INTRODUCTORY MICROBIOLOGY

MCB 201

KOLADAISI UNIVERSITY, IBADAN

Ecology of Microorganisms

Microbial Nomenclature

Cultivation and Isolation of Microorganisms

 Introduction Culture Media

 Types of Culture Media

 Preparation of culture media

 Isolation techniques

 Preservation of Cultured isolates

Microbial Growth

Control of Microbial Growth (Physical and Chemical Methods)

 Physical methods

 Chemical Methods

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 MICROBIAL ECOLOGY

Ecology is a branch of science, including human science, population, community, ecosystem and
biosphere. Ecology is the study of organisms, the environment and how the organisms interact
with each other and their environment. It is studied at various levels, such as organism, population,
community, biosphere and ecosystem.

An ecologist’s primary goal is to improve their understanding of life processes, adaptations and
habitats, interactions and biodiversity of organisms.

“Microbial ecology is the study of the behavior and activities of microorganisms in their natural
environments. Microbes usually live in communities and rarely as individuals. “Microbes are
small; their environments also are small.” In these small environments or “microenvironments,”
other kinds of microorganisms (and macroorganisms) often also are present.

The word Environment is derived from the French word “Environ” which means “surrounding”.
Our surrounding includes biotic factors like human beings, Plants, animals, microbes, etc and
abiotic factors such as light, air, water, soil, etc. Environment is a complex of many variables,
which surrounds man as well as the living organisms.

Environment includes water, air and land and the interrelation ships which exist among and
between water, air and land and human beings and other living creatures such as plants, animals
and microorganisms. The natural environment consist of four interlinking systems namely, the
atmosphere, the hydrosphere, the lithosphere and the biosphere.

These four systems are in constant change and such changes are affected by human activities and
vice versa.

Components of Environment

Environment has been classified into four major components:

1. Hydrosphere,

2. Lithosphere,

3. Atmosphere,

4. Biosphere.
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Hydrosphere includes all water bodies such as lakes, ponds, rivers, streams and ocean etc. It
functions in a cyclic nature, which is termed as hydrological cycle or water cycle.

Lithosphere means the mantle of rocks constituting the earth’s crust. The earth is a cold spherical
solid planet of the solar system, which spins in its axis and revolves around the sun at a certain
constant distance. It mainly contains soil, earth rocks, mountain etc. Lithosphere is divided into
three layers-crusts, mantle and core (outer and inner).

Atmosphere is the cover of the air, that envelopes the earth. It is a thin layer which contains gases
like oxygen, carbon dioxide etc. and which protects the solid earth and human beings from the
harmful radiations of the sun. There are five concentric layers within the atmosphere, which can
be differentiated on the basis of temperature and each layer has its own characteristics. These
include the troposphere, the stratosphere, the mesosphere, the thermosphere and the exosphere.

Biosphere it is otherwise known as the life layer, it refers to all organisms on the earth’s surface
and their interaction with water and air. It consists of plants, animals and micro-organisms, ranging
from the tiniest microscopic organism to the largest whales in the sea. Biology is concerned with
how millions of species of animals, plants and other organisms grow, feed, move, reproduce and
evolve over long periods of time in different environments. Its subject matter is useful to other
sciences and professions that deal with life, such as agriculture, forestry and medicine. The
richness of biosphere depends upon a number of factors like rainfall, temperature, geographical
reference etc. Apart from the physical environmental factors, the man made environment includes
human groups, the material infrastructures built by man, the production relationships and
institutional systems that he has devised. The social environment shows the way in which human
societies have organized themselves and how they function in order to satisfy their needs.

Organization of Ecology

Microorganisms serve essential roles in the complex nutrient exchange system that defines an
ecological community. There are five Levels of organization, and all levels are listed according to
their size in increasing order from smallest to the largest.

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Organism

It is the lowest level of organization, which includes both unicellular and multicellular organisms.
All the living species in this level exhibit all the characteristics required for the existence of life.

Population

A population is a group of individuals of a single species living together within a particular

geographic area. They interbreed and compete with each other for resources.

Community

It refers to the several populations that interact and inhabit a common environment and are
interdependent.

Ecosystem

It is a set of all living species and abiotic components existing and interacting in a given area.
There is an interaction with both living and non-living components of the environment.

Biosphere

It is the highest level of organization. It is the global ecological system which consists of all the
living organisms and other factors which support life. The biosphere mainly refers to the part of
the earth’s crust.

Why Study Microbial Ecology?

 Microbes live in all parts of the biosphere where there is liquid water.
 By virtue of their omnipresence, microbes impact the entire biosphere.
 Each microbial species in an ecosystem is thought to occupy a unique niche, which is a
complex description of the ways in which an organism uses its environment.
 The precise ecological niche of a microbe is primarily determined by the specific metabolic
properties of that organism.
 Present in every known ecosystem

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 Over 99% of microbes contribute to the quality of human life
 In terms of numbers, microbes represent most of the diversity of life on Earth and are found
in every environment
 Play a vital part of the ecosystem
 Major producers in aquatic environments
 Decomposers – bacteria and fungi – in many ecosystems
 Key role in Biogeochemical cycles to recycle carbon, nitrogen, carbon, water
 Natural pest killers in gardens and on crops
 Serving as natural water treatment
 Causing some ecological problems as red tide and algal blooms
 Involved in many symbiotic relations as lichens, human digestion, rumens of cows

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ECOSYSTEM AND BIODIVERSITY

Ecosystem

It deals with the entire ecosystem, including the study of living and non-living components and
their relationship with the environment. This science researches how ecosystems work, their
interactions.

Ecosystems have been defined as “communities of organisms and their physical and chemical
environments that function as self-regulating units.” These self-regulating biological units respond
to environmental changes by modifying their structure and function. It includes a wide range of
biological, physical, and chemical processes that connect organisms and their environment.

CONCEPT OF ECOSYSTEM:

In an ecosystem, the interaction of life with its environment takes place at many levels. A single
bacteria in the soil interacts with water, air around it within a small space while a fish in a river
interacts with water and other animals, rivals in a large space. Considering the operational point
of view; the biotic and abiotic components of an ecosystem are so interlinked such that their
separation from each other is practically difficult. So, in an ecosystem both organisms (biotic
communities) and abiotic environment (rainfall, temperature, humidity) each influence the
properties with other for maintenance of life.

STRUCTURE OF ECOSYSTEM

A structure of Ecosystem comprise of:

•The Composition of biological community including, species number, biomass, life history, and
distribution in space.

•The quantity and distribution of non-living material, such as nutrient water, etc.

•The rage of condition of existence such as temperature, light.

FUNCTION OF ECOSYSTEM:

•The rate of biological energy flow i.e. production & respiration rates of the community.

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•The rate of material or nutrient cycles

•Biological or ecological regulation including both regulation of organism by environment and

regulation of environment by the organisms.

COMPONENTS OF AN ECOSYSTEM:

There are two components of an ecosystem; Living components and non living components:

Non Living Components: (Abiotic) Non living components are the physical and chemical factors
that directly or indirectly affect the living components e.g. air, water, land, rock etc. Non living
components are also called Abiotic components. Physical factors include sunlight, water, fire, soil,
air, temperature etc. Chemical factors include moisture, salinity of water, soil nutrients, oxygen
dissolved in water.

Living Components: Living components in an ecosystem are either producers or consumers. They
are also called biotic components. Producers can produce organic components e.g. plants can
produce starch, carbohydrates, cellulose from a process called photosynthesis. Consumers are the
components that are dependent on producers for their food e.g. human beings and animals.

Biotic Components are further classified into 3 main groups

•Producers •Consumers •Decomposers or Reducers

1. Producer (Autotrophs): The green plants have chlorophyll with the help of which they trap
solar energy and change it into chemical energy of carbohydrates using simple inorganic
compound namely, water and carbon dioxide. This process is known as photosynthesis. The
chemical energy stored by the producers is utilized partly by the producers for their own growth
and survival and the remaining is stored in the plants for their future use. They are classified into
two categories based on their source of food.

a)Photoautotrophs: An organism capable of synthesizing its own food from inorganic substances
using light as an energy source. Green plants and photosynthetic bacteria are photoautotrophs.

b) Chemotrophs: Organisms that obtain energy by the oxidation of electron donors in their
environments. These molecules can be organic (chemoorganotrophs) or inorganic
(chemolithotrophs).

