MCB 101: INTRODUCTION TO MICROBIOLOGY

https://drive.google.com/file/d/1dLUBiRpS03jB0nFtPpC3gUJwF3Iu0bW9/view?usp=drive
_link
 COURSE OUTLINE/CONTENTS

MCB 101: Introduction to Microbiology 2 credit units

The objective of the course is to introduce the students to Microbiology. History and development

of Microbiology, microorganisms as geochemical agents, the growth of microbiology in the

twentieth century, theory of the cause of disease, equipment used in microbiology, pure culture

and media, control of microorganisms, e-microbiology.

1
INTRODUCTION TO MICROBIOLOGY

Microbiology is the scientific discipline dedicated to the study of microscopic life forms, that is
organisms that cannot be seen without the help of either magnifying glass or microscope. Such
organisms are called Microbes. Microbes encompasses a diverse range of organisms, including:
• Prokaryotes: Single-celled organisms lacking a true nucleus and membrane-bound
organelles (e.g., bacteria and archaea).
• Eukaryotes: Single-celled or multicellular organisms with a membrane-bound nucleus
and other membrane-bound organelles (e.g., protists and fungi).
• Viruses: Acellular entities consisting of genetic material (DNA or RNA) enclosed in a
protein coat, which replicate only inside a host cell.

Microbiology investigates the morphology, physiology, genetics, ecology, and evolutionary

relationships of these microorganisms. It also explores the interactions of the organisms with each
other, with humans, and with the environment as a whole.

Microbiology and its Historical Milestones

Period Development Scientist(s) Impact

- Concept of
"invisible
Early animates" in some
Observations ancient cultures Laid the groundwork
Various cultures
(Pre-17th (e.g., Jainism in for future discoveries
Century) India) suggesting
the existence of
microorganisms

Hans and
- Invention of the Enabled the first
Zacharias
17th Century compound observations of
Janssen (Dutch
microscope microorganisms
lensmakers)

2
 - Observation of
Pioneered the study
"animalcules" Antonie van
of microorganisms
(microscopic Leeuwenhoek
and laid the
organisms) in pond (Dutch
foundation for
water and other tradesman)
microbiology
materials

- Development of
John Needham
the concept of Sparked debate about
18th Century (English
spontaneous the origin of life
naturalist)
generation

- Challenge to
Lazzaro Contributed to the
spontaneous
Spallanzani foundation of the
generation through
(Italian priest germ theory of
experiments with
and biologist) disease
boiled broth

Revolutionized food
Louis Pasteur preservation
- Development of
(French chemist techniques and laid
19th Century the fermentation
and the groundwork for
process
microbiologist) the germ theory of
disease

Revolutionized our
understanding of
disease by
demonstrating the
- Germ theory of
Louis Pasteur role of
disease
microorganisms in
fermentation,
spoilage, and some
diseases

3
 Marked a significant
- Development of
leap forward in food
pasteurization Louis Pasteur
safety and public
process
health

First successful
- Identification of
Robert Koch isolation and
the bacterium
(German identification of a
responsible for
physician) specific bacterium
anthrax
causing a disease

Established a set of
criteria to link
specific
- Development of
Robert Koch microorganisms to
Koch's postulates
specific diseases, a
cornerstone of
microbiology

Ushered in the era of

Alexander
- Discovery of antibiotics and
Early 20th Fleming
penicillin, the first revolutionized the
Century (Scottish
antibiotic treatment of
physician)
infectious diseases

Various
- Development of
scientists (e.g., Demonstrated the
the first vaccines
Emil von effectiveness of
against bacterial
Behring, vaccination in
and viral diseases
Shibasaburo preventing infectious
(e.g., tetanus,
Kitasato, Jonas diseases
diphtheria, polio)
Salk)

- Development of Max von Laue Paved the way for

X-ray (German future techniques in
crystallography physicist) structural biology to

4
 study
microorganisms

Fritz Reiche and Enabled

- Development of
Alois Hubert visualization of
dark-field
(German unstained live
microscopy
scientists) microorganisms

James Watson
Revolutionized our
and Francis
Mid-20th - Discovery of the understanding of
Crick (British
Century structure of DNA genetics and its role
and American
in microorganisms
scientists)

Provided high-
resolution imaging
- Development of Ernst Ruska of microorganisms,
electron (German enabling detailed
microscopy physicist) studies of their
structure and
function

Enabled controlled
growth of
- Invention of the Jacques Monod
microorganisms for
continuous culture (French
research and
technique biochemist)
industrial
applications

