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Microbio Lab 8

Microbio Lab 8

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MICROBIOLOGY LAB 8USTMED ’07 S
EC
C - A
S
MS
ALMONELLA
T
YPHI
 
G
RAM
N
EGATIVE
B
ACILLI
 
IN
 
RANDOM
/
SINGLY
N
ON
-L
ACTOSE
F
ERMENTING
O
RGANISMS
E
NRICHMENT
 
MEDIUM
 
USED
: S
ELENITE
FColonies on Plated Medium:A.
Mac Conkey
A differential culture media to differentiatelactose from non-lactose fermentingorganisms.
Colorless colonies, sometimes black coloniesdue to H
2
S production (NLFO)B.
Eosin Methylene Blue Agar
A differential culture media to differentiatelactose from non-lactose fermenting organisms
Colorless colonies (NLFO)C.
Salmonella-Shigella Agar (SSA)
a selective medium for salmonella and shigella
colorless colonies, sometimes black coloniesdue to H
2
S production (black center w/ clearperiphery)D.
Bismuth-sulfite agar (BSA)
a selective medium for salmonella typhi only
jet black colonies, black sheen or with dottedblack or greenish grey.Biochemical Reactions:E.
TSIA:
K/A G+ (red/yellow with gas, with smallamount of H
2
S)
This reaction shows that only glucose/dextrosewas fermented by the organism and there is asmall 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 greenMotility 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-ShigellaAgar Plate (SSA)
. Salmonella typhi is aNon-lactose fermenting organism (NLFO)showing colorless colonies. May produceblack colonies due to hydrogen sulfide H
2
Sformation.
Salmonella typhi on MAC.
Salmonella typhi is NLFO showingcolorless colonies but sometimes may produce black colonies dueto H
w
S formation.
Salmonella typhi.
Jet black colonies, black sheen or with dottedblack or greenish gray colonies on Bismuth Sulfite Agar (BSA), aselective medium for Salmonella typhi only.
 
Salmonella typhi on EMB agar plate (EMB).
Salmonella typhi is anon-lactose fermenting organism (NLFO) showing colorlesscolonies.
TSIALIASCAUreaseS
HIGELLA
S
PECIES
 
G
RAM
N
EGATIVE
B
ACILLI
 
IN
 
RANDOM
 
OR
 
SINGLY
N
ON
-L
ACTOSE
F
ERMENTING
O
RGANISMS
I
NVOLVED
 
IN
G
ASTROINTESTINAL
T
RACT
I
NFECTION
Colonies on Plated Medium:A.
Mac Conkey-
colorless colonies (NLFO)B.
Eosin Methylene Blue Agar-
colorless colonies (NLFO)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 H
2
S)E.
LIA:-
K/A (purple/yellow)
-
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)
ii.Indole:
- Positive (presence of red ring)H.
Urease Test:-
negative (salmon pink)
P
SEUDOMONAS
A
ERUGINOSA
 
G
RAM
 
NEGATIVE
B
ACILLI
 
IN
 
RANDOM
/
SINGLY
N
ON
-
FERMENTATIVE
 
ORGANISM
Colonies on Plated Medium:A.
Mac Conkey
colorless colonies or blue green colonies dueto pigmentationB.
Eosin Methylene Blue Agar
colorless coloniesBiochemical Reactions:C.
TSIA:
K/N or K/K (red/orange or red/red)D.
LIA:
K/A (purple/yellow)
Alkaline slant – negative deamination
Acid butt – negative lysine decarboxylation
E.
Citrate Utilization Test (CUT)
:
positive (blue)Motility Tests
F.
Motility:
Motile (Growth away from the stabbing line orhaziness)
1.
Ornithine Decarboxylation
:
-
negative (yellow butt)
2.
Indole:-
negative (absence of red ring)G.
Urease Test:
negative (salmon pink)H.
Nutrient Agar Slant:
blue green colonies due to pigmentation
pigments produced: pyocyanin & pyoverdin(diffusible to medium)
 
