Clinical Microbiology Procedures Handbook

Section 3: Aerobic Bacteriology

3.17 Biochemical Tests for the Identification of Aerobic Bacteria

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Last Updated:2023

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3.17.1 Acetamide Utilization Test (Acetamide Agar)

Acetamide agar is used to test an organism’s ability to utilize acetamide by deamination. The medium contains acetamide as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth is indicative of a positive test for ...

3.17.2 Acetate Utilization Test (Acetate Differential Agar)

Acetate agar is used to test an organism’s ability to utilize acetate. The medium contains sodium acetate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth is indicative of a positive test for acetate ...

3.17.3 ALA (δ‐Aminolevulinic Acid) Test for Porphyrin Synthesis—Tube or Disk Test

The δ‐aminolevulinic acid (ALA) test is a rapid test used to determine the growth requirement for hemin (X factor) in the identification of Haemophilus spp. (1). Organisms that do not require hemin can produce porphobilinogen…

3.17.4 Antimicrobial Disk Tests for Identification

Antimicrobial disks dropped onto agar can help identify bacteria based on their susceptibility or resistance to certain antibiotics. Historically, most of the disks described here were used along with penicillin disks to help distinguish among…

3.17.5 Bile‐Esculin and Esculin Tests

The basis of the esculin test is the hydrolysis of esculin (a glucoside) into glucose and esculetin by a microorganism that has a constitutive (noninducible) β‐glucosidase or esculinase enzyme. When esculetin is produced by the hydrolysis of esculin…

3.17.6 Bile Solubility Test

The bile solubility test is used to differentiate Streptococcus pneumoniae from other alpha‐hemolytic Streptococcus spp. It may be performed in a test tube or by direct application of the reagent directly onto…

3.17.7 Butyrate Esterase Test

The butyrate test is a rapid test for the detection of the enzyme butyrate esterase. When used in conjunction with characteristic morphology on BAP, typical Gram stain, and a positive oxidase test…

3.17.8 CAMP Factor Tests (Standard/Rapid, Reverse, and Inhibition)

The CAMP test is used to identify microorganisms that have the ability to produce a thermostable, extracellular diffusible protein, CAMP factor, named for the initials of the authors of the article that first described the phenomenon…

3.17.9 Carbohydrate Utilization Tests

Carbohydrate utilization tests determine the ability of a bacterium to break down (by fermentation or oxidation) a specific carbohydrate incorporated into a basal medium and to produce acid or acid with visible gas. Patterns of carbohydrate utilization ...

3.17.10 Catalase Test

Bacteria that synthesize the enzyme catalase hydrolyze hydrogen peroxide into water and gaseous oxygen, which results in the liberation of gas bubbles (Figure 3.17.10–1). The catalase test…

3.17.11 Cetrimide Test

Cetrimide is a quaternary ammonium, cationic detergent that is toxic to most bacteria except Pseudomonas aeruginosa and a few others, including other pigment‐producing Pseudomonas

3.17.12 Citrate Utilization Test (Simmons)

Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen…

3.17.13 Coagulase Test—Protein A/Clumping Factor Agglutination Method

Staphylococcus aureus is separated from other species of staphylococci by the presence of coagulase, which is demonstrated in the 4‐ to 24‐h tube test to detect free coagulase. Clumping factor, termed bound coagulase, can be detected…

3.17.14 Coagulase Test—Rabbit Plasma Method

Staphylococcus aureus, the most pathogenic of the staphylococci, is separated from other species by the presence of coagulase. Coagulase is a thermostable…

3.17.15 Decarboxylase‐Dihydrolase Tests

The decarboxylase‐dihydrolase test is based on the principle that certain organisms remove a carboxyl or hydroxyl group (hydrolyze) from an amino acid to form an amine, resulting in an alkaline pH. The test medium contains…

3.17.16 DNase Test—Rapid Thermonuclease Test

DNases are enzymes that hydrolyze DNA and release free nucleotides and phosphate. The DNases produced by bacteria are extracellular endonucleases that cleave DNA, yielding a high concentration of oligonucleotides.…