Revolutionized our
Stanley Cohen ability to manipulate
Late 20th - Development of and Herbert and clone genes,
Century & recombinant DNA Boyer leading to
Beyond technology (American advancements in
biochemists) vaccine
development,

5
 production of
therapeutic proteins,
and microbial
engineering

Revolutionized the
field of molecular
biology by enabling
rapid and sensitive
- Development of Kary Mullis
detection and
Polymerase Chain (American
amplification of
Reaction (PCR) biochemist)
DNA sequences,
leading to improved
diagnosis of
infectious diseases

Marked a significant
- Sequencing of milestone in
Frederick Sanger
the first complete genomics and
(British
microbial genome opened doors for
biochemist) and
(Haemophilus large-scale
his team
influenzae) sequencing of
microbial genomes

- Continued
Provided even
advancements in
greater resolution
microscopy
and detail for
techniques (e.g., Various
studying
confocal scientists
microorganisms and
microscopy,
their interactions
atomic force
with other organisms
microscopy)

- Growing Unveiling the

understanding of Various complex
the human scientists communities of
microbiome and its microorganisms

6
 impact on health inhabiting the human
and disease body and their
potential roles in
various health
conditions

- Development of
new antimicrobial Crucial for
agents and maintaining public
strategies to Ongoing health and
combat emerging research addressing the
infectious diseases challenges of drug-
and antibiotic resistant microbes
resistance

COMMON EQUIPMENT USED IN MICROBIOLOGY

The microscopic world of microbes might seem invisible to the naked eyes, but it holds immense
power and significance. In this part of the lecture, we will embark on a journey to explore the
essential equipment and instruments that unlock the secrets of this fascinating realm.
These tools are the foundation of every microbiology laboratory, allowing use to:
i. Observe the diverse shapes and sizes of microorganisms using powerful microscopes.
ii. Grow and cultivate microbes in controlled environments to study their behaviour and
characteristics.
iii. Analyze their properties and interactions with other organisms.
iv. Maintain sterility to prevent contamination and ensure the accuracy of our
experiments.
By the end of this session, you will be familiar with the workhorses of the microbiology laboratory
and understand how they contribute to our ever-growing knowledge of the microbial world.

Essential Equipment in the Microbiology Laboratory: A Deep Dive

Let us take a look at each of the common equipment used in the microbiology laboratory and
explore their functionalities and applications:

7
1. Basic Laboratory Supplies

These form the foundation of a safe and well-organized laboratory environment.

• Safety Equipment
o Lab coat: This garment acts as a barrier, protecting your clothes from splashes,
spills, and accidental contact with microorganisms.
o Safety glasses: These shield your eyes from splashes, aerosols, and flying debris
during experiments.
o Gloves: Depending on the experiment, you might use different types of gloves
(e.g., latex, nitrile). They protect your hands from contact with potentially
hazardous materials and prevent contamination of samples.
o Biohazard waste container: This designated container is used for safe disposal
of materials that have come into contact with microorganisms or biological
materials.
• Bunsen burner (becoming less common due to safety concerns): This burner provides an
open flame historically used for sterilization procedures. In modern labs, alternatives like
autoclaves are often preferred due to safety concerns.
• Marking Pencils: Used for labelling glassware, test tubes, and other containers to ensure
proper identification and organization.
• Parafilm: This sealing film is a lifesaver in the laboratory. It creates an airtight seal on
plates and containers, preventing contamination and ensuring the integrity of your
experiment.
• Microscope slides and coverslips: These are the workhorses for microscopy. A smear of
your sample is placed on a slide and covered with a coverslip for examination under a
microscope.
• Forceps and Dissecting Scissors: These tools come in handy for handling and
manipulating specimens during dissections or transferring samples to different containers.
• Test tubes and Racks: These versatile containers are used for holding, storing, mixing,
and transporting liquid cultures and solutions. Test tube racks keep them organized and
upright.

8
 • Graduated cylinders and pipettes: Accurate measurement and transfer of liquids are
crucial in microbiology. Graduated cylinders measure volume, while pipettes allow for
precise transfer of small volumes.
• Markers: Permanent markers are used for labelling containers with essential information
like the contents and date.
2. Microbiology-Specific Equipment

These tools are specifically designed to work with the unique challenges and requirements of
studying microorganisms.

