GOOD MORNING

Shilpa.kMicrobiology Tutor

AIMSRC

Introduction

OBJECTIVES

–To impart sufficient basic science of the medically important microbes to assist you in diagnosing infectious diseases.–To impart sufficient clinical knowledge to raise your index of suspicion for

infectious diseases.

WELCOME TO THE MICROBIAL WORLD

Microbiology• Microbiology is the ‘biology of microscopic

organisms ’

Medical microbiologyStudy of microbes that

cause disease in humans.

TYPES OF MICROBES

Microbes vary in shape, size and structure and are categorized into,

1. Eukaryotic2. ProkaryoticFungi and Parasites

Bacteria

A cellular (noncellular)

Viruses and prions are incapable of independent life

Medical Microbiology comprises and integrates the fields of immunology, bacteriology, virology, mycology, and parasitology, each of which has seen

considerable independent development in the past few decades

IMMUNOLOGY

BACTERIOLOGY

MYCOLOGY

VIROLOGY

PARASITOLOGY

HISTORY

Antonie Philips van Leeuwenhoek was a Dutch tradesman and

scientist. He is commonly known as

"the Father of Microbiology", and considered to be the first microbiologist

Louis Pasteur was a French chemist and microbiologist

renowned for his discoveries of the principles of

vaccination, microbial fermentation and

pasteurization.

 ”Father of Medical Microbiology"

German physician and microbiologist.

The founder of modern

bacteriology.

Robert Koch

• Isolation of Anthrax, Tuberculosis and cholera• Isolating pure culture on solid media• Koch's Postulates

When a microorganism was accepted as the causative agent of infectious disease

1. The organism must always be present, in every case of the disease.

2. The organism must be isolated and grown in pure culture.

3. Samples of the organism taken from pure culture must cause the same disease when inoculated into a healthy, susceptible animal in the laboratory.

4. The same organism must be isolated again from the inoculated animal.

• These postulates are still used today to confirm the cause of an infectious disease

• Medical Microbiology comprises and integrates the fields of immunology, bacteriology, virology, mycology, and parasitology, each of which has seen considerable independent development in the past few decades.

• The common bond between them is the focus on the causes of infectious diseases and on the reactions of the host to the pathogens. Although the advent of antibiotics and vaccines has certainly taken the dread out of many infectious diseases, the threat of infection is still a fact of life: New pathogens are constantly being discovered; strains of „old“ ones have developed resistance to antibiotics, making therapy more and more difficult; incurable infectious diseases (AIDS, rabies) are still with us.

Definitions and terms• Infection is a generic term used to indicate invasion

of the host by a microorganism.• Infection may be subclinical or asymptomatic when

the patient is unaware of the infection.• Clinical infection is associated with the presence of

overt signs and symptoms of disease.• The term ‘colonization’ should be restricted to the

presence of a microbe at an expected site.• A pathogen is a microbe that potentially can cause

harm, i.e. tissue damage. An• opportunistic pathogen is a microbe that causes

infection in patients with impaired immunity, e.g. fungal infections in cancer patients.

Terminology

• Bacteria, fungi, protozoa, and helminths are named according to the binomial Linnean system that uses genus and species, but viruses are not so named.

• For example, regarding the name of the well-known bacteria Escherichia coli, Escherichia is the genus and coli is the species name. Similarly, the name of the yeast Candida albicans consists of Candida as the genus and albicans as the species.

• But viruses typically have a single name such as poliovirus, measles virus, or rabies virus. Some viruses have names with two words such as herpes simplex virus, but those do not represent genus and species.

Clinical Microbiology

• Infection• Nosocomial Infections• Systemic Infections

General Bacteriology• Morphology and Physiology• Culture Media• Culture Method• Bacterial genetics• Antimicrobial Agents and resistance

Systemic BacteriologyPathogenic organismsGram Positive – Cocci , BacilliGram Negative – Cocci, BacilliSpirocheatesMycoplasma, Chlamydiae and Rickettssiae

ImmunologyMycologyVirologyParasitology

Medical Microbiology

MICROSCOPY

MICROSCOPY• Microscope invented by Antony Van Leeuwenhoek in

17th century.

