CHAPTER

The Science
1 of Microbiology

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define Microbiology and give the importance of the study of Microbiology;
2. name important persons with significant contributions to the field of Microbiology;
3. differentiate the various types of microscopes and their uses;
4. compare the various staining methods used to visualize microorganisms; and
5. classify the different types of culture media based on their physical state, chemical
composition, and functional type.

Microbiology is derived from the Greek words mikros (“small”), bios (“life”), and logia or
logos (“study of”). It is therefore the study of organisms that are so small they cannot be seen
with the naked eye. These organisms are called microorganisms or microbes and are categorized
into two: (1) cellular, which may either be prokaryotes (bacteria, cyanobacteria, and archeans) or
eukaryotes (fungi, protozoa, and algae); and (2) acellular, which includes viruses. Microbiology
is further classified into different fields of study, namely: (1) bacteriology, the study of bacteria;
(2) virology, the study of viruses; (3) mycology, the study of fungi; (4) parasitology, the study
of protozoa and parasitic worms; (5) phycology, the study of algae; and, (6) immunology,
the study of the immune system and the immune response.
Why study microbiology? The study of microbiology is important for the following reasons:
1. Microbiology has an impact in the daily lives of humans. Microorganisms are
everywhere—in the air one breathes, in the environment, and even in one’s body.
About a thousand or more organisms inhabit the human body. These are collectively
called normal flora or indigenous flora which only produce disease in persons with
compromised immune systems
4 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Some microorganisms are essential in biotechnologyand a wide range of industries

which include food and beverage, pharmaceuticals, mining, genetics, and many more.
Much of the knowledge available in the study of genetics and biochemistry utilize
microorganisms as model organisms.
3. Some microorganisms, especially bacteria and fungi, are important sources of
antimicrobial agents. For example, penicillin was derived from the fungus Penicillium.
4. Some microorganisms act as saprophytes or decomposers of waste products and dead
organisms, making them essential in maintaining a balanced ecosystem.
5. The study of microorganisms has led to a better understanding of how
microorganisms produce disease, paving the way to better disease management and
control. This was further improved through the discovery of vaccines that helped
prevent sickness from infections diseases. By knowing the sources of disease producing
microbes, sanitation practices improved immensely, leading to better mitigation of
infectious diseases.
6. Certain diseases which were thought to have been eradicated are now re emerging.
Some have the potential as biological warfare agents. At the same time, there are now
a number of pathogens that are developing resistance to antibiotics. In this context,
the study of microbiology is relevant for better understanding of the negative instances
in which science can be used.

Evolution of Microbiology
Archaeologists and evolutionists have uncovered evidence demonstrating the existence of
primitive microorganisms. In Western Australia, as many as eleven different types of fossils of
primitive microorganisms have been found in ancient rock formations, dating back to as early as
3.5 billion years ago, long before the existence of animals and humans.
Infectious diseases have existed for thousands of years. In 3180 BC, an epidemic known
as the “plague” broke out in Egypt. In 1122 BC, an outbreak of a smallpox like disease that
originated in China spread worldwide. The exhumed mummified remains of Rameses V
showed skin lesions resembling smallpox.
In the mid 1600s, the microscope was discovered and with the use of this instrument,
Robert Hooke was able to discover the cell—the basic unit of living organisms. His discovery
heralded the cell theory that stated living organisms are made up of cells. Then in the 1670s,
Anton von Leeuwenhoek, a Dutch merchant, created a single lens microscope that he used
to make observations of microorganisms which he then called animalcules. Through his
observations, he became known as the “Father of Microbiology” and was the one who first
provided accurate descriptions of bacteria, protozoa, and fungi
 The Science of Microbiology

In the middle and late 1800s, Louis Pasteur performed countless experiments that led
to his germ theory of disease. He postulated that microorganisms were in the environment and
could cause infectious diseases. He also developed the process of pasteurization, which kills
microorganisms in different types of liquids, and which became the basis for aseptic techniques.
He also introduced the terms aerobes and anaerobes and developed the fermentation process.
Pasteur’s attempts to prove his germ theory of disease were unsuccessful. It took Robert
Koch to prove that microorganisms caused certain diseases through a series of scientific steps
which led to his formulation of the Koch’s postulates. This led to an increased effort by other
scientists to prove and illustrate further the germ theory that was initially formulated by
Louis Pasteur. Thus, the late 1800s and the first decade of the 1900s came to be known as the
Golden Age ofMicrobiology. Since then, numerous scientists have made significant contributions
to the field of Microbiology. Edward Jenner discovered the vaccine for smallpox. Joseph Lister
applied the theory to medical procedures paving the way for the development of aseptic surgery.
After World War II, antibiotics were introduced to the medical world. Paul Ehrlich
discovered Salvarsan for the treatment of syphilis. This drug was heralded the “magic bullet” of
chemotherapy, which is treatment of disease by using chemical substances. Alexander Fleming
discovered the antibiotic penicillin from the mold Penicillium notatum. With the discovery of
antibiotics, the incidence of infectious diseases like tuberculosis, pneumonia, meningitis, and
others was significantly reduced.
Most of the experiments conducted in the field of microbiology during the early 20th
century involved the study of bacteria. During this time scientists were not yet equipped
with advanced technology in their study of microorganisms. It was only in the 1930s when
the electron microscope was developed that experimentations in microbiology became more
complex. It was also during that time when viral culture was introduced paving the way for
rapid discoveries on viruses. The vast knowledge gained from the experiments performed by
microbiologists together with the discovery of other vaccines in the 1940s and 1950s have led to
better prevention and control of numerous potentially fatal infectious diseases.

Microscopy
Microorganisms are miniscule organisms that cannot be seen with the naked eye. The
discovery of the microscope has led to their close observation, allowing microbiologists and
other scientists to study them further.
A microscope is an optical instrument that can magnify organisms a hundredfold or even a
thousand fold. From the time of its initial discovery in the 1600s, the microscope has undergone
great revolutionary changes. Making it more advanced and complex throughout time. The
following are the different types of microscopes that have evolved from von Leeuwenhoek’s
simple prototype.
6 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Compound Microscope
The compound microscope is a type of microscope that contains more than one magnifying
lens. It can magnify objects approximately a thousand times their original size. Visible light is
its main source of illumination. As such, it is also known as the compound light microscope.
The compound microscope utilized today consists of two magnifying lens systems.
The eyepiece (or ocular) contains what is called the ocular lens that has a magnifying power
of 10x. The second lens system is located in the objective that is positioned directly above the
organism to be viewed.

Eyepiece (ocular lens)

Head
Diopter adjustment
Locking screw
Revolving nose piece
Arm
Objectives
Stage
Slide holder Coarse focus
Condenser
Fine focus
Iris diaphragm
Stage controls
Built in light source
Brightness adjustment
On/off switch Base
With built in light source

Eyepiece (ocular lens)

Body tube
Coarse focus
Fine focus
Revolving nose piece
Arm
Objectives
Stage clips
Stage
Condenser
Iris diaphragm

Mirror Base

With mirror to direct an external light source

Figure 1.1 Two compound light microscopes which differ in their light sourc
 The Science of Microbiology 7

Table 1.1 Components of the compound light microscope

Component Function
Ocular lens or Topmost part of the microscope which is the lens the viewer looks
eyepiece through to see the specimen.
Revolving nose piece Located above the stage, it holds the objective lenses.
Diopter adjustment It is used to change focus on one eyepiece in order to correct
any difference in vision between the two eyes.
Body tube or head It connects the eyepiece to the objective lenses.
Arm It connects the body tube to the base of the microscope.
Coarse adjustment It brings the specimen into general focus.
Fine adjustment It fine tunes the focus and increases the details of the specimen.
Objective lenses This is held in place above the stage by the revolving nosepiece and are
the lenses that are closest to the specimen. It contains 3 to 5 objectives
ranging in power from 4X to 100X.
Stage Located beneath the revolving nosepiece, it is the flat platform
on which the specimen is placed.
Stage clips Situated above the stage, these are metal clips that hold the slide
in place.
Stage control Found beneath the stage, these knobs move the stage either left
or right or forward and backward.
Aperture The hole in the middle of the stage that allows light from the illuminator
to reach the slide containing the specimen.
On/off switch The switch located at the base of the microscope that turns
the illuminator on or off.
Illuminator The light source of the microscope.
Iris diaphragm Found on the condenser, it is used to adjust the amount of light coming
through the condenser.
Condenser It is found beneath the stage and contains a lens system that focuses
light onto the specimen. It gathers and focuses light onto the specimen.
Base It supports the microscope and it is where the illuminator is found.

