Determining the nutritional and metaboliccapabilities of a bacterial isolate is the most common approach used for deter-mining the genus and species of an organism.

The methods available use a combination of teststo establish the enzymatic capabilities of a given bacterial isolate as well as the isolates ability to grow or survive the presence of certain inhibitors(e.g. salts, surfactants, toxins and antibiotics)

A.Establishing Enzymatic Capabilities

Enzyme based tests are designed to measure the presence of a single enzyme as well as a complete metabolic pathway.

SINGLE ENZYME TESTS

Catalase testCoagulase testPyrase testHippurate hydrolysis testOxidase testIndole testDnase testONPG(B-galactosidase)testUrease test

ASSAYS FOR METABOLIC PATHWAYS

Carbohydrate oxidation and fermentation

oxidation fermentation testscarbohydrate fermentation in TSIA methyl red testVoges Proskauer test

Amino acid degradation

decarboxylase-dihydrolase reactions deamination reactions decarboxylation and deamination reactions in LIA

Single substrate utilization

citrate utilization test acetate utilization test acetamide utilization test

B. Establishing Inhibitor Profiles

bacitracin susceptibility test bacitracin and sulfamethoxazole-trimethoprim susceptibility test novobiocin susceptibility test vancomycin susceptibility test antibiotic disks for presumptive identification of anaerobes

C. Other more specific tests growth in various NaCl concentrations - Enterococci and Vibrio species susceptibility to optochin and solubility in bile – Streptococcus pneumoniae ability to hydrolyze esculin in the presence of bile – Enterococcus spp.and Group D streptococcus CAMP – Streptococcus agalactiae

PURPOSE

To differentiate members of the family Microco- coccaceae (including Staphylococcus) which are catalase positive from Streptococcus species which are catalase negative.

To differentiate Listeria monocytogenes and corynebacteria(catalase positive) from other gram positive, non-sporeforming bacilli.

PRINCIPLE

The enzyme catalase catalyzes the release of water and oxygen from hydrogen peroxide. catalase 2 H202 -------------- 2 H20 + O2 bubbles or effervescence

INTERPRETATION

Positive – rapid and sustained appearance of bubbles or effervescence

Negative – lack of bubble formation 30 seconds later

A B

Catalase test

A.Positive – Staphylococcus aureus.B.Negative – Streptococcus pyogenes

PURPOSE

To determine the ability of the organism to produce coagulase which clots plasma.

To distinguish the pathogenic coagulase positive staphylococcus from the nonpathogenic coagulase negative staphylococcus.

Coagulase is an enzyme that converts soluble fibrinogen into soluble fibrin.

Two forms of coagulase

bound coagulase (clumping factor) – detected in the coagulase slide test

can directly convert fibrinogen to insoluble fibrin and causes the staphylococci to clump together

PRINCIPLE

free coagulase – detected in the coagulase tube test reacts with a globulin plasma factor(coagulase reacting factor-CRF) to form a thrombinlike factor, staphylothrombin--- catalyzes the conversion of fibrinogen to insoluble fibrin

INTERPRETATION

Slide Coagulase test

Positive – white fibrin clots in plasma Negative – smooth suspension

Tube Coagulase test

Positive – formation of fibrin clot Negative – no clot is formed

Slide coagulase test

A B

A. Negative – Staphylococcus epidermidisB. Positive – Staphylococcus aureus

Tube coagulase test

A B

A. Positive – Staphylococcus aureusB. Negative – Staphylococcus epidermidis

PURPOSE

To determine the ability of the organism to hydrolyze the substrate L-pyrrolidonyl-beta-napthylamide.

To differentiate the Enterococcus species from the nonenterococcus species.

Useful for presumptive identification of Group A beta hemolytic streptococcus(Streptococcus pyogenes)

PRINCIPLE

L-pyrrolidonyl-beta-napthylamide ------------hydrolysis

pyrrolidonylarylamidase

Beta napthylamide + p-dimethylaminocinnamaldehyde

Pink to cherry red color

(color developer)

INTERPRETATION

Positive – pink to cherry red color(after the addition of color developer)

Negative – no color change in inoculated portion of the disk

PYRase(PYR) test

A B

A.Positive – EnterococcusB.Negative – nonenterococcus

PURPOSE

To determine the ability of the organism to produce hippuricase which hydrolyzes the substrate hippurate.

Useful in the identification of Streptococcus agalactiae, Camphylobacter jejuni and Listeria monocytogenes.