• Petri dishes: These shallow, lidded dishes are the battlegrounds for microbial growth.
They contain solidified agar medium, which provides nutrients for microorganisms to grow
and form visible colonies.
• Inoculating loops and needles: These sterilized tools are used for transferring
microorganisms to culture media with precision. Loops are small circles of wire, while
needles are fine points for more delicate work.
• Spreaders: These L-shaped rods are used to gently spread microorganisms evenly across
the surface of an agar plate to obtain isolated colonies for further study.
• Anaerobic jars: Some microorganisms require an oxygen-free environment for growth.
These jars create an anaerobic atmosphere by removing oxygen and replacing it with inert
gasses like nitrogen.
• Colony counter: After incubation, agar plates will often be teeming with colonies. This
handheld device helps with the tedious but crucial task of accurately counting the number
of colonies on a plate.
• Incubators: These temperature-controlled chambers provide the optimal environment for
microbial growth. Different microorganisms have specific temperature requirements for
optimal growth and function.
• Centrifuge: This workhorse separates components in a suspension based on their size
and density through spinning. It is commonly used to separate cells from culture medium
or to pellet down bacterial cells for further analysis.

9
 • Haemocytometer: This specialized counting chamber allows for the accurate counting
and measurement of cells in a suspension. It's particularly useful for determining cell
concentration in cultures.
• Autoclave: This essential piece of equipment ensures sterility. It uses high-pressure
steam to kill all microorganisms, spores, and viruses on equipment and materials.
3. Advanced Instrumentation

These sophisticated instruments delve deeper into the world of microorganisms, allowing for
detailed analysis.

• Microscopes:
o Bright-field microscope: The workhorse of microscopy, it uses light and stains
to provide magnified views of microorganisms.
o Dark-field microscope: This microscope allows visualization of unstained live
microorganisms by illuminating them from the side, creating a bright image against
a dark background.
o Phase-contrast microscope: This technique offers enhanced contrast for
observing transparent cells, which can be difficult to visualize under bright-field
microscopy.
• Spectrophotometer: This instrument measures the concentration of a substance in a
solution by analysing how much light it absorbs at specific wavelengths. It's used to
quantify cellular components or measure microbial growth.
• Gel electrophoresis: This technique separates biomolecules (DNA, RNA, proteins)
based on their size and electrical properties. A gel acts as a sieving matrix, with smaller
molecules traveling farther through the gel than larger ones under an electric current. This
allows researchers to analyse the genetic makeup or protein profile of microorganisms.
• PCR (Polymerase Chain Reaction) machine: This ingenious invention amplifies
specific DNA sequences, creating millions of copies from a tiny sample. This allows for
the detection of specific pathogens or identification of microorganisms based on their
unique DNA sequences.

10
 • DNA sequencers: These powerful machines determine the order of nucleotides (the
building blocks of DNA) in a DNA molecule. This allows researchers to identify
microorganisms, study their genomes, and understand their genetic makeup in detail.
Additional Equipment:
• Magnetic stirrer: This device provides a controlled stirring action for mixing solutions.
It's particularly useful for keeping cultures homogenous or facilitating reactions requiring
constant mixing.
• Water bath: This provides a constant temperature environment for specific experiments.
Some experiments require incubation at a specific temperature outside the range of a
standard incubator. A water bath can precisely maintain that temperature.
• Shaking incubator: This specialized incubator combines temperature control with a
shaking mechanism. It's used for culturing microorganisms that require aeration or to keep
them in suspension during growth.
• Biosafety cabinet: This ventilated workspace provides a physical barrier and air
filtration system to protect researchers from handling hazardous biological materials. It's
essential for working with pathogens or other potentially risky microorganisms.
• Anaerobic chamber: While anaerobic jars can create an anaerobic environment for
limited use, an anaerobic chamber provides a larger workspace for manipulating
microorganisms in an oxygen-free environment for extended periods.
• Flow cytometer: This instrument analyzes the physical and chemical characteristics of
individual cells in a suspension. It can measure parameters like size, fluorescence, and
granularity, allowing researchers to distinguish different cell populations or assess cell
viability.
• Confocal microscope: This advanced microscopy technique provides high-resolution,
three-dimensional images of a sample. It allows researchers to visualize the spatial
distribution of structures within a cell or observe microorganisms in their natural
environment.

Understanding the functionalities and applications of this diverse array of equipment and
instruments, microbiologists can unlock the secrets of the microbial world. These tools enable us
to diagnose diseases, develop new drugs, understand ecological processes, and explore the vast

11
potential of microorganisms for various applications. As we continue our exploration of
microbiology, you will gain a deeper appreciation for the ingenuity and power of these tools in
shaping our understanding of the invisible world of microbes.