Brightfield Microscope
Made up of a series of lenses and utilizing visible light as its source of illumination, the
brightfield microscope can magnify an object 1,000 to 1,500 times. This is used to visualize
bacteria and fungi. Objects less than or thinner than 0.2 μm cannot be visualized by this type
of microscope. The term “brightfield” is derived from the fact that the specimen appears dark
against the surrounding bright viewer field of this microscope. However, it has very low contrast
and most of the cells need to be stained to be properly viewed
8 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Darkfield Microscope
This microscope utilizes reflected light instead of transmitted light, with a special
condenser that has an opaque disc that blocks the light, such that only the specimen is
illuminated. The specimen to be studied appears bright against a dark background. This type of
microscope is ideal for studying specimens that are unstained or transparent and absorb little or
no light. It is also useful in examining the external details of the specimen such as its outline or
surface. This type of microscope is used to view spirochetes.

Phase contrast Microscope

Phase contrast microscopy is based on the principle that differences in refractive
indices and light waves passing through transparent objects assume different phases.
This type of microscopy was first introduced by Frits Zernike, a Dutch physicist, in 1934.
The phase contrast microscope has a contrast enhancing optical technique in order to produce
high contrast images of specimens that are transparent which include thin tissue slices, living
cells in culture, and subcellular particles (such as nuclei and organelles).

Image Plane Digital

Camera
Diffracted System
Direct Light
Transmitted
(Surround) Observation Light
Light Biological
Microscope

Objective

Specimen
Phase
Plate
Condenser

Condenser
Annulus

Figure 1.2 Phase contrast microscope and its part

 The Science of Microbiology 9

Living Cells in Brightfield and Phase Contrast

Figure 1.3 a Appearance
of cells under brightfield
microscope where cells appear
semi transparent. The only
visible structures are the highly
refractive regions, such as
the membrane, nucleus, and
unattached cells (rounded or
spherical). b Same specimen
viewed using phase contrast
a b microscope showing significantly
more structural detail.

Differential Interference Contrast Microscope

The differential interference contrast microscope is similar to the phase contrast
microscope except that it utilizes two beams of light instead of one and therefore has higher
resolution. The resulting contrasting colors of the specimen being studied are due to the
prisms that split the light beam. It was developed by Georges Nomarski in 1952 as an
improvement to the phase contrast microscope. It is useful in examining living specimens
when normal biological processes might be inhibited by standard staining procedures. However,
the three dimensional image of the specimen produced may not be accurate since the enhanced
areas of light and shadow may distort the appearance of the image.

Fluorescence Microscope
The fluorescence microscope makes use of ultraviolet light and fluorescent dyes called
fluorochromes. The specimen under study fluoresces or appears to shine against a dark
background. Fluorescence microscopy is based on the principle that certain materials emit
energy that is detectable as visible light when they are irradiated with the light of a given
wavelength. It uses a higher intensity of light source and this in turn excites a fluorescent
species. The fluorescent species then emits a lower energy light of a longer wavelength which
produces the magnified image instead of the original light source. Fluorescence microscopy
can be used to visualize structural components of small specimens such as cells and to detect
the viability of cell populations. It may also be used to visualize the genetic material of the
cell (DNA and RNA)
10 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Confocal Microscope
Also known as the confocal laser scanning microscope (CLSM) or laser confocal scanning
microscope (LCSM), the confocal microscope uses an optical imaging technique that
increases optical resolution and contrast of the micrograph by using a spatial pin hole to
block out of focus light in image formation. The specimen is stained with a fluorescent dye
to make it emit or return light. The object is scanned with a laser into planes and regions. This
is used, together with computers, to produce a three dimensional image. It is also useful in the
study of cell physiology.

Electron Microscope
The electron microscope utilizes a beam of electrons to create an image of the specimen.
The electron beams serve as the source of illumination and magnets are used to focus the beam.
The first prototype of this microscope was built by the German Engineer Ernst Ruska in 1933,
which had a resolution power of up to 50 nm. Modern electron microscopes are capable of
magnifying objects up to 2 million times. It is used to visualize viruses and subcellular structures
of the cell. There are two types of electron microscopes—transmissionelectron microscope and
scanning electron microscope. The transmission electron microscope (TEM) is the original form of
the electron microscope. It produces two dimensional, black and white images, and magnifies
objects up to 200,000 times. The scanning electron microscope (SEM) relies on interactions at the
surface rather than transmission. It can magnify bulk samples with greater depth of view so that
the image produced represents the 3 D structure of the sample, but the image is still only black
and white. Generally, it can magnify the object 10,000 times.

Scanning Probe Microscope

The scanning probe microscope was developed in the 1980s by the Swiss scientists
Dr. Gerd Binnig and Dr. Heinrich Rohrer. It is used to study the molecular and atomic shapes
of organisms on a nanoscale. A physical probe is used to scan back and forth over the surface
of a sample. A computer then gathers data that are used to generate an image of the surface.
It can also be used to determine the variations in temperature inside the cell as well as its
chemical properties.

Staining
Most microorganisms besides being very tiny are also devoid of any color and are thus
difficult to see, even with the use of the microscope. To facilitate visualization, staining
procedures have been developed by various scientists. These staining procedures are meant to
give color to the organisms, making them easier to see under the microscope
 The Science of Microbiology 11

Simple Stains
Simple stains make use of a single dye which can either be aqueous (water based) or
alcohol based. This method of staining is a quick and easy way to visualize cell shape, size,
and arrangement of bacteria. It uses basic dyes such as safranin, methylene blue, or crystal violet.
These stains give up or accept hydrogen ion, leaving the stain positively charged. Most bacterial
cells and cytoplasm are negatively charged and since the dye is positively charged, it adheres
readily to the cell surface enabling the visualization of bacterial cell morphology.

a b

Figure 1.4 a Cocci in clusters and b Bacilli

Differential Stains
Differential stains are used to differentiate one group of bacteria from another. There are
two types of differential staining procedures commonly used, namely:
1. Gram stain – distinguishes gram positive bacteria from gram negative bacteria.
gram positive bacteria stain blue or purple, while gram negative bacteria stain red
or pink. As a general rule, all cocci are gram positive except Neisseria, Veilonella, and
Branhamella. On the other hand, all bacilli are gram negative except Corynebacterium,
Clostridium, Bacillus, and Mycobacterium.

Table 1.2 Reagents used in Gram staining and expected results

Reagent Function Result if Gram positive Result if Gram negative
Crystal violet Primary stain
Purple or blue Purple or blue
Gram’s iodine Mordant* Purple or blue Purple or blue
Acetone or 95% Decolorizer Purple or blue Colorless
alcohol
Safranin Counterstain or Purple or blue Red or pin
secondary stain
*A mordant enhances the uptake of the primary stain.
12 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Acid fast stain – stain used for bacteria with high lipid content in their cell wall,
hence cannot be stained using Gram stain. Two methods are used, namely:
a. Ziehl Neelsen stain – also known as the “hot method” because it requires steam
bathing the prepared smear after addition of the primary dye. This is because the
primary stain used is aqueous and will not bind to the cell wall of the organism.
Acid fast organisms will appear red on a blue background.
b. Kinyoun stain – also known as the “cold method” as it does not utilize
heat after addition of the primary stain, which is oil based. The acid fast
organisms will appear red on a green background.

Table 1.3 Reagents used in acid fast staining and the expected results
Reagent Result
Function
Ziehl Neelsen Kinyoun Acid fast Non acid fast
Carbol fuchsin Carbol fuchsin Primary stain Red or pink Red or pink
Acid alcohol Acid alcohol Decolorizer Red Colorless
Methylene Malachite Counterstain Ziehl Neelsen: Ziehl Neelsen:
blue green or secondary red organism/ blue organism/
stain blue background blue background
Kinyoun: red organism/ Kinyoun: green organism/
green background green background

Special Stains
These are used to demonstrate specific structures in a bacterial cell. For instance,
metachromatic granules can be visualized using the LAMB (Loeffler Alkaline Methylene Blue)
stain. Other special stains include Hiss stain (capsule or slime layer); Dyer stain (cell wall),
Fischer Conn stain (flagella), Dorner and Schaeffer Fulton stain (spores), and India ink
or nigrosine (capsule of the fungus Cryptococcus neoformans).

Capsule Staining
Capsules
Background Rods Flagella

a b

Figure 1.5 a Demonstration of the capsule using India ink and b flagella surrounding the
bacteria demonstrated using the Leifson method of stainin
 The Science of Microbiology 1

Culture Media
Staining procedures only give clues as to the probable organism being studied. To identify
a specific organism, culture using specific culture media is the most ideal. Media (sing. medium)
are used to grow microorganisms. A culture medium is basically an aqueous solution to which
all the necessary nutrients essential for the growth of organisms are added. These are classified
into three primary levels: physical state, chemical composition, and functional type.

