Microbiology_7_ID

MPharm Programme: Microbial Identification

• The presentation is about microbial identification techniques for the MPharm program.
• The presenter is Dr. Callum Cooper, from the University of Sunderland.

Learning Objectives

• Describe various microbiological techniques for identifying bacterial contaminants.
• Compare and contrast classical and molecular identification methods.
• Highlight the advantages and limitations of each method.
• Link the lecture content to practical laboratory work.
• Determine if the contaminant is causing infection.

What is Diagnostic Microbiology?

• Identification of microorganisms, usually to the species level.
• Determining the differences between strains within a species.
• Antimicrobial susceptibility testing.
• Characterization of pathogens.
• Includes applications in veterinary microbiology, plant pathology, food microbiology, and pharmaceutical microbiology (identifying contaminants).

Microbial Identification: Methods

• Culture-based methods:
  • Colony morphology analysis (shape, size, color, texture).
  • Use of selective media (e.g., Mannitol salt agar, MacConkey agar) to isolate specific types of bacteria.
  • Chromogenic media to identify particular microorganisms by the color change they generate.
• Microscopic examination:
  • Gram staining to differentiate Gram-positive and Gram-negative bacteria.
  • Electron microscopy for high-resolution images.
• Biochemical profiling:
  • Tests to identify bacteria based on metabolic properties.
  • Commercial kits (e.g., API strips, BD BBL Crystal) are used for fast testing.
• Molecular analysis:
  • Genomic analysis (e.g., sequencing).
  • Proteomic analysis.
  • Immunological methods (e.g., ELISA).

Colony Morphology

• Microbes often live in complex communities, not individual species.
• Colony morphology alone is not reliable in identifying species.
• Isolating individual colonies and purifying them is needed for accurate identification.

Selective and Differential Media

• Selective media:
  • Suppresses the growth of unwanted microorganisms.
  • Allows chosen species to grow.
  • Examples include MacConkey agar (for distinguishing Gram-negative bacteria), Sabouraud dextrose agar (for fungi), Baird-Parker agar (for staphylococci).
• Differential media:
  • Enables the differentiation of bacterial species on the same medium.
  • Examples include Mannitol salt agar (differentiating staphylococci based on fermentation of mannitol), Eosin Methylene Blue (EMB) agar (identifying lactose fermenters).

Microscopy

• Light microscopy and electron microscopy.
• Light microscopy types include dark field, transmission, and phase contrast.
• Electron microscopy types include transmission and scanning.
• Resolution and magnification capabilities of each type.

Light Microscopy - Sample Preparation

• Staining techniques to visualize microorganisms.
• Common stains for different types (bacteria, fungi, protozoa).
• Examples include Gram stain, Ziehl-Neelsen stain, Malachite Green stain.
• Periodic Acid-Schiff (PAS) stain detects polysaccharides.

Light Microscopy - Gram Staining

• Distinguishes bacteria based on cell wall composition (Gram-positive/Gram-negative).
• Detailed procedure for performing the gram stain.

Electron Microscopy

• Sample Preparation methods.
• Chemical fixation, dehydration, critical drying, metal coating etc.

Biochemical Profiling

• Identifying bacteria that are isolated in pure culture.
• Oxidase test (tests for cytochrome oxidase).
• Commercially available kits to analyze specific biochemical characteristics.

Molecular Identification

• Immunological Identification: ELISA (Enzyme-linked Immunosorbent Assay)
  • Used for medical diagnostics (detecting HIV, TB, etc).
  • Direct and Sandwich methods.
• Genomic Identification: PCR (polymerase chain reaction)
  • Amplifies DNA sequences using primers.
  • Used for identifying antibiotic resistance genes.
  • DNA Sequencing- Identifying changes and whole genomes.
  • Multilocus sequence typing methods to look for changes in key genes.
• Proteomic Identification (MALDI-TOF):
  • Identifies bacteria based on ionized sample properties and compared with references in a database.
  • Used to identify bacterial species from selective media.
  • Can also provide information about resistance.

Biochemical Profiling: ATP Determination

• Adenosine Triphosphate (ATP) is a key energy source for bacteria.
• Amount of ATP correlated with the amount of light produced.
• ATP determination can count bacteria.
• Not effective for evaluating the contaminated sample itself.

Practice: Classical Non-Sterile Pharmaceutical Micro

• Typical protocol for isolating and identifying contaminants.
• Includes serial dilutions, streaking to purify, Gram staining, and biochemical assays for species identification.

This quiz focuses on microbial identification techniques relevant to the MPharm program. Participants will explore classical and molecular methods for identifying bacterial contaminants, their advantages, and limitations. Link theory to practical laboratory skills through diagnostic microbiology applications, including antimicrobial susceptibility testing.