Overview of microbiology

Binish Arif Resident Microbiology Aga Khan University Hospital

Game plan
• Scope of Microbiology
– Extent of the microbial world

• Microbial diversity • History of Microbiology • Diagnosis of infection
– Techniques
• Microscopy and Staining • Pure culture methods • Quantitative methods

• Result reporting

What is microbiology?
• the study of microorganisms • organisms too small to be seen with the naked eye
– except in large groups

The universal tree of life

The universal tree of life

Microbial world
• • • • Viruses Bacteria (Eubacteria and Archaea) Fungi (Yeasts and Molds) Protozoa

• Maintain balance of environment (microbial ecology) • Basis of food chain • Nitrogen fixation • Photosynthesis • Digestion, synthesis of vitamins • Manufacture of food and drink

• • • • Genetic engineering Synthesis of chemical products Recycling sewage Bioremediation: use microbes to remove toxins (oil spills) • Use of microbes to control crop pests • Normal microbiota

Harmful effects
• Cause disease • Food spoilage

History of microbiology

Pioneers of microbiology
• Robert Hooke, (1665)
– Proposed the Cell Theory – Observed cork with crude microscope – All living things are composed of cells

• Antoni van Leeuwenhoek, (1673)
– First observed live microorganisms (animalcules)

• Schleiden and Schwann,
– Formulated Cell Theory: cells are the fundamental units of life and carry out all the basic functions of living things

• Pasteur, FR and Tyndall, (1861)
– Finally disproved S.G.

Pioneers of microbiology
• Louis Pasteur (1822-1895), Chemist
– Fermentation (1857) – Pasteurization: heat liquid enough to kill spoilage bacteria (1864) – Vaccine development – rabies – Proposed the germ theory of disease – Proposed aseptic techniques (prevent contamination by unwanted microbes) – Director of Pasteur Institute, Paris (1894)

Pioneers of microbiology
• Joseph Lister, (1867)
– Used phenol (carbolic acid) to disinfect wounds – First aseptic technique in surgery

• Robert Koch, (1876)
– Postulates – Germ theory (1876) – Identified microbes that caused anthrax (1876), tuberculosis (1882) and cholera (1883) – Developed microbiological media & streak plates for pure culture (1881)

Branches of microbiology
• Bacteriology: study of bacteria • Mycology: study of fungi • Immunology: study of immunity
– Edward Jenner : developed vaccination (1798) – Metchnikoff : discovered phagocytes (1884) – Paul Ehrlich : theory of immunity (1890)

• Virology: study of viruses
– Beijerinck : discovered intracellular reproduction of TMV; coined the term ―virus‖ (1899)

Microbiology lab pulling in all directions
Health care Providers Routine Service Keeping up with changing technology & innovation Other Clinical Labs: Reference work

Clinical Microbiology Lab

Administration (Budget, finance, QA & QQC, Accreditation, Licensing, HR, Institutional committees, etc)

Support to other partners/clients: Infection control Health Units Academic institutions, Pharmacy,, etc

Clinical Microbiology Labs
• Routine clinical microbiology testing
• Licensed test menu

• Reference microbiology
• (discipline speciality – centre for excellence)

• Quality assurance
• including accreditation

• Laboratory surveillance
• Infectious Diseases, Epidemiology

• • • •

Support to Infection Control program Emergency outbreak preparedness & management Teaching & training Research and development

Branches of microbiology
• Parasitology: study of protozoa and parasitic worms • Chemotherapy
– Treatment of disease by using chemicals – Antibiotics produced naturally – Synthetic drugs

Branches of microbiology
• Chemotherapy
– Alexander Fleming, (1928) discovered penicillin – Selman Waksman, (1944) discovered streptomycin

• Problems
– Toxicity of drugs => Selective toxicity – Resistance of bacteria to drugs

Branches of microbiology
• Recombinant DNA Technology
– Recombinant DNA – Genetic engineering/biotechnology – Microbial genetics – mechanism by which microbes inherit genes – Molecular biology – structure and function (expression) of genes – Molecular epidemiology/diagnostics

Role of Molecular diagnostic in Microbiology— the paradigm shift
• Traditional method have limited capability of providing timely information to physicians • Advantages of molecular infectious disease testing over conventional culture methods – Rapid test results (1–5 hr), – Relatively small sample size – High clinical sensitivity and specificity in the presence of antimicrobial therapy – Rapid identification of fastidious organisms – Direct detection of resistant strains

Branches of microbiology
• Biotechnology
– GMOs/GEMs for industrial, pharmaceutical and agricultural applications – Improvements of agriculture (plants and animals)
– Gene therapy: inserting a missing gene or replacing a defective one in human cells

Diagnosis of infection

The triad of infectious disease
1. The affected host 2. Infectious agent 3. The environment

The diagnostic cycle
Physician examines patient & makes a tentative clinical diagnosis Patient consults physician with signs/symptoms of infectious disease Pre-analytical Physician interprets reports and institutes appropriate therapy Final culture report is prepared and sent to the physician’s office, clinic or hospital Post-analytical Written orders are transcribed to a laboratory request form. Form and specimen are transported to the laboratory Upon receipt by the laboratory, data from the request form is entered into a computer file or log book. Analytical Specimen is directly examined. Microscopic mounts, smears, and stains may or may not be set up. Presumptive reports may or may not be issued. Subcultures are examined, and results of identification systems are examined.

