Biochemical TestsBiochemical Tests

ObjectivesObjectivesTests to knowTests to know Indole Indole Methyl Red/Voges ProskauerMethyl Red/Voges Proskauer CitrateCitrate HH22S production in SIMS production in SIM Urea hydrolysisUrea hydrolysis Sugar fermentationSugar fermentation Lactose fermentationLactose fermentation Oxidase test Catalase test

Indole Production TestIndole Production Test The amino acid tryptophan can be broken down by The amino acid tryptophan can be broken down by

enzyme tryptophanase to form indole.enzyme tryptophanase to form indole. Tryptophanase differentiates indole-positive enterics, Tryptophanase differentiates indole-positive enterics,

such as Escherchia coli and Proteus vulgaris from such as Escherchia coli and Proteus vulgaris from indole-negative enterics, such as Serratia marcescens.indole-negative enterics, such as Serratia marcescens.

Media and Reagents:Media and Reagents: SIM with tryptophan and SIM with tryptophan and Kovac’s reagent.Kovac’s reagent.

Method:Method: Inoculate medium and incubate at 37°C Inoculate medium and incubate at 37°C for for 24-48 hours. After incubation, add five drops of Kovac’s 24-48 hours. After incubation, add five drops of Kovac’s reagent to the surface. Do not stir or shake the tube.reagent to the surface. Do not stir or shake the tube.

Expected Results:Expected Results: Positive test :Positive test : Kovac’s reagent combines with indole and Kovac’s reagent combines with indole and

turns the surface red.turns the surface red. Negative test:Negative test: No red color development No red color development

Results for indole testResults for indole test

Methyl Red/Voges Proskauer Methyl Red/Voges Proskauer (MR/VP)(MR/VP)

How to Perform Tests:How to Perform Tests: Inoculate 2 glucose broths Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the few drops of MR to one tube, and VP reagents to the other tube.other tube.

Properties they test for:Properties they test for: Both tests are used to help Both tests are used to help differentiate species of the family differentiate species of the family Enterobacteriaceae. Enterobacteriaceae.

MR—tests for acid end products from glucose fermentation. MR—tests for acid end products from glucose fermentation. VP—tests for acetoin production from glucose fermentation.VP—tests for acetoin production from glucose fermentation.

Media and Reagents Used:Media and Reagents Used: Glucose BrothGlucose Broth Methyl Red indicator for acidMethyl Red indicator for acid Voges Proskauer reagents—A: Alpha-Naphthol, B: Potassium Voges Proskauer reagents—A: Alpha-Naphthol, B: Potassium

Hydroxide (KOH).Hydroxide (KOH).

MR/VP continuedMR/VP continued Reading Results:Reading Results:

MR— a + result is red (indicating pH below 6) and a – result is yellow MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production)(indicating no acid production)

VP—A + result is red pinkish after VP reagents are added (indicating VP—A + result is red pinkish after VP reagents are added (indicating the presence of acetoin) and a – result is no color change.the presence of acetoin) and a – result is no color change.

Methyl Red: left – and right + VP: left + and right –

Citrate UtilizationCitrate Utilization Citrate is an organic molecule that can be utilized Citrate is an organic molecule that can be utilized

by bacteria that produce the enzyme citrase. by bacteria that produce the enzyme citrase. Citrase is produced by some bacteria such as E. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coliaerogenes but not by others like E. Coli

Media and Reagent:Media and Reagent: Simmon’s Citrate Agar. It Simmon’s Citrate Agar. It has citrase as the only carbon source and PH has citrase as the only carbon source and PH indicator bromothymol blueindicator bromothymol blue

Method:Method: Inoculate the slant and incubate at Inoculate the slant and incubate at 37°C for 24-48 hours. 37°C for 24-48 hours.

Expected results:Expected results: Positive test:Positive test: Growth and color changes to blue Growth and color changes to blue Negative test:Negative test: No growth and color remains green No growth and color remains green

Results for Citrate TestResults for Citrate Test

NegativePositive

HH22S ProductionS Production

Bacteria use enzyme cysteine desulfurase to Bacteria use enzyme cysteine desulfurase to hydrolyze the amino acid cysteine, forming hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product. hydrogen sulfide as end-product.