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2. Consumers (Heterotrophs): The animals lack chlorophyll and are unable to synthesis their
own food therefore they depend on the producers for their food. •They are known as heterotrophs
(i.e. heteros= others, trophs= feeder).The Consumers are of 4 types:

(a) Primary Consumer: ( Herbivores) i.e. Animal feeding on plants, e.g. Rabbit, deer, goat etc.

(b) Secondary Consumers: The animal feeding on Herbivores are called as secondary consumer
or primary carnivores. e.g. Cats, foxes, snakes.

(c) Tertiary Consumers: These are large carnivores which feed on secondary

consumers. e.g. Wolves

(d) Quaternary Consumers: They are also called omnivores these are largest carnivores which
feed on tertiary consumers and are not eaten up by

any other animals. e.g. lion and Tiger.

3. Decomposers or Detrivores: Bacteria and fungi belong to this category. They break down the
dead organic matter of producers & consumers for their food and release to the environment the
simple inorganic and organic substance. These simple substances are reused by the producers
resulting in a cyclic exchange of material between biotic and abiotic environment. Eg: Bacteria,
Earth worms, Beetles

ECOLOGICAL SUCCESSION

Ecological Succession is the phenomenon or process by which a community progressively

transforms itself until a stable community is formed. It is a fundamental concept in ecology, refers
to more or less predictable and orderly changes in the composition or structure of an ecological
community. Succession may be initiated either by formation of new, unoccupied habitat (e.g., a
lava flow or a severe landslide) or by some form of disturbance (e.g. fire, severe wind throw,
logging) of an existing community.

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FOOD CHAIN, FOOD WEB AND ECOLOGICAL PYRAMIDS:
FOOD CHAIN:
In food chain each organism eats the smaller organisms and is eaten by the larger one. All those
organisms which are interlinked with each other through food to gather constitute the ecosystem.
The different levels in a food chain are called tropic levels, Each food chain has three main tropic
levels:- Producer level, Consumer level, and decomposer level. If any of the intermediate stage of
the food chain is removed, the succeeding links of the food chain will be affected.

Figure 2: showing a sample food

chain and their trophic levels

FOOD WEB:

The interconnected, interlocking pattern of food chain is known as food web. Under natural
condition of the linear arrangement of food chain hardly occurs and they remain interconnected
with each other through different types of organisms at different levels. Such an interconnected
and interlocking pattern of food chain is known as food web.

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ECOLOGICAL PYRAMIDS

The different species in a food chain are called tropic levels. Each food chain has 3 main trophic
level, producer, consumer, and decomposers. •Thus Graphical representation of these trophic
levels is called as Ecological Pyramids.

BIODIVERSITY

The word biodiversity is a combination of two words: “biological and diversity” and refers to the
variety of life on the Earth. Biodiversity is the degree of variation of life forms within a given
species, ecosystem, biome, or an entire planet. Biodiversity is a measure of the health of
ecosystems.

Biodiversity is usually considered at three different levels:

The following are different types of biodiversity

1. Genetic diversity: variety in the genetic makeup among individuals within a species

2. Species diversity: variety among the species or distinct types of living organisms found in
different habitats of the planet

3. Ecosystem or ecological diversity: variety of forests, deserts, grasslands, streams, lakes, oceans,
coral reefs, wetlands and other biological communities

4. Functional diversity: biological and chemical processes of functions such as energy flow and
matter cycling needed for the survival of species and biological communities

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RELATIONSHIPS BETWEEN MICROBES AND OTHER ORGANISMS

Mutualism – both the microbes and the other organism benefit

Commensalism - microbes that benefit from the relationship but the other organism is not helped
or harmed

Parasitism – the microbe benefits and the other organism is harmed. A successful parasite does
not harm its host enough to kill it or it loses its source of benefit. Disease is an example of microbe
parasitism.

Predation – the microbe attacks and kills the host. Sometimes there can be beneficial effects of
predation

Microbial Communities within Plants

Some examples of mutualism are:
• Methanotrophic organisms and shagnum moss – the methanotrophic bacteria are associated with
the moss – the bacteria oxidize methane and release carbon
dioxide the moss uses
• Mycorrhiza- Fungal symbiotic association with plants which helps plants to
absorb phosphorus from soil. A mycorrhiza is a symbiotic (generally mutualistic, but occasionally
weakly pathogenic) association between a fungus and the roots of a vascular plant. In a mycorrhizal
association, the fungus colonizes the host plant’s roots, either intracellularly as in arbuscular
mycorrhizal fungi (AMF or AM) or extracellularly as in ectomycorrhizal fungi.

Some examples of commensalism are where the waste products of one organism is utilized
by another
• Nitrosomonas oxidize ammonium to nitrite
• Nitrobacter oxidize nitrite to nitrate
• Bacteria alter their environment and allows other bacteria to grow – organisms in sauerkraut

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Microbial Communities within Animals
Some examples of mutualism are:
• Aphids and Buchnera aphidicola live in the cytoplasm of the aphid cells and provide 10 amino
acids to the aphid – neither will grow apart - coevolution
• Protozoan – termite relationship – the protozoa breakdown cellulose and release acetate which
can be absorbed and used by the termite
• Many marine invertebrates have endosymbiotic dinoflagellates
• Endosymbionts in tube worms fix carbon dioxide by the Calvin cycle and provide fixed carbon
for the worms
• The rumen of ruminants are filled

Aquatic and Marine Ecosystems

• There is a wide variety of microorganisms in aquatic systems:
o Viruses
o Bacteria
o Diatoms
o Algae
o Protozoa
o Some limited fungi - more in freshwater
Importance/Relevance
• Cyanobacteria, diatoms and algae serve as major producers and sources of oxygen
• Protozoa serve as consumers at the lower ends of the food web
• Bacteria and fungi serve as decomposers
• Viruses are parasites that can infect other cells
Soil Ecosystems
There are millions and millions of microbes naturally present in soil.
The types of microbes in soil:
o Bacteria -
o Archaea
o Fungi
-Algae
o Protozoa

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 Importance/Relevance
o Soil microbes play a major role in nutrient cycling
o Soil animals – insects and worms break down larger organic components and increase
availability for microbes
o The microbes become food for the small animals
o Breakdown rock particles in soil and convert nutrients such as Phosphorous, Calcium,
Manganese, Silica, Zinc, Copper etc. into plant soluble form using acid secretions and hold for
later use by the plant
o Fungal filaments called hyphae act as extensions to the roots. The surface area of the root system
becomes much greater allowing for far better access to moisture and nutrients from the soil. This
extended root system acts to aerate the soil
o A number of microbes produce natural plant stimulants like enzymes, vitamins and acids which
are necessary for growth and vigor. This reduces the need for Nitrogen.
o Secretions from different microbes help to form soil aggregates which improves soil structure
• Cyanobacteria or algae and fungi live in a mutualistic relationship to form lichens
• Lichens are the major produces in the tundra

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ROLE OF MICROBES IN BIOGEOCHEMICAL CYCLING

Key Points
 A biogeochemical cycle is a pathway by which a chemical element (such as carbon or
nitrogen) circulates through and is recycled by an ecosystem.
 Microorganisms play a primary role in regulating biogeochemical systems in virtually all
of our planet‘s environments.
 Microbes participate in essential biogeochemical cycling events such as carbon and
nitrogen fixation.

Phosphorus Cycle
• Microbes use phosphorus in the form of calcium phosphate, magnesium phosphate and iron
phosphate
• Phosphorous from these forms are assimilated as phosphate into DNA, RNA, and other organic
compounds
• When the organisms are used as food by other organisms, it is released into the food chain
• Decomposers release the phosphates into the soil so it can be absorbed by the plants
• Phosphate is often used in fertilizer and if excess phosphate from fertilizer or industry is drained
into water supplies, it can cause algal blooms

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The Nitrogen Cycle
Nitrogen is essential for all forms of life because it is required for synthesis of the basic building
blocks of life (e.g., DNA, RNA, and amino acids). The Earth’s atmosphere is primarily composed
of nitrogen, but atmospheric nitrogen (N ) is relatively unusable for biological organisms.
Consequently, chemical processing of nitrogen (or nitrogen fixation) is necessary to convert
gaseous nitrogen into forms that living organisms can use. Almost all of the nitrogen fixation that
occurs on the planet is carried out by bacteria that have the enzyme nitrogenase, which combines
N with hydrogen to produce a useful form of nitrogen (such as
ammonia). Thus, microorganisms are absolutely essential for plant and animal life forms, which
cannot fix nitrogen on their own.

