BACTERIOLOGY (LABORATORY)

BIOCHEMICAL TESTS K/K

●Glucose/sucrose/lactose
● TSI - stab-streak not fermented
● LIA - stab-streak-stab ● Typical among the Non
● IMViC - streak fermentative organisms
● Urease - streak ○ Pseudomonas
● SIM - stab ¾ aeruginosa
K/A, H2S (+)
TRIPLE SUGAR IRON . ● Glucose fermented
TSIA and KIA (Kligler Iron Agar) ● Lactose/sucrose not
● Useful in presumptive identification of fermented
gram-negative enteric bacteria ○ Salmonella
● Dispensed in tubes with equal butt and slant spp.
Contents ○ Proteus
● CHO ○ Edwardsiella
○ TSI - glucose (0.1% ○ Citrobacter
or 1 part), lactose K/A, H2S (-)
(1% or 10 parts) ● Glucose fermented
and sucrose (1% or ● Lactose/sucrose not
10 parts) fermented
○ KIA - glucose (0.1% ○ Yersinia
or 1 part), lactose ○ Escherichia
(1% or 10 parts) coli
● Others ○ Serratia
○ Peptone (0.1% or 1 ○ Shigella
part) ○ Providencia
H2S Indicator ○ Morganella
● Sodium thiosulfate: forms H2S gas upon ○ Citrobacter
interaction with the organisms (acid A/A, H2S (+)
environment) ● Glucose fermented
● Ferrous sulfate: bonds to H2S = forms a black ● Lactose/sucrose
precipitate fermented
pH Indicator ○ Salmonella
● Phenol red (7.4 pH) spp.
Medium: ○ Proteus
● Two reaction chamber with an aerobic slant ○ Citrobacter
portion and an anaerobic deep portion A/A, H2S (-)
Results ● Glucose fermented
● Yellow in acidic pH ● Lactose/sucrose
● Red in alkaline pH fermented
● Black precipitate (+) H2S production ○ Escherichia
● Bubbles/crack in medium (+) gas formation coli
Quality Control ○ Klebsiella
● A/A gas (+): Escherichia coli (ATCC 25922) ○ Enterobacter
● K/A H2S (+): Proteus mirabilis (ATCC 12453) ○ Serratia
● K/A gas (+/-): Salmonella serotype typhimurium
(ATCC 14028) ✓ Glucose, Sucrose, Lactose (All yellow)
● K/A: Shigella flexneri (ATCC 12022) ● Red > Consume glucose (6hrs); 1st because it's
● K/K: Pseudomonas aeruginosa (ATCC 27853) a monosaccharide > Yellow > ↓ Glucose (12hrs)
ROR > Consume > Yellow (18-24 hrs)
● Reaction; Gas (+/-); H2S (+/-) ✓ Glucose; X Sucrose, Lactose
Example (Slant-red;Butt-yellow)
1. A/A, Gas (+), H2S (-) ● Red > Consume glucose (6hrs) > Consume
2. K/A, Gas (-), H2S (-) peptone = alkaline > Return to red (Slant part)
3. K/K, Gas (-), H2S (-) X Glucose, Sucrose, Lactose (All red)
4. A/A, Gas (-), H2S (+) ● Consume peptone only
Note: H2S (all black) requires acidic environment

DECARBOXYLASE TEST .
Principle
● Determine whether the bacterial species
possess enzymes capable decarboxylating
specific amino acids in the test medium
Lysine and Ornithine

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● Amino acids commonly used to test for

decarboxylase activity
LYSINE IRON AGAR .
Principle
● Determine the ability of organisms to deaminase
or decarboxylase lysine and produce H2S
○ Deamination: slant
○ Decarboxylation: butt

Moeller Decarboxylase Base Medium contains:

● Glucose
● Peptones
● 2 pH indicators: Bromocresol blue and Cresol
red
● Specific amino acids at a concentration of 1%

