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. Mac Conkey
• A differential culture media to differentiate
lactose from non-lactose fermenting
organisms.
• Colorless colonies, sometimes black colonies
due to H2S production (NLFO)
B. Eosin Methylene Blue Agar
• A differential culture media to differentiate
lactose from non-lactose fermenting organisms Salmonella typhi on EMB agar plate (EMB). Salmonella typhi is a
• Colorless colonies (NLFO) non-lactose fermenting organism (NLFO) showing colorless
C. Salmonella-Shigella Agar (SSA) colonies.
• a selective medium for salmonella and shigella
• colorless colonies, sometimes black colonies
due to H2S production (black center w/ clear
periphery)
D. 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) TSIA
• Alkaline slant – negative deamination
• Alkaline butt – positive lysine decarboxylation
G. Citrate Utilization Test (CUT): LIA
• Negative (green)
• The uninoculated simmon citratei s green
• + is Prussian blue
• - is green SCA
Motility Tests
H. Motility: Motile (diffuse growth)
i. Ornithine Decarboxylation: Urease
- negative (yellow butt)
ii. Indole:
- negative (absence of red ring)
I. Urease Test: SHIGELLA SPECIES
• negative (salmon pink)
GRAM NEGATIVE BACILLI IN RANDOM OR SINGLY
Salmonella typhi on Salmonella-Shigella NON-LACTOSE FERMENTING ORGANISMS
Agar Plate (SSA). Salmonella typhi is a INVOLVED IN GASTROINTESTINAL TRACT INFECTION
Non-lactose fermenting organism (NLFO)
showing colorless colonies. May produce Colonies on Plated Medium:
black colonies due to hydrogen sulfide H2S
formation. A. Mac Conkey
- colorless colonies (NLFO)
Salmonella typhi on MAC. Salmonella typhi is NLFO showing B. Eosin Methylene Blue Agar
colorless colonies but sometimes may produce black colonies due - colorless colonies (NLFO)
to HwS formation. C. Salmonella-Shigella Agar (SSA)
- a selective medium for salmonella and shigella
(colorless colonies)

Biochemical Reactions:

D. TSIA:
- K/A (red/yellow no gas, no H2S)
Salmonella typhi. Jet black colonies, black sheen or with dotted E. LIA:
black or greenish gray colonies on Bismuth Sulfite Agar (BSA), a - K/A (purple/yellow)
selective medium for Salmonella typhi only. - Alkaline slant – negative deamination
- Acid butt – negative lysine decarboxylation
F. Citrate Utilization Test (CUT):
- Negative (green)

Motility Tests

G. Motility: Non-motile (growth along the stabbing)

 i. Ornithine Decarboxylation:
- negative (yellow butt) Pigment production by Pseudomonas aeruginosa. Two strains of P.
aeruginosa growing on MacConkey agar. The red pigment is due to the
ii. Indole: production of pyorubrin, a non-fluorescent, water-soluble pigment, and the
- Positive (presence of red ring) yellow-green color is due to pyocyanin, a fluorescent, water-soluble
pigment.
H. Urease Test:
- negative (salmon pink)

Pseudomonas aeruginosa. Non-lactose fermenting

organisms on EMB agar showing colorless colonies.

Pseudomonas aeruginosa in Nutrient agar. showing diffusible

pigments, pyocyanin & yoverdin.
PSEUDOMONAS AERUGINOSA TSIA

GRAM NEGATIVE BACILLI IN RANDOM/SINGLY

NON-FERMENTATIVE ORGANISM

Colonies on Plated Medium: LIA

A. Mac Conkey
• colorless colonies or blue green colonies due
to pigmentation MIO
B. Eosin Methylene Blue Agar
• colorless colonies

Biochemical Reactions:
SCA
C. TSIA:
• K/N or K/K (red/orange or red/red)
D. LIA:
• K/A (purple/yellow) Urease
• Alkaline slant – negative deamination
• Acid butt – negative lysine decarboxylation
E. Citrate Utilization Test (CUT):
• positive (blue)
PROTEUS MIRABILIS
Motility Tests
GRAM NEGATIVE BACILLI IN RANDOM/SINGLY
F. Motility: Motile (Growth away from the stabbing line or NON-LACTOSE FERMENTING ORGANISM
haziness)
Colonies on Plated Medium:
1. Ornithine Decarboxylation:
- negative (yellow butt)
A. BAP
2. Indole: • exhibit swarming
- negative (absence of red ring) B. Mac Conkey
• colorless colonies that may exhibit swarming
G. Urease Test:
(NLFO)
• negative (salmon pink)
C. Eosin Methylene Blue Agar
H. Nutrient Agar Slant:
• colorless colonies that may exhibit swarming
• blue green colonies due to pigmentation (NLFO)
• pigments produced: pyocyanin & pyoverdin
(diffusible to medium) Biochemical Reactions:

D. TSIA:
• K/A G+ (red/yellow with gas, with H2S)
E. LIA:
• R/A (red/yellow)
• Red slant – positive deamination
• Acid butt – negative lysine decarboxylation
F. Citrate Utilization Test (CUT):
• positive (blue)
A. Pseudomonas aeruginosa on 5% sheep blood agar. The colonies of
P.aeruginosa on blood agar are typically yellow-green and beta-hemolytic. Motility, ornithine decarboxylation & indole tests using MIO
Fluorescein pigments are produced by some species, but pyocyanin (blue- medium (do not shake)
green) 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.
G. Motility: Motile (diffused growth)
Pseudomonas aeruginosa on 5% sheep blood agar. Oblique lighting 1. Ornithine Decarboxylation:
demonstrates metallic sheen on the surface of colonies. - Positive (purple butt)
2. Indole:
Pseudomonas aeruginosa on - Negative (absence of red ring)
MacConkey agar. Flat, blue-green
colonies with very distinct feathered I. Urease Test:
edges growing on MacConkey agar. • Positive (fuschia pink)
Colonies grow well at 35oC aerobically.
They will also grow at 25oC.
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

Urease
Proteus mirabilis. On Blood Agar plate (BAP) showing
characteristic swarming phenomenon.

LIA MIO

MIO SCA

SCA

ESCHERICHI COLI
TSIA
GRAM NEGATIVE COCCOBACILLI IN RANDOM OR SINGLY
RAPID LACTOSE FERMENTER

Colonies on Plated Medium:

Urease
A. Mac Conkey
• Pink or colored colonies (rapid lactose
fermenter)
B. Eosin Methylene Blue Agar
KLEBSIELLA PNEUMONIAE • Greenish metallic sheen (rapid lactose
fermenter)
GRAM NEGATIVE COCCOBACILLI IN RANDOM/SINGLY
WIRE LOOP TEST POSITIVE Biochemical Reactions:
ENCAPSULATED ORGANISM
C. TSIA:
Colonies on Plated Medium: • A/A G (yellow/yellow with gas)
D. LIA:
A. Mac Conkey • K/K (purple/purple)
• Large, mucoid, pink or colored colonies (rapid • Alkaline slant – negative deamination
lactose fermenter) • Alkaline butt – positive lysine decarboxylation
B. Eosin Methylene Blue Agar
• Large, mucoid pink or colored colonies (rapid E. Citrate Utilization Test (CUT):
lactose fermenter) • Negative (green)

Biochemical Reactions: Motility Tests

C. TSIA: F. Motility: Motile (diffused growth)

• A/A G (yellow/yellow with gas) 1. Ornithine Decarboxylation:
D. LIA: - positive (purple butt)
• K/K (purple/purple) 2. Indole:
• Alkaline slant – negative deamination - positive (presence of red ring)
• Alkaline butt – positive lysine decarboxylation
J. Urease Test:
E. Citrate Utilization Test (CUT): • negative (salmon pink)
• positive (blue)

Motility Tests

F. Motility: Non-motile (Growth along the stabbing line)

1. Ornithine Decarboxylation:
- negative (yellow butt)
2. Indole:
- negative (absence of red ring)
Escherichia coli. EMB showing LFO with greenish metallic sheen.
G. Urease Test:
• positive (salmon pink)

Escherichia coli. (rapid lactose fermenting organism) on Mac

Conkey Agar Plate (MAC) showing pink, colored colonies due to
fermentation of lactose.

MIO

TSIA

Urease SERRATIA

LIA

SCA
MIO

LIA

SCA
VIBRIO CHOLERA

GRAM NEGATIVE CURVED BACILLI IN RANDOM/SINGLY

NON-LACTOSE FERMEINTING ORGANISMS (NLFO) TSIA
ENRICHMENT MEDIUM USED: ALKALINE PEPTONE WATER
OXIDASE TEST: POSITIVE
INVOLVED IN GASTROINTESTINAL TRACT INFECTION

Colonies on Plated Medium: Urease

A. Mac Conkey
- Colorless colonies (NLFO)
B. Eosin Methylene Blue Agar
- Colorless colonies (NLFO) ustmedc3@yahoogroups.com
C. Thiosulfate Citrate Bile Salt Sucrose Agar (TCBS) audrey_cl@yahoo.com
- a selective medium for vibrio
- medium sized, smooth, opaque, thin edge,
yellow colonies

Biochemical Reactions:

D. TSIA:
- A/A G (slant is slightly alkaline)
E. LIA:
- K/K (purple/purple)
- Alkaline slant – negative deamination
- Alkaline butt – positive lysine decarboxylation
F. 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.