According to Physical State

1. Liquid media – commonly called broths, milk, or infusions, these are water based solutions
that do not solidify at temperatures above the freezing point. These contain specific
amounts of nutrients but do not contain gelling agents such as gelatin or agar. Liquid
media are suited for the propagation of a large number of organisms, fermentation
studies, and other tests.
2. Semi solid media – exhibit a clot like consistency at ordinary room temperature and
contain agar at concentrations of 0.5% or less that allows thickening of the media without
producing a firm substance. They have a soft consistency similar to custard and are best
suited for culture of microaerophilic bacteria or for the study of bacterial motility.
3. Solid media – contain a solidifying agent such as 1.5%–2% agar, giving them a
firm surface on which cells can form discrete colonies. They are used for isolation
of bacteria and fungi or for determining the colony characteristics of the organism
under study. Solid media come in two forms: (a) liquefiable (or reversible) solid
media and (b) non liquefiable (or non reversible) solid media.

According to Chemical Composition

1. Synthetic media – contain chemically defined substances which are pure organic and/or
inorganic compounds. The precise chemical composition of a synthetic medium is known.
They may be simple or complex, depending on what supplement is added to it.
2. Non synthetic media – complex media that contain at least one ingredient that is not
chemically defined, which means that it is neither a simple or pure compound. It is not
representable by an exact chemical formula. Most are extracts of animals, plants, or yeasts.
Non synthetic media can support the growth of more fastidious organisms.
14 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

According to Functional Type

1. General Purpose media – are designed for primary isolation of a broad spectrum
of microbes and contain a mixture of nutrients that support the growth of both
pathogenic and non pathogenic organisms. Examples are peptone water, nutrient broth,
and nutrient agar.
2. Enrichment media – contain complex organic substances such as blood, serum, or special
growth factors, and are designed to increase the number of desired microorganisms
without stimulating the rest of the bacterial population. These are used to grow
fastidious or nutritionally exacting bacteria. There are two commonly used enrichment
media, namely:
a. Blood agar – contains general nutrients with 5%–10% (by volume) blood added
to a blood agar base. Certain gram positive bacteria produce exotoxins that cause
hemolysis of red blood cells contained in the blood agar. Their hemolytic reaction
is categorized into three, which is useful in the classification of these bacteria.
The hemolytic patterns are:
i. Beta hemolysis – shows complete lysis of red blood cells resulting in complete
clearing around the colonies.
ii. Alpha hemolysis – shows incomplete lysis of red blood cells, producing a
greenish discoloration of the blood agar around the colonies.
iii. Gamma hemolysis – shows no hemolysis, resulting in no change in the
medium.

a b c

Figure 1.6 Three types of hemolytic reactions seen in the culture: a beta hemolysis or complete
hemolysis; b alpha hemolysis or incomplete hemolysis; and c gamma hemolysis or no hemolysis

b. Chocolate agar – a type of nutrient medium that is used for the culture of fastidious
organisms such as Haemophilus sp. Heat is applied to lyse the red blood cells, causing
the medium to turn brown
 The Science of Microbiology 1

3. Selective media – contain one or more substances that encourage the growth of only a
specific target microorganism and inhibit the growth of others. It is designed to prevent
the growth of unwanted contaminating bacteria or commensals so only the target bacteria
will grow. Examples of approaches that will make the medium selective include changing
the pH of the culture medium or adding substances such as antibiotics, dyes, or other
chemicals. These are usually agar based solid media that allow isolation of individual
bacterial colonies. Examples of this type of culture medium include the following:
a. Thayer Martin agar – contains the antibiotics trimethroprim, nystatin, vancomycin,
and colistin. It is used for the isolation of Neisseria.
b. Mannitol Salt agar – contains 10% NaCl and used for the isolation of Staphylococcus
aureus.

c. MacConkey’s agar – promotes the growth of gram negative bacteria, primarily

those belonging to the family Enterobacteriaceae, and inhibits the growth of gram
positive bacteria through the addition of bile salts. It is both selective and differential.
d. Löwenstein Jensen medium – a selective medium used to recover Mycobacterium
tuberculosis. It is made selective by the incorporation of malachite green.

e. Saboraud’s dextrose agar – used for the isolation of fungi.

4. Differential media – allow the growth of several types of microorganisms. These are
designed to show visible differences among certain groups of microorganisms. The
differences may be in the form of variations in colony size or color, changes in color of
culture media, or formation of precipitates or gas bubbles. Differential media allow the
growth of more than one target microorganism that demonstrate morphologic variations
in colony morphology. Examples include MacConkey’s agar and Triple Sugar Iron agar.
5. Transport media – used for clinical specimens that need to be transported to the
laboratory immediately after collection. These media prevent the drying of specimen
and inhibit the overgrowth of commensals and contaminating organisms. Charcoal
is added to neutralize inhibitory factors. Examples are the Cary Blair transport medium
for transport of feces of suspected cholera patients and Pike’s medium which is used to
transport throat specimens of patients with streptococcal infection.
6. Anaerobic media – media used specifically for organisms that cannot survive in the
presence of oxygen and require reduced oxidation reduction potential and other nutrients.
These are supplemented with nutrients such as vitamin K and hemin. They undergo
boiling to remove dissolved oxygen. To reduce the oxidation reduction potential,
substances such as 1% glucose, 0.1% ascorbic acid, 0.1% thioglycolate, or 0.05% cysteine
are added. Methylene blue or resazurin is added as an indicator of the oxidation
reduction potential. Examples are chopped cooked meat and thioglycolate broth.
16 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Microbiology is the study of small, living microorganisms or microbes that cannot be seen
with the naked eye. These organisms may be cellular (prokaryotes, eukaryotes, and the
like) or acellular such as viruses.

• Microbiology is divided into several fields that deal with the study of bacteria
(bacteriology), viruses (virology), fungi (mycology), protozoa and parasitic worms
(parasitology), algae (phycology), and the immune system (immunology).

• Microorganisms may be beneficial or harmful. Some microorganisms are used in

different industries such as in food and beverage. Some microorganisms are sources of
antibiotics while some are used in the field of biotechnology and genetic engineering.
Microorganisms are also important in maintaining a balanced ecosystem.

• While some microorganisms are essential and have beneficial uses, there are also
numerous microorganisms that produce disease in humans, some of which are
potentially fatal.

• Some microorganisms have the potential to be used as biological warfare agents.

• Microorganisms are so miniscule that for them to be visualized, they need to be
stained and studied using the microscope. Several types of microscopes have been
developed for this purpose—from the compound microscope to the more sophisticated
electron microscopes.

• The use of various staining procedures has made visualization of microorganisms easier.
These stains may be classified into simple, differential, and special stains.
› Simple stains make use of a single water or alcohol based dye that is used to
demonstrate the shape and basic structures of the organism.
› Differential stains are used to distinguish one group of bacteria from another group.
These include the Gram stain and the acid fast stain.
› Special stains are mainly used to demonstrate specific bacterial structures such as the
spores (Dorner or Schaeffer Fulton), flagella (Fischer & Conn), capsule (Hiss
stain),
or the metachromatic granules (LAMB stain).

• Specific culture media are the most ideal in identifying specific organisms. Several
classes of culture media have been developed and these culture media can be classified
into three primary levels: physical state (liquid, semi solid, solid), chemical composition
(synthetic and non synthetic), and functional type (general purpose, enrichment, selective,
differential, transport, and anaerobic)
 CHAPTER
Prokaryotic and
2 Eukaryotic Cells

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. differentiate prokaryotes from eukaryotes; and
2. characterize the different medically important microorganisms.

Living Cells can be classified into two general categories—prokaryotesand eukaryotes.

