MICROBIOLOGY THEORY NOTES

1
© 2006 by THE MICHENER INSTITUTE for Applied Health Sciences 222 St. Patrick Street
Toronto. Ontario, Canada
M5T IV4
This material has been prepared and developed by The Michener Institute for Applied Health Sciences.
Reproduction of any part of this material, written, audio, visual or electronic, in any form, without the written
consent of The Michener Institute is forbidden.
2
ACCESS & OPTIONS
MICROBIOLOGY THEORY PACKAGE

TABLE OF CONTENTS

022-I-MI Introduction to Microbiology

014-I-MI Introduction to the Bacterial Cell
027-I-MI Bacterial Classification
840-279 I On Theory of the Gram Stain
840-279-I Gram Stain Methodology
024-I-MI Overview of Culture Media
025-I-MI Introduction to Bacterial Physiology and Growth
840-266 Overview of Principle of Biochemical Tests for Bacterial Identification
063-I-MI Introduction to the Laboratory Identification of COCCI
026-I-MI Colonial Appearance of Bacteria
060-I-MI Staphylococcus
06J-I-MI Streptococci
062-I-MI Nisseria and Moraxella catarrhalis
870-039 Microbial Antigen Identification and Detection
064-I-MI Enterobacteriaceae
065-I-MI I introduction to the Laboratory Identification of the Enterobacteriaceae
065-I-MI-I Supplement API 20E System
067-I-MI Pseudomonas and Related Organisms
069-I-MI Laboratory Identification of Gram Negative Bacilli Not Enterobacteriaceae
068-I-MI Campylobacter, Vibrio, Aeromonas and Similar Organisms
066-I-MI Fastidious Gram-Negative Bacteria
840-828 Aerobic and Facultative Gram-Positive Rods
072-I-MI Processing and Set-Up of Anaerobic Specimens
073-I-MI Diagnostics and Reporting of Anaerobic Specimens
041-I-MI Candida Species
042-I-MI Cryptococcus NCCLS Reference Antibiotics

3
4
 DIVISION OF CONTINUING EDUCATION MICROBIOLOGY
022-1-MI
INTRODUCTION TO MICROBIOLOGY

Table Of Contents

PAGE

OBJECTIVES 1

WHAT IS MICROBIOLOGY? 3

SIZES OF MICROORGANISMS 3

CLASSIFICATION OF LIVING THINGS 4

BACTERIOLOGY 6

MYCOLOGY 8

PARASITIOLOGY 9

VIROLOGY 9

IMMUNOLOGY 10

MICROBIAL INTERACTIONS 10

TRANSMISSION OF MICROBES 11

CONTROL OF MICROORGANISMS 12
Sterilization 12
Disinfectants 12
Antiseptics 13
Antimicrobial Agents 13

5
022-1-MI -1- 5237CEPC

OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon
completion of this module. The self-assessment question will enable you to judge your understanding of
the maternal.

Upon completion of this module, the student should be able to:

1. List the four major disciplines of microbiology

2. Differentiate between the four groups of microorganisms with respect to size (in micrometers),
nuclear characteristics and by what method(s)they can be observed.

3. Briefly describe the system for naming living things, using bacteria as an example.

4. Define and differentiate eukaryotic and prokaryolic microorganisms.

5. For bacteriology
a) State two common shapes of bacterial cells
b) Name a specific stain used to differentiate most bacteria
c) Name a disease caused by bacteria

6. For fungi:
a) State the two forms for fungi
b) State whether they are UIl1cellular or muilticellular
c) Name a disease caused by fungi

7. For parasitology
a) Name one example each of unicellular and multi-cellular parasites
b) Name a disease caused by a parasite

8. For Virology
a) Describe the basic structure of a virus
b) State the environment needed for Viruses to grow
c) Name a disease caused by a virus

6
022-1-MI -2- 5237CEPC

7
 MICROBIOLOGY
022-1-MI
INTRODUCTION TO MICROBIOLOGY

8
MICROBIOLOGY
022·1o
Ml INTRODUCTION TO MICROBIOLOGY

9
022-1-MI -3- 5237CEPC

MICROBIOLOGY
022·1-M1
INTRODUCTION TO MICROBIOLOGY

WHAT IS MICROBIOLOGY?

