Microbio Lab 8

MICROBIOLOGY LAB 8 USTMED ’07 SEC C - ASM SALMONELLA TYPHI GRAM NEGATIVE BACILLI IN RANDOM/SINGLY NON-LACTOSE FERMENTING ORGANISMS ENRICHMENT MEDIUM USED: SELENITE F Colonies on Plated Medium: A.

B.

C.

Mac Conkey • A differential culture media to differentiate lactose from non-lactose fermenting organisms. • Colorless colonies, sometimes black colonies due to H2S production (NLFO) Eosin Methylene Blue Agar • A differential culture media to differentiate lactose from non-lactose fermenting organisms • Colorless colonies (NLFO) Salmonella-Shigella Agar (SSA) • a selective medium for salmonella and shigella

Salmonella typhi on EMB agar plate (EMB). Salmonella typhi is a non-lactose fermenting organism (NLFO) showing colorless colonies.

•

D.

colorless colonies, sometimes black colonies due to H2S production (black center w/ clear periphery) Bismuth-sulfite agar (BSA) • a selective medium for salmonella typhi only • jet black colonies, black sheen or with dotted black or greenish grey.

Biochemical Reactions: E. TSIA: • K/A G+ (red/yellow with gas, with small amount of H2S) • This reaction shows that only glucose/dextrose was fermented by the organism and there is a small amount of hydrogen sulfide produced. F. LIA: • K/K (purple/yellow) • Alkaline slant – negative deamination • Alkaline butt – positive lysine decarboxylation G. Citrate Utilization Test (CUT): • Negative (green) • The uninoculated simmon citratei s green • + is Prussian blue • - is green Motility Tests H. Motility: Motile (diffuse growth) i. Ornithine Decarboxylation: - negative (yellow butt) ii. Indole: - negative (absence of red ring) I. Urease Test: • negative (salmon pink) Salmonella typhi on Salmonella-Shigella Agar Plate (SSA). Salmonella typhi is a Non-lactose fermenting organism (NLFO) showing colorless colonies. May produce black colonies due to hydrogen sulfide H2S formation. Salmonella typhi on MAC. Salmonella typhi is NLFO showing colorless colonies but sometimes may produce black colonies due to HwS formation.

TSIA

LIA

SCA

Urease

SHIGELLA SPECIES GRAM NEGATIVE BACILLI IN RANDOM OR SINGLY NON-LACTOSE FERMENTING ORGANISMS INVOLVED IN GASTROINTESTINAL TRACT INFECTION Colonies on Plated Medium: A. B. C.

Mac Conkey colorless colonies (NLFO) Eosin Methylene Blue Agar colorless colonies (NLFO) Salmonella-Shigella Agar (SSA) a selective medium for salmonella and shigella (colorless colonies)

Biochemical Reactions:

Salmonella typhi. Jet black colonies, black sheen or with dotted black or greenish gray colonies on Bismuth Sulfite Agar (BSA), a selective medium for Salmonella typhi only.

D.

TSIA:

E.

LIA:

F.

-

K/A (red/yellow no gas, no H2S)

K/A (purple/yellow) Alkaline slant – negative deamination Acid butt – negative lysine decarboxylation Citrate Utilization Test (CUT): Negative (green)

Motility Tests

G.

Motility: Non-motile (growth along the stabbing)

i. ii. H.

Ornithine Decarboxylation: - negative (yellow butt) Indole: - Positive (presence of red ring)

Urease Test: negative (salmon pink)

Pigment production by Pseudomonas aeruginosa. Two strains of P. aeruginosa growing on MacConkey agar. The red pigment is due to the production of pyorubrin, a non-fluorescent, water-soluble pigment, and the yellow-green color is due to pyocyanin, a fluorescent, water-soluble pigment.

Pseudomonas aeruginosa. Non-lactose fermenting organisms on EMB agar showing colorless colonies.

PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa in Nutrient agar. showing diffusible pigments, pyocyanin & yoverdin. TSIA

GRAM NEGATIVE BACILLI IN RANDOM/SINGLY NON-FERMENTATIVE ORGANISM Colonies on Plated Medium: A. B.

Mac Conkey • colorless colonies or blue green colonies due to pigmentation Eosin Methylene Blue Agar • colorless colonies

Biochemical Reactions: C. D.

E.

TSIA: • K/N or K/K (red/orange or red/red) LIA: • K/A (purple/yellow) • Alkaline slant – negative deamination • Acid butt – negative lysine decarboxylation Citrate Utilization Test (CUT): • positive (blue)

Motility Tests

F.

G. H.

Motility: Motile (Growth away from the stabbing line or haziness) 1. Ornithine Decarboxylation: negative (yellow butt) 2. Indole: negative (absence of red ring) Urease Test: • negative (salmon pink) Nutrient Agar Slant: • blue green colonies due to pigmentation • pigments produced: pyocyanin & pyoverdin (diffusible to medium)

LIA

MIO

SCA

Urease

PROTEUS MIRABILIS GRAM NEGATIVE BACILLI IN RANDOM/SINGLY NON-LACTOSE FERMENTING ORGANISM Colonies on Plated Medium: A. B. C.

BAP • exhibit swarming Mac Conkey • colorless colonies that may exhibit swarming (NLFO) Eosin Methylene Blue Agar • colorless colonies that may exhibit swarming (NLFO)

Biochemical Reactions: D.

TSIA:

E.