Prokaryotes are organisms that do not possess a true nucleus and membrane bound
organelles (e.g., bacteria). Eukaryotic organisms are those that possess a true nucleus and
membrane bound organelles. They are usually multicellular organisms and include plants,
animals, fungi, parasites, and algae. Viruses are acellular organisms that possess only DNA or
RNA. They are dependent on host cells for their replication and are considered as obligate
intracellular parasites
20 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Comparison between Prokaryotes and Eukaryotes

1–10 μm
10–100 μm

chloroplast

mitochondrion

circle of DNA
nucleus linear DNA

Typical Prokaryotic Cell Typical Eukaryotic Cell

Figure 2.1 Diagrammatic representation showing the difference between a prokaryotic cell and a
eukaryotic cell

Table 2.1 Comparison between prokaryotic and eukaryotic cells

Feature Prokaryotic Eukaryotic
Genetic material Not enclosed within a membrane; Enclosed within a membrane;
not associated with histones; usually associated with histones; usually
circular linear
Size Smaller (1–2 μm by 1–4 μm or less) Greater than 5 μm in diameter
Cell type Mostly unicellular Mostly multicellular
Nucleus No true nucleus and nuclear With true nucleus enclosed by
membrane; called nucleoid nuclear membrane
Cell wall Simple Complex
Cell division Budding or binary fission Mitosis
Sexual No meiosis; transfer of DNA only Meiosis
reproduction
Cytoskeleton Absent Present
Mesosome Functions as mitochondria and Golgi Absen
complex
 Prokaryotic and Eukaryotic Cells 21

Feature Prokaryotic Eukaryotic

Ribosomes 70S; located in cytoplasm 80S; located in membranes such
as in the endoplasmic reticulum
70S; found in organelles such as
mitochondria or chloroplast
Membrane bound Absent Present
organelles
Extrachromosomal Present Absent
plasmid
Duration of cell Short (20–60 minutes) Long (12–24 hours)
cycle
Adapted from [Link] prokaryotic and eukaryotic cells

Medically Important Microorganisms

Organisms that are considered medically important are those that have the potential or the
ability to produce significant clinical disease in humans. They may be part of the normal flora
of the body or are true pathogenic organisms. These may be categorized into bacteria, viruses,
fungi, algae, and parasites (protozoa and helminths).
Viruses are acellular organisms. Their outer surface is called capsid, which is composed of
repeating sub units called capsomeres. Viruses possess only a single nucleic acid, either DNA
or RNA, but never both. In addition, viruses lack the necessary cellular parts that can allow
them to replicate independent of the host cell. They also lack the genes and enzymes that are
necessary for energy production. They rely on the cellular machinery of the host cell for protein
and energy production. Hence, viruses are considered obligate intracellular parasites.
Viruses are classified based on the following: (1) type of nucleic acid they possess;
(2) shape of the capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres;
(4) size of the capsid; (5) presence or absence of an envelope; (6) type of host they infect
(humans, plants, or animals); (7) type of disease they produce; (8) target cell or tropism
(e.g., T helper cells for HIV); and (9) immunologic or antigenic properties
22 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Herpesvirus Orf virus

Vaccinia virus Paramyxovirus (mumps)

Adenovirus

Rhabdovirus
T even coliphage Influenza virus
Flexuous
tailed phage

Polyomavirus Picornavirus ΦX174 phage Tubulovirus

1 μm

Figure 2.2 Diagrammatic representation of various forms and sizes of viruses

Bacteriophages are a special type of viruses that primarily infect bacteria. They are similar
to other viruses in that: (1) they are obligate intracellular parasites; (2) they are similarly
shaped like other viruses; and (3) they may also be classified based on the type of nucleic
acid they possess. They play a role in the acquisition of virulence factors of certain bacteria
(e.g., diphtheria toxin of Corynebacterium diphtheriae), as well as in the transfer of genetic
material from one bacterium to another (as in transduction).
Bacteria are prokaryotic cells with majority having an outer covering called the
cell wall that is composed mainly of peptidoglycan. Unlike viruses, they possess both
DNA and RNA. Unlike eukaryotic organisms, bacteria possess a nucleoid instead
of a true nucleus, smaller ribosomes, and lack mitochondria. Based on their physical
characteristics, bacteria may be broadly categorized into (1) gram negative bacteria with
cell wall (e.g., Escherichia coli); (2) gram positive bacteria with cell wall (e.g., Staphylococcus
aureus); (3) acid fast bacteria with lipid rich cell wall (e.g., Mycobacterium tuberculosis);
and, (4) bacteria without cell wall (e.g., Mycoplasma)
 Prokaryotic and Eukaryotic Cells 2
Fungi are eukaryotic cells with an outer surface composed mainly of chitin. Their cell
membrane is made up mostly of ergosterol. Like bacteria, fungi possess both DNA and
RNA. Unlike bacteria, they possess a true nucleus that is enclosed by a nuclear membrane
and mitochondria that function for ATP production. Fungal ribosomes are also larger than
bacterial ribosomes (80 Svedberg units). Table 2.2 summarizes the major differences between
fungi and bacteria.
Protozoa are the representatives for parasites. Like bacteria and fungi, these are also
eukaryotic cells that have an outer surface called a pellicle. These are unicellular organisms that
usually divide through binary fission, similar to bacteria. Majority exist in two morphologic
forms—cysts and trophozoites. The infective stage is the cyst while the pathogenic stage is the
trophozoite. Protozoa possess both DNA and RNA as well as other cellular features seen in
typical eukaryotic cells.

Table 2.2 Comparison between fungi and bacteria

Features Bacteria Fungi
Cell type Prokaryotic; unicellular Eukaryotic; unicellular or
multicellular
Role in ecosystem Can be both producers and Mainly decomposers
decomposers
Optimal pH Neutral pH (6.5–7.0) Slightly acidic (4.0–6.0)
Cell structures No true nucleus and membrane Possess true nucleus and
bound organelles membrane bound organelles
Main component Peptidoglycan, except in Chitin
of cell wall archaebacteria
Sterols in cell Absent except in Mycoplasma Present
membrane
Mode of nutrition Heterotrophic, chemoautotrophic, Heterotrophic; majority aerobic
photoautotrophic, aerobic, and facultative anaerobic
anaerobic, facultative anaerobic
Reproduction Binary fission Sexual and asexual spores
Adapted from [Link] between bacteria [Link]

Algae are eukaryotic organisms whose outer surface consists primarily of cellulose. They are
described as plant like organisms because most of them have chlorophyll and are thus capable
of photosynthesis. Unlike plants, they do not possess true roots, stems, and leaves. Table 2.3
summarizes the major differences between algae and plants. Algae vary in size from the single
celled phytoplanktons to the large seaweeds found in the ocean floor.
Algae do not produce significant disease in humans. Most algae are beneficial in that they
are important sources of food, iodine, and other minerals. They may also be used as fertilizers,
emulsifiers for puddings, and stabilizers for ice cream and salad dressings.
24 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Table 2.3 Comparison between algae and plants

Features Algae Plants
Taxonomic classification Kingdom Protista Kingdom Plantae
Cellular structure Unicellular, multicellular or Multicellular
colony forming
Photosynthetic Yes Yes
Energy source Carbon dioxide Carbon dioxide
Storage form of energy Starch Starch
Vascular system* Absent Present
Habitat Mostly water Mostly rooted to the ground
Composed of roots, stems, No Yes
and leaves
Method of reproduction Both asexual and sexual Sexual (complex)
*Allow for dispersion of nutrients throughout the entire plant

Diatoms are unicellular algae that inhabit both fresh and saltwater. Their cell
wall contains silicone dioxide that may be utilized in filtration systems, insulation, and
as abrasives. Dinoflagellates are also unicellular algae that are important members of the
phytoplankton group. They contribute greatly to the oxygen in the atmosphere and serve
as important links in the food chain. On the other hand, they are also responsible for what
is known as “red tide.” These small organisms produce a powerful neurotoxin which, when
ingested in significant amounts, is responsible for the potentially fatal disease called paralytic
shellfish poisoning.

Cyanobacteria Diatom Dinoflagellate Green algae Coccolithophore

Figure 2.3 Various structures of phytoplanktons that are usually found floating on wate
 Prokaryotic and Eukaryotic Cells 25

CHAPTER SUMMARY

• Living cells can be classified as either prokaryotic or eukaryotic.

• Prokaryotic cells, as exemplified by bacteria, are usually unicellular, do not possess a true
nucleus and membrane bound organelles, and multiply by means of binary fission.

• Eukaryotic cells vary from unicellular (e.g., protozoa) to multicellular (e.g., fungi). They
possess a true nucleus surrounded by a nuclear membrane as well as membrane bound
organelles.

• Viruses are not classified as cells since they only possess an outer covering called capsid
and a nucleic acid (either DNA or RNA). As such, they are dependent on the host cell
machinery for their replication and are thus considered as obligate intracellular parasites.

• Medically important organisms are those which produce significant disease in humans.
These may take the form of viruses, bacteria, fungi, protozoa, and algae.

› Viruses
and may
are acellular, obligate intracellular parasites possessing only DNA or RNA
be classified based on: (1) type of nucleic acid they possess; (2) shape of the
capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres; (4) size
of the capsid; (5) presence or absence of an envelope; (6) type of host they infect
(humans, plants, or animals); (7) type of disease they produce; (8) target cell or tropism
(e.g., T helper cells for HIV); and (9) immunologic or antigenic properties.

› Bacteria are prokaryotic organisms that possess both DNA and RNA. Most possess a
of
cell wall composed predominantly peptidoglycan.

› Fungi are eukaryotic organisms with a cell wall composed mainly of chitin and cell
membrane that contains ergosterol.
› Protozoa are mostly unicellular parasites that are eukaryotic. Most divide by binary
fission similar to bacteria.

› Algae are eukaryotic, aquatic, plant like organisms. Similar to plants, they are
photosynthetic but unlike plants, they do not have true roots, stems, or leaves
 CHAPTER
Bacterial
3 Morphology

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. distinguish among the various general shapes of bacteria, citing examples for each; and
2. compare the external and internal structures of gram positive, gram negative and
acid fast bacteria.

