Overview of Bacterial Identification Methods and Strategies Flashcards
Differentiate microorganisms based on the ability to use acetamide as the sole source of carbon
Acetamide Utilization
Principle of acetamide utilization test
Bacteria capable of growth on this medium produce the enzyme acylamidase, which deaminates acetamide to release ammonia. The production of ammonia results in an alkaline pH, causing the medium to change color from green to royal blue
Expected results of acetamide utilization test
Positive: Deamination of the acetamide, resulting in a blue color
Negative: No color change
Quality control/s of acetamide utilization test
Positive: Pseudomonas aeruginosa—growth; blue color
Negative: Escherichia coli—no growth; green color
Differentiate organisms based on ability to use acetate as the sole source of carbon
Acetate Utilization
Differentiate Shigella sp. from Escherichia coli.
Acetate Utilization
Principle of acetate utilization test
Organisms capable of using sodium acetate grow on the medium, resulting in an alkaline pH, turning the indicator from green to blue
Expected results of acetate utilization test
Positive: Medium becomes alkalinized (blue)
Negative: No growth or growth with no indicator change to blue (green)
Quality control/s of acetate utilization test
Positive: Escherichia coli— growth; blue
Negative: Shigella sonnei— small amount of growth; green
This test is used for presumptive identification and differentiation of beta-hemolytic group A streptococci (Streptococcus pyogenes–susceptible) from other beta-hemolytic streptococci
Bacitracin Susceptibility
This test is used to distinguish staphylococci species (resistant) from micrococci (susceptible)
Bacitracin Susceptibility
Principle of bacitracin susceptibility test
The antibiotic bacitracin inhibits the synthesis of bacterial cell walls. It is placed on the agar plate and after incubation, the plate is examined for zone of inhibition
Expected result/s of bacitracin susceptibility test
Positive: Any zone of inhibition greater than 10 mm; susceptible
Negative: No zone of inhibition; resistant
Quality control/s of bacitracin susceptibility test
Positive: Streptococcus pyogenes—susceptible
Micrococcus luteus—susceptible
Negative: Streptococcus agalactiae—resistant
Staphylococcus aureus—resistant
This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group
Bile Esculin Test
This test differentiates enterococci and group D streptococci from non–group D viridans streptococci
Bile Esculin Test
Principle of bile esculin test
Gram-positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in this medium. Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin. Esculetin reacts with Fe3+ and forms a dark brown to black precipitate
Expected results of bile esculin test
Positive: Growth and blackening of the agar slant
Negative: Growth and no blackening of medium
No growth
Quality control/s of bile esculin test
Positive: Enterococcus faecalis — growth; black precipitate
Negative: Escherichia coli— growth; no color change
Streptococcus pyogenes—no growth; no color change
This test differentiates Streptococcus pneumoniae (positive–soluble) from alpha-hemolytic streptococci (negative–insoluble)
Bile Solubility Test
Principle of bile solubility test
Bile or a solution of a bile salt rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme, amidase. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process
Expected results of bile solubility test
Positive: Colony disintegrates; an imprint of the lysed colony may remain in the zone
Negative: Intact colonies
Quality control/s of bile solubility test
Positive: Streptococcus pneumoniae—bile soluble
Negative: Enterococcus faecalis—bile insoluble
This is a rapid test to detect the enzyme butyrate esterase, to aid identification of Moraxella (Branhamella) catarrhalis
Butyrate Disk
Principle of butyrate disk
Organisms capable of producing butyrate esterase hydrolyze bromochlorindolyl butyrate. Hydrolysis of the substrate in the presence of butyrate esterase releases indoxyl, which in the presence of oxygen spontaneously forms indigo, a blue to blue-violet color
Expected results of butyrate disk test
Positive: Development of a blue color during the 5-minute incubation period
Negative: No color change
Quality control/s of butyrate disk test