LIA contains:
● Lysine
● Glucose
● H2S Indicator: Sodium thiosulfate and ferric
ammonium citrate
● pH indicator: bromocresol purple
Quality Control
● Alkaline slant and butt: H2S positive: Citrobacter
Quality Control freundii (ATCC 8090)
Positive: ● Alkaline slant and butt: Escherichia coli (ATCC
● Lysine - Klebsiella pneumoniae (ATCC33495) 25922)
● Ornithine - Klebsiella aerogenes (ATCC13048) ● Alkaline slant and butt: H2S positive: Salmonella
● Arginine - Pseudomonas aeruginosa (ATCC enterica subsp. enterica serovar typhimurium
27853) (ATCC 14028)
Negative: ● Red slant, acid butt: Proteus mirabilis (ATCC
● Lysine - Citrobacter freundii (ATCC 331218) 12453)
● Ornithine - Proteus vulgaris (ATCC 6380) ROR
● Arginine - Escherichia coli (ATCC 25922) ● Deam (+/-); Decarb (+/-); H2S (+/-)
Note: H2S (all black) requires alkaline environment
Moeller
● Requires two conditions IMViC (INDOLE, METHYL RED,
1. Acidic environment VOGES-PROSKAUER, CITRATE) .
2. Anaerobic environment INDOLE .
● Result ● Principle
○ (+) Purple: original color
○ (-) Yellow: converted by Glucose, Tryptophanase
Peptone, 1% LOA Tryptophan Indole + Pyruvic acid + NH3
LIA + Aldehyde rgt.
● Result
○ Deam: (+) red slant Red color in upper
organic layer
○ Decarb: (+) purple butt: to meet
anaerobic environmentl ● Ehrlich's Method
○ Indole is extracted by the addition of 1ml
of Xylene
○ 0.5ml of Ehrlich's reagent:
p-dimethylaminobenzaldehyde (PDAB)
○ (+) red color

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○ Determines whether an organism can

use sodium citrate as a sole carbon
● Kovac's Method source
○ 5 drops of Kovac's reagent: ● Simmons' Citrate
p-dimethylaminobenzaldehyde (PDAB) Medium contains:
○ (+) red color ○ Sodium citrate
● Rapid Indole Test ○ Ammonium
○ Smeared onto filter paper that has been salts
moistened with ● pH Indicator:
p-dimethylaminocinnamaldehyde bromothymol blue
○ (+) blue-green color within 2 minutes ○ Uninoculated:
● Quality Control green
A. Ehrlich's Method ○ (+) blue
● Positive: Haemophilus
influenzae (ATCC 49766) ● Quality Control
● Negative: Haemophilus ○ Positive: Klebsiella aerogenes (ATCC
parainfluenzae (ATCC 76901) 13048)
B. Kovac's Method - growth; blue color
● Positive: Escherichia coli (ATCC ○ Negative: Escherichia coli (ATCC
25922) 259322
● Negative: Klebsiella - little to no growth; no color
pneumoniae (ATCC 13883) change

METHYL RED, VOGES-PROSKAUER .

Acidic Pathway Neutral Pathway
(Mixed Acid Fermentation) (Butylene Glycol Pathway)

Mixed acids (Lactic, Acetic, Acetyl methyl carbinol

Formic, & Succinic) (ACETOIN)
pH less than 4.5

Diacetyl (Dimethylcarbinol)
Red color + a-naphthol (Barritt's A)
+ KOH (Barritt's B) - 40%
Red color
(2,3 - Butanediol) ++-- --++ -+-+

Glucose metabolism and its by-product E. coli Klebsiella Citrobacter

Morganella Enterobacter Arizonae
● Medium Proteus Serratia Salmonella
○ MR-VP or the clark and lubs medium Edwardsiella Hafnia
● pH Indicator
○ Methyl red
● Results UREASE . .
○ (+) red Principle
○ (-) yellow ● Determines whether a
● Quality Control microorganism can hydrolyze
○ MR (+) VP (-): Escherichia coli (ATCC urea to form ammonia
25922) (produce a color change by a
○ MR (-) VP (+): Klebsiella aerogenes pH indicator), water, and
(ATCC 13048) CO2
Christensen's Urea Agar
● pH indicator: phenol red
Expected Results
● Positive: Bright pink
● Negative: No color change

RAPID UREASE PRODUCERS (w/in

2-4 hours)
● Proteus, Providencia, Morganella (PPM)
WEAK UREASE PRODUCERS (4 hours)
● Citrobacter, Klebsiella, Enterobacter, Yersinia,
and Serratia (CKEYS)
Quality Control
CITRATE . . .
● Positive: Proteus vulgaris (ATCC 13315)
● Principle

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● Weak Positive: Klebsiella pneumoniae (ATCC ● Negative: growth, but no change in color
13883) (medium remains purple)
● Negative: Escherichia coli (ATCC 25922) Quality Control
● Positive, with gas: Escherichia coli (ATCC
SIM (SULFIDE, INDOLE, MOTILITY) . 25922)
● Can be added with 1% triphenyltetrazolium ● Positive, with no gas: Shigella flexneri (ATCC
chloride to aid visualization (optimal) 12022)
Results
● Motile: cloudiness
spreading from the
inoculation line
● Non-motile: grow only along
the stab line
● Sulfide (+): black precipitate
● Indole (+): pink to red color
● Nonmotile at 37°C: Shigella,
Klebsiella, Yersinia
ROR
● Sulfide (+-); Indole (+/-);
Motility (motile/nonmotile)