Appropriate specimen(s) is/are collected for culture. All containers must be properly labelled.

Preliminary reports may or may not be issued.

After incubation, cultures are examined. Definitive identification are set up. Specimens are processed. Culture media are selected, inoculated and incubated.

Microscopy and staining
• Wet mounts • Staining
– – – – Gram’s staining (Hans Christian Gram) Acid fast staining (Ziehl-Neelsen stain) Fluorescent antigen/antibody stains Fluorochrome stains for mycobacteria (auramine and rhodamine)

Processing specimens
• Selection of primary culture media • Determine the temperature and atmosphere of incubation • Determine which of the isolates recovered on primary media require further characterization • Determine whether antimicrobial susceptibility testing is required

Quantitative culture methods
• Urine specimens, lower respiratory tract specimens • For urine --- 0.01µl or 0.001µl caliberated inoculating loop.
– 1 colony = 1000 CFU/ml – 10 colonies = 104 CFU/ml – 100 colonies = 105 CFU/ml

Interpretation of cultures
• Characteristics of colonies

Interpretation of cultures
• Reactions in agar media
– Haemolysis on blood agar: – Pigment production:

– Changes in differential media:

Result reporting
• ASAP the results are available, without any error • Electronic • Telephone • Paper

Environmental factors and the spread of communicable diseases in Pakistan
• Environmental factors that influence: – Waterborne diseases – From traditional to new ...Giardia to Naegleria – Food (Processed & Imported) Poultry industry ...Newcastle diseases and avian influenza viruses that has adversely affected the investment and growth rate of poultry industry in Pakistan – Climate • Flooding after heavy rains result in sewage overflow and widespread water contamination

Environmental testing Challenges & opportunities
• Govt labs – limited scope – Water, Food & Environmental testing • Fewer labs do: – Mould & spore testing – In door - Air quality monitoring – Monitoring sterilization & disinfection outcomes – Water testing for bottled water – Food testing (for imported foods)

• • • •

Scope of Microbiology Microbial diversity History of Microbiology Diagnosis of infection
– Techniques
• Microscopy and Staining • Pure culture methods • Quantitative methods

• Result reporting

Microbiology 2015 – Moving beyond PCR
• Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) – routinely identifying colonies of bacteria isolated from culture media or organisms recovered from blood culture. – could be used as parallel and complementary devices rather than as independent systems – Assist in unmasking multiple resistance factors and allowing targeted therapy

Future Staffing
• Staffing mix: – Tech group: Will need fewer ―speciality‖ trained and more generalist. – Lab supervisor/directors will be skilled more in molecular methods and less in classical microbiology. – There will be more specialty-trained pathologists and fewer PhD microbiologists. – Those who will be more financially savvy, basing administrative decisions on cost-effectiveness and evidence-based medicine
• Speculations on Microbiology Lab of the future: CID. Vol 35. Ellen Jo Baron

Future skills in interpreting complex diagnostic test results
• Skills will be required as we transition from classical, culture-based methods to automated molecular assays • Knowledge of human microbiome and the interactions of difficult-to-culture organisms and microbial flora and disease – real time antibiogram! • Knowledge in the fields of genomics and proteomics • Learning of multiplexed, specimen-specific molecular microbiology assays available for point-ofcare testing

Communication Future : Digital world
• Use of real-time digital graphics image capture to send slide images to an expert at a distant site for interpretation. • Same image can be easily included in the laboratory report, which can be instantly accessible at any location by use of a handheld device with wireless internet connectivity. • Advances in IT will allow immediate and global access to laboratory results for all physicians treating a patient.

Opportunities at AKUH-K
• • • • Facilitating R&D: Wet lab – rental Clinical trails – testing for physicians & industry Diagnostic instrument –testing for validation for industry Reference Centre for Excellence for SE Asia • Supplier of proficiency material for S.E Asia • Environmental testing for Industry using molecular diagnostics • Infection Prevention & Control – antimicrobial stewardship) • Training centre for lab personnel: – Molecular diagnostics – Lab Administration & Management – Infection Prevention & Control – (Distance ED) • Others

Thank you

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