Assignment:
Following the exact order the common microbiological equipment and instruments are listed,
generate a compendium of the images of the equipment and instruments.

12
PURE CULTURE AND MEDIA
Microorganisms are commonly found in soil, air, water, food, sewage and even on our
body surfaces. In short, every area of our environment is filled with them because they exist as a
mixture of many other cell types.
However, the microbiologist attempts to separate these mixed populations into individual
species for study. Therefore, a particular strain of organism growing in a laboratory medium is
called a pure culture.

Figure 1: Laboratory apparatus and culture techniques

How to Obtain a Pure Culture

To obtain a pure culture of an organism means to isolate identical species of the same cells.
Therefore, specific culture techniques, media and laboratory equipment are needed for the process
of isolation and study of microorganisms.
During the process of obtaining a pure culture, a process known as sub-culturing is
required. To maintain and study the cultural, morphological and biochemical properties of pure
cultures, microorganisms must be transferred from one medium to another a process known as
sub-culturing. Sub-culturing is important because it ensures that only the desired kind of
microorganism is allowed to grow.

13
 LABORATORY EQUIPMENT
Sterilising Equipment
i. Autoclave: An autoclave is a double-walled metal vessel that utilizes free-flowing steam and
is used for killing or removing viable microorganisms from growth media or apparatus. The
process of using autoclave for sterilizing is known as autoclaving. The standard laboratory
working condition for autoclave is at a pressure of 15psi, a temperature of 121°C for 15
minutes.
ii. Bunsen burner: is a laboratory apparatus that produces heated flame which is used for
sterilizing glass and metal equipment such as wire loop.
iii. Culture tubes: culture tubes are sterilized tubes 10ml in volume used for preparing culture
slants and agar deep (medium) for subculturing or during cultivation of pure cultures. A sterile
environment is maintained in culture tubes by various types of closure caps. Historically, the
first type was a cotton plug developed by Schroeder von Dusch has now been replaced by a
more conventional sleeve-like cap made of stainless steel or heat-resistant plastics.
iv. Petri dish: a Petri dish in culture cultivation is a circular container employed in culture
cultivation as agar plates (media). It is manufactured in various sizes to meet various
experimental requirements; it is usually about 15mm in diameter. Petri dishes provide a larger
surface area for growth and cultivation of microorganisms. They consist of a bottom dish
portion that contains the medium and larger top portion that serves as a loose cover.
v. Wire loops and needles: these are transfer instruments made from inert metals (metals that
are not conductors of electricity or without active chemicals) such as nichrome or platinum
and are inserted into metal shafts that serve as handles. They are usually used in transferring
culturing on to the agar plates.
vi. Pipettes: is an instrument used for aseptic transfers. Pipettes are similar in function to straws;
that is they draw up liquids. They are made of glass or plastic drawn out to a tip at one end
with a mouth piece forming the other end. They are calibrated to deliver different volumes
depending on requirements.
vii. Water baths: is a piece of apparatus used to cultivate microorganisms. It provides a rapid and
uniform heat transfer to the culture vessel. The thermostatically controlled shaking water bath
adds agitation by providing increased aeration which results in acceleration of growth. The

14
 disadvantage of this instrument is that it can be used only for cultivation of organisms in a bath
medium.
viii. Incubator: an incubator is another piece of equipment employed in the cultivation of
microorganisms. It is used to maintain optimum temperature during the necessary growth
period. It resembles an oven and is thermostatically controlled so that temperature can be
varied depending on the requirements of specific microorganisms.
ix. Refrigerator: a refrigerator is used for wide variety of purposes such as maintenance and
storage of stock cultures between sub-culturing periods and storage of sterile media to prevent
dehydration.
The survival and continued growth of microorganisms depend on adequate supply of
nutrients and a favourable growth environment. A solution containing these nutrients is called a
culture medium. Basically, all culture media are liquid, semi-liquid or solid. A liquid medium
that lacks a solidifying agent is called a “broth medium.” A broth medium supplemented with a
solidifying agent is called an “agar” and can result in a solid or semi-solid medium depending on
the concentration of the agar added.
Agar is an extract of seaweed. It is a complex carbohydrate composed mainly of galactose
and is without any nutritional value. Agar serves as an excellent solidifying agent because it
liquefies at 100°C and solidifies at 40°C. A completely solid medium requires an agar
concentration of about 1.5 – 1.8%.
A solid medium has the advantage that it presents a hardened surface on which
microorganisms can be grown using specialized techniques for isolation of colonies. Each colony
is a cluster of cells that originates from the multiplication of a single cell and represents the growth
of a single species of microorganisms. Such a defined and well isolated colony is a pure culture.
When in its liquefied state, solid media can be placed in test tubes which are then allowed to cool
and harden in a slanted position producing “agar slant.” Agar slants are useful for maintaining
pure cultures. Similar tubes that are allowed to harden in an upright position are called “agar deep
tubes,” they are used primarily for the study of the gaseous requirement of microorganisms.
Liquefied agar poured into a Petri dish and allowed to solidify is known as agar plate. Agar plates
provide large surface areas for the isolation and study of microorganisms.