CARBOHYDRATE FERMENTATION .
Lactose Utilization
● Carbohydrate determination test that is the most ONPG (Ortho-Nitrophenyl-B-D-Galactopyranoside) .
important Principle
● Lactose: disaccharide consisting of glucose and ● Determine whether the organism is a dLF (one
galactose that lacks the enzyme B-galactoside permease
2 Enzymes used in Lactose Degradation but possess B-galactosidase) or a true NLF
● B-galactoside permease (lactose permease) ONPG
● B-galactosidase ● Colorless compound
● Structurally similar to lactose
LF's possess both of the enzymes
● Does not require B-galactoside permease
NLF's do not possess both of the enzymes
ONPG
dLF's possess only B-galactosidase
pH Indicator
B-galactosidase
1. Bromocresol purple (6.3 pH)
2. Andrade (5.5 pH)
3. Phenol red (7.9 pH) galactose + O-Nitrophenyl
Base Medium
● Peptone broth
● Tryptic soy
Procedure
● Tryptophan
1. Add 1 drop brain heart infusion broth containing
the bacteria
2. Drop in disk
FERMENTATION MEDIA . .
3. Incubate at 18-28 hours @ 35°C
Principle
Expected Results
● Determine the ability of an organism to ferment
● Positive: yellow (presence of B-galactosidase)
a specific carbohydrate that is incorporated in a
● Negative: colorless (absence of enzyme)
basal medium, with or without visible base
Quality Control
Two Methods (35°C)
● Positive: Shigella sonnei (ATCC 9290)
● Andrade (7 days)
● Negative: Salmonella enterica subsp. enterica
● Bromocresol (4 days)
serovar typhimurium (ATCC 14028)
Peptone Medium contains:
● Pancreatic digest of casein (109)
NITRATE REDUCTION TEST .
● Beef extract (39)
Principle
● NaCl (59)
● Determines whether an
● Carbohydrate (109)
organism has the
● Peptone
ability to reduce nitrate
● pH indicator: Andrade's indicator [10mL, ph7.4]
to nitrite further to
● a Durham tube: for bubbles/gas presence
nitrogen gas (N2)
Expected Results
Medium
● Positive: indicator change to yellow

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● Nutrient broth with 0.1% potassium nitrate (can

have Durham tube)
Reagents
● Alpha-naphthylamine
● Sulfanilic acid
● Zinc dust: determine whether the test has
produced a true-negative result
Expected Result
● Positive: red color (with or without gas)

PHENYLALANINE DEAMINASE TEST .

Principle
● Determine whether an organism possess the
enzyme that deaminate phenylalanine
phenylpyruvic acid
Medium
● Agar slant containing a 0.2% concentration of
phenylalanine
Reagent
● 10% ferric chloride (FeCl3)

Phenylalanine

Phenylalanine deaminase

Phenylpyruvic acid

FeCl3

GREEN (+)

Note: (-) Yellow

Expected Results
● Positive: Green color develops on slant after
ferric chloride is added - Proteus, Morganella,
Providencia
● Negative: Slant remains original color after the
addition of ferric chloride
Quality Control
● Positive: Proteus mirabilis (ATCC12453)
● Negative: Escherichia coli (ATCC25922)

GELATIN HYDROLYSIS .
Quality Control Principle
● Positive: NO3+, no gas: Escherichia coli (ATCC ● Determine whether an organism produce
25922) gelatinases capable of hydrolyzing gelatin
● Positive: NO3, gas: Pseudomonas aeruginosa Procedure
(ATCC 17588) 1. Add 1 drop in brain heart infusion broth
● Negative: Acinetobacter baumannii (ATCC containing the bacteria
19606) 2. Incubate for 48 hours in 28°C
3. Remove in incubator and refrigerate at 4°C
4. Check if liquefaction (+) or solidification (-)
Expected Result
● Positive: Partial or total liquefaction of the
inoculated tube (the control tube must be
completely solidified) at 4°C within 14 days
● Negative: Complete solidification of the tube at
4°C
Quality Control

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● Positive: Bacillus subtilis (ATCC9372)

● Negative: Escherichia coli (ATCC25922)
● Uninoculated control tube: medium becomes
solid after refrigeration
Note
● Proteus and Serratia - only enterobacteriacae
that are capable of producing gelatinase
enzyme

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