Microbiology is the study of microscopic organism. There are four major groups of microorganisms winch
Impact humans, namely-bacteria, fungi, parasites, and Viruses. Certain members of these groups can cause
disease m humans and are termed pathogens. The study of human pathogens forms the core of clinical or
medical microbiology

Medical microbiology is composed of four major disciplines:

I. Bacteriology, the study of bacteria (singular bactarium)

2. Mycology, the study of fungi

3. Parasitology, the study of protozoa and metazoa (parasites)

4. Virology, the study of viruses

These groups of microorganisms vary considerably m structure and size. Bacteria are small unicellular
structures and must be observed under a light microscope. Skill With this instrument is a prerequisite for
bactecatrial study. Both fungi and parasites exist in unicellular or multicellular form. They may be studied, m
certain instances, With the naked eye (macroscopically) or With the microscope. Viruses, the smallest entities
studied, can only be observed with the electron microscope.

SIZES OF MICROORGANISMS

To fully appreciate the differences and similarities of microorganisms, you must be aware of the Importance
of organism Size.

Bacteria have been measured traditionally in microns (µ). However, the correct unit of measure m the metric
system is the micrometer (µ m). For reference, the common bacterium Staphylococcus aureus is

approximately one ~m m diameter. A light microscope with a magnification of 1000x is usually used for
examining bacteria.

Viruses are smaller than bacteria and are measured m nanometers (nm); a unit winch is 1000x smaller than
the micrometer. Since the light microscope is unable to resolve objects smaller than 200 nm (0.200 µm),
Viruses winch range m size from 10-300 run (0.010-0.300~) must be observed using the electron microscope.

10
022-1-MI -4- 5237CEPC

Limits of Resolution

The smallest size that each of the following 15 capable of observing:

 Human eye 0.2mm

 Light microscope 0.2 µ m
 Electron microscope 2.0 nm

For comparison, average sizes are given below. You should bear in mind that these sizes are rough guides and
there are variations in size within each population.
 Amoeba = 15 µm
 Yeast = 5 µm
 Bacteria= 1 µm l (1000 nm)
 Viruses = 0.1 µm ( 100 nm)

CLASSIFICATION OF LIVING THINGS

It is useful to see how microorganisms which cause disease are related to all other living organisms.

When living things are classified, most people think of plants and animals. Since plants and animals are
multicellular, their place in the scheme of living entities 15 easy to Visualization. However, the division line
between these two groups 15 often blurred when unicellular microscopic forms are considered.

Motile protozoans appear related to animals, whereas the chlorophyll-containing algae are plant-like. Where
does one put the unicellular fungi, such as yeasts, which are not easily recognizable as either plant or animal?
For one thing, all of these organisms are related In that they are eukaryotes. Eukaryotes have a "true nucleus",
i.e. a nucleus with a nuclear membrane, chromosomes, and mitotic apparatus for cell division. The questions
remain as to where the prokaryotes (bacteria and blue-green algae) belong. Prokaryotes have nuclear maternal
which Consists of a Single naked chromosome Without a nuclear membrane.

Five major kingdoms of living organisms s have been proposed. Table 1 outlines the relationships between
the five groups of life: plants, animals, protista, fungi and prokaryotes.

No kingdom has been assigned to Viruses since there is serious doubt about our ability to describe these
entitles as having life!

To facilitate our understanding of microorganisms, Scientists have given them a formal name according to the
binomial system of nomenclature. The genus name is applied to a number of related organisms. Micro-
organisms belonging to the same genus are then distinguished from each other with a species name. For
example, the common bacterium Eschenchria coli has as Its genus name Eschenchria and the specIes name,
coli. The complete name IS underlined (Eschenchria coli) when Witten or In Italics (Eschenchria coli when m
print. The first letter of the genus name 15 capitalized, while the first letter of the specific name 15 m lower
case.