LIA:

•

F. A. Pseudomonas aeruginosa on 5% sheep blood agar. The colonies of P.aeruginosa on blood agar are typically yellow-green and beta-hemolytic. Fluorescein pigments are produced by some species, but pyocyanin (bluegreen) pigments is produced by P.aeruginosa alone. Most colonies have a distinct grapelike odor due to aminoacetophenone. P.aeruginosa may be distinguished from other species by its ability to grow at 42 oC. B. Pseudomonas aeruginosa on 5% sheep blood agar. Oblique lighting demonstrates metallic sheen on the surface of colonies.

Pseudomonas aeruginosa on MacConkey agar. Flat, blue-green colonies with very distinct feathered edges growing on MacConkey agar. Colonies grow well at 35oC aerobically. They will also grow at 25oC.

K/A G+ (red/yellow with gas, with H2S)

• R/A (red/yellow) • Red slant – positive deamination • Acid butt – negative lysine decarboxylation Citrate Utilization Test (CUT): • positive (blue)

Motility, ornithine decarboxylation & indole tests using MIO medium (do not shake)

G.

Motility: Motile (diffused growth) 1. Ornithine Decarboxylation: Positive (purple butt) 2. Indole: - Negative (absence of red ring)

I.

Urease Test: • Positive (fuschia pink)

Proteus mirabilis. Non-lactose fermenting organisms on Eosin Methylene Blue Agar showing

colorless colonies that may exhibit swarming.

Klebsiella pneumoniae. On Mac Conkey agar Plate (MAC) showing large pink/colored mucoid colonies. Rapid Lactose Fermenting Organism.

TSIA

LIA

Proteus mirabilis. On Blood Agar characteristic swarming phenomenon.

plate

(BAP)

showing

Urease

LIA

MIO

MIO

SCA

SCA ESCHERICHI COLI TSIA

GRAM NEGATIVE COCCOBACILLI RAPID LACTOSE FERMENTER

IN RANDOM OR SINGLY

Colonies on Plated Medium: Urease

A. B.

KLEBSIELLA PNEUMONIAE GRAM NEGATIVE COCCOBACILLI WIRE LOOP TEST POSITIVE ENCAPSULATED ORGANISM

IN RANDOM/SINGLY

Colonies on Plated Medium: A. B.

Mac Conkey • Large, mucoid, pink or colored colonies (rapid lactose fermenter) Eosin Methylene Blue Agar • Large, mucoid pink or colored colonies (rapid lactose fermenter)

Biochemical Reactions: C. D.

E.

TSIA: • LIA: •

K/K (purple/purple) Alkaline slant – negative deamination

•

Alkaline butt – positive lysine decarboxylation

Citrate Utilization Test (CUT): • positive (blue)

lactose lactose

Biochemical Reactions: C. D.

E.

TSIA: • A/A G (yellow/yellow with gas) LIA: • K/K (purple/purple) • Alkaline slant – negative deamination • Alkaline butt – positive lysine decarboxylation Citrate Utilization Test (CUT): • Negative (green)

Motility Tests

F.

Motility: Motile (diffused growth) 1. Ornithine Decarboxylation: positive (purple butt) 2. Indole: positive (presence of red ring)

J.

Urease Test: • negative (salmon pink)

A/A G (yellow/yellow with gas)

•

Mac Conkey • Pink or colored colonies (rapid fermenter) Eosin Methylene Blue Agar • Greenish metallic sheen (rapid fermenter)

Motility Tests

F.

Motility: Non-motile (Growth along the stabbing line) 1. Ornithine Decarboxylation: negative (yellow butt) 2. Indole: negative (absence of red ring)

G.

Urease Test: • positive (salmon pink)

Escherichia coli. EMB showing LFO with greenish metallic sheen.

Escherichia coli. (rapid lactose fermenting organism) on Mac Conkey Agar Plate (MAC) showing pink, colored colonies due to

fermentation of lactose.

MIO

TSIA

SERRATIA

Urease

LIA SCA MIO LIA SCA VIBRIO CHOLERA GRAM NEGATIVE CURVED BACILLI IN RANDOM/SINGLY NON-LACTOSE FERMEINTING ORGANISMS (NLFO) ENRICHMENT MEDIUM USED: ALKALINE PEPTONE WATER OXIDASE TEST: POSITIVE INVOLVED IN GASTROINTESTINAL TRACT INFECTION Colonies on Plated Medium: A. B. C.

Mac Conkey Colorless colonies (NLFO) Eosin Methylene Blue Agar Colorless colonies (NLFO) Thiosulfate Citrate Bile Salt Sucrose Agar (TCBS) a selective medium for vibrio medium sized, smooth, opaque, thin edge, yellow colonies

Biochemical Reactions: D. E.

F.

TSIA: A/A G (slant is slightly alkaline) LIA: K/K (purple/purple) Alkaline slant – negative deamination Alkaline butt – positive lysine decarboxylation Citrate Utilization Test (CUT): Positive (blue)

Motility Tests

G.

Motility: Motile (diffused growth) i. Ornithine Decarboxylation: positive (purple butt) ii. Indole: positive (presence of red ring)

H.

Urease Test: - negative (salmon pink)

Positive oxidase test due to Vibrio sp. Oxidase test: Indophenol blue is produced when cytochrome oxidase in a sample colony reacts with tetramethyl phenylenediamine dihydrochloride. It is used to distinguish Enterobacteriaceae (negative) from Vibrio sp. (positive). Vibrio Parahaemolyticus. On thiosulfate citrate bile salt sucrose agar (RCBS), does not ferment sucrose, thus colonies are green.

TSIA

Urease

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