Exercise 7
Isolation and Detection of
Salmonella in Foods
Prepared by:Frances Grace N. OrdoñezMCB180 B-1L
Salmonella
SalmonellaFamily EnterobacteriaceaeGram negative rodsNon-sporeformerOptimum tempterature: 370CDoes not ferment lactose or sucroseMotile by peritrichous flagellaFacultatively anaerobePathogenic
◦gastroenteritis, enteric fever, and septicemia
Procedures and MediaPre-enrichment – Lactose BrothEnrichment – Selenite Cystine Broth and
Tetrathionate BrothSelection and Differentiation – Brilliant
Green Agar, Xylose Lysine Deoxycholate Agar, Bismuth Sulfite Agar
Confirmation using physiological tests –Triple Sugar Iron Agar, Lysine Decarboxylase Broth, Tryptone Broth, Urea Broth , Potassium Cyanide Broth (KCN)
Importance of pre-enrichment allows the recovery of injured cells
increases the numbers of Salmonella in proportion to other organisms
dilutes toxic substances in the food◦there are some foods with intrinsic
toxic substances which can hinder the growth of microorganisms
Lactose Broth (LB)Used in the pre-enrichment stepSalmonella does not ferment the lactose
in the LB ◦ it allows the optimal growth and multiplication
Accompanying bacteria in the food ferment the lactose◦ lowers the pH thereby inhibiting them but not
SalmonellaSalmonella utilizes the other metabolites
produced by lactose fermenting organisms
Selenite Cystine Broth (SCB)
Used for the selective enrichmentSelenite inhibits the growth of
coliform bacteria and enterococci in the first 6-12 hours of incubation◦inhibitory effect gradually declines
after this period. Salmonella, Shigella sonnei,
Proteus and Pseudomonas are however, only slightly inhibited
Tetrathionate Broth (TB)Used for the selective enrichment of
salmonellae from various materialsTetrathionate and excess thiosulfate
suppress coliform and other accompanying bacteria
tetrathionate-reducing bacteria (e.g., Salmonella and Proteus) can multiply more or less normally
Acidic tetrathionate decomposition products are formed which are neutralized by calcium carbonate
Tetrathionate Broth (TB)Bile salts largely inhibit all microorganisms
which do not normally live in the intestineThe addition of brilliant green suppresses
above all the gram-positive microbial floraThe resulting culture medium has a very
strong inhibitory action◦ it is sometimes better, therefore, to omit the
brilliant green in order to obtain satisfactory yields of Salmonella
Addition of iodine to thionate yields tetrathionate
Brilliant Green Agar (BGA)Selective culture medium for the isolation of
Salmonella with the exception of S. typhosa and Shigella from pathological material, feces, urine, foodstuffs, etc
contains lactose and sucrose◦ produce acids as indicated by phenol red that
changes color to yellowUnder alkaline conditions, the indicator exhibits
a deep red colorNutrient base is rich enough to improve the
growth of SalmonellaGrowth of accompanying organisms is inhibited
by brilliant green
Brilliant Green Agar (BGA)Salmonella and others =
pink/colorless with red zone◦Lactose – and sucrose – negative
E. coli and others = yellow green with yellow green zone◦Lactose – and sucrose – positive
Results in BGAColorless, pink to fuchsia, translucent
to opaque, with surrounding
medium pink to redSome salmonella appear as
transparent green colonies if surrounded by organisms fermenting lactose or sucrose, since these carbohydrate-fermenting organisms produce colonies and zones that are yellow green or green.
Xylose Lysine Deoxycolate Agar (XLD)
Degradation of xylose, lactose, and sucrose to organic acids causes phenol red to change its color yellow
Production of hydrogen sulfide from thiosulfate is indicated with iron(III) salt which react to form a precipitate of black iron sulfide in the colonies
Bacteria which decarboxylate lysine to cadaverine can be recognized by the appearance of a purple coloration around the colonies due to an increase in the pH value
Xylose Lysine Deoxycolate Agar (XLD)
These reactions can proceed simultaneously or successively◦may cause the pH indicator to
exhibit various shades of color from yellow to red on prolonged incubation
The culture medium is weakly inhibitory
Results in XLDAtranslucent sometimes
with black center
Bismuth Sulfite Agar (BSA) Brilliant green and bismuth largely
inhibit the accompanying bacterial floraColonies of H2S-positive salmonellae
exhibit blackening due to the formation of iron sulfide
Reduction of bismuth ions to metallic bismuth produces a metallic luster of the colonies
Freshly prepared medium is strongly inhibitory and is thus especially suitable for heavily contaminated samples
Results in BSABrown, black, sometimes
with metallic sheenSurrounding medium is
usually brown at first, turning black with increasing incubation time
Some strains produce green colonies with little or no darkening of surrounding medium.
Triple Sugar Iron Agar (TSI)
Test the ability of an organism to ferment glucose, lactose and sucrose and to produce hydrogen sulfide
Acids produced from the fermentation of these three sugars are detected by the indicator phenol red, which is yellow when acidic and red when alkaline
Thiosulfate is reduced to hydrogen sulfide by several species of bacteria, the hydrogen reacts with an iron salt to give black iron sulfide
Triple Sugar Iron Agar (TSI)
Formation of hydrogen sulfide causes blackening of the medium especially on the butt area of the slant
May also show gas production◦bubbles within the medium or◦entire agar slant is lifted from the
bottom of the tube
Triple Sugar Iron Agar (TSI)
Sugar fermented Butt Slant
None Red Red
Glucose only Yellow Red
Lactose or sucrose Yellow Yellow
Results in TSI
Uninoculated Escherichia coli Salmonella enteriditis Shigella flexnerri
Results in TSICarbohydrate fermentation is
indicated by a yellow coloration of the medium
If the medium in the butt of the tube becomes yellow (acidic), but the medium in the slant becomes red (alkaline), the organism being tested only ferments dextrose(glucose)
Results in TSIA yellow (acidic) color in the slant and
butt indicates that the organism being tested ferments dextrose, lactose and/or sucrose.
A red (alkaline) color in the slant and butt indicates that the organism being tested is a non – fermenter
Hydrogen sulfide production results in a black precipitate in the butt of the tube
Gas production is indicated by splitting and cracking of the medium
Urea Broth (UB)Differentiation medium for detecting
microorganisms which metabolize ureaOnly supports the growth of microorganisms
◦ Proteus which utilize urea as their sole carbohydrate source
Microorganisms, which produce urease, can metabolize urea to carbon dioxide and ammonia
When the medium becomes alkaline, the indicator phenol red changes its color to red and the medium may become turbid as a result of microbial growth
Urea Broth (UB)Red - Urease-positive
◦ Proteus (P. vulgaris, P. mirabilis)◦ Morganella◦ Rettgerella
Yellow - Urease-negative or weakly positive◦ Shigella,◦ Escherichia◦ Salmonella◦ Citrobacter◦ Enterobacter◦ Klebsiella◦ Serratia◦ Providencia
Results in UBPositive urease tubes turn the
phenol indicator a deep violet red color (alkalinization)
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