CONVENTIONAL METHODS CONVENTIONAL METHODS FOR FOR

BACTERIAL IDENTIFICATIONBACTERIAL IDENTIFICATION

Dr Mostafa Mahmoud Ahmed, Ph DDr Mostafa Mahmoud Ahmed, Ph DConsultant Microbiologist, GDHA, Riyadh, KSA.Consultant Microbiologist, GDHA, Riyadh, KSA.Associate Prof. of Microbiology & Immunology.Associate Prof. of Microbiology & Immunology.

Faculty of Medicine – Ain Shams UniversityFaculty of Medicine – Ain Shams University

Methods for IdentificationMethods for IdentificationA- Phenotypic Characteristics:

1- AppearanceColonial morphologyMicroscopic appearance

2- Biological activityEnzyme production, Sugar fermentation

3- Immunophenotyping (serotyping): presence of specific antigens (O, H, K,….)

B- Genotyping and Molecular identification

Macroscopic AppearanceMacroscopic Appearance

Growth on Growth on Culture MediaCulture Media

Growth in Fluid MediaGrowth in Fluid Media

Uniform

FlocculentSediment

Colonial MorphologyColonial Morphology

Colonial MorphologyColonial Morphology• Size• Shape• Surface• Pigment production People• Consistency Come• Edge (margin)• Elevation• Opacity Other• Effect on Blood agar are Badly• Lactose fermentation Late

Some

Early

SIZESIZE

• Pinpoint• Small• Medium• Large

The shapes of colonies describes the entire colony, while the margin describes the edges.

Colonies may be circular or regular, irregular and punctiform.

A punctiform colony is too small to describe as either regular or irregular.

                          

Circular

                              

Irregular

ShapeShape

Edge (Margin)Edge (Margin)

1- Entire • This means that the edge of the colony is

distinct all of the way around the colony, and many of the colonies found on plates will be described as circular with entire margins.

2- Serrate This means a saw-toothed edge to a colony.

3- Undulate Undulate margins are a regular pattern of waviness around the colony.

4- Lobate • This means the colony forms lobes at

irregular intervals. It may be difficult to distinguish between undulate and lobate at times – they are imprecise distinctions.

5- Diffuse edge (Swarmming growth)• This means the colony fades away from

the densest part making it difficult to tell where the edge of the colony is.

Swarming GrowthSwarming Growth

• Usually denotes highly motile organism (e.g. proteus)

Rhizoid and filamentous • Both are used to describe both the shape

and the margin of a colony. 6- Rhizoid • This means root-like, and describes thick

branchlike growths originating from the center of a colony becoming thinner as a result of sequential branching.

7- Filamentous • They are the result of growth filaments of

equal width that pile on top of one another forming a thicker part in the center.

ELEVATIONELEVATION

ConsistencyConsistency

Flat Raised Convex Umbonate

Soft, Mucoid, Sticky

Tough, Hard

SURFACESURFACESmooth, Shiny

Rough, Dull

Pigment ProductionPigment Production1- Non-pigmented (nonchromogenic)2- Pigmented (Chromogenic)

Endopigment Exopigment

Endopigment

Different Endopigments

Effect on Blood agar Effect on Blood agar (Hemolysis Types)(Hemolysis Types)

No hemolysis (No hemolysis (γγ))

Complete Hemolysis (Complete Hemolysis (ββ))

Partial Hemolysis (Partial Hemolysis (αα))

Identify??

Identify??

Growth on MacConkey’s mediaGrowth on MacConkey’s media

LFC

NLFC

Identify??

Identify??

Identify??

Microscopic ExaminationMicroscopic Examination• Cocci, Bacilli, comma-shape - Arrangement• Reaction to Gram stain (+ve, -ve, Variable) • Capsule, spore formation• Motility (motile, non-motile).

Motility testMotility test

Special tests for enzymatic Special tests for enzymatic productionproduction

1. Catalase test2. Oxidase test3. Gelatinase test4. Urease test5. Tryptophanase (Indole test)6. Cysteinase (H2S production)

CatalaseCatalase

Oxidase testOxidase test

Gelatinase (Proteinases)Gelatinase (Proteinases)

Positive

Negative

Urease ProductionUrease Production

Positive Negative

Indole testIndole test

PositiveNegative

H2S ProductionH2S Production

PositiveNegative

Citrate Utilization testCitrate Utilization test

Test for End product of Test for End product of fermentationfermentation

Litmus MilkLitmus Milk

Triple Sugar Iron MediaTriple Sugar Iron Media(TSI)(TSI)

Slant

Butt

Triple Sugar Iron MediaTriple Sugar Iron Media(TSI)(TSI)

Analytical Profile Index (API)Analytical Profile Index (API)SystemSystem

E.coliE.coli

KlebseillaKlebseilla

Salmonella typhiSalmonella typhi

Broad Spectrum antibioticBroad Spectrum antibiotic

Emergence of ResistanceEmergence of Resistance

Targeted therapyTargeted therapy

To the target

DISC Diffusion MethodDISC Diffusion Method

Highly Resistant BacteriaHighly Resistant Bacteria

Broth Dilution MethodBroth Dilution MethodDetermining MICDetermining MIC

MIC

Microdilution traysMicrodilution trays

Reading of OD for microdilution

E-testE-test

E-test

E-Test for exopigment producing organism

E-Test for fastidious microorganism

Identification of some microorganism

Identification of some microorganism

Identification of some microorganism

Identification of some microorganism

Identification of some microorganism

Identification of some microorganism

Thank You