PRINCIPLE

The end products of hydrolysis of the substrate hippurate by a constitutive enzyme hippuricase include glycine and benzoic acid. Glycine is deaminated by the oxidizing agent, ninhydrin, which is reduced during the process. The end products of ninhydrin oxidation react to form a purple colored product.

INTERPRETATION

Positive – deep purple color

Negative – slightly yellow pink or colorless

Hippurate hydrolysis test

A B

A. Positive – Streptococcus agalactiaeB. Negative- Enterococcus

PURPOSE

To screen colonies suspected of being one of the Enterobacteriaceae(all negative).

To identify colonies suspected of belonging to other genera such as Aeromonas, Pseudomonas, Neisseria, Camphylobacter and Pasteurella.

PRINCIPLE

The cytochrome oxidase test uses certain reagent dyes, such as p-phenylenediamine dihydrochloride that substitute for oxygen as artificial electron acceptors It is colorless in the reduced state. In the presence of cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized forming indophenol blue.

Tetramethyl-p-phenylene ----------- purple color diamine hydrochloride

Dimethyl compound(1%) ----------- black color

P-phenylenediamine -----------------dihydrochloride cytochrome oxidase + atmospheric air

Indophenol blueoxidation

INTERPRETATION

Positive – blue/ dark purple/black color

Negative – no color development

A B

Oxidase test

A. Positive – Pseudomonas aeruginosaB. Negative – Escherichia coli

PURPOSE

To distinguish Enterobacteriaceae based on the ability to produce indole from tryptophan.

To identify lactose fermenting members of Enterobacteriaceae, Escherichia coli(indol positive) from Klebsiella pneumoniae(indol negative).

To speciate Proteus: Proteus mirabilis – indole negative Proteus vulgaris – indole positive

PRINCIPLE

Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid and ammonia. A red complex is formed when indole reacts with the aldehyde group of p-dimethylaminobenzal- dehyde, the active chemical in Kovac’s and Ehrlich’s reagent.

Tryptophan ------------------indol + pyruvic acid + NH3 tryptophanase

Indol + p-dimethylaminobenzaldehyde -----red complex

Reagents used to detect indole

Ehrlich’s – to detect indol in anaerobic and nonfermentative bacteria Kovac’s – to identify members of Enterobacteriaceae

Media used with tryptophan

sulfide indol motility (SIM) motility indol ornithine(MIO) indole nitrate rapid spot tests – filter paper strips impregnated with p-diaminocinnamaldehyde reagent – useful in screening bacteria that are prompt indole producers

INTERPRETATION

Positive – red ring at the interface of reagent and broth (or reagent and xylene or chloroform) Negative – no color development Variable results – orange color, indicates products of skatole, a methylated intermediate that maybe a precursor to indole production

Rapid spot test paradimethylaminocinnamaldehyde – blue green paradimethylaminobenzaldehyde – bright pink color

A B

Indole test

A.Positive – Escherichia coliB.Negative – Klebsiella pneumoniae

Indole spot test

A B

A. Negative - Klebsiella pneumoniaeB. Positive - Escherichia coli

PURPOSE:

To detect Dnase activity in species of aerobic bacteria.

To differentiate nonfermenting gram-negative bacteria as well as Staphylococcus aureus and Serratia marcescens.

Metachromatic dyes

Toluidine blue is complexed with DNA. Hydrolysis of DNA by the inoculated microorganism causes changes of structure of the dye to yield a pink color. Methyl green is also complexed with DNA. If the organism growing on the medium hydrolyzes DNA, the green color fades and the colony is surrounded by a colorless zone.

PRINCIPLE

INTERPRETATION

Positive rose pink clear zoneNegative no change no clearing

Toluidine blue Methyl green

Deoxyribonuclease test

Positive – Staphylococcus aureus Serratia marcescens Negative – Staphylococcus epidermidis Enterobacter cloacae

Deoxyribonuclase test

A. Positive – Staphylococcus aureusB. Positive – Serratia marcescensC. Negative –Staphylococcus epidermidis

A

BC

PURPOSE

To determine the presence of late or slow fermenting strains. To detect the late lactose fermenting strains of Escherichia coli To distinguish some Citrobacter species and arizonae subspecies(ONPG positive) from similar Salmonella subspecies(ONPG negative) To speciate Shigella, since Shigella sonnei is the only ONPG-positive Shigella species.

