Biochemical TestsBiochemical Tests

Mohammed Laqqan

EnterobacteriaceaeEnterobacteriaceae

Classification – more than15 different genera• Escherichia• Shigella• Salmonella• Citrobacter• Klebsiella• Enterobacter• Serratia• Proteus• Providence• Morganella• Yersinia• Erwinia

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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.• 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

• 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 B.A they all produce similar colonies that are relatively large and gray. They may or may not be hemolytic.

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Mohammed Laqqan

Major Biochemical Reactions for Identification of the Enterobacteriaceae

1. Oxidase production.2. Voges-Proskauer fermentation reaction.3. Indole production from tryptophan.4. Utilization of citrate as a single carbon source. 5. Urease activity.6. Motility.7. Hydrogen sulfide (H2S) production.8. Decarboxylation of amino acids.

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Oxidase TestOxidase 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 procedureLimitations 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• Pseudomonas• Neisseria• Vibrio• 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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Tryptophan hydrolysis (Indole Production)Tryptophan hydrolysis (Indole Production)

• The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria. Tryptophan, for example, is hydrolyzed to indole, pyruvic acid and ammonia by tryptophanase.

• The pyruvic acid can be further metabolized to produce large amounts of energy. The ammonia is available for use in synthesis of new amino acids.

• Indole can be detected by reaction with Kovac's reagent (para-dimethylaminobenzaldehyde in alcohol) to produce a red color.

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• Inoculate Tryptone broth or SIM media with inoculating loop.

• Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan,

which makes by-products, of which, indole is one.

Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid,

dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.

Reading Results: After incubating the bacteria for at least 48 hours, Kovac’s reagent is added to the

media to detect if indole has been made by the bacteria. The development of a red/pink layer (Red Ring) on top of the media is a positive result (the bacteria can breakdown tryptophan to form indole). Failure to see a red layer is a negative result (indole was not formed from tryptophan).

How to Perform TestHow to Perform Test

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ResultResult

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The End