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Biochemical Tests for Enterobacteriaceae

Zhawaan Szepietowski

Esherichia Coli

  • aka E. Coli

Esherichia 0157:H7

Strains of E. Coli

Enterotoxigenic (ETEC)

Enteropathogenic (EPEC)

Enteroinvasive (EIEC)

Enterohemorrhagic (0157:H7) (EHEC)

Enteroaggregative (EAEC)

Strains of E. Coli

Enterotoxingenic

adults diarrhea in tropics; travelers diarrhea; high infectious dose

ETEC

Enteropathogenic

usually daycare centers and hospital nurseries

EPEC

Enteroinvasive

dysentery with destruction of mucosa; similar to shigella, but infectious dose is far higher

EIEC

Enterohemorrhagic

O157:H7;

watery diarrhea to bloody diarrhea;

HUS hemolytic uremic syndrome

EHEC

Enteroaggregative

Part of enteroadherent, adhere to intestinal mucosa

EAEC

Triple Sugar Iron Agar

Slant: Acid (A)

Butt: Acid (A)

Gas: Pos

H2S: Neg

TSI agar

Rapid Spot Indole Test

Positive Result

Rapid Spot Indole Test

Rapid Cytochrome Oxidase Test

Negative result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

MacConkey Agar

E.Coli 0157:H7 shows colorless on MacConkey Agar, other strains will be pink

SMAC- Enterohemorrhagic E. coli

E. coli 0157:H7: Colorless on SMAC

Other E. coli strains are pink

Voges Proskauer

Negative Result

Voges Proskauer

Citrate Utilization Test

Negative Result

Citrate Utilization

Urea test

Negative Result

Urea Test

Motility Media Test

Positive Result

Motility Media

Oxidative fermentative Test

Fermentative

Oxidative Fermentative

Nitrate Test

Positive Result

Nitrate Test

Shigella

  • gray entire non hemolytic smooth colonies on blood agar
  • catalse pos
  • colonies w/o yellow coloration on he agar
  • Shigella colonies appear green with the color fading to the periphery of the colony.
  • inert biochemical rxns
  • bacillary dysentery
  • characterized by blood and pus in stool
  • S. sonnei (most common in US)
  • S. flexneri
  • S. dysenteriae
  • S. boydii
  • Very low id50

Triple Sugar Iron Agar

  • Slant:Alkaline(K)
  • Butt:Acid (A)
  • Gas:Neg
  • H2S:Neg

TSI Agar

Rapid Spot Indole

Positive or Negative Result for groups A B and C

sonnei - negative result

Rapid Spot Indole

Rapid Cytochrome Oxidase

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

  • good growth
  • lack of red color
  • non lactose fermenter
  • produce colorless or transparent colonies with rare exceptions

Voges Proskauer

Negative Result

Voges Proskauer

Citrate Utilization Test

Negative Result

Citrate Utilization

Urea Test

Negative Result

Urea Test

Motility Media Test

Negative Result

Motility Media

Oxidative Fermentative Test

Fermentative

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Salmonella

Salmonella

  • gray entire nonhemolytic smooth colonies on blood ag
  • oxidase neg
  • catalse pos
  • black colonies with a red halo on xld agar
  • alk/acid rxn on kia w/h2s
  • lysine pos
  • common diseases
  • • Gastroenteritis
  • • Bacteremia or septicemia
  • • Enteric fever
  • • Carrier state

Triple Sugar Iron Agar

Slant: alkaline (K)

Butt: acid (A)

Gas: Pos

H2S: Pos

TSI Agar

Rapid Spot Indole Test

Negative Result

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

  • good growth
  • lack of red color
  • non lactose fermenter
  • produce colorless or transparent colonies with rare exceptions

