BACTE LAB ENTERO

• Enterobacteriaceae Laboratory Test

  Citrate utilization

  DNase hydrolysis

  Gelatin hydrolysis

  Indole production

  Lysine Iron Agar

  Methyl Red / Voges Proskauer

  Motility testing

  ONPG test

  Phenylalanine Deaminase Agar

  Triple Sugar Agar

  Urease test
• Enterobacteriaceae Laboratory Test Other Procedure

  Nitrate and Nitrite reduction

  Oxidation and Fermentation tests

  Malonate utilization

  Oxidase test

  MIO and SIM agar

  Manual Multi-test Systems

  Rapid and Automated Identification Systems
• Citrate Utilization Purpose

  To identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source.
• Citrate Utilization Principle

  Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of energy. Bacteria capable of growth in this medium use the citrate and convert ammonium phosphate to ammonia and ammonium hydroxide, creating an alkaline pH. The pH change turns the bromthymol blue indicator from green to blue.
• Citrate Utilization Expected Results and Bacteria

  (+) Growth (blue) (E. aerogenes)

  (0) No growth (green) (E. coli)
• DNA Hydrolysis (Dnase Test Agar) Purpose

  This test is used to differentiate organisms based on the production of deoxyribonuclease. It is used to distinguish Serratia sp. (positive) from Enterobacter sp., Staphylococcus aureus (positive) from other species, and Moraxella catarrhalis (positive) from Neisseria sp.
• DNA Hydrolysis (Dnase Test Agar) Principle

  The test is used to determine the ability of an organism to hydrolyze DNA. The medium is pale green because of the DNA–methyl green complex. If the organism growing on the medium hydrolyses DNA, the green color fades and the colony is surrounded by a colorless zone.
• DNA Hydrolysis (Dnase Test Agar) Expected Result

  (+) Colorless medium around the organism (S. aureus)

  (0) Green medium (E. coli)
• Gelatin Hydrolysis Purpose

  The production of gelatinases capable of hydrolyzing gelatin is used as a presumptive test for the identification of various organisms, including Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli.
• Gelatin Hydrolysis Principle

  This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate connective tissue.

  Nutrient gelatin medium differs from traditional microbiology media in that the solidifying agent (agar) is replaced with gelatin. When an organism produces gelatinase, the enzyme liquefies the growth medium.
• Gelatin Hydrolysis Expected Result and Bacteria

  (+) Partial or total liquefaction (B. subtilis)

  (0) Complete solidification of the tube (E. coli)
• Indole Production Purpose


  This test is used to identify organisms that produce the enzyme tryptophanase.
• Indole Production Principle

  The test is used to determine an organism’s ability to hydrolyze tryptophan to form the compound indole. Tryptophan is present in casein and animal protein. Bacteria with tryptophanase are capable of hydrolyzing tryptophan to pyruvate, ammonia, and indole. Kovac’s reagent (dimethylamine-benzaldehyde and hydrochloride), when added to the broth culture, reacts with the indole, producing a red color.
• Indole Production Expected Result and Bacteria

  (+) Pink to wine-colored ring after addition of reagent (E. coli)

  (0) No change in color (K. pneumoniae)
• Lysine Iron Agar (LIA) Purpose

  This test is used to differentiate gram-negative bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S).
• Lysine Iron Agar (LIA)

  A medium that contains lysine, peptones, a small amount of glucose, ferric ammonium citrate, and sodium thiosulfate
• LIA Principle

  1. Glucose fermentation makes the butt of the medium acidic (yellow)

  2. If lysine decarboxylase is produced, cadaverine is formed which neutralizes the organic acids and reverts the butt to alkaline (purple)

  3. If decarboxylase is not produced, the butt remains acidic (yellow)

  4. If oxidative deamination of lysine occurs, a compound is formed that creates a burgundy color on the slant

  5. If deamination does not occur, the LIA slant remains purple
• Bromocresol purple

  The pH indicator used, which is yellow at or below pH 5.2 and purple at or above pH 6.8
• Lysine Iron Agar (LIA) Mechanism

  SLANT: Lysine deamination (attacks the amino group instead of the carboxyl group)

  Lysine turns the light purple color slant to plum or reddish purple, the butt turns yellow because of glucose fermentation

  BUTT: Lysine decarboxylation

  * Decarboxylation happens only at an anaerobic environment

  * The black ppt (H2S) occurs only at an alkaline environment

  Organism (+) glucose fermentation - YELLOW;

  Organism (+) LDC - CADAVERINE - neutralize the organic acid formedà reverts to PURPLE
• Lysine Iron Agar Expected Result Quality Control

  Alkaline slant and butt: H2S positive: Citrobacter freundii

  Alkaline slant and butt: Escherichia coli

  Alkaline slant and butt: H2S positive: Salmonella typhimurium

  Red slant, acid butt: Proteus mirabilis
• Methyl Red/Voges Proskauer Test Purpose

  The combination test methyl red (MR) and Voges-Proskauer (VP) differentiates members of the Enterobacteriaceae family.
• Methyl Red/Voges Proskauer Test

  Used to determine the ability of an organism to:

  1. Produce and maintain stable acid end products from glucose fermentation
  2. Overcome the buffering capacity of the system
  3. Determine the ability of some organisms to produce neutral end products (e.g., 2,3-butanediol or acetoin) from glucose fermentation
• Methyl Red Test