• Bacteria and fungi act as decomposers of plant and animal matter to form ammonia. Also “•
Nitrogen fixation – converting nitrogen gas from atmosphere into ammonia and bacteria fix
atmospheric nitrogen in legume root nodules. Many bacteria and Cyanobacteria can live apart from
legumes and can fix nitrogen in the soil. “

• Nitrification – Conversion of ammonia to nitrate – Nitrification occurs in soils, • water and marine
environments where the nitrifying bacteria live.

• Denitrification – nitrate is converted into gaseous nitrogen. Numerous types of • denitrifying

bacteria are involved in reducing nitrates to nitrites to nitrous oxide and then to gaseous nitrogen

Key Terms

autotroph: Any organism that can synthesize its food from inorganic substances, using heat or
light as a source of energy.

heterotroph: An organism that requires an external supply of energy in the form of food as it
cannot synthesize its own.

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Producers (autotrophs): do not usually eat other organisms but pull nutrients from the soil or the
ocean and manufacture their own food using photosynthesis. In this way, it is the energy from the
sun that usually powers the base of the food chain.

Consumers (heterotrophs) cannot manufacture their own food and need to consume other
organisms.

Decomposers break down dead plant and animal material and wastes and release them into the
ecosystem as energy and nutrients for recycling.

Definition of ecosystem: is a unified system of exchange made up of autotrophic producers,

heterotrophic consumers, and decomposers.

A food web depicts a collection of heterotrophic consumers that network and cycle the flow of
energy and nutrients from a productive base of self-feeding autotrophs.

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 Taxonomy and Identification (Classification) of Microorganisms

Taxonomy – Taxonomy may be defined as the science or study of the classification of living
organisms. It involves separating living organisms (and fossil forms of preexisting organisms)
into groups or categories and developing the criteria to be used for determining which organisms
fit into which groups. Grouping or categorizing living organisms allows investigators to study and
understand them more readily. The categories used in the classification of organisms are intended
to show the natural relationships between organisms and to reflect phylogeny, i.e., the
evolutionary history of organisms.

Binomial Nomenclature – Binomial nomenclature refers to the two-part technical name applied
to each different type of living organism. It is important to biologists because it provides a system
for communicating information about specific organisms named in a language universally
recognized and accepted.
Each organism (species) has a two part name (Genus & species). Names are either italicized or
underlined.
Genus name: Always capitalized, always a noun. May use initial.
species name: Always lower case, usually an adjective.
Names are usually derived from Latin (or Greek) or may have latinized endings.
Examples: Escherichia coli, Lactococcus lactis

The development of the binomial system of nomenclature (binomial nomenclature) is credited to

Carolus Linnaeus (a botanist/naturalist), in association with his Systema Natura, a manuscript
containing a classification of living organisms, first published in 1735. Linnaeus's text contained
lengthy descriptions of multiple living organisms, but also included a two-part name for each one,
based on key characteristics.

Currently the two-part technical name applied to each different type of living organism includes
the genus name (which is capitalized) and the specific epithet (all lower case). Both names are
Latinized and include either Latin or Greek roots providing descriptive information. For example

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the name Staphylococcus aureus, describes a type of organism forming grape-like clusters of
spherical-shaped cells, and golden or yellow-colored colonies. Linnaeus's text was in Latin
because it was the language used in universities at the time; however, since Latin is no longer a
spoken language, terms and their meanings remain stable and provide the basis for universally
accepted scientific communication. Currently, the binomial names of organisms are italicized
when in print and underlined when written by hand, a convention allowing for easy
recognition. The two-part name applied to each type of organism indicates where that organism
fits into a larger taxonomic schema as indicated below.

Taxonomic Ranks – Taxonomic ranks are the categories used in the classification of living
organisms. These are nested ranks, with each successively lower level being contained within the
one above. A group of organisms occupying a specific rank is called a taxon (pleural = taxa) or
taxonomic group.

The original taxonomic ranks were as follows:

Kingdoms (singular = Kingdom, the largest or most encompassing)
Phyla (singular = phylum) Sometimes called Divisions
Classes (singular = Class)
Orders (singular = Order)
Families (singular = Family)
Genera (singular = Genus)
Species (the most specific category)

One of several mnemonic forms = Kids playing chase on freeways go splat!

At the time of Linnaeus’s work, living organisms were grouped into two broad categories, the
Plantae (plants) and the Animalia (animals). These broad categories were called Kingdoms.
Since then, a number of classification categories have been added between the levels of kingdom
and genus. Similar organisms with the same genus name, or genera, are grouped within the same
family, similar families are grouped within the same order, similar orders are grouped within the
same class, similar classes are grouped within the same phylum (or division), and similar phyla

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are grouped within the same kingdom. The criteria or rules used for the classification of living
organisms into taxonomic ranks are quite specific and are determined by groups of biologists from
around the world. These international groups called congresses meet at varying intervals to
determine how plants and animals are to be categorized.

Although most macroscopic organisms can be readily classified into two kingdoms (Plantae and
Animalia), microscopic organisms cannot. Following Van Leeuwenhoek’s discovery of
microscopic life forms, many new organisms were identified that did not meet the criteria for either
kingdom. One way to solve this problem was to establish a new kingdom. In 1866, Ernst Haeckel
proposed a third kingdom be established which he called Protista. This kingdom would include
all single-celled organisms and those multicellular forms not developing complex tissues. A
diverse group of organisms including protozoa, algae, fungi, sponges and slime molds were to be
classified within this kingdom, but their relatedness was minimal. In 1957, Roger Stainer and his
associates used electron microscopy to demonstrate significant differences between prokaryotic
and eukaryotic cells, suggesting more than three kingdoms were required. In 1969, R.H.
Whittaker proposed a five-kingdom system to improve classification. This system included three
kingdoms of more complex organisms based on three modes of nutrition. The Animalia ingest
food, the Plantae make their own food via photosynthesis, and the
Fungi (Myceteae) absorb food in a liquid form. The other two kingdoms, Protista and Monera,
include organisms without complex structures that are separated based on their cell types. Monera
are prokaryotic and Protista are eukaryotic. Although the Whittaker five-kingdom system is
included in many modern textbooks, it is not without problems. Recent studies based on
biochemical analyses indicate considerable variation among eukaryotic microorganisms, and the
need for multiple additional kingdoms. In 1978, Carl Woese and his associates using biochemical
analyses demonstrated significant differences within the kingdom Monera; and prompted the
addition of a new taxonomic rank called Domain above kingdoms in the taxonomic hierarchy.
This work provided evidence that organisms now recognized as Archaea (formerly
Archaeobacteria) have multiple important characteristics unlike either bacteria or eukaryotic
organisms. The three domains of life currently accepted by most biologists include the Eukarya
(all organisms with eukaryotic cells), the Bacteria and the Archaea.

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Adding domains to the previously established taxonomic ranks generates a slightly modified
hierarchy as shown below:
Domain (pleural = Domains, the largest or most encompassing)
Kingdom (pleural = Kingdoms)
Phylum (pleural = Phyla) Sometimes called Divisions
Class (pleural = Classes)
Order (pleural = Orders)
Family (pleural = Families)
Genus (pleural = Genera)
Species (the most specific category)

Mnemonic form = Dumb kids playing chase on freeways go splat!

