GW

LQB362 Lecture Week 3_25se1 (1)

School of Biomedical Sciences Overview

Lecture Information

  • Instructor: Dr. Eva Hatje

  • Course: LQB362 Fundamentals of Microbiology

  • Contact Email: e.hatje@qut.edu.au

Acknowledgements

  • Acknowledgment of the Turrbal and Yugara people, the traditional owners of the land where QUT is located.

  • Respect paid to their Elders, customs, and oral traditions.

Learning Resources

Textbook References

  • Microbiology: an Introduction (Thirteenth Edition) by Tortora, G. J., Funke, B. R., & Case, C. L.

    • Chapter 3: Focus on Gram staining

  • Brock Biology of Microorganisms (Sixteenth Edition) by Madigan, M. T., et al.

    • Chapter 29: Identification of microorganisms

  • Acknowledgment: A/Prof Francesca Frentiu for slide contributions.

Learning Outcomes

  1. Steps in identifying infectious agents from clinical specimens, focusing on collection and transport considerations.

  2. Microbial identification methods: traditional culture techniques, biochemical tests.

  3. Methods for microbial identification: nucleic acid detection, microbial antigens, and host antibodies.

  4. Overview of MALDI-TOF technology and its application in diagnostic microbiology.

  5. Timeframes for microbiological testing and diagnosis of infectious diseases.

Laboratory Diagnosis of Infectious Diseases

Methods of Diagnosis

  • Microscopy

    • e.g., Gram staining

  • Culture Techniques

    • Detection and isolation of infectious agents

  • Serology

    • Antigen and antibody detection

    • IgM and IgG interpretation for infection status.

  • Diagnostic Tests

    • ELISA, PCR processes, fluorescent antibody assays.

Specimen Types and Collection

  • Types of Specimens: Blood, urine, CSF, sputum, etc.

    • Collected based on the infection suspected.

  • Essential Identifiers on Specimens:

    • Patient’s Full Name, Date of Birth, Collection Time & Date, and Specimen Type.

  • Collection Considerations:

    • Aseptic technique to prevent contamination, especially for body sites with resident microbiota.

  • Storage and Transport Conditions:

    • Timeliness in processing samples is crucial (e.g., swabs <24 hours; blood <2 hours).

Microbial Identification Techniques

Conventional Techniques

  • Gram Staining

    • Classifies bacteria based on cell wall properties.

    • Provides information on Gram reaction, shape, and size.

  • Culture Media:

    • Selective and differential media can assist in presumptive or confirmatory identification.

Biochemical Tests

  • Several specific tests include:

    • Catalase Test: Differentiates staphylococci (positives) from streptococci (negatives).

    • Oxidase Test: Identifies the presence of cytochrome C oxidase.

    • Indole Test: Differentiates certain bacteria within Enterobacteriaceae.

    • Coagulase Test: Differentiates S. aureus from other staphylococci.

    • Urease Test: Differentiates members of the Proteeae family.

    • Citrate Test: Examines the organism's ability to utilize citrate.

    • Triple Sugar Iron (TSI) Test: Determines fermentation capabilities and sulfur reduction.

Advanced Identification Methods

Automated Systems

  • Vitek 2 Identification

    • Automated system using 'cards' for rapid biochemical tests and identification.

Recent Technologies

  • MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry)

    • Identifies microorganisms by analyzing the ions of their chemical structure against a database for accurate identification.

Nucleic Acid Detection Techniques

Methods of Detection

  • Conventional PCR

    • Extract DNA and amplify for identification.

  • 16S rRNA PCR

    • Targets the ribosomal RNA gene for species identification.

  • Real-Time PCR (qPCR)

    • Combines detection with amplification to quantify microbial loads.

  • Reverse Transcriptase PCR

    • For RNA viruses, converts RNA into cDNA for analysis.

Diagnosis in Absence of Culture

  • Serological Tests: Detection of antigens/antibodies via assays like ELISA.

  • Detection of DNA: Capture specific sequences without needing the organism to be cultured.

Summary of Key Points

  • Identification of infectious agents requires systematic collection, transport of samples, and a variety of microbial identification techniques (biochemical, serological, nucleic acid detection).

  • Advances like MALDI-TOF MS and automated systems enhance accuracy and efficiency in microbial diagnosis.