15
PURE CULTURE TECHNIQUES
The techniques commonly used for isolation of pure culture colonies requires that the
number of organisms in the inoculums be reduced. The resulting reduced population size ensures
that following inoculation, individual cells will be sufficiently far apart on the surface of the agar
medium to separate the different species.
There are three techniques that can be used to accomplish this necessary dilution:
a. Streak plate method
b. Spread plate method
c. Pour plate method
Streak Plate Method
This is a fast qualitative isolation method. It is an important dilution technique that involves
spreading a loopful of culture over the surface of an agar plate. It involves a four-way or quadrant
procedure which is the most common streak procedure used. Streak plate technique is used to grow
bacteria on a growth medium surface so that individual bacterial colonies are isolated and sampled.
Isolated colonies indicate a clone of cells, being derived from a single precursor cell.
Purpose of streak plate: the purpose of the streak plate is to obtain isolated colonies from an
inoculum by creating areas of increasing dilution on a single plate. Isolated colonies represent a
clone of cells, being derived from a single precursor cell. When culture media is inoculated using
a single isolated colony, the resulting culture grows from that single clone.
Theory: one bacterial cell will create a colony as it multiplies. The streak process is intended to
create a region where the bacteria are so diluted that when each bacterium touches the surface of
the agar, it is far enough away from other cells so that an isolated colony can develop. In this
manner, spreading an inoculum with multiple organisms will result in isolation of the different
organisms. This procedure is described below:

Figure 1: A quadrant (4-way) streak-plate technique of isolation of pure cultures

16
Figure 2: Four-way streak-plate inoculation with Serratia marcescens

Figure 3: Simplified diagram of streak-plate technique

17
Figure 3: A detailed diagrammatic presentation of streak-plate technique. (A) A loop is sterilized,
(B) a sample of cells is obtained from a mixed culture, and (C) streaked near one edge of the plate
of medium. (D) Successive streaks are performed, and the plate is incubated. (E) Well-isolated
and defined colonies illustrate a successful isolation

1. Flame the wire loop and cool it by touching an unused part of the agar close to the edge of
the plate. Then drag it several times across the surface of area 1 as shown in the diagram.
2. Re-flame and cool the loop, and turn the Petri dish 90°. Then touch the loop to a corner
near culture area 1 and drag it several times across the agar in area 2. The loop should never
enter area 1 again.
3. Re-flame and cool the loop again and turn the dish about 90°. Streak area 3 in the same
manner.
4. Without re-flaming the loop, again turn the dish and then drag the culture from a corner of
area 3 across area 4 using a wider streak. Do not let the touch any of the previously streaked
areas.
NOTE: The flaming of the loop at points 1, 2, and 3 is to dilute the culture so that fewer
organisms are streaked in each area resulting in the final desired separation.

18
Tutorial Question 2: Justify the need to streak a mixed sample over four areas on a culture
plate.

Spread Plate Method

In the spread plate technique, the diluted mixture of microorganisms is used. During
inoculation, the cells are spread over the surface of a solid agar medium with a sterile L-shaped
bent glass rod when the Petri dish is spun, usually on equipment known as “lazy Susan” turn table.
Principle of the spread plate: In natural habitats, bacteria usually grow together in populations
containing a number of species. In order to adequately study ad characterize an individual bacterial
species, one needs a pure culture. The spread plate technique is an easy, direct way of achieving
this result. In this technique, a small volume of dilute bacterial mixture containing 100 to 200 cells
or less is transferred to the centre of an agar plate and is spread evenly over the surface with a
sterile, L-shaped glass rod. The glass rod is normally sterilized by dipping in alcohol and flame to
burn off the alcohol. After incubation, some of the dispersed cells developed into isolated colonies.
A colony is a large number of bacterial cells on solid medium, which is visible to the naked eyes
as a discrete entity. In this procedure, one assumes that a colony is derived from one cell and
therefore represents a clone of a pure culture. After a well-isolated colony has been identified, it
can then be picked up and streaked onto a fresh medium to obtain a pure culture.
The step-by-step procedure is described below:
1. Place the bent glass rod into a beaker containing 95% ethyl alcohol to cover the lower bent
portion.
2. Place the nutrient agar plate on the turntable.
3. Using a sterile pipette, place a drop of sterile water on the centre of the plate followed by
a sterile loopful of the organism. Mix gently with the loop and replace the cover.
4. Remove the glass rood from the beaker and pass it through the blue Bunsen burner flame.
Make sure the bent portion of the rod is pointing downward to prevent the alcohol from
running down your arm.
5. Allow the alcohol to burn off the rod completely. Cool the rod for about 10 seconds.
6. Remove the Petri dish cover and spin the turn table.
7. While the turn table is spinning, lightly touch the sterile bent rod to the surface of the agar
and move it back and forth. This will spread the culture over the agar surface.