In preparing a classification scheme, one places the microorganism within a small, homogeneous
group ( rank or category) that is itself a member of larger groups in a non-overlapping hierarchical
arrangement.
In prokaryotic taxonomy the most commonly used levels or ranks (in ascending order) are:
Species: a group of related isolates or strains.
Genera: a collection of related species.
Families: a collection of similar genera. In prokaryotic nomenclature, the name of the family ends
in the suffix (aceae).
Orders: a collection of similar families. In prokaryotic nomenclature, the name of the order ends
in the suffix (ales).
Classes: a collection of similar orders. In prokaryotic nomenclature, the name of the class ends in
the suffix (ia) .
Phylum or Division: collection of similar classes.
Kingdom : collection of similar phyla or divisions.
Domain: collection of similar kingdoms.

Since understanding the phylogeny (evolutionary history) of life on earth is a major goal
of taxonomists, numerous methods have been employed to determine the evolutionary

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relationships between organisms. Since the 1970s, computer technology and a method called
cladistics have provided considerable information relative to evolutionary trends. In cladistics,
specific features of organisms are used to determine relatedness. A feature that is common to
several different types of organisms, but shows variation within them is assigned a value or form
called a character state. Analysis of the character is then conducted to determine which state is
primitive (ancestral) and which is derived (evolved from something else). Finally, the
evolutionary relationships determined are portrayed as straight-line diagrams (evolutionary trees)
called cladograms. Many changes in taxonomy, including the addition of the new taxonomic rank
(domain), are due to studies involving cladistics.

Although the primary features used to determine the relatedness between macroscopic organisms
were initially based on morphology (the study of external features) and mode of reproduction,
these are not as useful for the classification of microorganisms. New features such as types of
nutrition and metabolism, temperature requirements, gas requirements, pH and salinity
preferences, and biochemical properties have proven to be much more useful. Taxonomy is an
ongoing science, and despite multiple new discoveries, a complete “picture” of the relatedness
between all living organisms has yet to be developed.

Because viruses are non-cellular entities, they are not included in any of the taxonomic schemas
described above. Viruses are often categorized according to the organisms they infect, but new
taxonomic schemes are being developed to better demonstrate the natural relationships between
viruses.

Criteria Useful in the Identification and/or Classification of Microorganisms

Some of the information/terminology included in this section relates to microbial growth and the
culture of microorganisms; however, since it also relates to identification and classification, it will
be presented here.

1. Morphology – Although highly valuable in the classification of multicellular organisms,

morphology has limited usefulness when applied to prokaryotes. Many different types of

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 bacteria form colonies and cells with similar morphology even when subjected to various
stain techniques. Recall information presented on colony and cell morphology in the
laboratory.
2. Mode of Reproduction – Variation in reproductive structures/methods is of primary
consideration in the classification of plants, animals and fungi; but somewhat less useful in
the classification of single-celled organisms. Most single-celled eukaryotes, bacteria and
archaea reproduce by means of fission, i.e., one cell divides itself into two daughter cells.
3. Nutrition and Metabolism – All living organisms can be categorized on the basis of their
nutritional requirements and type of metabolism.
A. Nutritional categories are based on energy source and carbon source. Organisms can
obtain the energy they require either from light or from chemicals. Those using light energy
are called phototrophs (photo = light) and those using chemical energy are called
chemotrophs (Chemo = chemical). In this case the root word troph refers to activity and
organisms can be activated either by light or by chemicals.
Organisms can obtain the carbon they need either from inorganic or organic carbon
compounds. Organisms using inorganic compounds as carbon sources are called autotrophs
(auto = self) while those using pre-formed organic compounds as their source of carbon are
called heterotrophs (hetero = different). The term troph in this case refers to feeding, so
organisms are either feeding themselves or feeding on a variety of different organic materials.
By combining energy source and carbon source, we obtain four nutritional categories:
Photoautotrophs = Organisms using light energy and inorganic compounds for carbon.
Photoheterotrophs = Organisms using light energy and organic compounds for carbon.
Chemoautotrophs = Organisms using chemical energy and inorganic compounds for
carbon.
Chemoheterotrophs = Organisms using organic compounds for both energy and carbon.
Plants, algae and some bacteria are photoautotrophs, but only prokaryotic cells function as
photoheterotrophs or chemoautotrophs. Animals (including humans), fungi, protozoa and
many prokaryotes function as chemoheterotrophs, so this category is often subdivided.
Saprotrophs = Chemoheterotrophs using dead or decaying organic materials for nutrients.
These are sometimes called saprophytes or decomposers.

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 Parasites = Chemoheterotrophs using living organisms as their source of nutrients (some
living inside their host and others living outside).
Hypotrophs = Obligate intracellular parasites, i.e., organisms able to grow and reproduce
only when inside a living cell. Viruses, some protozoa and some bacteria are hypotrophs.
Carnivores = Chemoheterotrophs obtaining nutrients from meat.
Herbivores = Chemoheterotrophs obtaining nutrients from plant material.
Omnivores = Chemoheterotrophs able to obtain nutrients from both meat and plant
material.

B. Metabolism – Metabolism includes all the chemical reactions occurring within living
organisms (anabolism and catabolism), and can be categorized as either fermentative or
respiratory (oxidative).
Fermentative organisms use organic compounds (usually pyruvic acid) as the final electron
acceptors in their metabolic processes.
Respiratory (oxidative) organisms use inorganic compounds (usually molecular oxygen) as
the final electron acceptors in their metabolic processes.

4. Gas Requirements – The gas requirements of organisms (based on oxygen utilization) can
be useful in their classification as indicated below:
Obligately aerobic organisms (obligate aerobes) = Organisms requiring molecular oxygen
for growth and reproduction (metabolic processes).
Obligately anaerobic organisms (obligate anaerobes) = Organisms unable to tolerate
exposure to molecular oxygen; oxygen is often toxic to these and they cannot grow in its
presence.
Facultatively anaerobic/aerobic organisms (facultative anaerobes/aerobes) = Organisms
able to grow and reproduce with or without oxygen available to them.
Microaerophiles = Organisms able to grow best in environments with limited oxygen, as
might occur in the mud at the bottom of a pond, lake, sea, etc., or within the gastrointestinal
tract.

23
 Although obligately aerobic organisms typically have a respiratory or oxidative metabolism
and require oxygen as a final electron acceptor; not all obligately anaerobic organisms are
fermentative. Many types of bacteria can use inorganic compounds other than molecular
oxygen as final electron acceptors for their respiratory metabolic processes.

5. Temperature Requirements – The temperatures required for optimum growth are variable and
can be used to categorize microorganisms as follows:
Psychrophiles = Psychrophiles are cold-loving organisms (psychro = cold, phil = love).
These organisms grow best at cold temperatures (between –5 and 20 degrees C).
Mesophiles = Mesophiles are moderate-loving organisms (meso = medium or intermediate)
and grow best at moderate temperatures (between 20 and 45 degrees C).
Thermophiles = Thermophiles are warm-loving organisms (thermo = warm) and grow best
at warm temperatures (between 45 and 60 degrees C).
Hyperthermophiles = Hyperthermophiles are hot-loving organisms and grow best at hot
temperatures, e.g., above 60 degrees C. Hyperthermophiles living in hot springs grow at
temperatures above 90 degrees C.
Organisms can also be described relative to their temperature tolerance, i.e., ability to survive
or tolerate exposure to temperature extremes. Organisms that can tolerate exposure to
extreme cold are said to be psychroduric. They cannot grow at these temperatures, but do
not die either. Most bacteria are psychroduric and can be maintained in a viable state at –70
degrees C. Organisms that can tolerate exposure to heat are said to be thermoduric. They
cannot necessarily grow in hot environments, but are not killed by exposure to them.
Endospores are thermoduric.

6. Acidity Vs Alkalinity or pH Requirements – Although most organisms grow best in neutral

environments (pH between 6.5 and 7.5), some prefer acidic environments, and others prefer
alkaline. Many types of culture media contain buffers (substances that resist pH change) to
help stabilize the pH or pH indicators (substances that change color in response to changes
in pH) to indicate the presence of acidic or alkaline metabolic end products. Organisms that
grow best in acidic environments are called acidophiles, but are relatively rare. Highly acidic

24
 or alkaline environments tend to inhibit microbial growth because cellular enzymes fail to
function under these conditions.