PRINCIPLE

Two enzymes required for lactose fermentation

lactose permease – actively transfers lactose into the bacterial cell beta galactosidase- degrades lactose into glucose and galactose

Lactose fermenters – possess both enzymesSlow or late lactose fermenters – no permease ; only beta galactosidaseNon lactose fermenters – lack both enzymes

ONPG(o-nitrophenyl-beta-D-galactopyranoside) is useful in detecting late lactose fermenters, since ONPG molecule is structurally similar to lactose. It can enter the bacterial cell without a permease. In the presence of galactosidase, ONPG(colorless) is converted into galactose and o-nitrophenyl, which is a yellow chromogen and the alkaline end product.

INTERPRETATION

Positive – yellow color within 20 minutes to 24 hours

Negative- no color change or colorless after 24 hours

ONPG(O-nitrophenyl-beta-D-galactopyranoside) test

A B

A. Negative – Salmonella typhimuriumB. Positive – Escherichia coli(EHEC)

PURPOSE

To determine the ability of an organism to produce the enzyme, urease, which hydrolyzes urea.

To identify the rapid urease producers(Proteus and Morganella) and weak urease producers(Klebsiella pneumoniae and species of Enterobacter)

PRINCIPLE

Urease splits the urea molecule into ammonia(NH3), CO2 and water(H20). Ammonia reacts in solution to form an alkaline compound, ammonium carbonate, which results in an increased pH of the medium and a color change in the indicator to pink red. Urea + 2H2O --------------- CO2 + H2O +2NH3 urease (NH4)2CO3

INTERPRETATION

Christensens agar Positive – rapid urease activity; red throughout the medium Positive – slow urease activity: red in slant initially gradually converting the entire tube Negative – no urease activity; medium remains yellow

Stuart (urea) brothPositive - red color in the mediumNegative – no color change(buff to pale yellow)

A B C

A. Positive – Proteus spp.B. Positive - Klebsiella spp.C. Negative – Escherichia coli

Urease test(Christensens agar)

Urease test Stuart Urea broth

A B C

A. Uninoculated B. Strong positive reaction- Proteus spp.C. Negative – Escherichia coli

PURPOSE:

To determine whether a substrate utilization is an oxidative or fermentative process for the identification of several different bacteria

To separate organisms into two major groups: Enterobacteriaceae – fermentative Pseudomonas – oxidative

COMPOSITION

high concentration of carbohydrates (1%) small concentration of peptone(2%) Indicators bromcresol purple – purple to yellow Andrade’s acid fuchsin – pale yellow to pink phenol red – red to yellow bromthymol blue – green to yellow

Principle of glucose oxidative fermentation test

INTERPRETATION

glucose fermenter – when acid production is detected on both tubes since fermentation can occur with or without oxygen glucose oxidizer – acid is detected by the open aerobic tube Nonutilizer – some bacteria do not use glucose as a substrate

Open tube Closed tube Metabolism

Acid(yellow) alkaline(green) oxidative

Acid(yellow) acid(yellow) fermentation

Alkaline(green) alkaline(green) nonsaccharolytic (nonutilizer)

Oxidative-Fermentation Medium of Hugh and Leifson

Oxidative Fermentative medium(CDC method)

A. Fermenter – Escherichia coli

B. Oxidizer – Pseudomonas aeruginosa

Oxidative fermentative medium (CDC method)

Oxidative fermentative medium (CDC method)

C. Nonutilizer- Alcaligenes faecalis

1. As an initial step in the identification of Enterobacteriaceae

PRINCIPLE:

1. The action of many species of microorganisms on a carbohydrate substrate results in the acidification of the medium with or without gas formation.2. Iron salts(ferrous sulfate and ferric ammonium citrate) reacts with H2S to produce an insoluble black precipitate(ferrous sulfide).

PURPOSE

TSIA – two reaction chamber

Aerobic slant portionAnaerobic deep portion

Protein sources – beef extract, peptone, yeast extract, proteose peptone Sugars(lactose, sucrose, glucose) Indicators a. phenol red – carbohydrate fermentation b. ferrous sulfate – hydrogen sulfide production Sodium thiosulfate – source of sulfur atoms Sodium chloride – osmotic stabilizer

COMPOSITION

BIOCHEMICAL REACTIONS

carbohydrate fermentation acid production yellow deep – glucose fermented yellow slant – lactose and/ or sucrose fermented gas formation bubble formation cracking or splitting of the agar upward displacement of the agar pulling away of the medium from the walls of test tube H2S production blackening of the butt(FeS – black precipitate)