Positive: Moraxella catarrhalis— formation of blue color
Negative: Neisseria gonorrhoeae—no color change
Test used to differentiate group B streptococci (Streptococcus agalactiae– positive) from other streptococcal species
CAMP Test
What does CAMP Test means
Christie, Atkins, and Munch-Peterson (CAMP) test
Reaction of Listeria monocytogenes in CAMP Test
Positive CAMP reaction
Principle of CAMP test
Certain organisms (including group B streptococci) produce a diffusible extracellular hemolytic protein (CAMP factor) that acts synergistically with the beta-lysin of Staphylococcus aureus to cause enhanced lysis of red blood cells. The group B streptococci are streaked perpendicular to a streak of S. aureus on sheep blood agar. A positive reaction appears as an arrowhead zone of hemolysis
Expected results of CAMP test
Positive: Enhanced hemolysis is indicated by an arrowhead-shaped zone of beta-hemolysis at the juncture of the two organisms
Negative: No enhancement of hemolysis
Quality control/s of CAMP test
Positive: Streptococcus agalactiae —enhanced arrowhead hemolysis
Negative: Streptococcus pyogenes —beta-hemolysis without enhanced arrowhead formation
This test differentiates catalase-positive micrococcal and staphylococcal species from catalase-negative streptococcal species
Catalase Test
Principle of catalase test
Catalase, is capable of converting hydrogen peroxide to water and oxygen. The presence of the enzyme in a bacterial isolate is evidenced when a small inoculum introduced into hydrogen peroxide (30% for the slide test) causes rapid elaboration of oxygen bubbles
Expected results of catalase test
Positive: Copious bubbles are produced
Negative: No or few bubbles are produced
Quality control/s of catalase test
Positive: Staphylococcus aureus
Negative: Streptococcus pyogenes
This test is primarily used to isolate and purify Pseudomonas aeruginosa from contaminated specimens
Cetramide Agar
Principle of cetramide agar
The test is used to determine the ability of an organism to grow in the presence of cetrimide, a toxic substance that inhibits the growth of many bacteria by causing the release of nitrogen and phosphorous, which slows or kills the organism. P. aeruginosa is resistant to cetrimide
Expected results of cetramide agar test
Positive: Growth, variation in color of colonies
Negative: No growth
Quality control/s of cetramide agar test
Positive: Pseudomonas aeruginosa—growth and color change; yellow-green to blue-green colonies
Negative: Escherichia coli—no growth and no color change
The purpose of this test is to identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source
Citrate Utilization
This test is part of a series referred to as IMViC (indole, methyl red, Voges-Proskauer, and citrate), which is used to differentiate Enterobacteriaceae from other gram-negative rods
Citrate Utilization
Principle of citrate utilization test
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
Expected results of citrate utilization test
Positive: Growth on the medium, with or without a change in the color of the indicator. Growth typically results in the bromthymol blue indicator turning from green to blue
Negative: Absence of growth
Quality control/s of citrate utilization test
Positive: Enterobacter aerogenes—growth, blue color
Negative: Escherichia coli—little to no growth, no color change
This test is used to differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci (negative)
Coagulase Test
Principle of coagulase test
S. aureus produces two forms of coagulase, bound and free. Bound coagulase, or “clumping factor,” is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in precipitation of fibrinogen on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. The presence of bound coagulase correlates with free coagu- lase, an extracellular protein enzyme that causes the formation of a clot when S. aureus colonies are incubated with plasma. The clot- ting mechanism involves activation of a plasma coagulase-reacting factor (CRF), which is a modified or derived thrombin molecule, to form a coagulase-CRF complex. This complex in turn reacts with fibrinogen to produce the fibrin clot
Expected results of coagulase test (Slide test)
Positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop
Negative: No clumping in either drop.