7. Osmotic Pressure Requirements – The effective osmotic pressure (tonicity) of an

environment is influenced by the solute concentration present, and can significantly impact
microbial growth.
Isotonic environments (iso = same) contain solute levels similar to protoplasm, so cells
placed into them will experience neither a net gain nor net loss of water.
Hypotonic environments (hypo = under, beneath, less than or too little) contain lower levels
of solute than protoplasm and will cause cells placed into them to gain water. Microorganisms
equipped with cell walls (e.g., algae, fungi, bacteria and archaea) or contractile vacuoles
(many types of fresh water protozoa) can live comfortably in hypotonic environments.
Organisms lacking these protective structures will tend to take on water (via osmosis) until
they explode.
Hypertonic environments (hyper = over, above, too much or excessive) contain higher levels
of solute than protoplasm and will cause cells placed into them to lose water. Hypertonic
environments containing high levels of salt or sugar are often used to preserve foods, i.e.,
inhibit microbial growth within those foods.
Organisms capable of growing and reproducing in environments containing high levels of salt
are called halophiles. These may be categorized as extreme halophiles/obligate halophiles
(those requiring high levels of salt for growth) or facultative halophiles (those capable of
growing with or without salt).

8. Environmental Relationships – The types of environmental relationships microorganisms

form with other organisms can be useful as criteria for classification; however, these
relationships are often not thoroughly documented nor understood.
Symbiosis – Symbiosis is a condition or circumstance existing when two or more different
types of organisms are living together in a close association. Although once thought to be
unusual, symbiosis is now recognized as a common occurrence, essential to ecosystem
function.

25
 Pathogen Vs Host – Pathogens growing within a host benefit from host resources, but the
host is harmed, and sometimes killed. Microorganisms capable of causing infection and
disease in humans, domestic animals and plants used in agriculture have been extensively
studied, but represent an extremely small percentage of the total.
Parasite Vs Host – Parasites also benefit from their hosts without giving in return. Organisms
capable of parasitizing humans and other animals have been studied extensively because some
cause disease and others serve as vectors involved in the transmission of disease-causing
agents.
Mutualistic relationships (mutualism), i.e., those involving organisms in mutually beneficial
arrangements are the most common form of symbiotic relationships. Even pathogens and
parasites can be considered beneficial in the sense that they help prevent population
overgrowth and maintain balance within ecosystems, a concept foreign/repugnant to most
humans.

9. Biochemical Analysis – Biochemical analysis allows for a more technical evaluation of the
relationships existing between organisms and has become the method of choice for the
classification of bacteria and archaea. Various subcategories exist as follows:

A. Enzymatic Testing – The types of enzymes organisms produce can be determined by testing
their ability to catabolize various materials and/or to form specific end products. Enzymatic
testing will be used extensively during the identification of Physiological Unknown No. 1.
B. Chromatography – Various applications of chromatography can be used to identify specific
chemical constituents of cells, e.g., cell wall lipid or amino acid content, membrane protein
content, or the presence of specific pigments.
C. Serology – Serology is the science or study of antibody and antigen interactions in vitro, and
has multiple applications in the detection, identification and classification of microorganisms.
Microorganisms are antigenic, i.e., are perceived by the body as foreign agents (antigens),
and typically stimulate the production of immune proteins called antibodies. Because the
interactions between antigens and antibodies are quite specific, and because antibodies can
bind with antigens, it is possible to use known types of antibodies to detect or identify specific

26
 types of antigens. Several different types of serological reactions will be explained and
demonstrated in the laboratory.
D. Phage Typing – Phage typing (bacteriophage typing) involves the use of viruses called
bacteriophages. Like antibodies, these will recognize and bind with specific types of bacteria;
however, unlike antibodies, they cause infection typically resulting in cell death. Because
these viruses are host-specific, known types of virus particles can be used to identify unknown
types of bacteria. Phage typing will be explained and demonstrated in the laboratory.
E. Nucleic Acid Analysis – The analysis of nucleic acids, DNA and RNA, can provide
considerable information useful in the identification and classification of microorganisms.
Techniques commonly used in nucleic acid analysis include:
1. Percent base composition (G + C or A + T) – Organisms with identical percentages
in base composition may or may not be closely related, but organisms with very
different percentages in base composition are not related.
2. Nucleic Acid Hybridization – Hybridization, the ability of two nucleic acid strands to
form hydrogen bonds with one another, has multiple applications including PCR and
DNA chip technology.
3. Polymerase Chain Reaction (PCR) – The polymerase chain reaction involves
hybridization and can be used to amplify DNA or RNA in vitro.
4. Gel Electrophoresis – Gel electrophoresis can be used to separate DNA or RNA
fragments on the basis of size by exposing them to an electric field.
5. DNA Fingerprinting or RFLP analysis – Fragments of DNA generated by restriction
endonuclease digestion will form patterns when subjected to electrophoresis. These
patterns are called DNA fingerprints or RFLP patterns.
6. Nucleotide sequencing – Determining the sequence of nucleotides in a strand of DNA
or RNA can yield information highly significant to identification and classification.

Note – Methods 2, 3, 4, 5 and 6 will be covered more extensively in the laboratory where
they will be used in the identification of Physiological Unknown No. 2.

F. Protein analysis – The analysis of proteins other than antibodies can also be useful in the
identification and classification of microorganisms. Some methods involved include:

27
 1. Gel electrophoresis – Similar to methods used with nucleic acids.
2. Amino acid sequencing – Determining the sequence of amino acids present in a
protein can be useful in determining protein function and sometimes protein origin.
For example, the origin of prions (infectious protein particles) was determined using
amino acid sequencing in conjunction with nucleic acid analysis.

According to the taxonomic system currently used by biologists, living organisms are grouped
relative to similar characteristics, i.e., they are categorized according to specific criteria as
described above. Under the binomial system of nomenclature (binomial nomenclature), each
different type of organism is identified with a two-part technical name (scientific name) indicating
its genus and species. In the case of multicellular, eukaryotic organisms, a species is defined as a
group of closely related organisms that will breed among themselves. The classification of such
organisms is therefore based largely on morphology and mode of reproduction. In the case of
prokaryotes a species can be defined simply as a population of cells with similar characteristics (a
bacterial culture containing only one population of organisms is considered a pure culture and
contains only one species). Because most prokaryotes have similar morphology and mode of
reproduction, prokaryotic taxonomy is based primarily on other criteria, with biochemical analysis
being most important.

Armed with new methods of biochemical analysis and computer technology, modern biologists
are attempting to use the information to reconstruct the pattern of events leading to the distribution
of life on our planet. They are attempting to understand the evolutionary relationships between all
living organisms and to determine the mechanisms of evolution involved in their origins. This
branch of science is called phylogenetic systematics, and has been applied extensively to the
classification of prokaryotic organisms, i.e., archaea and bacteria.

28
 CULTURE MEDIA

General and specialized media are required for bacterial growth and for characterization. The
media you prepare are, in fact, research tools. Peruse this section and use it as a reference as
needed. The basic procedures can be applied to almost any type of assay or culture requirement
for propagation of obligate aerobes or facultative anaerobes. Obligate anaerobes are poisoned by
oxygen, and specialized procedures are needed for their maintenance.

Media requirements

Bacteria display a wide range of nutritional and physical requirements for growth including

 Water
 A source of energy
 Sources of carbon, nitrogen, sulfur, phosphorus
 Minerals, e.g., Ca2+, Mg2+, Na+,
 Vitamins and growth factors

Microorganisms may be grown in liquid, solid or semisolid media. Liquid media are utilized for
growth of large numbers of organisms or for physiological or biochemical studies and assays.
Some species, such as Streptococcus or Staphylococcus, often demonstrate typical morphologies
only when grown in liquid media. Solid media are useful for observations of characteristic
colonies, for isolation of pure cultures and for short-term maintenance of cultures. Usually, the
preparation of a solid medium for growth simply includes the addition of 1 to 2% agar to a solution
of appropriate nutrients. Agar is a complex carbohydrate extracted from marine algae that
solidifies below temperatures of 450C. It is not a nutritional component. Usually, bacteria are
grown in complex media, because we simply do not know enough about the organism or organisms
to define all of their requirements for growth and maintenance. Neither the chemical composition
nor the concentration of substrates are defined. Media frequently contain nutrients in the form of
extracts or enzymatic digests of meat, milk, plants or yeast. For fastidious organisms we must often
use delicious-sounding concoctions such as tomato juice agar or chocolate agar, or something less
appetizing (but nutrient-rich) such as brain-heart infusion broth or blood agar.