Media and Reagent:Media and Reagent: SIM with cysteine and SIM with cysteine and ferrous sulfate (detects H2S)ferrous sulfate (detects H2S)

Method:Method: Inoculate the media and incubate at Inoculate the media and incubate at 37°C37°C for 24-48 hours. for 24-48 hours.

Expected Results: Expected Results: Positive Test:Positive Test: H2S production = Black H2S production = Black Negative Test:Negative Test: No H2S production = No blackening of No H2S production = No blackening of

medium medium

Results of HResults of H22S S productionproduction

PositiveNegative

Urea UtilizationUrea Utilization Some bacteria produce urease, an enzyme Some bacteria produce urease, an enzyme

capable of breaking down urea and capable of breaking down urea and produce alkaline end products. This produce alkaline end products. This distinguishes Proteus from other bacteriadistinguishes Proteus from other bacteria

Media and Reagent:Media and Reagent: Urea Broth with Urea Broth with phenol redphenol red

Method:Method: Inoculate the media with a loop Inoculate the media with a loop and incubate at and incubate at 37°C37°C for 24 hours. for 24 hours.

Expected Results: Expected Results: Positive test:Positive test: production of alkaline end production of alkaline end

products = pinkish red colorproducts = pinkish red color Negative test:Negative test: No color change No color change

Results for Urea TestResults for Urea Test

NegativePositive

Glucose Fermentation & Gas Glucose Fermentation & Gas ProductionProduction

How to Perform Test:How to Perform Test: Inoculate broth with inoculating loop.Inoculate broth with inoculating loop.

Property it tests for:Property it tests for: This tests for the bacteria’s ability to This tests for the bacteria’s ability to ferment glucose and produce gas and/or an acid end-product..ferment glucose and produce gas and/or an acid end-product..

Media and Reagents Used:Media and Reagents Used: Glucose broth contains phenol Glucose broth contains phenol red as indicator to indicate an acid end product. A Durham tube is red as indicator to indicate an acid end product. A Durham tube is added to indicate gas production.added to indicate gas production.

ResultsResults A positive result for acid is yellow after indicator is added A positive result for acid is yellow after indicator is added

(indicating glucose fermentation)(indicating glucose fermentation) A positive result for gas is a bubble in the Durham tube.A positive result for gas is a bubble in the Durham tube. A completely negative result has no color change or reddish A completely negative result has no color change or reddish

color and no bubble.color and no bubble.

Sugar Fermentation TestsSugar Fermentation Tests

Tube 1: Negative acid /Negative gasTube 2A: Must incubate longer (ambiguous result)Tube 2B: Positive acid /Negative gasTube 3A: Positive acid/ Positive gas

Lactose FermentationLactose Fermentation MacConkey Agar differentiates lactose-fermenting MacConkey Agar differentiates lactose-fermenting

bacteria, such as E. Coli from non-lactose bacteria, such as E. Coli from non-lactose fermenting bacteria.fermenting bacteria.

Media and Reagent:Media and Reagent: MacConkey Agar and MacConkey Agar and neutral red dyeneutral red dye

Method:Method: Streak MAC plate and incubate at 37°C Streak MAC plate and incubate at 37°C for 2 days.for 2 days.

Expected results: Expected results: Positive test:Positive test: Lactose fermentation = Growth and color Lactose fermentation = Growth and color

change to pinkchange to pink Negative test:Negative test: No lactose fermentation = May or may No lactose fermentation = May or may

not grow and no color changenot grow and no color change

Results of Lactose Results of Lactose FermentationFermentation

- Oxidase test: Some bacteria produce Oxidase enzyme (Pseudomonas ) Detection by adding few drops of colorless oxidase reagent Colonies turn deep purple in color (positive)

Catalase TestThe Catalase test determines if the organism produces the enzyme “Catalase”, which breaks down hydrogen peroxide (H2O2) to water and oxygen (O2).

Catalase2 H2O2 2 H2O + O2 (gas)

and may be seen in aerobic and facultatively anaerobic organisms like Staphylococcus bacteria.

Addition of H2O2 lead to production of gas Addition of H2O2 lead to production of gas bubblesbubbles (O2 production)(O2 production)

Examples of Biochemical TestsExamples of Biochemical Tests

API 50 Test