3.17.17 Fluorescent‐Pigment Agars for Pseudomonas Identification

Characteristic pigments produced by Pseudomonas aeruginosa, including blue‐green pyocyanin and yellow, fluorescent pyoverdine, can be enhanced by growing isolates on media containing different formulations of peptones…

3.17.18 Gelatin Liquefaction

Gelatinase production is useful as a taxonomic aid in classification and identification of both Gram‐negative bacilli and Gram‐positive bacilli (1, 2). Gelatinases…

3.17.19 Gram Reaction Enzymatic Test

In the Gram stain, some Gram‐positive bacilli appear Gram variable or Gram negative, especially members of the genera Bacillus, Erysipelothrix, Lactobacillus, and Listeria or related genera, which may…

3.17.20 Hippurate Hydrolysis Rapid Test

The hippurate test is used in the identification of Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis, and Streptococcus agalactiae

3.17.21 Hydrogen Sulfide Production

Certain organisms are capable of enzymatically liberating sulfur from inorganic sulfur, as hydrogen sulfide (H2S) compounds, or from…

3.17.22 Indole Test

The ability of an organism to split indole from the amino acid tryptophan is due to the presence of tryptophanase. Indole, if present, combines with the aldehyde in the reagent to produce a pink to red‐violet quinoidal compound…

3.17.23 Indoxyl Acetate Disk Test

The presence of bacterial enzyme production of indoxyl can be confirmed in vitro by the bacterial hydrolysis of indoxyl acetate. Indoxyl then combines with oxygen to spontaneously form a characteristic indigo color…

3.17.24 Kligler’s Iron Agar Test and Triple Sugar Iron Agar Test

Kligler’s iron agar (KIA) and triple sugar iron agar (TSI) are used to detect the production of acid from carbohydrates. Both media contain glucose and lactose. KIA contains casein and meat peptones, phenol…

3.17.25 Leucine Aminopeptidase Test

Detection of the enzyme leucine aminopeptidase (LAP) is one of the tests in the definitive identification of catalase‐negative, Gram‐positive cocci. Specifically, it differentiates Leuconostoc and…

3.17.26 Lecithinase and Lipase Detection

Lecithinases or phospholipases are enzymes released by bacteria that destroy animal tissues. Lecithin is a normal component of egg yolks. In egg yolk agar (EYA), the lipoprotein component, lecithovitellin, can be split by lecithinase…

3.17.27 Lipophilism Test for Corynebacterium

Some species of Corynebacterium grow better in the presence of added lipid. Such strains are called lipophilic or, historically, rabbit serum stimulated. They may also require lipid for growth…

3.17.28 Malonate Test

The medium for the malonate test contains sodium malonate (). Malonate is an enzyme inhibitor and inhibits utilization of succinic acid by bacteria, shutting down the Krebs and glyoxylic cycles. Growth is indicative of…

3.17.29 Methyl Glucopyranoside (MGP) Test

MGP broth is used to differentiate enterococci based on the ability to acidify the carbohydrate methyl‐α‐d‐glucopyranoside (MGP). Vancomycin‐resistant…

3.17.30 Motility Tests

The motility test is used to detect the presence of flagella on bacteria, allowing them to travel in and out of the microscopic field or beyond their initial inoculation in agar (1, 2). For the wet preparation…

3.17.31 MRS Broth

Gas production by intrinsically vancomycin‐resistant Gram‐positive coccobacilli is tested in glucose‐containing medium called lactobacillus MRS broth, named for the authors of the publication…

3.17.32 Methyl Red–Voges‐Proskauer (MR‐VP) Tests

The methyl red (MR) test is used to determine if an organism can produce stable acid end products from glucose fermentation (1). MR indicator (red color below pH 4.4; yellow color at pH 5.8) is used to determine the…

