ENTEROBACTERIACEAE

(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
Escherichia coli

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: ++--
• TSIA (Triple Sugar Iron Agar) reaction: A/A (acidic slant/acidic butt), (+) gas, (-) hydrogen sulfide
or H2S
• Lysine decarboxylase reaction: (+)

For table :

Biochemical test

• Note:
Pathogenic
Enteropathogenic E.coli, Enterotoxingenic E.coli, EnteroinvasiveE.coli,
Enterohemorrhagic E.coli serotype 0157:H7, Enteroadherent E.coli
• Disease:
Nosocomial Urinary tract infection
Diarrhea

Klebsiella

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: --++
• TSIA (Triple Sugar Iron Agar) reaction: A/A (acidic slant/acidic butt), (+) gas, (-) hydrogen sulfide
or H2S
• Differential Test: (+) String test

For table :

Biochemical test K. pneumoniae subsp. K. oxytoca

pneumoniae
IMVIC --++ --++
Urease + +
Lysine decarboxylase (LDC) + +
Different test +string test
Neufeld-Quellung test + +
Hydrogen Sulfide or H2S - -
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST

• Note:
Culture and biochemical tests reaction are the same with K.pneumoniae
• Disease:
Agent of Granuloma inguinale or donovanosis
Urinary tract infection
Pneomonia
bacteremia

Enterobacter

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction:--++

• TSIA (Triple Sugar Iron Agar) reaction: A/A (acidic slant/acidic butt), (+) gas, (-) hydrogen sulfide
or H2S
• Lysine decarboxylase (LDC) test: (+) positive

For table:

Biochemical test E. aerogenes E. cloacae

Urease - + (w)
Lysine decarboxylase (LDC) + -
IMVIC ++- -++
Hydrogen Sulfide or H2S - -

• Note:
Exhibit pink color and are sometimes mucoid (LF)
• Disease:
Bacteremia
Nosocomial pneumonia
UTI

Serratia

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: --++
• TSIA (Triple Sugar Iron Agar) reaction: K/A or A/A, (+) gas, (-) hydrogen sulfide or H2S
• DNAse: Positive
• Gelatinase: Positive
• LATE LACTOSE FERMENTERS → Can be seen in K/A, H2S- algorithm

For table:

Biochemical test S. marcecens

Urease +
Gelatinase +
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
ONPG +
Aribinose fermentation -
IMVIC --++ --++

• Note:
Some strain may exhibit late lactose fermentation
• Disease:
Pneumonia

Proteus

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: P.mirabilis (-+vv) / P.
vulgaris (++-v)
• TSIA (Triple Sugar Iron Agar) reaction: P.mirabilis (K/A, (+) gas, (+) hydrogen sulfide or H2S)
P. vulgaris (K/A, (+/-) gas, (+) hydrogen sulfide or H2S)
• LIA (Lysine iron agar) reaction: R/A (red/slant/acid butt)

For table:

Biochemical test P. mirabilis P. vulgaris

Indole - +
Phenylalanine deaminase + +
LIA R/A R/A
IMViC -+VV -+VV
TSIA K/A, gas+, H2S - K/A, gas+, H2S -

• Note:
Colonies are clean and colorless (NLF)
Swarming phenomenon
Burnt-Chocolate
• Disease:
UTI
Infect proximal kidney tubules
Acute glomerulonephritis (AGN)

Citrobacter

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: C.freundii: -+-+ /
C.koseri: ++-+
• TSIA (Triple Sugar Iron Agar) reaction: C.freundii: A/a, gas +, H2S + / C.koseri: K/A, gas +, H2S –
• Urease test: +
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST

For table:

Biochemical test C.freundii C.koseri

Indole - +
H2S + +
Growth in KCN + -
ONPG + -
IMVIC -+-+ ++-+
TSIA A/a, gas +, H2S + K/A, gas +, H2S –

• Note:
It become clear and colorless (NLF) after 24 hours and may appear light pink color (LF)
after 48 hours
• Disease:
False-positive agglutination

Salmonella

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: -+-+ / -+-- (Salmonella
serotype Typhi)
• TSIA (Triple Sugar Iron Agar) reaction: K/A, gas +, H2S + / : K/A, gas - , H2S + (Salmonella
serotype Typhi)
• Urease test: -

For table:

Biochemical test S. serotype Typhi S. serotype S. serotype Other sterotypes

Paratyphi Cholerasuis
Citrate utilization - - v* +
Gas production - + + +
H2S + - v +
Lysine
decarboxylase + - + +
(LDC)
Ornithine - + + +
decarboxylase
Aribinose - + - +
fermentation
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST

• Note:
MAC - clear and colorless (NLF)
SSA – colorless with black centers

• Disease:
Acute gastroenteritis
Enteric fever
Bacteremia

Shigella

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: v+--
• TSIA (Triple Sugar Iron Agar) reaction: K/A, gas -, H2S –
• Urease test: -
• Lysine decarboxylase test: -

For table:

S. dysenteriae A + - -
S.fleneri B + - +
S.boydii C + - +
S.connie D + + +

• Note:
MAC- clear and colorless NLF
SSA- clear without black center
• Disease:
Bacillary dysentery

Yersinia

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: -+--
• TSIA (Triple Sugar Iron Agar) reaction: K/A, gas -, H2S –
• Urease test: -

For table:

Biochemical test Y.pestis Y.enterocolitica

Indole - v
Methy Red + +
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
Voges- Proskeur - v
Urease - +
Ornithine Decarboxylase - +
Sucrose Fermentation - +
Beta- galactosidase + +
Motility
25 degree celsius - +
37 degree celsius - -

• Note:
Y.pestis MAC- NLF,
Y.pestis BAP- Colonies are pinpoint at 24 hour
Y.enterocolitica MAC- clear and colorless NLF
Y.enterocolitica CIN-exhibit a “bulls-eye” appearance

• Disease:

Bubonic plague
Waterborne gastroenteritis

Providencia

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: ++-+
• TSIA (Triple Sugar Iron Agar) reaction: K/A, gas -, H2S –
• Lysine decarboxylase test: R/A

For table:

Biochemical test P. rettgeri P. stuartti

Arbitol Fermentation + -
Phenylalanine deaminase + +
(PAD)
Urease + -

• Note:
It becomes clear and colorless NLF

• Disease:
Nosocomial outbreaks involving burn units
UTI

Edwardsiella

• IMVIC ( Indole, Methy Red, Voges- Proskeur, and Citrate) reaction: ++--
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
• TSIA (Triple Sugar Iron Agar) reaction: K/A, gas +, H2S +
• Lysine decarboxylase test: +
• Urease test: -

For table:

Biochemical test E. tarda E. hoshinae

• Note:
It becomes clear and colorless NLF

• Disease:

Isolated mostly in cold-blooded and warm-blooded animals including reptiles

E.tarda- Human pathogen
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST

BIOCHEMICAL TEST

 CARBOHYDRATE FERMENTATION
 Principle: the conversion of carbohydrates, such as glucose or lactose, into other products,
such as organic acids, gases, or alcohol, by microorganisms like bacteria or yeast in the
absence of oxygen.
 pH indicators:
- Bromcresol purple
- Andrade's fuchsin
- Phenol red
 Organisms
-Lactobacillus
Streptococcus
Leuconostoc
Enterobacteriaceae (Escherichia coli and Salmonella).
 TRIPLE SUGAR IRON (TSI)
 Principle: used to differentiate bacteria based on their ability to ferment different sugars,
produce gas, and produce hydrogen sulfide.
 Indicators
- pH indicator: Phenol Red
- H2S indicator: ferrous sulfate & sodium thiosulfate
 Organisms
Enterobacteriaceae
-Escherichia coli
-Salmonella
-Shigella
-Proteus.
 Reaction
A. K/K = NO FERMENTATION
ALKALINE SLANT/ALKALINE BUTT
Glucose/Lactose/Sucrose NOT fermented
Reaction is common to NON-fermentative organisms

B. K/A = GLUCOSE FERMENTATION

ALKALINE SLANT/ACID BUTT
Glucose is fermented, with acid
Lactose/Sucrose NOT fermented

C. A/A = LACTOSE FERMENTATION

ACID SLANT/ACID BUTT
All sugars are fermented
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST

 KLIGLER IRON AGAR

 Principle: a differential and selective medium used in microbiology to differentiate bacteria
based on their ability to ferment glucose and lactose, produce hydrogen sulfide gas, and
their ability to reduce sulfur compounds.
 pH Indicator: Phenol Red
 Organisms
- Escherichia coli
-Klebsiella pneumoniae
-Enterobacter aerogenes
-Salmonella typhimurium
-Shigella flexneri
-Proteus vulgaris.
-Pseudomonas
-Staphylococcus
-Streptococcus
 LYSINE IRON AGAR
 Principle:differentiate bacteria based on their ability to produce lysine decarboxylase and
lysine deaminase enzymes.
 Indicator
pH indicator: Bromcresol purple
H2S indicator: Ferric Ammoniucitratete
 Organisms
Enterobacteriaceae
Pseudomonas
Proteus
Escherichia coli
Salmonella enterica
Shigella flexneri
Proteus mirabilis
Klebsiella pneumoniae
Pseudomonas aeruginosa.
 SPOT INDOLE TEST
 Principle: based on the reaction of the amino acid tryptophan with the enzyme
tryptophanase, which is present in some bacteria. Tryptophanase catalyzes the conversion
of tryptophan to indole, pyruvic acid, and ammonia.
 Indole indicator: p-dimethylaminocinnamaldehyde/PDAMCA
 Organisms
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
Enterobacter aerogenes.

 METHYL RED TEST

 PRINCIPLE: some organisms produce large amounts of acid from dextrose while others
produce less. This test is based upon the final hydrogen ion concentration (acidity) reached
by a culture.
 pH indicator: Phenol Red
 Organisms
Escherichia coli
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus mirabilis
Acetobacter
Pseudomonas
Streptococcus

 VOGES PROSKAUER TEST

 PRINCIPLE: Some bacteria have the ability to produce acetoin (acetylmethylcar-binol) from
glucose. In an alkaline pH, acetoin is oxidized to diacetyl (dimethylcarbinol), which reacts
with guanidine compounds present in the broth to give a red-colored complex.
 REAGENTS:
- 5% alpha-naphthol in 95% ethanol
- 40% KOH in distilled water (provides alkalinity)
- 0.5% creatine in distilled water (prevents false negative results)
 Organisms
Escherichia coli
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus mirabilis
Streptococcus
Enterococcus
Lactobacillus.

 •CITRATE
 Principle: used to determine the ability of bacteria to utilize citrate as a sole source of
carbon and energy for growth. The test is based on the ability of certain bacteria to produce
an enzyme called citrate permease, which allows them to transport citrate into the bacterial
cell and use it as a carbon source.
 pH indicator: Bromthymol Blue
 Organisms
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
Escherichia coli - Negative for citrate utilization
Klebsiella pneumoniae - Positive for citrate utilization
Enterobacter aerogenes - Positive for citrate utilization
Salmonella typhimurium - Negative for citrate utilization
Citrobacter freundii - Positive for citrate utilization
Proteus vulgaris - Negative for citrate utilization
 Reaction
Positive: Growth with an intense Prussian blue
Negative: No color change- Green

 •UREASE
 Principle
 pH indicator: Phenol Red
 Organisms
Helicobacter pylori
Proteus mirabilis
Klebsiella pneumoniae
Staphylococcus saprophyticus
Yersinia enterocolitica
Citrobacter
Morganella
Providencia
 Reaction
Original Color: Yellow
Positive result: PINK
 •DECARBOXYLASE TEST
 Principle
 Indicator
 Organism
Escherichia coli
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus mirabilis
Salmonella enterica
Citrobacter
Morganella
Serratia
 •PHENYLALANINE DEAMINASE TEST
 Principle: this test is based on the enzymatic activity of phenylalanine deaminase, an
enzyme that catalyzes the removal of the amine group (-NH2) from phenylalanine,
producing phenylpyruvic acid and ammonia.
 REAGENT: 10% aqueous ferric chloride (FeCl3)
 ENTEROBACTERIACEAE
(GRAM-NEGATIVE BACILLI)

BIOCHEMICAL TEST
pH indicator: Cresol Red
 Organisms
Proteus vulgaris
Morganella morganii
Providencia
Pseudomonas aeruginosa
Klebsiella
 INTERPRETATION:
Decarboxylase (+) = purple/purple
Decarboxylase (-) = purple/yellow
Deaminase (+) = red/yellow
H2S (+) = blackening
Gas (+) = cracks, bubble formation
 •MISCELLANEOUS TEST
 Principle: each miscellaneous test varies depending on the specific test being performed.
However, in general, these tests are based on the detection of certain enzymes,
metabolites, or other products produced by the bacterial cells during metabolic reactions.
 Indicator
 Organisms
 •DNASE TEST
 Principle: the medium is pale green because of the DNA-Methyl green complex. If the
organism growing on the medium hydrolyses DNA, green color fades and colony is
surrounded by colorless zone
 Indicator
 Organism