29
TYPES OF MEDIA

All-purpose Media
There is no media that is considered universal in that it can be used for all microbial growth.
Conditions of incubation come into play as well as the nutrients needed. There are, however, all-
purpose media. Tryptic Soy Agar and Broth, Nutrient Agar and Broth, Blood Agar, and several
others are classified as such and can be used to grow a wide variety of soil microbes, pathogens,
and human body flora.

Enrichment Media
Fluid media that increases the number of a pathogen by containing enrichments and/or substances
that discourage the multiplication of unwanted bacteria. E.g Selenite F broth media, Alkaline
peptone water

Enriched Media
Media that are enriched with whole blood, lyzed blood, serum, special extracts or vitamins to
support the growth of pathogenic bacteria. E.g Blood Agar, Chocolate Agar.

Differential Media
Media to which indicator substances are added to differentiate bacteria. E.g McConkey Agar,
TCBs Agar differentiates sucrose fermenting yellow colonies of Vibrio cholerae to non sucrose
fermenting blue colonies other than Vibrio species. Most differential media distinguish between
bacteria by an indicator which changes color when acid is produced following carbonhydrate
fermentation.
Selective Media
Media which contain substances like antibiotics which that prevent or slow down the growth of
bacteria other than pathogens for which the media are intended. E.g Modified Thayer-Martin Agar,
Salmonella-Shigella (SS) Agar.

30
Media sterilization

When fungal spores or bacteria-laden microscopic particles make contact with your plates, broths,
and tubes colonies happily reproduce and your precious media eventually resemble something out
of an abandoned full refrigerator. One can't recognize individual colonies when the plates are
covered with fuzz. No untreated surface in the lab is sterile, and nearly all dust and other particles
have spores or active cells on their surfaces. Obviously, then, all labware and all media must be
sterilized before use. We sterilize most media and supplies using a steam autoclave to produce
moist heat. Other methods, including filtration, ethylene oxide, radiation, or ultraviolet light, may
be necessary if components are heat-labile or materials are not heat-resistant. An autoclave is
designed to deliver steam into a pressure chamber, generating high heat and pressure at the same
time. Heating media to above 1210C for 4 to 20 min. destroys nearly all living cells and spores.
High pressure (typically 20 lbs/sq. in) allows the temperature to exceed 100 0C, which can't be
accomplished with steam at one atmosphere. We use an autoclave that starts timing when the
temperature reaches 1210C, and exhausts the steam slowly after the prescribed time above 121 0C
(to prevent exploding bottles!). The autoclave is effectively a giant pressure cooker.

To properly use an autoclave

 Know the instrument - some are fully automatic, some are fully manual
 Prepare supplies properly - the more layers or greater the volume, the longer it will take
for the interior to heat up
 Check the steam pressure and ensure that the instrument is set for slow exhaust if liquids
are to be sterilized
 Ensure that the door is closed properly and securely
 Check that the time and/or automatic cycle are set properly
 Ensure that the temperature is well below 100 degrees before attempting to open the door
 Crack the door to allow steam to vent, keeping face and hands well away from the opening
 ***CAUTION*** Exposing tightly stoppered bottles to variable pressures invites
explosion and injury. When heating any liquids using any method, take care disturbing the
flask or bottle. Material near the bottom may be superheated and boil over when moved.
Stoppers, caps, covers, must be vented - never make them fit tightly.

31
General Procedure for Media Preparation

AGAR PLATES

1. Suspend desired grams of the media in appropriate quantity of deionized water according
manufacturers specifications.
2. Swirl to mix properly.
3. Sterilize by autoclaving or boiling at 1210C at 15psi for 10-15minutes.
4. Allow to cool to 50oC before dispensing into Petri dishes.
5. Allow to solidify and store plates inverted in a cool place.

Broth tubes

The only difference between broth and agar media is that broths do not contain an agar component.
We use broth tubes primarily for specific assays, or (rarely) for bacteria that will not form colonies
on a solid surface. In broth a species may display motility and/or a characteristic pattern of
association among individual cells, such as chains or clusters that is not as obvious in agar cultures.
To prepare broth a dry medium is layered onto the surface of a measured volume of water as with
agar media, mixed, and distributed into individual loosely capped or vented capped tubes in racks.
Heating to dissolve components is sometimes required, but not always. Racks are steam sterilized
and then allowed to cool, and caps tightened to prevent evaporation. Unlike preparation of agar
plates, tubes are prepared with media already in the incubation vessel. A large volume syringe can
facilitate distribution of media into individual tubes.

Agar tubes and agar slant tubes

Prepare agar for a tube as you would agar for pouring plates, but use an open vessel, not a bottle.
Beakers are most appropriate. Medium must be uniformly distributed after melting the agar. As
with broth tubes, it is easiest to use a syringe or some other repeating dispenser to deliver media
to individual tubes. Some applications call for a tube that is partially filled with agar to give a level
surface. For maintaining stocks of isolates or to prepare material for assays, slant tubes are helpful.

32
A slant is simply a tube placed at an angle during cooling to give a large slanted surface for
inoculation. The tube can be tightly capped for relatively long term storage of an isolate with low
risk of contamination or drying out of the culture. A large "butt," that is, the depth of agar below
the start of the surface area, helps prevent drying out. Some liquid near the bottom of the surface
also helps serve that purpose. To prepare an agar slant each tube should be filled sufficiently to
allow the agar to flow to just below the neck when the neck is laid over a horizontal 10 ml glass
pipet. The tubes are sterilized with caps loose as with all media, then laid on their sides using a
pipet to keep them tilted up just enough to create a long slanted surface. After cooling, the caps
are tightened and the tubes are ready for use.

CULTIVATION OF MICROORGANISMS
CULTURE TECHNIQUES
Introduction
Microorganisms are everywhere (air, food, beverages, etc.) therefore they are termed ubiquitous.
The microbiologist must be able to identify organisms that appear quickly and easily. If molds or
other microbes are present other than bacteria, specific characteristics are observed that will be
covered in later lab sessions. This lab deals specifically with the growth characteristics of bacterial
organisms.

Broth
Bacteria, when suspended in broth, may cause cloudiness or turbidity. If there are floating clumps
it is known as flocculent. Often bacteria will form a ring at the top or will float like a heavy island
known as a pellicle. Cells that sink to the bottom of the tube become sediment and those that float
in wisps down from the surface are termed streamers.

Plates
On plates, an isolated colony is best used for descriptive purposes. The following are commonly
used terms to describe colonies as viewed on an agar plate: Size - diameter of colony measured in
mm or cm Whole colony shape - circular, rhizoid, punctiform, irregular, lenticulate Edge or margin
- lobate, entire, undulate, filamentous, erose Elevation - raised, flat, umbonate, convex, pulvinate,

33
crateriform Surface - wrinkled, rough, concentric rings, dull, waxy, glistening, etc. Pigmentation:
Color - red, cream, white, yellow, none, etc.
Opacity - transparent, translucent, opaque. Odor can also be used as a diagnostic tool, however,
this characteristic should only be used sparingly and if the odor is distinctive - sweet, putrefying,
fruity, etc.
Preparing a Spread (Lawn) Plate
The exact process requires an inoculation from the original culture to be spread over the primary
area. The loop is flamed and cooled and, after turning the plate 90 degrees, a few bacteria are
spread over in the secondary area from the primary area. The loop is flamed, cooled, and the plate
again turned and a few bacteria are spread from the secondary area into the tertiary area. If the
inoculum is heavy, the primary area should be made smaller so fewer organisms are distributed.
If the inoculum is lighter, the primary area should be made larger so that more organisms might
be introduced. Spread plates are often used when test reactions are needed. Testing antiseptics,
antibiotics, and infecting the bacteria with a bacteriophage (virus that infects bacteria) are just
some of the examples in which spread plates are employed.