11
022-1-MI -5- 5237CEPC

12
022-1-MI -6- 5237CEPC

BACTERIOLOGY

For bacteria to be easily Visible under the microscope, they must be stained. Many different stains are
available, but the most important one is the Gram's stain. The Gram stain helps m differentiating bacteria.
Bacteria subjected to Gram staining usually stain either blue or pink (red). Those staining blue are classified
as gram-positive, whereas those staining pink are classified as gram-negative.

In addition to the Gram stain reaction, bacteria can be categorized according to their cellular morphology
(shape) and arrangement.

Most bacteria fall into one of four basic morphologies: spherical cells or cocci, rod-shaped cells or bacilli,
spiral-shaped cells or spirilla, and comma-shaped cells or vibrios. Most bacteria encountered in the climcal
laboratory are either coccal or bacillary m shape (Figures I and 2).

Bacteria that occur as spherical gram-positive COCCI m clusters are often members of the Staphylococcus
genus. Spherical gram-positive COCCI that are found In short or long chains are charlatanistic of members of
the genera Streptococcus or Enterococcus. Sometimes COCCI may become slightly elongated as seen In
some Streptococcus species. One Streptococcus species, Streptococcus pneumoniae, is known for Its elliptical
or "lanceolate-shaped" COCCI that occur m pairs (diplococci). Gram-negative kidney bean-shaped cocci in
pairs (diplococci) are characteristic of members of the genus Nelsseria.

Bacilli may be long with square ends (i.e., Bacillus species) or short with rounded ends (i.e., Haemophilus
species). If the organisms are so short as to appear coccoid, they are referred to as coccobacillary

13
P 022-1-MI -7- 5237CEPC

14
022-1-MI -8- 5237CEPC

Preliminary Identification of bacteria may also be made by growing them on nutritive substrates called
culture media (singular: medium). As a bacterium grows, It forms a colony or a mass of bacterial cells winch
is Visible macroscopically (Figure 3). The Size, shape, colour, and texture of the colonial contribute to the
Identification of the bacterium. Tins macroscopic appearance is often called a bacterium's colonial
morphology Once a bacterium has grown enough on a culture medium, It may be Identified using
biochemical tests.

MYCOLOGY

Medical mycology is the study of fungi winch can Infect humans. Fungal Infections may range from those
caused by unicellular yeasts (e.g. thrush) to those caused by multicellular molds (e.g. athlete's foot). Again,
specimens suspected of containing fungal agents are placed on one or more suitable culture media. Growth is
then Identified macroscopically, microscopically, and sometimes with the aid of physiologlca1 /biochemical
tests.

15
022-1-MI -9- 5237CEPC

PARASITOLOGY

Parasites vary markedly m Size from unicellular protozoan’s, which may cause diseases such as malaria and
amebiasis, to multlcellular helminthes, such as tapeworms and roundworms, which cause a number of
different diseases m humans (Figure 5). Microscopic techniques are primarily used for identification of
parasites.

VIROLOGY

Viruses are not cells. Viruses contain a central core of nucleic acid, either RNA or DNA, that is covered by a
protein coat (Figure 6). Because of this Simple structure, they are unable to live on their own and require a
living cell, either m the body or laboratory, in which to grow and reproduce. There is even controversy
whether a virus can be considered a living entity because of Its lack of biochemical complexity Some say that
Viruses are merely opportunistic groups of chemicals With bits of genetic informal On. Once the virus’s
genetic information enters a host cell, the Virus is able to take over the host's cell machinery As a result the
host cell makes many copies of the virus. Some Viral diseases include: polio, German measles, hepatitis,
common colds, influenza, rabies, measles, mumps, chickenpox and smallpox.

16
022-1-MI -10- 5237CEPC

IMMUNOLOGY

The study of the body's response to invasion by microorganisms is included m the field of Immunology.

