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

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

Benefits
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 (15 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 physicians 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
Grams 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 --- 0.01l or 0.001l 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 ionizationtime 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