Bacteria, which are prokaryotic, have simpler structures compared to eukaryotic organisms.
In terms of morphology, bacteria may be classified into three basic shapes: coccus (pl. cocci),
bacillus (pl. bacilli), and spiral shaped or curved. Cocci can be described as spherical or round
shaped organisms (e.g., Staphylococcus, Streptococcus). They may be arranged singly, in pairs
(diplococci), in chains (streptococci), in clusters (staphylococci), in groups of four (tetrad),
or in groups of eight (octad). Rod shaped organisms are called bacilli (e.g., Escherichia
coli, Salmonella). Some may be very short, resembling elongated cocci called coccobacilli
(e.g., Haemophilus influenzae). Curved and spiral shaped organisms may show variations in
their morphology. Vibrio cholerae, the organism causing cholera, is described as comma shaped.
The causative agent of syphilis, Treponema pallidum, is spiral in shape while the causative agent
of diphtheria, Corynebacterium diphtheriae, is club shaped
30 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Fundamental Shapes of Bacteria

Flagella

Pseudomonas Salmonella typhi

Pneumococci Streptococci

Treponema
Spores

Mycobacterium Clostridium
tuberculosis tetani
Staphylococci Leptospira

Spheres (Cocci) Rods (Bacilli) Spirals (Spirochetes)

Figure 3.1 Fundamental shapes of bacteria

Envelope Structures
Prokaryotic cells are surrounded by a complex envelope that may vary in composition.
The envelope serves to protect the bacteria from harsh environmental conditions.

Glycocalyx
This is the outermost covering of some bacteria. It is a gelatinous substance that is located
external to the cell wall, composed of polysaccharide or polypeptide, or both. It is called capsule
if it is strongly attached to the cell wall and slime layer if it is loosely attached. The presence of
the capsule is indicative of the virulence of an organism, aiding the organism in the evasion
of phagocytosis. It can stimulate an antibody response from the immune system. The capsule
serves to protect the organism from dehydration.

Cell Wall
The bacterial cell wall is sometimes called the murein sacculus. Its principal component is
peptidoglycan, which is also called murein or mucopeptide. It is multi layered in gram positive
bacteria and single layered in gram negative bacteria. The cell wall provides rigid support and
gives shape to the bacteria. It protects the bacteria from osmotic damage and plays an important
role in cell division
 Bacterial Morphology 31

Special components of gram positive cell walls

1. Teichoic acids – comprise major surface antigens of gram positive organisms and can
elicit antibody response. In some gram positive organisms such as Staphylococcus aureus,
teichoic acids function for the attachment of the organism to the host cell. These also
provide tensile strength to gram positive bacterial cell walls.
2. Polysaccharides – polysaccharide molecules include neutral sugars such as mannose,
arabinose, rhamnose, and glucosamine. It also includes some acidic sugars such as
glucuronic acid and mannuronic acid.

Teichoic acid
Wall associated protein

Lipoteichoic
acid

Peptidoglycan

Cytoplasmic
membrane

Figure 3.2 Diagrammatic representation of a typical gram positive bacterial cell wall

Special components of gram negative cell walls

1. Outer membrane – a bi layered structure where the inner leaflet is composed of a
lipopolysaccharide (LPS). It has special protein channels that allow the passage of
small or low molecular weight hydrophilic substances such as sugars and amino acids.
LPS has a complex glycolipid called lipid A, responsible for its endotoxin activity. It is
located in the outer leaflet of the outer membrane. The inner core is a polysaccharide
made up of repeat units. This repeat unit is also called the O antigen, which is unique for
every species of bacteria.
2. Lipoprotein – functions to anchor the outer membrane to the peptidoglycan layer and
stabilizes the outer membrane.
3. Periplasmic space – a fluid filled space between the outer membrane and the
inner plasma membrane. It contains enzymes for the breakdown of large non
transportable molecules into transportable ones and enzymes that serve to detoxify and
inactivate antibiotics
 32 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Lipoteichoic acid
Teichoic acid Porin
O specific side chains
Lipopolysaccharide
Outer membrane
Peptidoglycan
Broun's lipoprotein
Periplasmic space Peptidoglycan
Periplasmic space
Plasma membrane Plasma membrane
and integral proteins and integral proteins
Gram (+) cell wall Gram (–) cell wall
Figure 3.3 A comparison between gram positive and gram negative cell walls showing the
differences in their constituents

Acid fast cell wall

Unlike gram positive and gram negative bacteria, acid fast organisms such as
Mycobacterium tuberculosis possess an outer layer that is lipid rich. The cell wall of acid fast
organisms is composed of large amounts of waxes that are known as mycolic acids. The inner
layer of the cell wall is also made up of peptidoglycan but because the outermost layer is lipid
rich, cell walls of acid fast organisms are hydrophobic. This is the reason why they cannot be
stained using the reagents used in gram staining. The hydrophobic nature of their cell wall
protects them from harsh chemicals such as strong acids and detergents.

LAM
Lipoteichoic acid Glycolipid
LPS Mycolic acid
Lipoprotein Porin

PeptidoglycanPeptidoglycan Peptidoglycan Galactan

Mannophosphoinositide
Gram positive Bacteria Gram negative Bacteria Mycobacteria
Figure 3.4 Schematic representation comparing gram positive, gram negative, and acid fast
cell wal
 Bacterial Morphology 33

Projecting Structures
Flagella
These are thread like structures made up entirely of molecules of the protein sub unit
flagellin. They project from the capsule and are organs for motility. Flagella are classified into
four types, namely: (a) monotrichous (single polar flagellum); (b) lophotrichous (a tuft of flagella
at one end of the bacterium); (c) amphitrichous (flagella at both ends of the bacterium); and
(d) peritrichous (flagella all around the bacterium). Bacteria without flagella are called atrichous.

a b

c d

Figure 3.5 Typical arrangement of bacterial flagella. a Peritrichous, b monotrichous and polar,
c lophotrichous and polar, and d amphitrichous and polar.

Pili or Fimbriae
These are rigid surface appendages found on many gram negative bacteria. They are fine
and short in comparison with flagella. Their structural protein sub units are called pilins.
Pili may also function for motility. They function for adherence to cell surface (common pili)
or attachment to another bacterium during a form of bacterial gene exchange called
conjugation (sex pili).

Axial Filaments
Axial filaments are also called endoflagella and are found in spirochetes (e.g., Treponema
pallidum causing syphilis). These are composed of bundles of fibrils, the structures of which
are similar to flagella. They arise from the ends of the bacterial cell and spiral around the cell.
The filaments rotate producing movement of the outer sheath of the spirochetes propelling
them forward
34 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Cytoplasmic Membrane
Also called cell membrane or plasma membrane, the cytoplasmic membrane is located beneath
the cell wall. It is sometimes called the cell sac because it encloses the cytoplasm of the cell.
The cytoplasmic membrane is a selectively permeable membrane that allows for transport of
selected solutes. In aerobic organisms, it is the site of the electron transport chain and serves as
the site of ATP production. It therefore serves the function of the mitochondria, which are not
found in prokaryotic cells. The cytoplasmic membrane also contains the enzymes needed for
the biosynthesis of DNA, cell wall components, and membrane lipids.

Internal Structures
Nucleoid
Bacteria have no true nucleus that is surrounded by a nuclear membrane. Its genetic
material is packaged in a structure called the nucleoid. Bacteria possess a single, circular, double
stranded DNA.

Mesosomes
The mesosome functions for cell division. It is also involved in the secretion of substances
produced by bacteria.

Ribosomes
The ribosomes function for protein synthesis. Unlike eukaryotic ribosomes, bacterial
ribosome is smaller (70S).

Granules or Inclusion Bodies

These are found in certain bacteria and serve for storage of food and energy
(e.g., metachromatic granules of Corynebacterium diphtheriae or Much granules of
Mycobacterium tuberculosis)
 Bacterial Morphology 35

Endospores
Endospores are structures produced by many bacteria when they are placed in a hostile
environment. It is composed of dipicolinic acid which confers resistance to heat, drying, chemical
agents, and radiation; making it very difficult to destroy. The process of spore production
is called sporulation, and this occurs when the environmental conditions are detrimental to
the bacteria. When environmental conditions become favorable, the endospores revert to
their vegetative state through a process called germination. Some gram positive, but never
gram negative, bacteria form spores.
Pilus

Capsule
Cytoplasm
Inclusion Ribosomes
Cell wall
Capsule Plasma
membrane
Nucleoid
Cell wall containing DNA
Plasmid
Plasma
membrane

Fimbriae

Flagella

Figure 3.6 Parts of a typical prokaryotic cell

a b c

Figure 3.7 Spores showing a terminal and b central location, c as well as metachromatic
granules of Corynebacterium diphtheria
36 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• There are three basic shapes of bacteria: (a) spherical or cocci; (b) rod shaped or bacilli;
and (c) curved or spiral.

