Biochemical Tests Used for the Identification of Gram Negative Rods by Cyndie J. Hobson, MLS(ASCP)cmSMcm Carolinas College of Health Sciences School of Clinical Laboratory Sciences Medical Laboratory Science Program

Kligler Iron Agar (KIA) OR Triple Sugar Iron Agar (TSI)* Principle: Medium used to differentiate gram negative rods based on their ability to ferment glucose, lactose and produce hydrogen sulfide. A/A = Lactose Fermenter K/A = Glucose Fermenter K/K = Non-Fermenter (Group IV) H2S detected in the butt of the slant. *TSI: has third sugar - sucrose eplantscience.com

Lysine Iron agar Principle: to determine whether an organism can decarboxylate or deaminate the amino acid lysine and produce hydrogen sulfide. Decarboxylation is the anaerobic process whereby bacteria that posses specific decarboxylase enzymes are capable of attacking amino acids at their carboxyl end (-COOH) yielding an amine or a deamine and CO2.

LIA continued Interpretation: Purple slant/purple butt = POSITIVE Decarboxylation Purple slant/yellow butt = NEGATIVE Decarboxlation Burgundy slant/yellow butt = POSITIVE Deamination

MIO Media Motility,Indole, Ornithine Inoculation: Very Carefully inoculate the agar making a straight, vertical stab MOTILITY Motility: Motile organisms move away from the line of inoculum = Positive Non-motile organisms grow only along the line of inoculation = Negative Other means of detecting motility are: Direct wet mount Hanging drop method Flagellar stain _ +

Indole Principle: to determine the ability of an organism to split typrophan to form the compound indole Reagent: para-dimethylaminobenazldehyde Interpretation: Positive: Cherry-red Negative: No change/yellowish _ +

Ornithine Principle: to determine if the organism can decarboxylate the amino acid ornithine (see Lysine handout) Interpretation: Positive: Purple butt Negative: Yellow butt

Phenylalanine Deaminase Principle: to determine if the organism has the ability to deaminate phenylalanine resulting in the production of phenylpyruvic acid. Reagent Needed: 10% Ferric Chloride Interpretation: Positive: Dark green on surface of slant Negative: No color change _ +

Simmon’s Citrate Agar (Tartrate, Malonate, Acetamide) Principle: to determine if the organism can utilize citrate as its sole source of carbon Inoculation: Streak Slant Indicator: Brom-thymol blue Interpretation: Positive: Blue Negative: Green

Sugar Fermentation Test Principle: to determine the ability of an organism to ferment a carbohydrate incorporated into a basal medium, thereby producing acid. Indicator: Phenol red Interpretation: Positive: Yellow Negative: Red, orange, peach _ +

Urea Principle: to determine if the organism has the ability to split urea, forming two molecules of ammonia by the action of the enzyme urease with resulting alkalinity Inoculation: Streak slant Interpretation: Positive: Hot Pink Negative: Amber/Neutral Positive Negative

Nitrate Reduction Test Principle: to determine the ability of an organism to reduce nitrates to nitrites or free nitrogenous gas. Inoculation: Emulsify organism in broth Reagents Needed: Nitrate A = Sulfanilic acid Nitrate B = Alpha-naphthylamine Zinc Dust

Nitrate Test continued: Interpretation: Positive: Pink/Red color immediately after the addition of Nitrate A & B Positive: No change after the addition of Nitrate A & B and zinc dust = N2 Gas Negative: Red after the addition of Nitrate A, B & zinc dust + +

Gelatin Liquefaction Test Principle: to determine the organisms ability to produce proteolytic-like enzymes (gelatinases) which in turn are detected by the digestion (liquefying) of gelatin Inoculation: Stab agar several times Interpretation: Positive: Partial or total liquefaction Negative: Solidification of tube at 4 C +

MR-VP Methyl-Red/Vogues Proskauer Methyl-Red Principle: to see if the organism can produce large quantities of organic acids (mixed acid fermentation) such as lactic, formic and succinic. Reagent Needed: Methyl Red Interpretation: Positive: Cherry Red Negative: Yellow uwyo.edu

MR-VP Continued: Vogues-Proskauer Principle: to see if the organism can produce large quantities of neutral products like “acetyl-methyl-carbinol” or “acetoin” (2,3-butylene glycol and di-acetyl) Reagents Needed: 5% alpha-naphthol 40% KOH (potassium hydroxide) Interpretation: Positive: Pink to Red Negative: Neutral to Clear +

Oxidase Test Principle: to see if the organism can oxidize certain aromatic amines, for example, p-aminodimethylaniline, to form colored end products. This oxidation correlates with the cytochrome oxidase activity of some bacteria, including the genera Pseudomonas and Neisseria. While a positive oxidase test is important in the identification of these genera, the test is also useful in characterizing the enteric bacteria (Family Enterobacteriaceae), which are oxidase negative.

