Biochemical Tests

Enterobacteriaceae Classification – more than15 different genera Escherichia Shigella Edwardsiella Salmonella Citrobacter Klebsiella Enterobacter Hafnia Serratia Proteus Providence Morganella Yersinia Erwinia Pectinobacterium Mohammed Laqqan

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 Mohammed Laqqan

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. Mohammed Laqqan

Voges-Proskauer fermentation reaction Phenylalanine deaminase activity Major Biochemical Reactions for Identification of the Enterobacteriaceae 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 Mohammed Laqqan

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. Mohammed Laqqan

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. Mohammed Laqqan

Reading Results: Observe for a color change 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. Mohammed Laqqan

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. Mohammed Laqqan

Oxidase producing bacteria 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. Mohammed Laqqan

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 Mohammed Laqqan

Mohammed Laqqan

Media and Reagents Used: Urea Disks or Tablets 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: 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. Mohammed Laqqan

Mohammed Laqqan

Limitations of the procedure Some bacteria have a delayed urease reaction that may require an incubation period longer than 48 hours. Mohammed Laqqan

Urease-Producing Enterobacteriaceae Proteus Klebsiella pneumoniae Enterobacter cloacae Yersinia enterocolitica Mohammed Laqqan

End of lecture Mohammed Laqqan