• Awithtypical prokaryotic cell is composed of three major components—an outer envelope

its projecting structures, the cell membrane, and the internal structures.

• The envelope is composed of the following:

» The outermost covering is the glycocalyx, also known as the capsule if it is adherent to
the cell wall and slime layer when it is loosely attached to the cell wall.
» The cell wall or the murein sacculus provides rigid support and shape to the bacteria.
Its main component is peptidoglycan, which is multilayered in gram positive bacteria
and monolayered in gram negative bacteria.
› Gram positive cell wall contains teichoic acids which may function for the
attachment of the bacterium to the host cell, as well as polysaccharide molecules.
› Gram negative cell walls contain lipopolysaccharide made of a lipid A molecule
A is
and polysaccharides. The lipid component responsible for the endotoxic
activity of gram negative bacteria. The lipopolysaccharide is an integral part of the
outer membrane of gram negative bacteria. Gram negative bacteria also have a
periplasmic space where important enzymes are found.
› Acid fast organisms possess a cell wall that is also made up of an inner layer of
peptidoglycan and an outer layer rich in waxes composed of mycolic acid and
other lipids. This is responsible for the hydrophobic nature of its cell wall and
the main reason why acid fast organisms cannot be stained using the reagents
for Gram staining.

• Structures projecting from the bacterial capsule include pili or fimbriae of gram negative
organisms, flagella, and axial filaments of spirochetes.
» There are two types of pili: common pili which functions for attachment and sex pili
which participates in gene exchange among bacteria in a process called conjugation.
» Flagella may be of four patterns: (1) lophotrichous (a tuft of flagella on one end of
the bacterium), (2) amphitrichous (a single flagellum on each end of the bacterium),
(3) peritrichous (flagella surrounding the bacterium), and (4) monotrichous (only one
flagellum at one end of the bacterium).
» Axial filaments are similar in structure to flagella and help propel the spirochetes
forward
 Bacterial Morphology 3

• Bacterial cytoplasmic membrane is the functional analogue of the mitochondria. It is

selectively permeable and is the site of ATP production of aerobic bacteria.

• Bacteria do not have a true nucleus. Its genetic material is packaged in a structure called
nucleoid. Bacterial ribosome is smaller than a typical eukaryotic ribosome.

• Bacteria possess structures that enable them to withstand adverse environmental

conditions. These structures are the endospores which are mainly composed of dipicolinic
acid.

• Other structures found in bacterial cells are the mesosomes, which play a role in cell
division, and inclusion bodies or granules in some bacteria which serve as storage for food.
 CHAPTER
Bacterial Growth
4 Requirements

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define microbial growth;
2. discuss the various nutritional and physical requirements of bacteria for growth; and
3. illustrate the bacterial growth curve with explanation of the events occurring in each
phase of the bacterial growth curve.

Growth as defined in medical dictionaries involves an orderly and organized increase in

the sum of all components of the organism. The process entails the replication of all cellular
structures, organelles, and components. Microbial growth is concerned with the increase in the
number of cells and not an increase in the size of the organism. A bacterial colony is composed
of thousands of cells; hence, colonies in culture are actually composed of billions of cells. As
in any living organism, bacteria require certain nutrients and physical conditions that will
promote their growth. This chapter discusses the various nutritional and physical requirements
of bacteria for growth.

Nutritional Requirements
Carbon
Carbon makes up the structural backbone or skeleton of all organic molecules. Based
on their carbon source, microorganisms may be classified into autotrophs (lithotrophs) and
heterotrophs (organotrophs)
42 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Autotrophs are microorganisms that utilize inorganic compounds (e.g., carbon dioxide) and
inorganic salts as their sole carbon source. Organotrophs are organisms that make use of organic
substances like sugars or glucose as their carbon source. For both autotrophs and heterotrophs,
their energy may be derived from either light (photolithotrophs and photoorganotrophs) or the
oxidation of inorganic substances (chemolithotrophs and chemoorganotrophs).Most medically
important bacteria are chemoorganotrophs.

Nitrogen, Sulfur, Phosphorus

These are necessary for the synthesis of cellular materials like proteins and nucleic acids.
Nitrogen and sulfur are required for the synthesis of proteins. Nitrogen and phosphorus are
essential for the synthesis of nucleic acids and ATP. Approximately 14% of the dry weight
of a bacterial cell is nitrogen and about 4% is sulfur and phosphorus.

Inorganic Ions
These include magnesium, potassium, calcium, iron, and trace elements (e.g., manganese,
zinc, copper, cobalt). Magnesium stabilizes ribosomes, cell membranes, and nucleic acids.
It also serves as a co factor in the activity of many enzymes. Potassium is required for the
normal functioning and integrity of ribosomes and participates in certain enzymatic activities
of the cell.
Calcium is an important component of gram positive bacterial cell wall and contributes
to the resistance of bacterial endospores against adverse environmental conditions. Iron is
a component of cytochrome, a component of the electron transport chain, and functions as
a co factor for enzymatic activities. Trace elements are components of enzymes and function
as co factors. Some are necessary for the maintenance of protein structure.

Growth Factors
Growth factors are essential to promote the growth and development of the bacterial cell.
These include vitamin B complex and amino acids
 Bacterial Growth Requirements 4

Physical Requirements
Moisture/Water
The bacterial cell is composed mainly of water. It serves as the medium from which bacteria
acquire their nutrients.

Oxygen
Oxygen is used by aerobic bacteria for cellular respiration and serve as the final electron
acceptor. Microorganisms are classified as either aerobes or anaerobes based on their oxygen
requirements.
Microorganisms that utilize molecular oxygen for energy production are referred to as
aerobes. Strict aerobes are organisms that strictly require oxygen for growth. Microbes that
cannot survive in the presence of oxygen are called obligate anaerobes. These organisms do
not have the enzymes that break down free radicals produced in the body (i.e., catalase and
superoxide dismutase).
There are organisms that can grow and survive under both aerobic and anaerobic
conditions. These are called facultative organisms. Most medically important bacteria are
facultative. Some organisms are able to grow at low oxygen tension but their rate of growth is
diminished. These are called microaerophiles. There are some organisms though that may require
the addition of carbon dioxide to enhance their growth. These are called capnophiles.

Temperature
Enhanced enzyme activity requires certain temperatures. Microbes are classified into
three groups based on their temperature requirements, namely: (1) thermophiles, which grow
best at temperatures higher than 40 °C; (2) mesophiles, which require an optimal temperature
of 20 °C–40 °C; and, (3) psychrophiles, which require an optimum temperature of 10 °C–20 °C.
Most medically important bacteria are mesophiles.
44 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

90 °C

80 °C
Thermophile

70 °C

60 °C Pasteurization (62.8 °C)

50 °C

40 °C
Mesophiles

30 °C Human body (37 °C)

Psychrophiles
20 °C

10 °C

0 °C Refrigerator (4 °C)

Figure 4.1 Classification of bacteria into three groups based

on their optimum temperature requirements

pH
Another requirement of bacteria is the extent of acidity or alkalinity of their environment,
which is referred to as the pH. Microorganisms that grow best in pH 8.4–9.0 are called
alkalophiles. Those that grow best in pH 6.5–7.5 are called neutrophiles. Most medically
important bacteria are neutrophiles. The pH of most human tissues are 7.0–7.2. Certain
bacteria require a pH less than 6.0. These bacteria are called acidophiles.

Osmotic Conditions
Most organisms grow best under ideal conditions of osmotic pressure, which is determined
by the salt concentration. The normal microbial cytoplasmic salt concentration is approximately
1%. The optimum condition is if the external environment also has the same salt concentration.
If the extracellular salt concentration is increased (e.g., when food is salted), water will flow out
of the microbial cell and the organism will shrink and die. On the other hand, if the external
environment does not contain salt, water will flow into the bacterial cell causing the organism
to swell and rupture. Organisms that require high salt concentrations for growth are called
halophiles (e.g., diatoms and dinoflagellates) and those that require high osmotic pressure for
optimal growth are called osmophiles.
 Bacterial Growth Requirements 45

Bacterial Growth Curve

The bacterial growth curve illustrates the phases in the growth of the population of
bacteria when they are grown in a culture of fixed volume. It reflects the different stages in the
growth of the organism and is divided into four phases: lag phase, log phase, stationary phase,
and death or decline phase.

microorganism
Stationary
Log, or phase
exponential
growth, Death, or
phase decline, phase

of
Numbers

Survival
Lag phase
phase

Time

Figure 4.2 Bacterial growth curve

Lag Phase
This is the period of adjustment for the bacteria in the new environment. During this
phase, there is no appreciable increase in the number of microorganisms. The organisms will
show increased metabolic activity in order to synthesize DNA as well as secrete enzymes which
might not be present in their new environment but which are needed by the organism. Bacteria
attain their maximum size toward the end of the lag phase. This phase may last for 1 to 4 hours.