Note: All negative slide tests must be confirmed using the tube test
Expected results of coagulase test (Tube test)
Positive: Clot of any size
Negative: No clot
Quality control/s of coagulase test
Positive: Staphylococcus aureus
Negative: Staphylococcus epidermidis
This test is used to differentiate decarboxylase-producing Enterobacteriaceae from other gram-negative rods
Decarboxylase Tests (Moeller’s Method)
Principle of Decarboxylase Tests (Moeller’s Method)
This test measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine. Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH and a color change from orange to purple
Expected results of Decarboxylase Tests (Moeller’s Method)
Positive: Alkaline (purple) color change compared with the control tube
Negative: No color change or acid (yellow) color in test and control tube. Growth in the control tube
Quality control/s of Decarboxylase Tests (Moeller’s Method)
Positive:
Lysine—Klebsiella pneumoniae
Ornithine—Enterobacter aerogenes
Arginine—Enterobacter cloacae
Base—
Negative:
Lysine—Enterobacter cloacae Ornithine—Klebsiella pneumoniae
Arginine—Klebsiella pneumoniae
Base—Klebsiella pneumoniae
This test 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
Principle of DNA Hydrolysis Test
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
Expected results of DNA Hydrolysis Test
Positive: COLORLESS around the test organism
Negative: Medium remains green
Quality control/s of DNA Hydrolysis Test
Positive: Staphylococcus aureus
Negative: Escherichia coli
This test is used for the presumptive identification and differentiation of Enterobacteriaceae
Esculin hydrolysis
Principle of Esculin Hydrolysis Test
This test is used to determine whether an organism can hydrolyze the glycoside esculin. Esculin is hydrolyzed to esculetin, which reacts with Fe3+ and forms a dark brown to black precipitate
Expected results of Esculin Hydrolysis Test
Positive: BLACKENED MEDIUM
Negative: No blackening
Quality control/s of Esculin Hydrolysis Test
Positive: Enterococcus faecalis
Negative: Escherichia coli
Media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium
Fermentation media
Expected results of Fermentation test using Andrade’s formula and Bromcresol purple
Andrade’s Formula
Positive: Indicator change to PINK
Negative: Growth, but no change in color
Bromcresol Purple
Positive: Indicator change to YELLOW
Negative: Growth, but no change in color
Quality control/s of Fermentation Test using Andrade’s formula and Bromcresol purple
Andrade’s Formula
Positive, with gas: Escherichia coli
Positive, no gas: Shigella flexneri
Bromcresol Purple
Positive, with gas: Escherichia coli
Negative, no gas: Moraxella osloensis
This technique is used to visualize the presence and arrangement of flagella for the presumptive identification of motile bacterial species
Flagella stain
Principle of Flagella stain test
A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria
Quality control/s of Flagella stain test
Peritrichous: Escherichia coli
Polar: Pseudomonas aeruginosa
Negative: Klebsiella pneumonia
Presumptive test for the identification of various organisms, including Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli
Gelatin Hydrolysis Test
Principle of Gelatin Hydrolysis Test
This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin
Expected results of Gelatin Hydrolysis Test
Positive: Partial or total liquefaction at 4°C within 14 days
Negative: Complete solidification
Quality control/s of Gelatin Hydrolysis Test
Positive: Bacillus subtilis
Negative: Escherichia coli
This test is used to differentiate a pyocyanogenic pseudomonads from other Pseudomonas sp.
Growth at 42 C
Principle of Growth at 42 C
The test is used to determine the ability of an organism to grow at 42°C. Several Pseudomonas species have been isolated in the clinical laboratory that are capable of growth at elevated temperatures
Expected results of Growth at 42 C
Positive: Good growth at both 35°and 42°C
Negative: No growth at 42°C but good growth at 35°C
Quality control/s of Growth at 42 C
Positive: Pseudomonas aeruginosa
Negative: Pseudomonas fluorescens
Production of the enzyme hippuricase is used for the presumptive identification of a
variety of microorganisms
Hippurate Hydrolysis
Principle of Hippurate Hydrolysis
The end products of hydrolysis of hippuric acid by hippuricase include glycine and benzoic acid. Glycine is deaminated by the oxidizing agent ninhydrin, which is reduced during the process. The end products of the ninhydrin oxidation react to form a purple-colored product. The test medium must contain only hippurate because ninhydrin might react with any free amino acids present in growth media or other broths
Expected results of Hippurate Hydrolysis Test
Positive: Deep purple color
Negative: Colorless or slightly yellow pink color
Quality control/s of Hippurate Hydrolysis Test
Positive: Streptococcus agalactiae
Negative: Streptococcus pyogenes
This test is used to identify organisms that produce the enzyme tryptophanase