  1. Methyl red indicator is added after incubation

  2. Methyl red is red at pH 4.4 and yellow at pH 6.2

  3. A clear red is a positive result

  4. Yellow is a negative result

  5. Various shades of orange are negative or inconclusive
• Voges Proskauer Test

  1. Detects the organism's ability to convert the acid products to acetoin and 2,3-butanediol

  2. Organisms capable of using the VP pathway produce a smaller amount of acid during glucose fermentation and therefore do not produce a color change when the methyl red indicator is added

  3. A secondary reagent, alpha-naphthol, is added, followed by potassium hydroxide (KOH)

  4. A positive test result is indicated by a red color complex
• Methyl Red/Voges Proskauer Test Mechanism

  MR: Glucose - Pyruvic acid - Mixed acid fermentation (ph 4.4) - Red color with methyl indicator

  VP: Glucose - Pyruvic acid - Acetoin - Diacetyl + KOH + Alpha-Naphthol - Red Complex - 2,3-Butanediol
• MR/VP Expected Result and Bacteria

  MR Positive: Bright red color - acid fermentation (Escherichia coli)

  VP Positive: Red color - acetoin production (Enterobacter aerogenes)

  Negative: Yellow Color
• Motility Test Purpose

  These tests are used to determine whether an enteric organism is motile. An organism must have flagella to be motile.
• Motility Test Principle

  The inoculum is stabbed into the center of a semisolid agar deep. Bacterial motility is evident by a diffuse zone of growth extending out from the line of inoculation. Some organisms grow throughout the entire medium, whereas others show small areas or nodules that grow out from the line of inoculation.
• Motility Test Expected Result

  (+) Motile organisms will spread out into the medium from the site of inoculation (E. coli)

  (0)Non-motile organisms remain at the site of inoculation (S. aureus)
• o-NITROPHENYL-beta-D-GALACTOPYRANOSIDE (ONPG) TEST Purpose

  This test is used to determine the ability of an organism to produce β-galactosidase, an enzyme that hydrolyzes the substrate ONPG to form a visible (yellow) product, orthonitrophenol. The test distinguishes late lactose fermen- ters from non–lactose fermenters of Enterobacteriaceae.
• o-NITROPHENYL-beta-D-GALACTOPYRANOSIDE (ONPG) TEST Principle

  Lactose fermenters must be able to transport the carbohydrate (β-galactoside permease) and hydrolyze (β-galactosidase) the lactose to glucose and galactose. Organisms unable to produce β-galactosidase may become genetically altered through a variety of mechanisms and be identified as late-lactose fermenters. ONPG enters the cells of organisms that do not produce the permease but are capable of hydrolyzing the ONPG to galactose and a yellow compound, o-nitrophenol, indicating the presence of β-galactosidase.
• o-NITROPHENYL-beta-D-GALACTOPYRANOSIDE (ONPG) TEST Expected Result

  (+) Yellow/Orange (presence of beta-galactosidase) (S. sonnei)

  (0) Colorless/No change in color (absence of the enzyme) (S. typhimurium)
• Phenylalanine Deaminase Agar (PAD) Purpose

  This test is used to determine the ability of an organism to oxidatively deaminate phenylalanine to phenylpyruvic acid. The genera Morganella, Proteus, and Providencia can be differentiated from other members of the Enterobacteriaceae family.
• Phenylalanine Deaminase Agar (PAD) Principle

  Microorganisms that produce phenylalanine deaminase remove the amine (NH2) from phenylalanine. The reaction results in the production of ammonia (NH3) and phenylpyruvic acid. The phenylpyruvic acid is detected by adding a few drops of 10% ferric chloride; a green- colored complex is formed between these two compounds.
• Phenylalanine Deaminase Agar (PAD) Expected Result and Bacteria

  (+) Green color on the slant after adding FeCl3 (P. mirabilis)

  (0) No change in color in the slant (E. coli)
• Triple Sugar Iron Agar (TSI) Purpose

  TSI is used to determine whether a gram- negative rod ferments glucose and lactose or sucrose and forms hydrogen sulfide (H2S). The test is used primarily to differentiate members of the Enterobacteriaceae family from other gram-negative rods.
• Triple Sugar Iron Agar (TSI) Principle

  The composition of TSI is 10 parts lactose:10 parts sucrose:1 part glucose and peptone. Phenol red and ferrous sulfate serve as indicators of acidification and H2S formation, respectively.

  The formation of CO2 and hydrogen gas (H2) is indicated by the presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube. The production of H2S (sodium thiosulfate reduced to H2S) requires an acidic environment, and reaction with the ferric ammonium citrate produces a blackening of the agar butt in the tube.
• Triple Sugar Iron Agar Expected Result 1

  Alkaline slant/no change in the butt (K/NC): glucose, lactose, and sucrose nonutilizer; this may also be recorded as K/K (alkaline slant/ alkaline butt).

  Alkaline slant/acid butt (K/A): glucose fermentation only.
• Triple Sugar Iron Agar Expected Result 2

  Acid slant/acid butt (A/A): glucose, sucrose, and/or lactose fermenter Note: A black precipitate in the butt indicates

  production of ferrous sulfide and H2S gas (H2S+).

  Bubbles or cracks in the tube indicate the production of CO2 or H2.  Drawing a circle around the A for the acid butt; that is, A , usually indicates this means the organism ferments glucose and sucrose, glucose and lactose, or glucose, sucrose, and lactose, with the production of gas.
• Urease Test Purpose

  This test is used to determine an organism’s ability to produce the enzyme urease, which hydrolyzes urea. Proteus sp. may be presumptively identified.