Log/Logarithmic/ExponentialPhase
This period is characterized by rapid cell division, resulting in an increase in the number
of bacteria. The organism exhibits high metabolic activity. This is the period when the
generation time or doubling time of the organism (i.e., the time required for the bacterial cells to
double in number) is determined. A generation time of 10 minutes means that the bacteria will
double in number every 10 minutes showing exponential growth. The average duration of this
phase is about 8 hours.
46 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Stationary Phase
This is considered as the period of equilibrium. During this period, the rate of growth slows
down, nutrients start to deplete, and toxic wastes begin to accumulate. As a consequence, some
bacterial cells may die. However, since there are still bacterial cells undergoing cell division, the
number of living cells equals the number of dead cells. Gram positive organisms may become
gram negative organisms in this phase. Sporulation occurs towards the end of this phase, or in
the case of spore forming organisms, during the beginning of this phase.

Death or Decline Phase

This is the period of rapid cell death where the number of dead cells is greater than the
number of living cells. This is due to the continuous depletion of nutrients and accumulation
of waste materials. Sporulation continues to occur during this stage. The duration of this phase
varies from a few hours to a few days
 Bacterial Growth Requirements 47

CHAPTER SUMMARY

• Bacteria require optimum nutrient and physical conditions for their growth.
• Nutritional requirements of bacteria include adequate supply of carbon, nitrogen, sulfur,
phosphorus, inorganic ions, and growth factors.

• Bacteria are classified into two groups based on their carbon source: autotrophs/
lithotrophs and heterotrophs/organotrophs.
» Autotrophs utilize inorganic compounds for their carbon source while organic
compounds such as glucose serve as the carbon source of heterotrophs.

• Bacteria derive energy by two means: from sunlight or from oxidation of inorganic
substances.

• Physical requirements of bacteria include moisture, oxygen, temperature, pH, and osmotic
conditions.
» Bacterial cell is made up mostly of water, which serves as the medium from which
bacteria derive their nutrients.
» Organisms that require oxygen for optimal growth are called aerobes while those that
cannot survive in the presence of oxygen are called anaerobes.
» Facultative organisms are those which can grow in the presence or absence of oxygen.
» Bacteria may be grouped into three based on their temperature requirements: (1) those
that require high temperature (thermophiles); (2) those that require temperature of
20 °C–40 °C (mesophiles); and (3) those that require temperature of 10 °C–20 °C
(psychrophiles).
» Acidophiles are organisms that grow best in pH < 6.0. Neutrophiles grow best
at pH of 7.0–7.2 while alkalophiles are those that grow best at pH of 8.4–9.0.
» Organisms that require salt for growth are called halophiles. Osmophiles are those that
need high osmotic pressure for maximal growth.
• Based on their nutritional and physical requirements, most medically important bacteria
are chemoorganotrophs,facultative, mesophiles, and neutrophiles.

• The bacterial growth curve illustrates the phases of growth of a bacterial population
grown in culture of fixed volume. It is divided into a lag phase, log phase, stationary
phase, and death or decline phase
 5 Normal Flora
CHAPTER

of the Human Body

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define “normal flora;”
2. differentiate between resident flora and transient flora;
3. explain the role of normal flora in the body; and
4. give examples of organisms that normally inhabit different sites in the body.

Microbial Ecology is the study of the relationships between microorganisms and their
environment. Among these relationships is the relationship of microbes with humans, and such
include the normal flora (or indigenous flora) of the human body. Normal flora consists of the
group of organisms that inhabit the body of a normal healthy individual in the community.
These indigenous flora may be non pathogenic or pathogenic and may at times behave as
opportunistic pathogens.
There are two types of flora, namely: (1) resident flora and (2) transient flora. Resident flora
are organisms that are relatively of fixed types and are regularly found in a given area of the
body at a given age. Transient flora are those that inhabit the skin and mucous membrane
temporarily for hours, days, or weeks and are derived from the environment. Normal flora are
beneficial to the human body because they can inhibit the growth of pathogenic organisms
by priming the immune system of newborns. At the same time, normal flora protects the
body’s organs and systems that are in direct contact with the external environment and are
therefore subject to the attack of invasive organisms. Normal flora do this by either competing
with invasive organisms for nutrients essential for their growth or by producing substances
that can kill them. Normal flora synthesize important vitamins that are essential to humans
52 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Normal intestinal flora secrete vitamin K that is needed for the activity of some clotting factors.
Other beneficial effects of normal flora include the following:
1. Normal flora can prevent pathogenic organisms from attaching to and penetrating the
skin and other tissues by producing mucin which make it difficult for the pathogenic
organisms to attach to the tissues to produce disease.
2. Normal flora in the intestines aid in the digestion of food by producing enzymes such
as cellulase, galactosidase, and glucosidase.
3. Intestinal flora also help in the metabolism of steroids.
The healthy fetus is normally sterile until birth, following the rupture of the bag of
water. Once born, the newborn normal flora is derived from the mother’s genital tract during
delivery, from the skin and respiratory tract of individuals who handled the newborn, and
from the environment.
There are certain body tissues and fluids that are normally sterile. Body fluids that are
sterile include the cerebrospinal fluid (CSF), synovial fluid, and blood. In the blood, there may
be low transient bacteremia brought about by physiologic trauma. The sterile tissues include the
urinary bladder, uterus, fallopian tubes, middle ear, and paranasal sinuses. Presence of bacteria
in these tissues and body fluids may lead to serious infections in these areas. For example,
bacteria in the CSF can gain entry into the central nervous system, leading to a potentially
fatal encephalitis.

Normal Flora on Different Sites of the Body

Skin
The skin is the part of the human body that is in constant contact with the environment,
making it the most exposed to microorganisms. There are certain factors that eliminate
non resident flora from the skin, namely: (1) lysozyme in the skin; (2) acidic pH of the skin
due to sweat; (3) free fatty acids in sebaceous secretions; and (4) the constant sloughing off of
the skin.
The normal flora of the skin consists mainly of bacteria and fungi. The microorganisms
vary depending on the region of the skin. The skin may be divided into three regions: (1) axilla,
perineum, and toe webs; (2) hand, face, and trunk; and (3) upper arms and legs. Skin of the
axilla, perineum, and toe webs is characterized by having higher moisture levels, higher body
temperature, and higher levels of surface lipids. These regions have more microorganisms
compared to the others and are predominantly inhabited by gram negative bacilli. Dry sites
(e.g., hands, forearms, feet, legs) have diverse flora because of their exposure to the environment.
Predominant flora in these areas include Staphylococcus epidermidis and Staphylococcus hominis
 Normal Flora of the Human Body 53

Most microorganisms in the skin are found in its superficial layers (stratum corneum)
and hair follicles. Anaerobes inhabit the deeper structures and layers of the skin, such as hair
follicles, sebaceous glands, and sweat glands. Table 5.1 summarizes the various microorganisms
that inhabit the skin.

Table 5.1 Normal flora found on the skin

Organism Remarks
Staphylococcus epidermidis Major skin inhabitant, comprising approximately 90% of
resident aerobic flora
Staphylococcus aureus Most commonly found in nose and perineum; in the nose,
number varies with age (greater in newborns than in
adults)
Micrococci (Micrococcus luteus) Accounts for 20%–80% of micrococci in the skin
Diphtheroids (Coryneforms) Classified into: lipophilic (common in axilla)
or non lipophilic (more common on glabrous or hairless
skin such as palms of hands)
Anaerobic diphtheroids (Propionibacteriumacnes) – areas
rich in sebaceous glands
Gram negative Bacilli Seen in moist intertriginous areas such as toe webs and
(Enterobacter, Klebsiella, axilla
Escherichia coli, and Proteus spp.)
Nail Flora Similar to that of the skin
Fungi may also be present (Aspergillus, Penicillium,
Cladosporium, Mucor)

Mouth and Respiratory Tract

The tongue and buccal mucosa are inhabited mostly by Streptococcus viridans group, which
includes S. mutans, S. milleri, S. salivarius, and S. sanguis. Although they are part of the normal
flora of the mouth, the viridans streptococci have been implicated in the pathogenesis of dental
caries. The gingival crevices and the tonsillar crypts are primarily inhabited by anaerobic flora.
The normal flora of the pharynx and trachea are similar to those found in the oral cavity.
However, there may be transient carriage in the pharynx of potentially pathogenic organisms.
These include Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis
and Mycoplasma.
54 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

In the upper respiratory tract, initial colonization by pathogenic organisms may be seen.
These include Neisseria meningitidis, Corynebacterium diphtheriae, and Bordetella pertussis.
The lower respiratory tract is usually sterile and organisms that reach this region are usually
destroyed by the defense mechanisms of the body such as the alveolar macrophages.

