clinical microbiology

1. Clinical Microbiology
Dr. Dhanji P. Rajani
Microbiologist

2. Clinical Diagnostic
Microbiology
All aspects of infection
Initial isolation/diagnosis
Treatment
Infection control
Surveillance
Infection
Antimicrobial
Clinical management
Public health

3. Microcare Laboratory
 General Microbiology
– Routine Culture & Sensitivity
– Molecular Diagnostics
– Media Preparation
– Fungal Culture & Sensitivity
– Anaerobic Culture
 National TB Ref Lab

4. Specimen Investigation
 Direct
– Microscopy
– Culture & AST
– PCR

5. Direct methods
1. Macroscopic examination
2. Microscopic examination
1. Direct
2. Stain
3. Molecular methods
4. Specimen Culture

6. Microscopy
 Direct
– WCC
– Parasites
– Bacteria
 Stain
– Simple
– Differential
– Structural
– Flourescent

7. Staining
 Increase contrast of microorganisms
– Identify organism
– Structural characteristics
 Classified into types of stains
– Simple stain:
– Differential stain:
– Structural or special stains

8. 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

9. 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

10. 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.

11. Bacterial Culture
 Media
– Solid
– Liquid
25 different types of solid & liquid media
used routinely in Microcare Laboratory

12. Culture Media
– Nutrient
 BA
 CA
– Selective
 SS Agar
 XLD Agar
– Differential
 MacC
– Chromogenic

13. Incubation
 Temperature
– 37oC, 30oC,22oC,40oC
 Time
– 18 hours
 Atmospheric conditions
– Air
– CO2 / Microaerophilic
– Anaerobic

14. Bacterial Identification
 Fermentation
 Nutritional requirements
 Enzyme detection
 Metabolic activity
 Antigenic determinants
 Genomic
– PCR

15. Identification
Isolation (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)

16. Automated BC
 Continuous monitoring
 Early detection
 CO2 production
 Closed system
 Reduced risk of laboratory
contamination

17. 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

18. 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)

19. Disc Diffusion
 Classic Microbiology Technique
 Standardised suspension swabbed
onto plate
 Discs placed on the surface
 Zones read and compared to standard

20. Microbiology, St. James's Hospital

21. 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

22. 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
MIC
Bacterial growth Inhibition

23. E-test