Voges Proskauer Test

Negative Result

Voges Proskauer

Citrate Utilization Test

Positive Result

Citrate Utilization

Urea Test

Negative Result

Urea Test

Motility Media Test

Positive Result

Motility Media

Oxidative Fermentative Test

Fermentative

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Citrobactor

slow/weak lactose fermenter

Citrobactor

Triple Sugar Iron Test

Alk/A or alk/a + H2S

TSI Agar

Rapid Spot Indole Test

Positive or Negative Result

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

may appear colorless or slightly pink after 24 hours

MacConkey Agar Apperance

Voges Proskauer

Negative Reaction

Voges Proskauer

Citrate Utilization Test

Positive Result

Citrate Utilization

Urea Test

Positive and Negative Result

Urea Test

Motiltity Test

Positive Result

Motility Media

Oxidative Fermentative Test

Fermentative

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Klebsiella

Triple Sugar Iron Agar

Slant: Acid (A)

Butt: Acid (A)

Gas: Pos

H2S: Neg

TSI Agar

Rapid Spot Indole Test

kliebsiella pneumoniae - neg

klebsiella oxytoca - pos

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

Strong rxn, produces pink colonies with pink/red pigment diffsusing surrounding agar

MacConkey Agar Apperance

Voges Proskauer

Positive Result

Voges Proskauer

Citrate Utilization Test

Positive Result

Citrate Utilization

Urease Test

Positive Result

Urea Test

Motility Test

Negative Result

Motility Media

Oxidative Fermentative Test

Fermenter

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Enterobacter

Triple Sugar Iron Agar

Slant: Acid (A)

Butt: Acid)9A)

gas pos

h2s neg

TSI Agar

Rapid Spot Indole Test

Negative Result

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar AppAppearance

MacConkey Agar Apperance

Pink colored after 24 hr incubation; pale-colored colonies turn pink after further 24 hr incubation.

mucoid colonies but smaller than Klebsiella.

Voges Proskauer Test

Positive Result

Voges Proskauer

Citrate Utilization Test

Positive Result

Citrate Utilization

Urease Test

Positive or Negative Result

Urea Test

Motility Test

Positive Result

Motility Media

Oxidative Fermentative Test

Fermenter

Oxidative Fermantative

Nitrate Test

Positive result

Nitrate Test

Serratia

Triple Sugar Iron Agar

Slant alkaline

butt acid

gas neg

h2s neg

TSI Agar

Rapid Spot Indole Test

Negative Result

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

growth appears red w/o pigment color leaching into surrounding agar

MacConkey Agar Apperance

Voges Proskauer Test

Positive Result

Voges Proskauer

Citrate Utilization

Positive Result

Citrate Utilization

Urease Test

Negative Result

Urea Test

Motility Test

Postitive Test

Motility Media

Oxidative Fermentative Test

Fermenter

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Proteus

  • oxidase negative
  • catalse pos
  • black pigment
  • h2s pos colonies on xld agar w/o surrounding halo
  • pad tda pos

Triple Sugar Iron Agar

slant alkaline

butt acid

gas pos

h2s pos

TSI Agar

Rapid Spot Indole Test

certain strains may give false negatives with this test

proteus mirabilis - neg

proteus vulgaris - pos

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance Test

MacConkey Agar Apperance

  • Spreading colonies on blood agar
  • no swarming on MacConkey agar specifically
  • colorless or transparent colonies with rare exceptions
  • foul smell

Voges Proskauer

Negative Result

Voges Proskauer

Citrate Utilization Test

Positive or Negative Result

Citrate Utilization

Urea Test

strong positive

Urea Test

Motility Test

Positive Test

Motility Media

Oxidative Fermentative test

Fermenter

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Morganella

Triple Sugar Iron Agar

A/A or ALK/A

TSI Agar

Rapid Spot Indole Test

Positive Result

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

Flat colorless non-lactose fermenters

MacConkey Agar Apperance

Voges Proskauer

Negative Result

Voges Proskauer

Citrate Utilization Test

Negative Result

Citrate Utilization

Urease Test

Positive Result

Urea Test

Motility Test

Positive result

Motility Media

Oxidative-Fermentative Test

Fermenter

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Yersinia

  • relatively small flat irreugular gray/yellow smooth colonies on blood agar
  • optimal growth at 25C
  • oxidase neg
  • catalase pos
  • indole pos
  • lysine neg
  • ornithine pos

