Identification/Typing Methods:

Microbial Diagnosis

Dr Rajiv Mohan Gupta

MD (Microbiology),DNB(Pathology),Ph.D (Virology),M.Phil, MNAMS
Professor & HoD, Department of Microbiology
 Methods for Microbial Typing/Classification

• What is Bacterial Typing/Classification

• Types of Methods for Bacterial Typing/ Classification

– Phenotypic Methods

– Genotypic Methods

• Polymerase Chain Reaction

• Uses and applications of Bacterial typing in clinical setting

 Bacterial Classification/Typing : Aim

• An organism identified by one individual (scientist, clinician,

epidemiologist), is recognized as the same organism each &
every time.
• Study evolution of Organisms over time.
• Detection and identification of new organisms.
• Documentation of non-cultivable organisms.
• Assign Vectors for Infection transmission.
• Identify Reservoirs of Infection.
 Classification Methods

• Phenotypic Methods
– Gram Stain
– Oxygen Requirement for growth
– Biochemical Reactions (Biotyping)
– Serological characteristics (Serotyping)
– Antibiograms
– Bacteriocin Typing
– Phage Typing
 • Gram Stain
Hans Christian • Morphology/ Differential Staining Character

Gram • Gram Positive (Blue-Purple)

• Gram Negative (Red)
• Property of Cell - Wall

Gram Stain:
1884
M.tuberculosis C.tetani Staphylococci Lactobacilli Campylobacter spp
N.meninigitidis C.perfringens Enterobacteriaceae
Biochemical
Typing/Classification

• Lactose Fermentation

(Utilization of Substrate)
• Oxidase

(Production of Enzyme)
• Nitrate Reduction

(Production of Nitrase
Reductase Enzyme :Nitrate
to Nitrite)
 Serotyping
• Based on cell wall & other
components
• Enterobacteriaceae
• Streptococci
– Lancefield Grouping
– Griffith Typing
Antibiograms
 Bacteriocin Typing

• Bacteriocins
– synthesized antimicrobial peptides
• Kill or inhibit bacterial strains
– closely-related
– non-related to produced bacteria
• Colicins (Gram Negative Bacteria)
– Pyocin (Psuedomonas spp)
– Shigella sonnei (23 Colicin Types)
• Diphthericin (Corynebacterium diphtheriae)
 Phage typing
• Viruses that infect bacteria
– λ phage of E.Coli
– T4 phage of E.coli
• Uses
– Relatedness of organism strains
– Source of infections
– Route of transmission
– Outbreaks and epidemics
• Staphylococcus aureus, Salmonella typhi
Genotypic/Molecular Typing Methods
Central Dogma Molecular Biology
Polymerase Chain Reaction
• Molecular Xeroxing
Detection of PCR Product: Gel
Electrophoresis
 Types of PCR
• Conventional PCR

• Reverse Transcriptase PCR (RNA To cDNA)(RT-PCR)

– RNA Viruses

• Multiplex PCR (Multiples Genetic Fragments in One Reaction

Simultaneously)

– Respiratory Panel, Meningitis Panel, Diarrhea Panel, PUO Panel

• Real Time PCR (rt-PCR)

– Viral Loads Estimation (HBV, HCV, HIV, CMV etc)

 Applications of PCR in Clinical setting

• Disease Diagnosis
– Viral Diseases
– Follow-up of Treatment Response
– Syndromic Diagnosis
– Uncultivable Agents/ Difficult to Culture
• Screening for Blood Safety
• Drug Resistance Testing & Documentation
• Classification of Micro-organisms
• Typing of Micro-organisms (Molecular Methods)
 Genotypic Typing Methods

• RFLP (Restriction Fragment Length Polymorphism)

• PFGE (Pulse field Gel Electrophoresis)
• Sequence Based Methods
– Nucleotide Sequencing
• Single Gene Sequencing (SNPs)
• Multiple Genes Sequencing (MLST)
– Whole Genome Sequencing (NGS)
 RFLP
(Restriction Fragment Length Polymorphism)
• Use of
Restriction
Enzymes
• Patterns
Produced
• Cutting sites

• Size of
fragments
generated
 PFGE

Uses

• Genotyping

• Genetic Fingerprinting

• Gold Standard in

Epidemiology

• Strain Differentiation
Thank You

https://ellipse.prbb.org/viruses-adaptation-to-the-
tissues-they-infect/