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 (blue-green) pigments is produced by P.aeruginosa alone. Most colonies have adistinct grapelike odor due to aminoacetophenone. P.aeruginosa may bedistinguished from other species by its ability to grow at 42
o
C.
B.Pseudomonas aeruginosa on 5% sheep blood agar.
Oblique lightingdemonstrates metallic sheen on the surface of colonies.
Pseudomonas aeruginosa onMacConkey agar.
Flat, blue-greencolonies with very distinct featherededges growing on MacConkey agar.Colonies grow well at 35
o
C aerobically.They will also grow at 25
o
C.
Pigment production by Pseudomonas aeruginosa.
Two strains of P.aeruginosa growing on MacConkey agar. The red pigment is due to theproduction of pyorubrin, a non-fluorescent, water-soluble pigment, and theyellow-green color
is
due to pyocyanin, a fluorescent, water-solublepigment.
Pseudomonas aeruginosa.
Non-lactose fermentingorganisms on EMB agar showing colorless colonies.
Pseudomonas aeruginosa in Nutrient agar
. showing diffusiblepigments, pyocyanin & yoverdin.
TSIALIAMIOSCAUreaseP
ROTEUS
M
IRABILIS
 
G
RAM
N
EGATIVE
B
ACILLI
 
IN
 
RANDOM
/
SINGLY
N
ON
-
LACTOSE
F
ERMENTING
O
RGANISM
Colonies on Plated Medium:
A.BAP
exhibit swarming
B.Mac Conkey
colorless colonies that may exhibit swarming(NLFO)
C.Eosin Methylene Blue Agar
colorless colonies that may exhibit swarming(NLFO)Biochemical Reactions:
D.TSIA:
K/A G+ (red/yellow with gas, with H
2
S)
E.LIA:
R/A (red/yellow)
Red slant – positive deamination
Acid butt – negative lysine decarboxylation
F.
Citrate Utilization Test (CUT)
:
positive (blue)Motility, ornithine decarboxylation & indole tests using MIOmedium (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-lactosefermenting organisms on EosinMethylene BlueAgar showing
 
colorless colonies that may exhibit swarming.
Proteus mirabilis.
On Blood Agar plate (BAP) showingcharacteristic swarming phenomenon.
LIAMIOSCATSIAUrease
LEBSIELLA
P
NEUMONIAE
 
G
RAM
N
EGATIVE
C
OCCO
B
ACILLI
 
IN
 
RANDOM
/
SINGLY
W
IRE
 
LOOP
 
TEST
 
POSITIVE
E
NCAPSULATED
 
ORGANISM
Colonies on Plated Medium:A.
Mac Conkey
Large, mucoid, pink or colored colonies (rapidlactose fermenter)B.
Eosin Methylene Blue Agar
Large, mucoid pink or colored colonies (rapidlactose fermenter)Biochemical Reactions:C.
TSIA:
A/A G (yellow/yellow with gas)D.
LIA:
K/K (purple/purple)
Alkaline slant – negative deamination
Alkaline butt – positive lysine decarboxylation
E.
Citrate Utilization Test (CUT)
:
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)G.
Urease Test:
positive (salmon pink)
Klebsiella pneumoniae.
On Mac Conkey agar Plate (MAC) showinglarge pink/colored mucoid colonies. Rapid Lactose FermentingOrganism.
TSIALIAUreaseMIOSCAE
SCHERICHI
C
OLI
 
G
RAM
N
EGATIVE
C
OCCO
B
ACILLI
 
IN
 
RANDOM
 
OR
 
SINGLY
R
APID
L
ACTOSE
F
ERMENTER
Colonies on Plated Medium:A.
Mac Conkey
Pink or colored colonies (rapid lactosefermenter)B.
Eosin Methylene Blue Agar
Greenish metallic sheen (rapid lactosefermenter)Biochemical Reactions:C.
TSIA:
A/A G (yellow/yellow with gas)D.
LIA:
K/K (purple/purple)
Alkaline slant – negative deamination
Alkaline butt – positive lysine decarboxylation
E.
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)
Escherichia coli.
EMB showing LFO with greenish metallic sheen.
Escherichia coli.
(rapid lactose fermenting organism) on MacConkey Agar Plate (MAC) showing pink, colored colonies due tofermentation of lactose.

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