General Microbiology Laboratory
Biochemical Tests
Enterobacteriaceae
 Classification – more than15 different genera
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Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Klebsiella
Enterobacter
Hafnia
Serratia
Proteus
Providence
Morganella
Yersinia
Erwinia
Pectinobacterium
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 Morphology and General Characteristics
• Gram-negative, non-sporing, rod shaped bacteria
• Oxidase –
• Ferment glucose and may or may not produce gas in the
process (aerogenic vs anaerogenic)
• Reduce nitrate to nitrite (there are a few exceptions)
• Are facultative anaerobes
• If motile, motility is by peritrichous flagella
• Many are normal inhabitants of the intestinal tract of man and
other animals
• Some are enteric pathogens and others are urinary or
respiratory tract pathogens
• Differentiation is based on biochemical reactions and
differences in antigenic structure
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Most grow well on a variety of lab media including a lot
of selective and differential media originally developed
for the selective isolation of enteric pathogens.
• Most of this media is selective by incorporation of
dyes and bile salts that inhibit G+ organisms and
may suppress the growth of nonpathogenic species
of Enterobacteriaceae.
• Many are differential on the basis of whether or not
the organisms ferment lactose and/or produce H2S.
• On BA they all produce similar colonies that are
relatively large and gray. They may or may not be
hemolytic.
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 Major Biochemical Reactions for Identification of the
Enterobacteriaceae
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Oxidase production
Urease activity
Voges-Proskauer fermentation reaction
Phenylalanine deaminase activity
Indole production from tryptophan
Utilization of citrate as a single carbon source
Motility
Hydrogen sulfide (H2S) production
Decarboxylation of amino acids
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Oxidase Test
 Oxidase enzymes play an important role in the operation of the electron
transport system during aerobic respiration. Cytochrome oxidase uses O2
as an electron acceptor during the oxidation of reduced cytochrome c to
form water and oxidized cytochrome c.
 For example, in most gram positive bacteria and many gram negative
bacteria cytochrome oxidase performs the final step in electron transport,
reducing oxygen to water.
 Other bacteria, such as the Enterobacteriaceae, do not reduce oxygen using
this enzyme.
 Thus detection of cytochrome oxidase is a valuable tool in differentiating
among bacteria.
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 How to Perform Test:
• The ability of bacteria to produce cytochrome oxidase can be determined
by the addition of the oxidase test reagent or test strip to colonies that have
grown on a plate medium.
• Using a sterile swab, transfer the bacteria to the filter paper.
• (A platinum loop may be used to transfer organisms but iron
in a nichrome loop may interfere with the reaction).
 Media and reagent: oxidase test reagent or test strip
(tetramethyl-p-phenylenediamine dihydrochloride or an Oxidase
Disk, p aminodimethylaniline)
 Property it tests for: This test is done to determine a bacteria’s
ability to produce cytochrome oxidase enzyme.
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Reading Results: Observe for a color change. The light pink oxidase test
reagent (Disk, strip, or Slide) serves as an artificial substrate, donating
electrons to cytochrome oxidase and in the process becoming oxidized to
a purple and then dark purple compound in the presence of free O2 and
the oxidase. (figure 1). The presence of this dark purple coloration
represents a positive test. No color change or a light pink coloration on the
colonies indicates the absence of oxidase and is a negative test.
This chemical in the presence of oxygen and an oxidase enzyme will form
a colored compound.
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Limitations of the procedure
 We keep the Oxidase reagent either frozen or unopened in
tubes until needed. If old reagent is sitting out on the bench
and is PURPLE.
 Use a young culture, preferably less than 24 hrs old.
 Use a culture growing on an agar plate or agar slant.
 Use FRESH reagent, less than a couple of hours old (it is
taken out of the freezer).
 Pick your inoculum, not with a metal loop (reagent may react
with the metal), but with a wooden stick.
 Read the reaction within 20 seconds (NOT after), usually it
will change in less than 15 seconds. The oxygen will change
the reagent color as time passes, so it must be read quickly.
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 Oxidase producing bacteria
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Pseudomonas
Neisseria
Vibrio
Aeromonas
 Pseudomonas aeruginosa is a gram-negative,
aerobic rod having a strictly respiratory type of
metabolism with oxygen as the terminal
electron acceptor and thus is oxidase positive.
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Urease Test
 Some bacteria are able to produce an enzyme called urease that attacks the
nitrogen and carbon bond in amide compounds such as urea, forming the
end products ammonia, CO2, and water (figure 1).
 Urease activity (the urease test) is detected by growing bacteria in medium
containing urea and using a pH indicator such as phenol red. When urea is
hydrolyzed, ammonia accumulates in the medium and makes it alkaline.
This increase in pH causes the indicator to change from orange-red to deep
pink or purplish red and is a positive test for urea hydrolysis.
 This test is particularly useful in distinguishing the genus Proteus from
other enteric bacteria
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Mohammed Laqqan
 How to Perform Test: Inoculate Urea broth or urea slant agar with
inoculating loop.
 Property it tests for: This test is done to determine a bacteria’s ability to
hydrolyze urea to make ammonia using the enzyme urease.
 Media and Reagents Used:
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Urea Disks or Tablets
Urea broth or Urea slants contains a yeast extract, monopotassium
phosphate, disodium phosphate, urea, and phenol red indicator.
 Reading Results: Urea slant is a yellow color. The enzyme urease will
be used to hydrolyze urea to make ammonia. If ammonia is made, the
broth turns a bright pink color, and is positive. If test is negative, broth has
no color change and no ammonia is made.
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Mohammed Laqqan
Limitations of the procedure
Some bacteria have a delayed urease reaction
that may require an incubation period longer
than 48 hours.
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Urease-Producing Enterobacteriaceae
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Proteus
Klebsiella pneumoniae
Enterobacter cloacae
Yersinia enterocolitica
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End of lecture
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