3.17.33 MUG (4‐Methylumbelliferyl‐β‐D‐Glucuronide) Test

Escherichia coli and rare other Enterobacterales (Salmonella, Yersinia, and Shigella) produce the enzyme β‐glucuronidase. This enzyme hydrolyzes the substrate 4‐methylumbelliferyl‐β‐D‐glucuronide…

3.17.34 Nitrate/Nitrite Reduction Test

Nitrate broth and nitrite broth are used to determine if an organism can reduce nitrate (NO3) to nitrites (NO2) and reduce nitrites to nitrogen gas (N2). The reduction of nitrate…

3.17.35 O/129 Disk Susceptibility Testing for Vibrio and Aeromonas spp.

Disks containing 150 μg of O/129, the vibriostatic agent 2,4‐diamino‐6,7‐diiso‐propylpteridine phosphate, can be dropped onto a lawn of plated organism to help differentiate Vibrio spp. from Aeromonas spp…

3.17.36 ONPG (o‐Nitrophenyl‐β‐D‐Galactopyranoside) Test

Late lactose fermenters are very difficult to distinguish from lactose nonfermenters because both appear as colorless colonies on MacConkey agar. The o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG)…

3.17.37 Optochin Susceptibility Test

Streptococcus pneumoniae is found commonly in the human respiratory tract, as are other streptococci, and has a hemolytic pattern indistinguishable from that of other alpha‐hemolytic streptococci and…

3.17.38 Oxidase Test

Certain bacteria possess either cytochrome oxidase or indophenol oxidase, which catalyzes the transport of electrons (hydrogen) from donor compounds (NADH) to electron receptors (usually oxygen) with the formation of water…

3.17.39 Phenylalanine Deaminase Test

The phenylalanine deaminase (PDA) test is used to determine the ability of a microorganism to oxidatively deaminate the amino acid l‐phenylalanine to phenylpyruvic acid, a keto acid. The deamination of phenylalanine…

3.17.40 PYR (L‐Pyrrolidonyl‐β‐Naphthylamide) Test

L‐Pyrrolidonyl‐β‐naphthylamide (PYR) serves as a substrate for the detection of pyrrolidonyl peptidase. Following hydrolysis of the substrate by the peptidase, the resulting β‐naphthylamide…

3.17.41 Quellung Reaction for Streptococcus pneumoniae (Neufeld Test)

In Streptococcus pneumoniae, antigen‐antibody reactions between antiserum and the capsule result in distinctive capsular swelling known as the Quellung reaction (, ). This swelling…

3.17.42 6.5% Salt and Temperature Tolerance Test

As part of the differentiation of catalase‐negative, Gram‐positive cocci, growth in 6.5% salt and growth at low (10°C) and high (42 to 45°C) temperatures are used (1). Salt tolerance broth, containing heart infusion (HI), glucose, and…

3.17.43 Satellite Test

Haemophilus influenzae requires two factors, hemin and NAD, for growth. Hemin is referred to as the X factor and NAD as the V factor. BAP supplies hemin but not NAD. Although H. influenzae

3.17.44 SPS (Sodium Polyanethol Sulfonate) Disk Test

Organism susceptibility to sodium polyanethol sulfonate (SPS), a common anticoagulant, can be determined using disks containing SPS and observing cultures of a standardized inoculum for zones of inhibition. Gardnerella vaginalis

3.17.45 Starch Hydrolysis Test

Some bacteria excrete an amylase, endoamylase, which hydrolyzes amylose to maltose and glucose. Detection of this amylase via starch hydrolysis can be done by culturing organisms being tested on starch‐containing agar…

3.17.46 Urease Test

Urea medium, whether a broth or agar, contains urea and the pH indicator phenol red. Many organisms, especially those that infect the urinary tract, have a urease enzyme, which can split urea in the presence of water to release two molecules of ammonia ...

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Section Editors: Amanda T. Harrington and Melanie L. Yarbrough

Editors in Chief: Amy L. Leber and Carey-Ann D. Burnham

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