19
Figure 5: Spread plate technique
8. When the turn table comes to a stop, replace the cover,. Dip the rod in alcohol and flame
again.

Pour Plate Method

This technique requires a serial dilution of the mixed culture by means of a loop or pipette.
The dilution inoculum is then added to a molten agar medium in a Petri dish. The solution is then
mixed and allowed to solidify.
Procedure:
1. Prepare and/or dilute the sample.
2. Place an aliquot of the prepared sample in a labelled empty sterile plate.
3. Pour 15 ml of molten agar, cooled to 45°C, into the plate, swirl to mix well.
4. Let it cool to solidify (without disturbance).
5. Invert and incubate to develop colonies (24 – 48 hours).

20
Purpose of pour plate technique: the pour plate technique is used to determine the number of
isolated colonies per milliliter of a specimen. It has the advantage of not requiring previously
prepared plates and is often used to assay bacterial contamination of food-stuff. One disadvantage
of pour plate is that embedded colonies are much smaller than those which happen to be on the
surface. Also, obligate aerobes may grow poorly if deeply embedded in the agar.

Figure 6: Summary of the methods of isolating pure cultures

21
CONTROL OF MICROORGANISMS

Methods used to control the growth of microorganisms and their transmission of infectious
disease involve stopping the growth of the microorganism for a period of time, reducing the
number of microorganisms to a safe level, or destroying the microorganisms. The degree of
effectiveness depends on the number of microorganisms, the type of microorganism, their
physiological state, such as the stage of growth or formation of endospores, and the environment
in which they are growing (glassware, instruments, tissue, food, etc.).

Terms Related to Destruction of Microorganisms

1. Sterilization - the destruction of all microorganisms, including endospores, on an object

or in a material.
2. Disinfection - the destruction of pathogens, but not endospores, on an object or in a
material. The number of pathogens is reduced or growth is inhibited to a level that does not
produce disease. Theses chemical substances that kill or inhibit the growth of microbial
forms on non-living materials are called disinfectants.
3. Antisepsis - chemical disinfection of the skin, mucosal membranes, or other living tissues.
These chemical substances used on “living tissues” that kill or inhibit the growth of
microbes on living forms are called antiseptics.
4. Germicide ("cide" = kill) - a chemical agent that rapidly kills microorganisms.
Specific germicides include:
a. sporicide - kills spores
b. bactericide - kills bacteria
c. viricide - kills viruses
d. fungicide - kills fungi

TERMS RELATED TO SUPPRESSION OF MICROORGANISMS

1. Asepsis ("without infection") - the absence of pathogens from an object or area. Aseptic
techniques prevent the entry of pathogens into the body.
There are two types of asepsis:
a. Surgical asepsis - techniques designed to exclude all microorganisms. It prevents
infectious agents from reaching a wound. Prevention begins before the patient enters

22
 the operating room and includes removal of hair and cleansing and disinfecting the skin
with an antiseptic that removes skin oils and microorganisms from the area of the skin
to be opened. Substances include Betadine, Isodine, Ioprep, and Surgidine.
b. Medical asepsis - techniques designed to exclude microorganisms associated with
communicable diseases. It includes dust control, hand-washing, use of individualized
equipment and instruments, waste disposal, care of instruments, syringes, needles,~
thermometers, and dressings.
2. Sanitization - the reduction or removal of pathogens on inanimate objects by chemical or
mechanical cleansing.
3. Bacteriostasis - ("static" = halt) - bacterial growth and multiplication are inhibited, but the
bacteria are not killed.

Terms for Destruction or Suppression of Microorganisms

1. Antimicrobial - agents that destroy or suppress any microorganism.
2. Antibiotic - a product formed by microorganisms that destroy or suppress microorganisms.
3. Chemotherapeutic agents – these are chemical substances that destroy or inhibit the growth
of microorganisms inside living tissues.

