenting 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
 Inhibitory effect declines after this period
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
 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
 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
 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
• 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
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 (yellow-acid 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)

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