• Required for the morphological study of micro-organisms.

• USES - to magnify the image. - to achieve maximum resolution. - to provide sufficient contrast for observation

Resolution : the extent to which details in the magnified object are maintained.

Resolving Power (RP) : the smallest distance bywhich 2 points can be separated and still be observed as 2 distinct / different points. RP (eye) – 200 µm RP (visible light) – 300 nm RP (electron microscope) – 0.1 nm

Types of Microscopes

• Optical or light microscope - Simple - Compound• Phase contrast microscope• Dark field (dark ground) microscope• Fluorescent microscope• Electron microscope

Optical (Light) Microscope

Optical (Light) Microscope

• Principle : when visible light passes through the specimen & then through a series of lenses, the light gets reflected in such a manner that it results in magnification of the organisms present in the specimen.

• Magnification = objective x eyepiece

• To achieve maximum resolution with 1000x magnification, oil immersion must be used

• Oil prevents dispersion of light after light passes through the specimen.

• Images produced have very little contrast, therefore dyes are used to stain the specimen.

Phase Contrast Microscope

• Improves the contrast

• Makes the structures within the cells evident that differ in their thickness or refractive index.

• Also, differences in the refractive index of cell & the surrounding medium make them clearly visible.

Phase Contrast Microscope-Principle

• When beams of light pass through the specimen, it is partially scattered by the microbial cells or cell structures. Scattering depends on the thickness/ refractive indices of various structures

• High refractive index - more scattering of light. A scattered light also loses its velocity when travelling through the object and is not in phase with the unaltered light. Therefore appears as dark spot whereas the unobstructed light appears as bright spot. These differences in the intensity provide light & dark contrast to the image.

Advantage : since staining is not involved, live organisms can be observed.

Dark Field (Ground) Microscope

• Specimens appear as bright images against a nearly black background.

• Dark field condenser with a central circular stop – does not allow light to directly fall on the specimen.

• Light passes only around the edges of the condenser.

• Light rays which hit the object in the specimen are deflected upwards into the objective for visualization, rest will not enter and give a dark background.

Spirochetes under dark ground illumination

Uses of Dark Field microscopy

• Very useful in finding extremely small, unstained and / or moving objects.

• Organelles like cilia, flagella, vacuoles and cell nuclei can be clearly seen.

Fluorescent Microscope• Microorganisms or tissue cells are stained with

dyes or compounds called fluorochromes.

• Examined under microscope with ultra – violet radiation instead of visible light.

• They convert light of shorter (UV) wavelength into visible light and so become luminous – Fluoresce.

• Wavelengths absorbed & emitted are specific for specific fluorochromes.

Fluorochromes

• Acridine orange : Orange• Auramine-Rhodamine : Yellow • Calcofluor white : White• Fluorescein Isothiocyanate (FITC) : Green

Modification of Fluorescent Microscope

• Immunofluorescence : Antibodies labeled with fluorochrome used to specifically stain a particular bacterial species.

• Uses of IF : viruses, direct examination of C.trachomatis, B.pertussis

Electron Microscope

• Invented by Knoll & Ruska in 1936.• Uses electrons in place of light.• Electrons are focussed by electromagnetic

field.• Image is formed on a fluorescent screen or is

taken on a photographic material.• Resolving power is 100,000 times more than

light microscope.

Types of EM• 2 types : Transmission EM ( TEM )

Scanning EM ( SEM )SEM allows the study of cell surfaces with greater contrast & higher resolution than TEM.

• Disadvantages : Only dead & dried objects can be examined, since the medium is vacuum.

Cell morphology is distorted.

• Uses : for viruses, microbes less than 0.1 to 0.2.

Any Queries??????

THANK YOU!