When the body is invaded by a potentially pathogenic microorganism, an immune response is started. The
body attempts to protect itself, often by producing antibodies. The production of antibodies m the blood helps
to halt the disease process. If antibodies are already present through a previous contact with the pathogen or
through a prophylactic (protective) Immunization, they will prevent the microorganisms from gaining a
foothold.

It is frequently necessary to measure the level of antibodies m the body to determine If an active
Inaction is occurring or If an adequate level of immunity is being maintained. These tests are usually carried
out on serum, the straw-coloured, cell-free liquid portion of the blood which appears after clotting has
occurred. The sub-division of immunology which deals with the testing of serum for presence of antibodies is
termed serology An example of the common use of serological testing is the routine screening of blood for
evidence of hepatitis antibodies.

MICROBIAL INTERACTIONS

The interaction between a microbe (e.g. microorganism) and a host (e.g. human) IS a dynamic process m
which each participant acts to enhance Its Survival. There are a number of interactions which can be
described as follows:

Parasitism: When one organism lives on or m another organism (the host). The parasite benefits at the
expense of the host. The most successful parasite does not quickly destroy the host, unless It has evolved a
survival mechanism. The Survival mechanism IS usually a cyst stage that will infect another host The multi-
cellular tapeworm is an example of a well-adapted parasite that extracts nutrition from the intestinal tract of
the human host.

Commensalism: When an organism lives on or m a host without causing It harm. Non-pathogenic bacteria
found on normal healthy skin (normal flora) are commensals m that their presence causes no harm to the
host.

Symbiosis: When two organisms living together both benefit from the association, the relationship ia termed
symbiosis. For example, Lactobacilli m the vagina of humans produces acid end-products that keep the
vagina acidic. Vaginal acidity protects the female from the acquisition and growth of pathogenic organisms,
such as Neisseria gonorrhoeae. Thousands of bacterial species occupy the human landscape and contribute to
human health and Survival.

Normal flora: The human body is home to thousands of species of bacteria, fungi, and protozoa Not all of
these organisms are m place all of the time, but at anyone tlme the host will possess a spectrum of
microorganisms. The spectrum of resident flora that is common to most people is called normal flora.

Opportunistic: When an organism is normally non-pathogenic but has the capacity to produce disease m a
host, the organism is referred to as opportunistic. Often for this interaction to occur,

17
022-1-MI -11- 5237CEPC
The host's immune system IS weakened or the host has some other debilitating disease. Such persons are
frequently referred to as compromised hosts.

Infection: The successful multiplication of a microbe on or within a host, which mayor may not produce
disease, is called infection. In presence, however, an infection is considered to be present when invading
organisms elicit symptoms of disease from the host.

Colonization: Colonization is Said to occur when the microbe occupies the host Without development of
disease. Colonization does not necessarily lead to infection.

Pathogenicity: The ability of a microorganism to cause disease is called pathogenicity

Virulence: The term Virulence is a measure of the disease-producing capability or pathogenicity of a

microorganism. Certain microorganisms have extensive disease-podiuing weapons or Virulence factors,
which make them formidable pathogens (e.g. the agents of plague, cholera, and tuberculosis). Virulence
factors include: adherence structures like fimbriae that allow the organism to colonize, enzymes that
inactivate antibiotics, and a protective coating or capsule, which Inhibits the neutralizing effects of white
blood cells.

Host defenses: A number of barriers and biochemical mechanisms combine to form the host defense system.
Intact skin, ciliated cells in the respiratory and alimentary tracts, antibacterial substances in tears (lysozyme)
and mechanical flushing constitute the primary defense system; protects the host from invasion and
colonization by microorganisms. The normal bacterial flora of the host also prevents the colonization of
pathogens, by competing for growth resources or by producing antibacterial substances (e.g. bacteriocins).
Secondary host defenses include: complement, lysozyme, and antibodies; the phagocytic action of
neutrophils and macrophages; and cell-mediated immune responses (e.g. killer T lymphocytes).

