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)