Pour plate method

Another method of separating bacteria is the pour plate method. With the pour plate method the
bacteria are mixed with the melted agar until evenly distributed throughout the liquid. The melted
agar is then poured into an empty plate an allowed to solidify. After incubation, discrete bacterial
colonies can then be found growing both on the agar an in the agar.

Streak plate method

The most common way of separating bacterial cells on the agar surface to obtain isolated colonies
is the streak plate method, it provides a simple an rapid method of diluting the sample by
mechanical means as the loop is streaked across the agar surface. After incubation the area at the
beginning of the streak pattern will show confluent growth while the area near the end of the
pattern should show discrete colonies. Below is an example of a streaked agar plate. The figure
below shows streak plate culture technique.

34
Figure 3: showing streak plate culture method

35
 MYCOLOGY
Identification

A) FILAMENTOUS FUNGI

Introduction:

Most filamentous fungi can be identified based on a combination of colonial morphology and
microscopic features. Pathogenic dimorphic fungi such as Blastomyces, Histoplasma, Sporothrix,
etc., can often be presumptively identified by the presence of their characteristic conidia seen on
Lactophenol Aniline Blue (LPAB) preparations of culture isolates. The extent to which a
filamentous fungus is identified in the laboratory will depend on several factors. The following
should be used as a guide.

Procedure:

Examine the culture plates and record the macroscopic and microscopic findings.

Macroscopic Examination:

1. Colonial morphology
2. Surface pigment on non-blood containing medium
3. Reverse pigment on non-blood containing medium
4. Growth on cycloheximide containing medium

Microscopic Examination:
For coloured filamentous fungus
1. Prepare a tease mount or scotch tape preparation of each fungus colony type from each
media using Lactophenol Aniline Blue (LPAB).

2. Under the light microscope, examine the slide(s) for the presence, shape, size and
attachment of conidia. Compare and match the above features with those described in
a reference textbook.

36
 3. If the filamentous fungus can be identified from the LPAB preparation, mark the
identified colony (ies) with an “X” on the back of the culture plate(s) [if more than one
type of fungus is identified, place number (e.g. 1, 2, 3, etc) beside the “X” which
matches the number and identification entered into the LIS]. Re-incubate the original
culture plates for the remaining incubation period and examine plates for additional
growth.
4. Report the identification according the instructions.

B. IDENTIFICATION OF YEASTS

Yeasts are a heterogenous group of fungi that superficially appear to be homogeneous. Yeasts
grow in a conspicuous unicellular form that reproduces by fission, budding, or a combination of
both. True yeasts reproduce sexually, developing ascospores or basidiospores under favorable
conditions. The majority of ascomycetous and basidiomycetous yeasts isolated by the lab go
unrecognized because most of them are heterothallic. In most instances, only one of the mating
types is isolated and therefore no asci or basidia are produced.
B. Yeast-like fungi (imperfect yeasts) reproduce only by asexual means. The identification of these
fungi is based upon a combination of morphological and biochemical criteria. Morphology is
primarily used to establish the genera, whereas biochemical assimilations are used to differentiate
the various species.
C. Principal Criteria and Tests for Identifying Yeasts
1. Culture characteristics - Colony color, shape, texture
2. Asexual structures
a. Shape and size of cells
b. Bipolar, fission, multipolar or unipolar "budding"
c. Absence or presence of arthroconidia, ballistoconidia, blastoconidia, clamp
connections, endoconidia, germ tubes, hyphae, pseudohyphae, or sporangia and
sporgangiospores.
3. Sexual structures - Arrangement, cell wall ornamentation, number, shape and size of
ascospores or basidiospores
4. Physiological studies

37
a. Assimilation
b. Cycloheximide resistance
c. Fermentation
d. Nitrogen utilization
e. Urea hydrolysis
f. Temperature studies

38
 MICROBIAL GROWTH
Bacterial growth is the division of one bacterium into two daughter cells in a process called binary
fission. Providing no mutational event occurs the resulting daughter cells are genetically identical
to the original cell. Therefore, “local doubling” of the bacterial population occurs. Both daughter
cells from the division do not necessarily survive. The doubling time is the generation time of the
bacteria. If the number surviving exceeds unity on average, the bacterial population undergoes
exponential growth. The measurement of an exponential bacterial growth curve in batch culture
was traditionally a part of the training of all microbiologists. The basic means requires bacterial
enumeration (cell counting) by direct and individual (microscopic, flow cytometry), direct and
bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most probable
number, turbidity, nutrient uptake) methods. Bacterial growth in batch culture can be modeled with
four different phases: lag phase, exponential or log phase, stationary phase, and death phase.

Figure: Bacterial Growth Curve: This chart shows the logarithmic growth of bacteria. Note the
Y-axis scale is logarithmic meaning that the number represents doubling. The phases of growth
are labelled on top.

39
Lag phase
Number of cells does not increase
Begin synthesizing enzymes required for growth
Delay depends on conditions

Exponential (log) phase

Cells divide at constant rate
Generation time measured
Most sensitive to antibiotics
Production of primary metabolites
Important commercially
Secondary metabolite production occurs as nutrients are depleted and wastes accumulate

Stationary phase
Nutrient levels too low to sustain growth
Total numbers remain constant
Some die, release contents; others grow

Death phase
Total number of viable cells decrease
Cells die at constant rate
Exponential, but usually much slower than cell growth

40
 ENVIRONMENTAL FACTORS THAT INFLUENCE MICROBIAL GROWTH
Prokaryotes inhabit nearly all environments, Some live in comfortable habitats favored by humans
while some live in harsh environments termed extremophiles; most are Archaea
Major conditions that influence growth:
Oxygen Requirements
Temperature
pH
Osmotic pressure
Oxygen Requirements – The gas requirements of organisms (based on oxygen utilization) can be
useful in their classification as indicated below:
Obligately aerobic organisms (obligate aerobes) = Organisms requiring molecular oxygen
for growth and reproduction (metabolic processes).
Obligately anaerobic organisms (obligate anaerobes) = Organisms unable to tolerate
exposure to molecular oxygen; oxygen is often toxic to these and they cannot grow in its
presence.
Facultatively anaerobic/aerobic organisms (facultative anaerobes/aerobes) = Organisms
able to grow and reproduce with or without oxygen available to them.
Microaerophiles = Organisms able to grow best in environments with limited oxygen, as
might occur in the mud at the bottom of a pond, lake, sea, etc., or within the gastrointestinal
tract.
Although obligately aerobic organisms typically have a respiratory or oxidative metabolism and
require oxygen as a final electron acceptor; not all obligately anaerobic organisms are
fermentative. Many types of bacteria can use inorganic compounds other than molecular oxygen
as final electron acceptors for their respiratory metabolic processes.

5. Temperature Requirements – The temperatures required for optimum growth are variable and
can be used to categorize microorganisms as follows:
Psychrophiles = Psychrophiles are cold-loving organisms (psychro = cold, phil = love).
These organisms grow best at cold temperatures (between –5 and 20 degrees C).
Mesophiles = Mesophiles are moderate-loving organisms (meso = medium or intermediate)
and grow best at moderate temperatures (between 20 and 45 degrees C).

41
 Thermophiles = Thermophiles are warm-loving organisms (thermo = warm) and grow best
at warm temperatures (between 45 and 60 degrees C).
Hyperthermophiles = Hyperthermophiles are hot-loving organisms and grow best at hot
temperatures, e.g., above 60 degrees C. Hyperthermophiles living in hot springs grow at
temperatures above 90 degrees C.
Organisms can also be described relative to their temperature tolerance, i.e., ability to survive
or tolerate exposure to temperature extremes. Organisms that can tolerate exposure to
extreme cold are said to be psychroduric. They cannot grow at these temperatures, but do
not die either. Most bacteria are psychroduric and can be maintained in a viable state at –70
degrees C. Organisms that can tolerate exposure to heat are said to be thermoduric. They
cannot necessarily grow in hot environments, but are not killed by exposure to them.
Endospores are thermoduric.

6. pH Requirements (Acidity Vs Alkalinity) – Although most organisms grow best in neutral

environments (pH between 6.5 and 7.5), some prefer acidic environments, and others prefer
alkaline. Many types of culture media contain buffers (substances that resist pH change) to
help stabilize the pH or pH indicators (substances that change color in response to changes
in pH) to indicate the presence of acidic or alkaline metabolic end products. Organisms that
grow best in acidic environments are called acidophiles, but are relatively rare. Highly acidic
or alkaline environments tend to inhibit microbial growth because cellular enzymes fail to
function under these conditions.