Oxidase continued: Reagent Needed: a) tetramethyl-p-phenylenediamine dihydrochloride or dimethylsulfoxide Interpretation: Positive: Immediately purple (within 10 seconds) Negative: Yellow to no color change

O.N.P.G. Test (Ortho-NitroPhenyl-beta-D-Galactopyranoside) Principle: to determine the ability of an organism to ferment lactose (by means other than permease) Lactose fermentation depends on permease, which allows lactose to enter the cell and an enzyme galactosidase, which breaks the molecule into its’ component parts – glucose and galactose Slow lactose fermenters are deficient in permease and the demonstration of Beta-galactosidase provides a rapid means of detecting slow/late-lactose fermenters A positive ONPG test shows the organism contains the intracellular enzymes necessary for the fermentation of lactose and may be classified as a lactose fermenter Determine the ability of an organism to produce Beta-galactosidase, an enzyme that hydrolyzes ONPG to orthonitrophenol Some organisms ferment lactose slowly Lactose fermentation depends on permease, which allows lactose to enter the cell and an enzyme galactosidase, which breaks the molecule into its’ component parts – glucose and galactose Slow lactose ferementers are deficient in permease and the demonstration of Beta-galactosidase provides a rapid means of detecting late-lactose fermenters A positive ONPG test shows the organism contains the intracellular enzymes necessary for the fermentation of lactose and may be classified as a lactose fermenter

ONPG continued: This test uses beta-galactosidase (an enzyme that hydrolyzes the substrate O.N.P.G. to orthonitrophenol) to provide a means of detecting lactose fermentation. Reagents Needed: ONPG disk in .85% Saline Interpretation: Positive: any shade of yellow Negative: clear or colorless . +

O-F Dextrose Basal Media Principle: to determine the fermentative or oxidative properties of various gram negative rods Usually fermentation is stronger than oxidation. The small amount of acid produced by oxidation may be masked by other reactions. In ordinary carbopeptone media, these substances may completely neutralize the oxidative reaction. O-F media contains a high carbohydrate:low peptone concentration for optimum oxidative conditions. Fermentative: these organisms will produce an acid reaction in both the covered and uncovered media Oxidative: these organisms will produce an acid reaction in the uncovered tube only and no growth in the covered tube Non-Utilizer: these organisms are not classified as oxidative or fermentative and yield no change in the covered tube and an alkaline reaction in the uncovered tube

Inoculation: Stab 2 tubes; Overlay 1 with oil Oxidation-Fermentation of Dextrose Continued: Inoculation: Stab 2 tubes; Overlay 1 with oil Interpretation: a) Fermenter: Yellow in the “Closed” tube b) Oxidizer: Yellow in the “Open” tube c) Non-Utilizer: Green in both tubes Y Y G Y G G a b c famsbc.wordpress.com

Growth at 42O C Principle: to see if the organism can grow at increased temperatures Supplies Needed: 42O C Water Bath or Incubator Interpretation: Positive: Growth or turbid broth Negative: No growth or Clear broth +

Cetrimide Principle: to determine the ability of an organism (especially Pseudomonas) to grow in the presence of Cetrimide, a toxic substance that inhibits the growth of many bacteria. Inoculation: Streak slant/broth with Cetrimide Interpretation: Positive: Growth Negative: No Growth +

REFERENCES Pictures Obtained from: Hardy Diagnostics Studyblue REFERENCES Pictures Obtained from: Hardy Diagnostics Studyblue.com Faculty.lacitycollege.edu Quizlet.com Famsbc.wordpress.com Microbiology Media Images Blinn.edu Phobos.ramapo.edu Rci.rutgers.ecu Faculty.ccbcmd.edu Karen M. Kiser Difco Laboratories