Clinical Microbiology

Dr. Dhanji P. Rajani

Microbiologist

Clinical Diagnostic

Microbiology

All aspects of infectionInitial isolation/diagnosisTreatmentInfection controlSurveillance

InfectionAntimicrobial

Clinical managementPublic health

Microcare Laboratory

General Microbiology

– Routine Culture & Sensitivity

– Molecular Diagnostics

– Media Preparation

– Fungal Culture & Sensitivity

– Anaerobic Culture

National TB Ref Lab

Specimen Investigation

Direct

– Microscopy

– Culture & AST

– PCR

Direct methods

1. Macroscopic examination

2. Microscopic examination

1. Direct

2. Stain

3. Molecular methods

4. Specimen Culture

Microscopy

Direct

– WCC

– Parasites

– Bacteria

Stain

– Simple

– Differential

– Structural

– Flourescent

Staining

Increase contrast of microorganisms

– Identify organism

– Structural characteristics

Classified into types of stains

– Simple stain:

– Differential stain:

– Structural or special stains

Gram stain

Most common stain

Valuable first step in identification

Differentiates into two groups

Physicochemical cell wall properties

– crystal violet to a heat-fixed smear

– Lugol’s iodine as a mordant

– rapid decolorization with alcohol /acetone

– counterstaining with safranin/carbol fuchsin

Gram stain morphology

Shape

– cocci (round)

– diplococci

– bacilli (rods)

– spiral or curved (spirochetes)

Single or multiple cells

– clusters (staph)

– chains (strep)

Gram positive or negative

Bacterial

isolation and identification

Samples

streaked on culture plates

isolated colonies of bacteria appear after incubation.

Key step in identification – colonial morphology

size,

texture,

colour,

haemolysis ,

smell.

Incubation temperature, time and atmospheric conditions

important characteristic.

Bacterial Culture

Media

– Solid

– Liquid

25 different types of solid & liquid media used routinely in Microcare Laboratory

Culture Media

– Nutrient

BA

CA

– Selective

SS Agar

XLD Agar

– Differential

MacC

– Chromogenic

Incubation

Temperature

– 37oC, 30oC,22oC,40oC

Time

– 18 hours

Atmospheric conditions

– Air

– CO2 / Microaerophilic

– Anaerobic

Bacterial Identification

Fermentation

Nutritional requirements

Enzyme detection

Metabolic activity

Antigenic determinants

Genomic

– PCR

IdentificationIsolation (culture)

Agar

plate/colonies

Liquid media

test tube - bulk

Identification & taxonomy

Family

Genus

Species

Type

Strain

Biochemical (physiological) tests

Fermentation

Metabolic characteristics

Molecular tests

DNA-DNA homology

16S rRNA sequencing

Chemical profiling

Mass Spectrometry

Non culture based detection

Polymerase chain reaction- (PCR)

Agglutination (antigen detection)

Stain

Serology (antibody detection)

Automated BC

Continuous monitoring

Early detection

CO2 production

Closed system

Reduced risk of laboratory contamination

Antimicrobial susceptibility test

Minimum inhibitory concentration [MIC]

– The smallest concentration of antibiotic that

inhibits the growth of organism

Liquid media (dilution) allows MIC estimation

Solid media (diffusion)

– Disk diffusion CLSI/EUCAST

– E-tests

– Allows MIC estimation

Identification of resistance determinants

Natural & acquired resistance

Natural resistance

– Affect almost all species strains

– Existed before antibiotic use (Enterobacter sp. -amoxicillin)

Acquired resistance (mutation)

– Chromosomic, plasmidic

– Affects a fraction of strains

– Increased with antibiotic use(extended spectrum beta-lactamase producing E. coli)

Disc Diffusion

Classic Microbiology Technique

Standardised suspension swabbed onto plate

Discs placed on the surface

Zones read and compared to standard

Microbiology, St. James's Hospital

Common problems

Problem with the size of the inoculum

Depth of medium

Type of medium

Moisture content

Solution:

Use McFarland 0.5 photometer

Scale -> same tubes

Dilution in liquid broth

increasing antibiotic concentrations

Standard concentration

Incubation for 18 hr at 37°C

(Control 0,25 0,50 1 2 4 8 mg/l

MICBacterial growth Inhibition

E-test