Second year Lab Medicine (Females)1433- 1434 A.H. / 2012- 2013

Microbiology Practical (Course I)

Mrs. Hibah Abdul-Wahab I. Abu-Sulaiman

How do you think you could distinguish easily between two similar people?!

Fingerprint

That was among humans

Okay, What about bacteria?!!!

Different kinds of bacteria are distinguished through….

Enzymes using biochemical tests

Biochemical Tests

Test the ability, activity or the presence of an enzyme

Objectives:• Introduction.• Biochemical Tests.• Antibiotic Sensitivity Test.

• Catalase test.• Coagulase test.• Oxidase test.• Urease test.• Indole test.• API 20E.

Biochemical ID Testing

Catalase TestReagents: H2O2

Reaction (Principle) : 2H2O2 2H2O + O2 (bubbles)

Aim: to differentiate bacteria that produce catalase enzyme.Results:

◦ bubbles +ve (Staphylococcus)◦ No bubbles -ve (Not Staph.)

Test QC:◦ +ve cont.: Staphylococci spp.◦ -ve cont.: Streptococci spp.

catalase

Catalase testPositive Negative

Coagulase TestTypes of Coagulase:

◦Free coagulase.◦Bound coagulase.

Aim: to ID S. aureusReagent:

◦Free coagulase: plasma containing coagulase reacting factor (CRF).

◦Bound coagulase: plasmaPrinciple:

fibrinogen fibrin clot

coagulase

• Results:– Free coagulase: fibrin clot (within 2hrs, 4hrs and

24 hrs) +ve– Bound coagulase: fibrin clot 10 sec +ve.– No fibrin clot: -ve

• Test QC:– +ve Cont.: Staph. aureus– -ve Cont.: Staph. epidermidis

Coagulase Test (Cont. …)

Free Coagulase

Bound CoagulasePosetive Negative

Oxidase TestAim: to ID cytochrome oxidase producing bacteria.Reagent:1% tetramethyl-p-phenylenediamine

dihydrochloride.Principle: tetramethyl-p-phenylenediamine dihydrochloride +

reduced cytochrome c oxidized cytochrome c (Purple) + H2O

Results : Dark purple: +ve PseudomonasColorless: -ve Others

oxidase

Oxidase Test• QC: +ve Cont. Pseudomonas

-ve Cont. E. coli

Neg. Pos.

Urease TestReagent: Urea slant mediaPrinciple:

◦Urea ammonia (alkaline pH) + CO2

◦alkaline pH + Phenol red Pink slantAim: to ID urease producing bact.Results: Pink Slant: +ve

Pale Slant: -veTest QC:

◦+ve Cont. …: Proteus◦-ve Cont. …: E. coli

Urease

Indole Test• Reagent: Tryptophan broth cultures and Kovac’s

reagent (4(p)-dimethylaminobenzaldehyde)• Principle: – tryptophan pyrovic acid+ ammonia+ indole.– 4(p)-dimethylaminobenzaldehyde + indole red ring

• Aim: to ID tryptophanase producing bact.

Indole Test (Cont. …)• Results: red ring +ve

yellow ring -ve• Test QC: +ve cont.: E. coli

-ve cont.: Klebsiella

Negative Pos.

API 20E• Analytical Profile Index (API) 20E: is a

miniturized version of conventional tests that is used for the ID of members of the family Enterobacteriaceae and other gram negative bacteria.

• It utilizes plastic cups with 20 separate compartments. Each containing a capsule or dehydrated medium.

API 20E

Antibiotic Sensitivity Testing (AST)

• Once the causative agent of a specific disease in a patient has been isolated, it is up to the attending physician to prescribe a chemotherapeutic agent that will kill or inhibit the pathogen without causing serious harm to the patient.

Chemotherapeutic agents vary in their effectiveness against various pathogenic bact.

• Antibiotics: are compounds, usually of low molecular weight and produced by microorganisms.

AST

ANY QUESTIONS?!!Thank you