Indole Production Test
This test is used to help distinguish Escherichia coli from Enterobacter and Klebsiella
Indole Production Test
Principle of Indole Production Test
The indole test detects tryptophanase production and determines the ability of a
microorganism to produce indole from the degradation of the amino acid tryptophan. The test is used to determine an organism’s ability to hydrolyze tryptophan to form the
compound indole
Expected results of Indole Production Test
Positive: PINK- TO WINE-COLORED RING
Negative: No color change
Quality control/s of Indole Production Test
A. KOVAC’S METHOD
- Positive: Escherichia coli
- Negative: Klebsiella pneumoniae
B. EHRLICH’S METHOD
- Positive: Haemophilus influenzae
- Negative: Haemophilus parainfluenzae C. EHRLICH’S METHOD (ANAEROBIC)
- Positive: Porphyromonas asaccharolytica
- Negative: Bacteroides fragilis
Test used for the presumptive identification of catalase-negative gram-positive cocci
Leucine Aminopeptidase (LAP) Test
Principle of Leucine Aminopeptidase (LAP) Test
The LAP disk is a rapid test for the detection of the enzyme leucine aminopeptidase. Leucinebeta- naphthylamide– impregnated disks serve as a substrate for the detection of leucine aminopeptidase. After hydrolysis of the substrate by the enzyme, the resulting beta- naphthylamine produces a red color upon addition of cinnamaldehyde reagent
Expected results of Leucine Aminopeptidase (LAP) Test
Positive: Development of a red color within 1 minute
Negative: No color change or development of a slight yellow color
Quality control/ of Leucine Aminopeptidase (LAP) Test
Positive: Enterococcus faecalis
Negative: Aerococcus viridans
This test differentiates microorganisms based on various metabolic reactions in litmus
milk
Litmus Milk Medium
Principle of Litmus Milk Medium
Fermentation of lactose is demonstrated when the litmus turns pink as a result of acid production. If sufficient acid is produced, casein in the milk is coagulated, solidifying the milk. With some organisms, the curd shrinks, and whey is formed at the surface. Some bacteria hydrolyze casein, causing the milk to become straw colored and resemble turbid serum. Additionally, some organisms reduce litmus, in which case the medium becomes colorless in the bottom of the tube
Quality control/s of Litmus Milk Medium
Fermentation: Clostridium perfringens
Acid: Lactobacillus acidophilus
Peptonization: Pseudomonas aeruginosa
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)
Principle of Lysine Iron Agar (LIA)
When glucose is fermented, the butt of the medium becomes acidic (yellow). If the organism produces lysine decarboxylase, cadaverine is formed. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to the alkaline state (purple). If the decarboxylase is not produced, the butt remains acidic (yellow). If oxidative deamination of lysine occurs, a compound is formed that, in the presence of ferric ammonium citrate and a coenzyme, flavin mononucleotide, forms a burgundy color on the slant. If deamination does not occur, the LIA slant remains purple.
A combination test use to differentiate members
of the Enterobacteriaceae family
Methyl Red / Voges-Proskauer (MRVP) Test
Principle of Methyl Red / Voges-Proskauer (MRVP) Test
This test is used to determine the ability of an organism to produce and maintain stable acid end products from glucose fermentation, to overcome the buffering capacity of the system, and to determine the ability of some organisms to produce neutral end products (e.g., 2,3-butanediol or acetoin) from glucose fermentation
Expected results of Methyl Red / Voges-Proskauer (MRVP) Test
Positive: Red color, indicative of acetoin production
Negative: Yellow color
Quality control/s of Methyl Red / Voges-Proskauer (MRVP) Test
MR positive/VP negative: Escherichia coli
MR negative:/VP positive: Enterobacter aerogenes
This test is used to differentiate gram-positive, catalase-positive cocci (micrococci from staphylococci)
Microdase Test (Modified Oxidase)
Principle of Microdase Test
The microdase test is a rapid method to differentiate Staphylococcus from Micrococcus spp. by detection of the enzyme oxidase. In the presence of atmospheric oxygen, the oxidase enzyme reacts with the oxidase reagent and cytochrome C to form the colored compound, indophenol
Expected results of Microdase Test
Positive: Development of BLUE TO PURPLE-BLUE COLOR
Negative: NO COLOR CHANGE
Quality control/s of Microdase Test
Positive: Micrococcus luteus
Negative: Staphylococcus aureus
These tests are used to determine whether an enteric organism is motile
Motility Testing
Principle of Motility Testing
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
Expected results of Motility Testing
Positive: Spread out from the site of inoculation
Negative: Remain at the site of inoculation
Quality control/s of Motility Testing
Positive: Escherichia coli
Negative: Staphylococcus aureus
This test is used to determine whether an organism forms gas during glucose fermentation.