PHYSICAL AGENTS
1. Heat
Heat is the most common, inexpensive, simplest, reliable, and effective method used to
destroy microorganisms. Heat denatures the proteins and enzymes of the microorganisms. Most
pathogens will be destroyed at temperatures between 50° and 70°C for a duration of 10 minutes,
except endospores which may survive 1 to 2 hours at 100°C.
The heat used in sterilization is either moist or dry heat.
a. moist heat
i. boiling - Bacteria, fungi, and many viruses are destroyed by boiling at 100°C for 10 to 30
minutes. Some viruses and endospores may require boiling for up to 20 hours. Sterilization
is accomplished by denaturing the proteins and enzymes of the microorganisms.
ii. steam under pressure - Water is heated under pressure which raises the temperature above
100°C which denatures proteins and amino acids. The most common device used is an
autoclave for sterilizing surgical bandages, instruments, media, and contaminated material.

23
 At a pressure of 15 pounds/square inch and a temperature of 121°C for 15 to 20 minutes it
will destroy microorganisms and endospores.
iii. pasteurization - It is mainly used in the food and dairy industries and it involves raising
the temperature high enough to destroy pathogens or inhibit their growth without affecting
the quality of the product.
b. dry heat
Dry heat penetrates substances more slowly than moist heat.
i. incineration - The burning of disposable substances in a chamber.
ii. direct flaming - Sterilization of inoculating loops, needles, and rims of test tubes with a
bunsen burner. Wire heated to a red glow is 100% effective.
iii. hot-air oven - Sterilization of glassware, test tubes, petri dishes, instruments, syringes, and
needles requires higher temperatures for a longer period of time than other methods. Most
endospores will be destroyed at 160-165°C for a period of 2 hours.
2. Cold
The effect of low temperature on microorganisms depends on the type of microorganism
and the intensity of the application. Temperature in a refrigerator ranges from 0-8°C and has a
bacteriostatic effect which reduces the metabolic rate of most organisms so that they cannot
reproduce or synthesize toxins.
Freezing at -20°C kills most bacteria, but some may survive in a frozen state.
3. Drying or desiccation
To grow and multiple, microorganisms require water. The removal of water by evaporation
or freeze-drying (solid to gas) inhibits growth and reproduction of microorganisms by inhibiting
enzymes. They may be viable for years so when water is made available they resume growth and
reproduction.
4. Ultra-violet radiation and ionizing radiation (x-rays and gamma rays)
Both types of radiation damage the DNA of microorganisms and denatures their proteins.
U-V radiation cannot penetrate materials such as glass, clothing, dirt, paper, or pus and is used to
kill microorganisms on surfaces. Germicidal lamps in operating rooms, nurseries, and
communicable disease wards reduce the number of bacteria in the air, but they do not sterilize.

24
5. Filtration
Filtration is the passage of material or liquids through a filter containing small pores that
retain microorganisms. Membrane filters of cellulose acetate are the most common, but gauze,
cotton, and paper serve as filters for air. Vaccines that require the presence of live viruses, such as
polio, are passed through filters which prevent bacteria from going through the pores. Air filters
are used in operating rooms to lower the number of airborne microorganisms.

CHEMICAL AGENTS
Chemical agents are used to control the growth of microorganisms on inanimate objects
and on living tissue. Most chemical agents reduce the number of microorganisms (disinfect), but
do not achieve sterility. Selection of a disinfectant depends on the mode of action, concentration
necessary, the type of microorganism, the number of microorganisms present, the type of material
to be disinfected, temperature, and pH.
Disinfectants are classified based on their chemical structure and activity.
1. Phenols (carbolic acid)
Phenol and derivatives called phenolics disrupt the plasma membrane, denature proteins,
and inactivate enzymes. Phenol is rarely used anymore because it causes skin irritation and has a
disagreeable odor.
Common phenolics are cresols such as that found in Lysol, and hexachlorophene which
is used as an antiseptic.
2. Alcohols
Ethanol and isopropanol are widely used as skin antiseptics, but they are not effective
against enveloped viruses or bacterial endospores. They effectively destroy bacteria and fungi by
disrupting the lipids in the plasma membrane resulting in lysis, and denatures proteins.
3. Surface-active agents (surfactants)
They include soaps and detergents that lower the surface-tension of liquid molecules and
make microorganisms accessible to other agents. The pH of soaps is usually alkaline which
destroys some bacteria. Detergents are more effective against gram-positive than gram-negative
bacteria and they disrupt the plasma membrane.