Yersinia

Triple Sugar Iron

A/A or ALK/A

TSI Agar

Rapid Spot Indole Test

postive and negative Result

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Negative Result

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

  • Colorless to peach pigmented colonies
  • nonlactose fermenter

MacConkey Agar Apperance

Voges Proskauer Test

Negative Result

Voges Proskauer

Citrate Utilization Test

Negative Result

Citrate Utilization

Urea Test

Positive Result

Urea Test

Motility Media

Motile at 25 C

Motility Media

Oxidative Fermentative Test

Fermenter

Oxidative Fermantative

Nitrate Test

Positive Result

Nitrate Test

Test Explanations

  • what each test actaully tests for
  • what a postive and negative demonstrates

Test Explantions

Triple Sugar Iron Agar

  • Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
  • Alkaline slant/alkaline butt (K/K) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
  • Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only, gas (+ or -), H2S (+ or -)
  • Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+ or -), H2S (+ or -).

TSI Agar

Rapid Spot Indole Test

  • The Indole Test is a test that demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indole, which accumulates in the medium. This test is important in the identification of Enterobacteria. The test is performed by adding Kovac’s reagent to a broth culture containing tryptophan. If indole is present, the solution will turn from yellow to cherry red
  • designed to detect Gram-negative bacilli (Oxidase-negative).

Rapid Spot Indole

Rapid Cytochrome Oxidase Test

Rapid Cytochrome Oxidase

MacConkey Agar Apperance

Voges Proskauer Test

Voges Proskauer

  • The Voges-Proskauer (VP) Test is a biochemical test used in microbiology to determine if an organism produces acetylmethyl carbinol from glucose fermentation. This test was first observed by Voges and Proskauer in 1898
  • Positive test:
  • The medium changes color from yellow to pink-red, indicating that the organism can produce acetylmethyl carbinol1.
  • Negative test:
  • The medium remains yellow or fails to turn pink-red within 48 hours, indicating that the organism cannot produce acetylmethyl carbinol.
  • identification of several genera and species of Enterobacteriaceae, including Enterobacter, Klebsiella, Serratia

Citrate Utilization

Urea Test

Urea Test

  • used in microbiology to determine if an organism can produce the enzyme urease. This enzyme hydrolyzes urea into ammonia and carbon dioxide.
  • Positive test:
  • The medium changes color from light orange to magenta, indicating that the organism can produce urease12.
  • Negative test:
  • The medium remains light orange, indicating that the organism cannot produce urease12.
  • The Urease Test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease1. This test can be used as part of the identification of several genera and species of Enterobacteriaceae, including Proteus, Klebsiella, and some Yersinia and Citrobacter species

Motility Media test

Motility Media

  • The Motility Test is a method used in microbiology to determine if an organism can move by itself. This ability is mostly demonstrated in a semi-solid agar medium. In semi-solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that is easily recognized by the naked eye.
  • Positive test:
  • Diffuse, hazy growths that spread throughout the medium rendering it slightly opaque.
  • Negative test:
  • Growth that is confined to the stab-line, with sharply defined margins and leaving the surrounding medium clearly transparent.
  • used particularly with proteus, morganella and providencia

Oxidative Fermantative

Nitrate Test

Nitrate Test

  • Positive test: The medium changes color from yellow to red, indicating that the organism can produce nitrate reductase1.
  • Negative test: The medium remains yellow, indicating that the organism cannot produce nitrate reductase1.
  • The Nitrate Reduction Test is used to differentiate organisms based on their ability to produce nitrate reductase1. This test can be used as part of the identification of several genera and species of Enterobacteriaceae, including Proteus, Morganella, and Providencia2.

Dichotomyous Key

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