De Man, Rogosa and Sharpe (MRS) Broth
Principle of MRS Broth
The MRS broth contains sources of carbon, nitrogen, and vitamins to support the growth of lactobacilli and other organisms. It is a selective medium that uses sodium acetate and ammonium citrate to prevent overgrowth by contaminating organisms. Growth is considered a positive result
Expected results of MRS Broth
Positive: Leuconostoc sp.: Growth, gas production indicated by a bubble in the Durham tube
Positive: Lactobacillus spp.: Growth, no gas production
Negative: No growth
Quality control/s of MRS Broth
Positive: Lactobacillus lactis
Negative: Escherichia coli
This test is used to presumptively identify various genera of Enterobacteriaceae and
verotoxin-producing Escherichia coli
4-Methylumbelliferyl-β-D-Glucuronide (MUG) Test
Principle of MUG Test
E. coli and other Enterobacteriaceae produce the enzyme β-d-glucuronidase, which hydrolyzes β-d-glucopyranosid-uronic derivatives to aglycons and d-glucuronic acid. The substrate 4-methylumbelliferyl-β-d-glucuronide is impregnated into the disk and is hydrolyzed by the enzyme to yield the 4-methylumbelliferyl moiety, which fluoresces blue under long wavelength ultraviolet light. However, verotoxin producing strains of E. coli do not produce MUG, and a negative test result may indicate the presence of a clinically important strain
Expected results of MUG Test
Positive: Electric blue fluorescence
Negative: Lack of fluorescence
Quality control/s of MUG Test
Positive: Escherichia coli
Negative: Klebsiella pneumoniae
This test is used to determine the ability of an organism to reduce nitrate to nitrite
Nitrate Reduction Test
Expected results of Nitrate Reduction Test
Positive: RED
Negative: No color change
Quality control/s of Nitrate Reduction Test
Positive: NO3+, no gas: Escherichia coli
Positive: NO3+, gas: Pseudomonas aeruginosa
Negative: Acinetobacter baumannii
This test is used to determine whether an organism can reduce nitrites to gaseous nitrogen or to other compounds containing nitrogen
Nitrite Reduction Test
Principle of Nitrite Reduction Test
Microorganisms capable of reducing nitrite to nitrogen do not turn color and do produce gas in the nitrate reduction test
Expected results of Nitrite Reduction Test
Positive: No color change to red 2 minutes; gas production in Durham tube
Negative: Broth becomes red; no gas production is observed
Quality control/s of Nitrite Reduction Test
Positive: Proteus mirabilis
Negative: Acinetobacter baumannii
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
O-NITROPHENYL-Β-D-GALACTOPYRANOSIDE (ONPG) TEST
This test distinguishes late lactose fermenters from non–lactose fermenters of Enterobacteriaceae
O-NITROPHENYL-Β-D-GALACTOPYRANOSIDE (ONPG) TEST
Principle of O-NITROPHENYL-Β-D-GALACTOPYRANOSIDE (ONPG) TEST
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
Expected results of O-NITROPHENYL-Β-D-GALACTOPYRANOSIDE (ONPG) TEST
Positive: YELLOW (presence of β-galactosidase)
Negative: Colorless
Quality control/s of O-NITROPHENYL-Β-D-GALACTOPYRANOSIDE (ONPG) TEST
Positive: Shigella sonnei
Negative: Salmonella typhimurium
The test used to differentiate Streptococcus pneumoniae from other alpha-hemolytic streptococci
Optochin (P disk) Susceptibility Test
Principle of Optochin (P disk) Susceptibility Test
Optochin is an antibiotic that interferes with the ATPase and production of adenosine triphosphate (ATP) in microorganisms. The Optochin impregnated disk (TaxoP) is placed on a lawn of organism on a sheep blood agar plate, allowing the antibiotic to diffuse into the medium. The antibiotic inhibits the growth of a susceptible organism, creating a clearing, or zone of inhibition, around the disk. A zone of 14 to 16 mm is considered susceptible and presumptive identification for Streptococcus pneumoniae
Expected results of Optochin (P disk) Susceptibility Test
Positive: ZOI ≥ 14 mm in diameter, with 6-mm disk
Negative: No zone of inhibition
Quality control/s of Optochin (P disk) Susceptibility Test
Positive: Streptococcus pneumoniae
Negative: Streptococcus pyogenes