25
4. Halogens
The major halogens are iodine and chlorine. Iodine is an effective antiseptic for the skin
and superficial wounds. It destroys many kinds of bacteria, some endospores, fungi, and some
viruses. Iodine inhibits protein function in the microorganism. Chlorine is used as a disinfectant
and it is a strong oxidizing agent which affects functions of the microorganisms’ enzymes.
5. Ions of heavy metals
The major metal ions are mercury and silver, and they function in denaturing proteins and
enzymes. Both are used in combination with other substances as antiseptics.
6. Chlorhexidine
Chlorhexidine is used as an antiseptic on skin and mucosal membranes and functions in
disruption of the plasma membrane of gram-positive and gram-negative bacteria resulting in lysis.
7. Alkylating agents
Alkylating agents disrupt the structure of proteins and nucleic acids. Formaldehyde (0.2 -
0.4%) is used to inactivate viruses and toxins used in vaccines, it also destroys spores in bacteria
and fungi. Glutaraldehyde is less irritating and more effective than formaldehyde. It kills many
microorganisms, viruses, and endospores. Ethylene oxide is a gas used in a closed chamber to
sterilize materials. It kills all bacteria, including endospores. Betapropiolactone kills spores in
concentrations not much more than required for killing cells. The effect is rapid and it disappears
in a few hours. It is used to sterilize bone, cartilage, and artery grafts.

MODES OF ACTION OF AGENTS OF MICROBIAL CONTROL

The modes of action of different chemical and physical agents of control vary but they all produce
lethal effects to one or more cellular structures in order to cause cell death (microbicidal) or
inhibition of cell growth (microbistatic). Understanding the mode of action of the physical and
chemical agents of control is essential for their proper selection and application in microbial
control. The following are ways structures and function adversely suffer during the action of these
control agents:
1. Cell Wall Injury
The adverse effect of cell wall injury can occur in two ways:
a. Lyses of the cell wall will leave the wall-less cell called the protoplast susceptible to
osmotic damage creating a hypotonic environment which may cause lyses/burst of the
vulnerable protoplast.

26
 b. Certain agents inhibit cell wall synthesis a mandatory process of microbial cell
reproduction. Failure to synthesis a missing segment of the cell wall results in an
unprotected protoplast.
2. Disruption of Cell Membrane
This results in the lyses of the membrane that causes immediate cell death. Control agents
also affect the selective nature of membrane often leading to the complete disruption of
metabolic processes. As a result, there may be a loss of essential cellular molecule or
interference with the uptake of nutrients.
3. Denaturation of Cytoplasm
Certain control agents alter the colloidal state of cytoplasmic proteins. Denaturing
processes are responsible for enzyme inactivation and cellular death by irreversibly destroying
the molecular bonding of these proteins rendering them biologically inactive, a process known
as denaturation.
4. Inactivation of Cellular Enzymes
Control agents may inactivate enzymatic reactions competitively or non-competitively.
Non-competitive inhibition is irreversible a d occurs following the application of some agents
such as mercuric chloride (HgCl2) that results in the uncoiling of the protein molecule
rendering it biologically inactive. Competitive inhibition occurs when a natural substrate is
forced to compete for the active site of an enzyme surface with a chemically similar molecular
substrate. The chemical blocks the enzyme’s ability to create end products Competitive
inhibitions are reversible. The whole idea is that these chemical agents compete with
chemically similar substrates blocking enzyme’s ability to create end products.
5. Interference with the Structure and Function of the DNA Molecule
The DNA molecule is the control of the cell and also represents a cellular target area for
the destruction or inhibition. Some agents have an affinity for DNA and can cause breakage or
distortion of the molecule thereby interfering with its replication and role in protein synthesis.

27
 CONTROL OF MICROORGANISMS

PHYSICAL AGENTS MECHANICAL REMOVAL CHEMICAL AGENTS

METHODS

Heat Radiation
Gases Liquid
Filtration

Moist Ionising Non-ionising

Dry Sterilisation Disinfection On inanimate
objects
Boiling water, On animate
hot water, Ultra-Violet
light objects
Dry oven Pasteurisation Sterilisation
Stream under X-ray, cathode, Antisepsis
Incineration
pressure gamma
Disinfection Disinfection
Sterilisation Sterilisation
Sterilisation Disinfection
Sterilisation

Air Liquid

Decontamination Sterilisation

Figure 7: Microbial Control Methods

28