Exercise 7

Isolation and Detection of Salmonella in Foods

Prepared by: Frances Grace N. Ordoez MCB180 B-1L

Salmonella

Salmonella
Family Enterobacteriaceae y Gram negative rods y Non-sporeformer y Optimum tempterature: 370C y Does not ferment lactose or sucrose y Motile by peritrichous flagella y Facultatively anaerobe y Pathogenic
y

gastroenteritis, enteric fever, and septicemia

Procedures and Media

Pre-enrichment Lactose Broth y Enrichment Selenite Cystine Broth and Tetrathionate Broth y Selection and Differentiation Brilliant Green Agar, Xylose Lysine Deoxycholate Agar, Bismuth Sulfite Agar y Confirmation using physiological tests Triple Sugar Iron Agar, Lysine Decarboxylase Broth, Tryptone Broth, Urea Broth , Potassium Cyanide Broth (KCN)
y

Importance of pre-enrichment prey

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 step y Salmonella does not ferment the lactose in the LB
y

it allows the optimal growth and multiplication

y

Accompanying bacteria in the food ferment the lactose

lowers the pH thereby inhibiting them but not Salmonella

Salmonella utilizes the other metabolites produced by lactose fermenting organisms

Selenite Cystine Broth (SCB)

Used for the selective enrichment y Selenite inhibits the growth of coliform bacteria and enterococci in the first 6-12 hours of incubation
y

inhibitory effect gradually declines after this period.

y

Salmonella, Shigella sonnei, Proteus and Pseudomonas are however, only slightly inhibited

Tetrathionate Broth (TB)

Used for the selective enrichment of salmonellae from various materials y Tetrathionate and excess thiosulfate suppress coliform and other accompanying bacteria y tetrathionate-reducing bacteria (e.g., Salmonella and Proteus) can multiply more or less normally y Acidic tetrathionate decomposition products are formed which are neutralized by calcium carbonate
y

Tetrathionate Broth (TB)

Bile salts largely inhibit all microorganisms which do not normally live in the intestine y The addition of brilliant green suppresses above all the gram-positive microbial flora y The resulting culture medium has a very strong inhibitory action
y

it is sometimes better, therefore, to omit the brilliant green in order to obtain satisfactory yields of Salmonella
y

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 y contains lactose and sucrose
y

produce acids as indicated by phenol red that changes color to yellow

Under alkaline conditions, the indicator exhibits a deep red color y Nutrient base is rich enough to improve the growth of Salmonella y Growth of accompanying organisms is inhibited by brilliant green
y

Brilliant Green Agar (BGA)

y

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 BGA
Colorless, pink to fuchsia, translucent to opaque, with surrounding medium pink to red y Some 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.
y

Xylose Lysine Deoxycolate Agar (XLD)

Degradation of xylose, lactose, and sucrose to organic acids causes phenol red to change its color yellow y 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 y 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
y

Xylose Lysine Deoxycolate Agar (XLD)

y

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 XLDA
y

translucent sometimes with black center

Bismuth Sulfite Agar (BSA)

Brilliant green and bismuth largely inhibit the accompanying bacterial flora y Colonies of H2S-positive salmonellae exhibit blackening due to the formation of iron sulfide y Reduction of bismuth ions to metallic bismuth produces a metallic luster of the colonies y Freshly prepared medium is strongly inhibitory and is thus especially suitable for heavily contaminated samples
y

Results in BSA
Brown, black, sometimes with metallic sheen y Surrounding medium is usually brown at first, turning black with increasing incubation time y Some strains produce green colonies with little or no darkening of surrounding medium.
y

Triple Sugar Iron Agar (TSI)

Test the ability of an organism to ferment glucose, lactose and sucrose and to produce hydrogen sulfide y 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 y Thiosulfate is reduced to hydrogen sulfide by several species of bacteria, the hydrogen reacts with an iron salt to give black iron sulfide
y

Triple Sugar Iron Agar (TSI)

Formation of hydrogen sulfide causes blackening of the medium especially on the butt area of the slant y May also show gas production
y

bubbles within the medium or entire agar slant is lifted from the bottom of the tube

Triple Sugar Iron Agar (TSI)

Sugar fermented None Glucose only Lactose or sucrose

Butt Red Yellow Yellow

Slant Red Red Yellow

Results in TSI

Uninoculated

Escherichia coli

Salmonella enteriditis

Shigella flexnerri

Results in TSI
Carbohydrate fermentation is indicated by a yellow coloration of the medium y 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)
y

Results in TSI
A yellow (acidic) color in the slant and butt indicates that the organism being tested ferments dextrose, lactose and/or sucrose. y A red (alkaline) color in the slant and butt indicates that the organism being tested is a non fermenter y Hydrogen sulfide production results in a black precipitate in the butt of the tube y Gas production is indicated by splitting and cracking of the medium
y

Urea Broth (UB)

Differentiation medium for detecting microorganisms which metabolize urea y Only supports the growth of microorganisms
y

Proteus which utilize urea as their sole carbohydrate source

Microorganisms, which produce urease, can metabolize urea to carbon dioxide and ammonia y 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
y

Urea Broth (UB)

y

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 UB
y

Positive urease tubes turn the phenol indicator a deep violet red color (alkalinization)