This test determines the presence of cytochrome oxidase activity in microorganisms for the identification of oxidase negative Enterobacteriaceae, differentiating them from other gram-negative bacilli
Oxidase Test (Kovac’s Method)
This test is used to differentiate oxidase positive microorganisms such as Aeromonas spp., Pseudomonas spp., and Haemophilus spp. from the oxidase negative Enterobacteriaceae
Oxidase Test (Kovac’s Method)
Principle of Oxidase Test (Kovac’s Method)
To determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate tetramethyl-p-phenylenediamine dihydrochloride to indophenol, a dark purple colored end product. A positive test (presence of oxidase) is indicated by the development of a dark purple color. No color development indicates a negative test and the absence of the enzyme
Expected results of Oxidase Test (Kovac’s Method)
Positive: Development of a dark purple color within 10 seconds
Negative: Absence of color
Quality control/s of Oxidase Test (Kovac’s Method)
Positive: Pseudomonas aeruginosa
Negative: Escherichia coli
This test is used to differentiate microorganisms based on the ability to oxidize or ferment specific carbohydrates
Oxidation/Fermentation (of) Medium (CDC Method)
Expected results of Oxidation/Fermentation (of) Medium (CDC Method)
Positive: Acid production (A) indicated by the color indicator changing to yellow
Weak-positive (Aw): Weak acid formation
Negative: Red or alkaline (K) color in the deep
No change (NC) or neutral (N)
Quality control/s of Oxidation/Fermentation (of) Medium (CDC Method)
Fermenter (Glucose): Escherichia coli
Oxidizer (Glucose): Pseudomonas aeruginosa
This test is used to determine the ability of an organism to oxidatively deaminate phenylalanine to phenylpyruvic acid
Phenylalanine Deaminase Agar
Test used to differentiate Morganella, Proteus, and Providencia from other members of the Enterobacteriaceae family
Phenylalanine Deaminase Agar
Principle of Phenylalanine Deaminase Agar
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
Expected results of Phenylalanine Deaminase Agar
Positive: GREEN COLOR on slant
Negative: Slant remains original color
Quality control/s of Phenylalanine Deaminase Agar
Positive: Proteus mirabilis
Negative: Escherichia coli
This test is used for the presumptive identification of group A streptococci (Streptococcus pyogenes) and enterococci by the presence of the enzyme L-pyrrolidonyl arylamidase
L-Pyrrolidonyl Arylamidase (PYR) Test
Principle of L-Pyrrolidonyl Arylamidase (PYR) Test
The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl-β-naphthylamide substrate to produce a β-naphthylamine. The β-naphthylamine can be detected in the presence of N,N-methylaminocinnamaldehyde reagent by the production of a bright red precipitate
Expected results of L-Pyrrolidonyl Arylamidase (PYR) Test
Positive: Bright red color within 5 minutes
Negative: No color change or an orange color
Quality control/s of L-Pyrrolidonyl Arylamidase (PYR) Test
Positive: Enterococcus faecalis, Streptococcus pyogenes
Negative: Streptococcus agalactiae
This test is used to determine the ability of an organism to utilize pyruvate
Pyruvate Broth
This test aids in the differentiation between Enterococcus faecalis (positive) and Enterococcus faecium (negative)
Pyruvate Broth
Principle of Pyruvate Broth
Pyruvate broth is a carbohydrate-free, nutrient limited medium. Pyruvic acid is added to the broth to determine whether the microorganism is able to use pyruvate, resulting in the formation of metabolic acids. Bromthymol blue indicator changes from blue to yellow in the presence of acid as a result of the decrease in pH
Expected results of Pyruvate Broth
Positive: Indicator changes from green to yellow
Negative: No color change
Quality controls of Pyruvate Broth
Positive: Enterococcus faecalis
Negative: Streptococcus bovis
This test is used to determine the ability of an organism to grow in high concentrations of salt
Salt Tolerance Test
This test is used to differentiate enterococci (positive) from nonenterococci (negative)
Salt Tolerance Test
Principle of Salt Tolerance Test
