Summary of Biochemical Tests in Microbiology Better living through chemistry.

Summary of Biochemical Testsin Microbiology

“Better living through chemistry”

Catalase Test

SummaryTests for presence of catalase.

BackgroundCatalase is an enzyme that breaks down hydrogen peroxide into water and oxygen gas. Evolution of oxygen gas bubbles is positive.All + except EF –

MethodsLoop of bacteria added to a drop of Hydrogen Peroxide H2O2

Blood Agar Test

SummaryTests for presence of hemolysin.

BackgroundHemolysin is an enzyme that destroys red blood cells.

MethodsBacteria streak and loop stabs to blood agar.

3 Types of Blood Agar Results

SummaryAlpha – Partial hemolysisBeta – Complete hemolysisGamma – No hemolysis

BackgroundAlpha hemolysis partially oxidizes hemoglobin changing it to green.All B+ except SE and EF

Beta hemolysis lyses RBC completely making a clear zone

Gamma hemolysis – no activity

Mannitol Salts Agar (MSA) TestSummarySelective for gram +Differential for fermentation of mannitol sugar.

BackgroundGram + bacteria tolerate sodium chloride (NaCL) salt.SA + EF + SE - and others –

MethodsStreak surface of MSA Agar. Watch for change from pink to yellow as a +

McConkeys Test

SummaryTests for fermentation of lactose.

BackgroundSelects for gram - , Crystal violet inhibits gram +Lactose and neutral red produce a dark pink differentiating lactose +EC, CLO, KP are +

MethodsLoop of bacteria streaked on Mac agar

EMB Agar Test

SummaryTests for fermentation of lactose.

BackgroundSelects for gram - , Eosin and methylene blue inhibit gram +Lactose and dyes produce a dark pink differentiating lactose + in CLO and a green sheen in ECEC, CLO, KP are +

MethodsLoop of bacteria streaked on EMB agar

Nitrate Reduction Test

SummaryTests for nitrate reductase and nitrite reductase.

BackgroundPotassium Nitrate KNO3 as substrate. Nitrate Nitrite N2

Methods. Add Nitrate A & B and cherry red means + for nitrate reductaseAll +Follow up with Zinc = grey = + for nitrite reductasePA +

Triple Sugar Iron (TSI) TestSummaryDifferentiates gram – enterics by fermentation patterns. BackgroundLactose and glucose are substrates. Phenol red is the indicator. pH of 6.8 or lower turns yellow.MethodsStab and streak TSI slant. Red slant & yellow but = glucose fermentation. ST SF PA

Yellow slant and yellow butt = lactose and glucose fermentation. SA SE EF BS EC CLO KP PV(false)

Red slant & red butt = no fermentation.

Black butt = Hydrogen sulfide production

Urease TestSummaryDifferentiates organisms using urease to break down urea into ammoniaBackgroundSubstrate is urea. pH indicator is phenol red (pH 6= yellow, 6.8 topaz, 8 = fuchsia)

MethodsLoop inoculation of urea broth.SA SE KP PV are +

IMViC Indole TestSummary and BackgroundDifferentiates enteric organisms such as EC and PV which are able to metabolize the amino acid tryptophan into indole and pyruvic acid. This test also detects motility of bacteria and H2S2

production.

MethodsNeedle inoculation of Sufide-Indole-Motility (SIM) media. Substrate is tryptophan. Add Kovac’s indole reagent. A red color is + for indole production.

Black = + for H2S2

Motility is observed as turbidity radiating out from the inoculation line.

IMViC Methyl RedSummary and BackgroundDifferentiates enteric organisms such as EC from CLO using the timing of glucose fermentation products. Both produce acids but CLO further converts to non acidic ethanol and then elevates the pH.

MethodsLoop inoculation of MR VP broth. Add methyl red indicator. Red = + for acid conditions. SA SE BS EC KP PV ST SF

Yellow is – indicating more basic conditions. EF CLO PA

Citrate TestSummaryDifferentiates organisms using citrate as a carbon source. BackgroundOrganisms using citrate must also have nitrogen found in the medium as ammonium dihydrogen phosphate. Citrate fermentation produces ammonia causing the indicator (bromothymol blue) to turn deep blue.

MethodsLoop inoculation of citrate slant.Prussian blue = + for citrate fermentation and alkaline conditions.Green or yellow = - (low pH)