A/@H2S(-) Acid slant; acid butt; gas formation; no H2S

all sugars fermented; with gas formation; no blackening of the butt

Escherichia Klebsiella Enterobacter

K/@H2S+ alkaline slant; acid butt; with gas formation with H2S

glucose fermented; lactose and or/sucrose not fermented; with gas formation and black precipitate

Salmonella Proteus Citrobacter

K/A H2S( –) alkaline slant; acid butt; no gas; no H2S

glucose is fermented; lactose and/or sucrose not fermented; no gas formation; no black precipitate

Shigella Providencia Serratia anaerogenic Escherichia coli

K/KH2S(-) alkaline slant; alkaline butt; no gas; no H2S

no sugars fermented; no gas; no black precipitate in the butt

Pseudomonas Alcaligenes

A/@H2S+ acid slant;acid butt; with gas; with H2S

all sugars fermented; with gas formation; with black precipitate in the butt

Citrobacter freundii

PURPOSE:

To identify the lactose fermenting Enterobacte- riaceae such as Escherichia coli (MR positive and VP negative) whereas most members of the Klebsiella-Enterobacter-Serratia-Hafnia group are VP positive.

Metabolism of glucose using MR and VP pathways

Glucose

Acetoin Pyruvic acid Mixed acid fermentation

KOH + air pH less than 4.4(red) Diacetyl Napthol + creatine pink red complex Positive VP

In the first pathway, mixed acid products (lactic, acetic, formic and succinic) result, leading to a decrease in the pH of the medium and a positive MR test. The pH must drop to 4.4 or less for the MR indicator to take on its acidic red color.

PRINCIPLE – METHYL RED TEST

In the second pathway, acetylmethyl carbinol acetoin is an intermediate product to butylene glycol. It is the neutral product detected in the VP reaction. In the presence of oxygen and 40% potassium hydroxide, acetoin is converted to the diacetyl form, which results in a red color in the presence of alpha-napthol.

PRINCIPLE – VOGES PROSKAUER TEST

INTERPRETATION

Methyl red test Positive – distinct red color at surface of the medium Negative – yellow color at the surface of the medium

Voges Proskauer test Positive – pink red color at surface of the medium Negative – yellow color at surface of the medium

A B

Methyl Red test

A. Positive – Escherichia coliB. Negative – Klebsiella pneumoniae

A B

Voges Proskauer test

A. Positive – Klebsiella pneumoniaeB. Negative – Escherichia coli

PURPOSE:

To determine the production of decarboxylase by bacteria(Enterobacteriaceae).

Composition – Moeller decarboxylase medium

1. Glucose2. Amino acid substrate(1% lysine, 1% arginine 1% ornithine)3. pH indicator a. bromcresol purple 1. alkaline pH- purple 2. acid ph-yellow b. phenol red 1. alkaline pH– red 2. acid pH-yellow

PRINCIPLE Decarboxylase enzyme - removes carboxyl groups from the amino acids lysine and ornithine. Dihydrolase enzyme - removes a carboxyl group group from arginine. Glucose base without the amino acid and tubes containing glucose plus the amino acid substrates are inoculated. Decarboxylation and dihydrolation are anaerobic reactions so overlay the inoculated tubes with mineral oil to exclude air.

Lysine ----------------- cadaverine

Ornithine-------------- putrescine

Arginine--------------- citrulline----------- ornithine dihydrolase reaction decarboxylation putrescine

Specific amine products

Early incubation – both tubes yellow due to acidification of the indicator (bromcresol purple) by the acid end products of glucose fermentation.

If amino acid is decarboxylated, alkaline amines are formed and cause the indicator to revert to an alkaline pH.

INTERPRETATION

Control tube – yellow- glucose fermentation; viable organisms; pH of the medium has been lowered sufficient to activate the decarboxylase enzyme Positive test – purple – decarboxylation; formation of the amino acids from the decarboxylation

A B

A. Positive – purple; decarboxylationB. Negative – yellow; no decarboxylation; only glucose fermentation

Moeller decarboxylase medium

A B C D

Decarboxylase-dihydrolase reactions – Enterobacter cloacae)

A. Control – without amino acid C. lysine-negativeB. Arginine – positive D. ornithine-positive

Enterobacter cloacae Klebsiella pneumoniae

Arginine +(purple)alkaline -(yellow) acid

Lysine -(yellow)acid +(purple)alkaline

Ornithine +(purple)alkaline -(yellow)acid