The salt tolerance test is a selective and differential medium. Enterococci are resistant to high salt concentration. A heart infusion broth containing 6.5% NaCl is used as the test medium. This broth also contains a small amount of glucose and bromcresol purple as the indicator for acid production
Expected results of Salt Tolerance Test
Positive: Visible turbidity in the broth, with or without a color change from purple to yellow
Negative: No turbidity and no color change
Quality control/s of Salt Tolerance Test
Positive: Enterococcus faecalis
Negative: Streptococcus bovis
This test is used to determine the presence of the enzyme tryptophanase
Spot Indole Test
Principle of Spot Indole Test
Tryptophanase breaks down tryptophan to release indole, which is detected by its ability to combine with certain aldehydes to form a colored compound. For indole-positive bacteria, the blue-green compound formed by the reaction of indole with cinnamaldehyde is easily visualized. The absence of enzyme results in no color production (indole negative
Expected results of Spot Indole Test
Positive: Development of a blue color within 20 seconds
Negative: No color development or slightly pink color
Quality control/s of Spot Indole Test
Positive: Escherichia coli
Negative: Klebsiella pneumoniae
This test 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 (TSI) Agar
Composition of TSI Agar
Triple sugar iron agar contains three sugars (lactose, sucrose, and glucose), ferrous sulfate, and a pH indicator.
COMPOSITION: 10 parts Lactose, 10 parts sucrose, 1 part glucose and peptone
Phenol Red: pH indicator
Ferrous Ammonium Sulfate: H2S Indicator
CO2 and hydrogen gas (H2): presence of bubbles or cracks or by separation of the agar
Reactions included in Triple Sugar Iron Test
Acid reaction (A): yellow color
Alkaline reaction (K): red color
Hydrogen sulfide production (H2S): black color or precipitate
Gas production (G): bubbles, cracks, or media displacement
Expected results of Triple Sugar Iron Test
Alkaline slant/ Alkaline butt (K/K): glucose, lactose, and sucrose nonutilizer
Alkaline slant/Acid butt (K/A): glucose fermentation only
Acid slant/Acid butt (A/A): glucose, sucrose, and/or lactose fermenter
Black Precipitate: production of ferrous sulfide and H2S gas (H2S+)
Bubbles or cracks: gas production
Quality control/s of Triple Sugar Iron Test
A/A gas production: Escherichia coli
K/A, +/− gas production, H2S+: Salmonella typhimurium
K/K: Pseudomonas aeruginosa
K/A, H2S+: Proteus mirabilis
K/A: Shigella flexneri
This test is used to determine an organism’s ability to produce the enzyme urease, which hydrolyzes urea
Urea Test (Christensen’s Method)
Proteus sp. may be presumptively identified by the ability to rapidly hydrolyze urea. True or False?
True
Principle of Urea Test (Christensen’s Method)
Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours. Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production
Expected results of Urea Test (Christensen’s Method)
Positive: Change in color of slant from light orange to magenta
Negative: No color change
Quality control/s of Urea Test (Christensen’s Method)
Positive: Proteus vulgaris
Weak positive: Klebsiella pneumonia
Negative: Escherichia coli
Test used to differentiate Haemophilus species
X and V Factor Test
Principle of X and V Factor Test
Members of the genus Haemophilus require accessory growth factors in vitro. Some Haemophilus spp. require X factor (hemin) alone, V factor (nicotinamide adenine dinucleotide [NAD]) alone, or a combination of the two
The organisms will grow only around the disk that provides the appropriate factor for growth of the organism
Expected results of X and V Factor Test
Positive:
Growth around the XV disk: requirement for both factors
Growth around the V disk, no growth around the X disk, and light growth around the XV disk shows a V factor requirement
Negative: Growth over the entire surface of the agar indicates no requirement for either X or V factor
Quality control/s of X and V Factor Test
Haemophilus influenza: halo of growth around the XV disk, no growth on the rest of the agar surface
Haemophilus parainfluenzae: halo of growth around the XV and V disks
Haemophilus ducreyi: halo of growth around the XV and X disks