Conjunctiva
The normal flora in the conjunctivae are very scanty because they are held in check by the
flow of tears that contain lysozyme. The lysozyme may interfere with the cell wall synthesis of
organisms. However, some bacteria may transiently colonize the conjunctiva including Neisseria,
Moraxella, and Corynebacterium. Staphylococci and streptococci may also be present.

Digestive Tract
The esophagus contains transient mouth flora. Minimal bacteria may be found in the
stomach due to the relatively hostile environment in the stomach. Bacteria that may be found
in the stomach are those that may be swallowed with the food or those that are dislodged from
the mouth. The acidity in the environment of the stomach is further increased after meals
because of the release of gastric acid. However, there are certain bacteria that are able to survive
in the acidic environment of the stomach. One of these is Helicobacter pylori, the most common
cause of duodenal ulcer. This organism produces urease that causes alkalinization of gastric acid
thereby enabling it to colonize the stomach.
The number of bacterial flora differs between the small intestine and large intestine. In the
small intestine, scanty flora may be found due to the constant peristaltic movement of the
intestines. Most of the bacteria cultured in the small intestine include streptococci, lactobacilli,
and Bacteroides which are all transient.
The number of bacterial flora in the large intestine is far greater than in the small intestine.
The colon is inhabited predominantly by anaerobes (95%–99%) which includes Bacteroides
fragilis (most common), Bifidobacterium/Lactobacillusbifidum (predominant in breast fed
infants), Eubacterium, Peptostreptococcus, and Clostridium. In bottle fed infants, the predominant
intestinal flora is Lactobacillus acidophilus. About 1%–4% of the flora of the colon are facultative
aerobes, predominantly Escherichia coli and other Enterobacteriaceae.
Intestinal flora play important roles in the body, namely: (1) synthesis of vitamin B complex
and vitamin K; (2) conversion of bile into bile acids; (3) competition with transient flora for
nutrients; (4) prevention of colonization of the intestines by transient flora; and (5) production
of potentially pathogenic end products of metabolism that are toxic to transient flora
 Normal Flora of the Human Body 55

Esophagus
Major bacteria present Organ Major physiological
processes

Secretion of acid (HCl)

Stomach Digestion of
macromolecules
pH 2
Duodenum
Enterococci Continued digestion
Lactobacilli Small Absorption of
Jejunum intestine monosaccharides, amino
acids, fatty acids, water
Enterobacteria pH 4–5
Enterococcus faecalis Ileum
Bacteroides
Bifidobacterium Absorption of bile acids,
Eubacterium vitamin B12
Peptococcus Colon Large pH 7
Peptostreptococcus intestine
Ruminococcus
Clostridia
Lactobacilli
Streptococcus
Staphylococcus
Anus

Figure 5.1 Normal flora of the digestive tract

Genitourinary Tract
The urinary tract is sterile above the distal 1 cm of the urethra. In the anterior
urethra, the predominant flora isolated are S. epidermidis, enterococci, and diphtheroids. In both
males and females, Mycobacterium smegmatis may be found as normal commensals in
their secretions. In addition, Gardnerella vaginalis, bacteroides, and alpha streptococci
may be found in penile urethra. The female urethra is either sterile or contains
Staphylococcus epidermidis.
Vaginal flora varies depending on the age, hormonal levels, and vaginal pH of the host. In
female infants, the predominant vaginal flora is Lactobacillus spp. From 1 month of age until
puberty, there is cessation of glycogen secretion making the vaginal pH higher (around 7.0).
The microorganisms that may inhabit the vagina at this time include Staphylococcus epidermidis,
Streptococci, diphtheroids, and Escherichia coli. At the onset of puberty, there is resumption
of glycogen secretion making the vaginal pH acidic. Predominant flora include Lactobacillus
acidophilus, corynebacteria, peptostreptococci, streptococci, Bacteroides, and staphylococci.
Lactobacillus plays a crucial role in preventing gonococcal infection by producing lactic acid that
adds to the acidity of the vagina. Young girls are more prone to the development of gonococca
56 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

infection compared to adult women because the normal acidic pH of the vagina as well as the
normal vaginal flora are not yet fully developed.
After menopause, the vaginal pH increases once more due to the lessened production
of glycogen. Normal flora that predominate during this period are similar to those found
during pre puberty. Most of these flora are derived from the skin and from the colon. Fungi
such as Torulopsis and Candida may also be found (10%–30%). Conditions that will allow the
overgrowth of these fungi (e.g., intake of antibiotics) can lead to vaginal infections such as
vaginitis.

Bacterial Flora in Normal

Bacterial Flora in Normal
Persons in Hospital
Persons in the Community
or Long term Care Facilities
Trac
• Staphylococcus sp. • Staphylococcus sp.
• Streptococcus pneumoniae •
Streptococcus sp. Anaerobes
UpperRespiratory
– •Enterobacteriaceae
– Viridans Streptococcus – Escherichia coli
• Haemophilus sp. – Klebsiella sp.
• Anaerobes • Candida sp.
• Pseudomonas sp.
• Staphylococcus sp. • Staphylococcus sp.
Skin
• Coryneform bacteria • Enterobacteriaceae
or "Diptheroids" – Escherichia coli
• Propionibacterium sp. – Klebsiella sp.
• Anaerobes • Anaerobes sp.
• Enterococcus sp. • Enterococcus
• Enterobacteriaceae • Enterobacteriaceae
Gastrointestinal

Tract –Escherichia coli –

Escherichia coli
– Klebsiella sp. – Klebsiella sp.
• Streptococcus sp. • Candida sp.
–Streptococcus anginosus
(milleri) group
• Pseudomonas sp.
• Lactobacillus sp.
• Candida sp.
Genital
Tract
• Lactobacillus sp. • Candida sp.
• Streptococcus sp.
– Streptococcus agalactiae

Figure 5.2 Comparison of bacterial flora of persons who are healthy and those that are confined
in hospitals or long term care facilities
 Normal Flora of the Human Body 57

CHAPTER SUMMARY

• Normal or indigenous flora refers to organisms that inhabit the body of a normal healthy
individual.

• Resident flora, also known as normal flora, refers to microorganisms that are regularly
found in a given area at a given age.

• Transient flora are those organisms that inhabit the skin and mucous membrane
temporarily for a few hours, days, or weeks. They do not establish themselves permanently
in the body tissues.

• Normal flora have important roles in the body which can be beneficial or harmful.
» Advantages of normal flora include:
1. Inhibition of growth of pathogenic organisms by priming of the immune system
2. Synthesis of vitamin B12 and vitamin K in the intestines.
3. Synthesis of substances that may inhibit growth of pathogenic organisms
(e.g., enzymes, fatty acids, bacteriocins).
» Disadvantages of normal flora include:
1. Production of disease if the individual becomes immunocompromisedor if they
change their usual anatomic location.
2. Production of disease since most of them are pathogens or opportunistic
pathogens.

• Most of the normal flora in the skin are found in moist, intertriginous areas.
Diphtheroids and epidermidis are the predominant flora of the
Staphylococcus skin.

• The tongue and buccal mucosa are inhabited mostly by viridans group, which
Streptococcus
includes S. mutans, S. milleri, S. salivarius, and S. sanguis. The gingival crevices and the
tonsillar crypts are primarily inhabited by anaerobic flora.

• There may be transient carriage in the pharynx of potentially pathogenic organisms.

These include Haemophilus influenzae, pneumoniae, Neisseria meningitidis, and
Streptococcus
Mycoplasma.

• InThese
the upper respiratory tract, initial colonization by pathogenic organisms may be seen.
include Neisseria meningitidis, Corynebacterium diphtheriae, and Bordetellapertussis.

• Most of thewhich
Bacteroides
bacteria cultured in the small intestine include streptococci, lactobacilli, and
are all transient
58 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

• The colonfragilis
is inhabited predominantly by anaerobes (95%–99%) which includes
Bacteroides (most common), Bifidobacterium/Lactobacillusbifidum (predominant in
breastfed infants), Eubacterium, Peptostreptococcus, and Clostridium.

• Vaginal flora varies depending on the age, hormonal levels, and vaginal pH of the host.
» In female infants, the predominant vaginal flora is Lactobacillus spp.
» From 1 month of age until puberty, microorganisms which may inhabit the vagina
include Staphylococcus epidermidis, Streptococci, diphtheroids, and Escherichia coli.
» At puberty the predominant flora include Lactobacillus acidophilus, corynebacteria,
peprostreptococci, streptococci, Bacteroides, and staphylococci.
» Fungi such as Torulopsis and Candida may also be found (10%–30%)