Saprophytes: Free-liVIng organisms found m soil, water or m dead or decaying matter are called saprophytes.
They do not usually cause disease.

TRANSMISSION OF MICROBES

The movement of microorganisms from a source to a susceptible host is termed transmission. The
transmission of infectious agents IS central to the study of epidemiology. Epidemiology is the study of the
occurrence; spread and etiology (cause) of disease m a specific human population (e.g. hospital or
community). Environmental bacteriology i.e. the study of bacteria m food, water, milk, and sewage aIds the
public health epidemiologist m the control of communicable diseases.

An epidemic is the Simultaneous occurrence in a human population of a larger-than-expected number of

cases of a specific disease. A pandemic is an epidemic that has spread world-Wide. For example, an influenza
epidemic may develop into a pandemic (e.g. the influenza outbreak after World War I).
An endemic disease, on the other hand, is one which is in a population at all times but occurs m only a small
number of cases. Individuals who harbor a pathogenic organism Without clinical symptoms of disease are
called carriers.

Some infections arise when members of the normal flora at one particular body site gain access to a sterile
body site. The source of these infections is Said to be endogenous or from within the host. For example,
lower genitourinary flora may ascend the urinary tract and Cause an infection of the bladder.

18
022-1-MI -12- 5237CEPC
Other infections occur when a microorganism from outside the host, an exogenous organism gains entry.
These organisms enter the host via one of three routes: the gastrointestinal tract (ingestion), the respiratory
tract (inhalation), or breaks in the skin. For example, the source of pulmonary tuberculosis is usually another
infected patient and the mode of transmission is Vla inhalation of infectious droplets. There are many
Infection Control strategies to disrupt the transmission of bactria.

Zoonoses are diseases caused by infectious agents which originate from animals (e.g. plague, brucellosis and
rabies).

CONTROL OF MICROORGANISMS

An infection, which develops while a patient is in a health care facility, is called a nosocomial infection.
Nosocomial infections are of primary concern to all health care providers m acute and long-term care
facilities. An Infection Control Committee devises and Implements policies that will halt the transmission of
microorganisms Within their facility The microbiology laboratory is an essential component of this multi-
disciplinary group. The laboratory must provide reliable Identification and susceptibility test results for
nosocomial agents.

All front-line health care workers must be aware of the basics of Infection Control (some of which are
discussed below) m order to protect both themselves and patients.

Sterilization

Sterilization is the complete destruction of all living forms of microorganisms. Chemicals, moist heat under
pressure, radiation or filtration are all methods that maybe used for sterilization. One of the fastest and most
efficient methods involves the use of an autoclave. The principle of operation of the autoclave is similar to
that of a pressure cooker: microorganisms are destroyed by steam under pressure.

Disinfection

Disinfection is the process of killing or destroying most microorganisms on inanimate objects.

Disinfectants are not capable of killing all bacterial spores. One common disinfectant, sodium
hypochlorite or household bleach, IS used to disinfect bench tops and floors.
022-1-MI -13- 5237CEPC
Antisepsis

Antisepsis is the eradication or inhibition of micro-orgasnis ( With the exception of bacterial spores) on living
tissue using special chemical agents. For example, surgeons often apply an alcohol-based Iodine antiseptic to
skin prior to surgery to reduce the chances of a post-surgical infection.

Aseptic technique is a term which describes a number of work practices which create and maintain sterility or
prevent the contamination of controlled culture systems. Students should refer to module 880-058 for aseptic
techniques used m the transfer of microbial cultures.

Antimicrobial Agents
Chemotherapeutic agents are chemicals (e.g. sulphonamides) which are used to kill or inhibit the growth of
pathogenic organisms within the body Ideally these agents cause no (or minimal) harm to the
patient.Antibiotics are substances produced by organisms which prevent the growth of other organisms. The
mold Penicillium notatum produces penicillin which has been very potent antibiotic versus many pathogens
for years.
19