7. Osmotic Pressure Requirements – The effective osmotic pressure (tonicity) of an

environment is influenced by the solute concentration present, and can significantly impact
microbial growth.
Isotonic environments (iso = same) contain solute levels similar to protoplasm, so cells
placed into them will experience neither a net gain nor net loss of water.
Hypotonic environments (hypo = under, beneath, less than or too little) contain lower levels
of solute than protoplasm and will cause cells placed into them to gain water. Microorganisms
equipped with cell walls (e.g., algae, fungi, bacteria and archaea) or contractile vacuoles
(many types of fresh water protozoa) can live comfortably in hypotonic environments.

42
Organisms lacking these protective structures will tend to take on water (via osmosis) until
they explode.
Hypertonic environments (hyper = over, above, too much or excessive) contain higher levels
of solute than protoplasm and will cause cells placed into them to lose water. Hypertonic
environments containing high levels of salt or sugar are often used to preserve foods, i.e.,
inhibit microbial growth within those foods.
Organisms capable of growing and reproducing in environments containing high levels of salt
are called halophiles. These may be categorized as extreme halophiles/obligate halophiles
(those requiring high levels of salt for growth) or facultative halophiles (those capable of
growing with or without salt).

43
 MEASUREMENT OF MICROBIAL GROWTH

Studying growth of a microorganism is the basis of biotechnological exploitation of microflora for

production of desired product. Despite this, some microorganisms have specific requirement and
they grow in a particular growth media. Optimization of growth of microorganism in a particular
media is desirable due to economical reasons.

Measurement of Bacterial Growth

 Measurement of Bacterial Growth
 Why Should We Measure Bacterial Growth
 Cell Number count
 Cell Mass Measurement
 Cell Activity Measurement
Measurement of Bacterial Growth
 Bacterial Growth is a biological process that involves increasing cell number, cell mass,
and cell activity.
 We can thus measure the bacterial growth through measuring cell activity, cell mass and
counting cell numbers.
 Two methods can be used to count cells: directly by microscopy, using an electronic
particle counter or indirectly using a colony counting.
 You can measure cell mass directly by using weighing, measuring cell nitrogen or
indirectly by turbidity.
 You can measure cell activity indirectly by relating the level of biochemical activity with
the size of the population.

Why Should We Measure Bacterial Growth?

The following are reasons we measure bacterial growth:
 To count the number of cells within a media.
 To measure cell mass.
 To measure cell activity.

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Methods used for Measurement of Bacterial Growth
There are many methods available to measure bacterial growth. Each one is discussed below.
Cell Number count
The cell number counting is done by two following methods;
1. Direct Method
2. Indirect Method
A. Direct Method
The following methods are used for direct count of bacterial cells;
1. Direct Microscopic Count/Counting chamber
2. Electronic Enumeration of Cell Numbers
a. Direct Microscopic Count/Counting chamber
 Direct counts in a counting chamber, such as the Petroff-Hausser chamber, are the best way
to determine microbial populations.
 Counting chambers are made up of specially designed slides, coverslips, and a space
between them that creates a chamber with known depth.
 This slide is precisely ruled into squares of 1/400 mm2 area. A glass cover slip rests
1/50mm above the slide so that the volume is 1/20,000mm3 (1/20.000.000cm3).
 A suspension of unstained bacteria can also be counted inside the chamber using a phase
contrast microscope.
 You can calculate the number of microorganisms present in a sample by taking the volume
of the chamber and the dilutions of the sample prior to counting.

 An example: Five bacteria are found in every square ruled. This is 5 X 20,000,000 or 108
bacteria per milliliter.
Advantages of Direct Microscopic Count
 This method is simple, cheap, and quick.
 It also provides information on the size and morphology microorganisms.
 The Petroff-Hausser counting chamber makes it easy to count Bacteria accurately and
quickly.
 It is possible to quickly and easily perform direct microscopic measurements using very
little equipment.

45
  If they are properly diluted, very dense suspensions can still be counted.

Disadvantages of Direct Microscopic Count

 It is impossible to count the number of bacteria in a suspension, such as at the beginning
or end of a growth curve.
b. Electronic Enumeration of Cell Numbers
 Electronic counters, such as the Coulter counter, can also be used to count microorganisms.
 This method involves placing the bacterial suspension in an electronic particle counter. The
bacteria are then passed through a small orifice measuring 10-30 um in diameter.
 This orifice connects two counter compartments that contain an electrically conductive
solution.
 Each bacterium that passes through the orifice experiences a temporary increase in the
electrical resistance.
 This creates an electric signal that is counted automatically.

Advantages of Electronic Enumeration of Cell Numbers

 This is a fast method.
 It is easy to use

Disadvantages of Electronic Enumeration of Cell Numbers

 This requires advanced electronic equipment.
 Clogged orifices are more common.
 It is impossible to tell if the cells being counted have survived.

B. Indirect Method
The Indirect Method of bacterial cell count is done by this following method;
a. The Plate-Count Method
 This allows you to determine the number of cells which will multiply under certain
conditions.
 In a Petri dish, a measured amount of the bacterial suspension will be added.

46
  The agar medium, which is kept at 45°C, is then added to the mixture. Finally, the plate is
rotated to mix the two.
 The medium will solidify and the organisms will be trapped in the gel.
 Each organism reproduces itself, growing until there is a visible colony.
 A colony count on the plate shows the viable microbial population.
 The original sample is often diluted to reduce the number of colonies that form on the plate.
This usually results in a range of 30-30%. This range allows for accurate counting and
minimizes the chance of interference with the growth of other organisms.
 Colonies can be counted by lighting them from below (dark field illumination). This makes
them easily visible and often a magnifying lens is used. There are many electronic methods
that can be used to count colonies.
 The plate-count method is routinely used with satisfying results to estimate bacterial
populations in milk, food, and other materials.

Advantages of Plate-Count Method

 It is simple to use and can be used to measure populations of any size.
 Because it can count very few organisms, it has the advantage that it is sensitive. If a
specimen has less than one bacterium per liter, then one colony should form upon plating
1 ml.
 Can differentiate between dead and alive cells. Colonies cannot be formed by dead cells.

Disadvantages of Plate-Count Method

 The plate-count method has one limitation. Only bacteria that can grow in the medium and
conditions of incubation used will be counted. This is an important point to remember if
you are trying to count a mixture of bacteria.
 One limitation is that not all viable organisms that are capable of growing in the provided
culture conditions will necessarily produce one colony. One colony can be formed from
one cell if the bacterial suspension contains no aggregates. However, this is possible if
there are cells that have a tendency for accumulation. The resulting counts, such as cocci
in clusters or chains (staphylococci), or pairs (diplococci), will be lower than the individual

47
 cells. This is why “counts” are sometimes reported as colonies per milliliter, rather than
the number of bacteria per liter.

b. Membrane-Filter Count
 Direct counts of bacteria found in aquatic samples are often determined after they have
been captured on membrane filters.
 The membrane filter technique involves filtering the sample first through a black
membrane filter made of polycarbonate.
 Next, the bacteria is stained with fluorescent nucleic acids such as DAPI or acridine orange
and microscopically observed.
 You can also use fluorescently-labeled dyes, which are specific to members of a taxon.
 The membrane filter’s black background makes it easy to see the stained cells and you can
count them with an epifluorescence microscope.
 Direct cell count of environmental samples almost always results in higher cell densities
that methods that rely upon culturing. Because only a small fraction (about 1%) of cells
that grow in nature can be grown in the laboratory, this is why it is so important.
Advantages of Membrane-Filter Count
 It is easy to use.
 Not expensive.
Disadvantages of Membrane-Filter Count
 It cannot distinguish between live and dead cells.

Cell Mass Measurement

The measurement of cell mass can be done by two methods such as;

1. Direct Methods
2. Indirect Methods
Direct Methods of Cell Mass Measurement
It is accomplished by this following methods;
a. Determination of Nitrogen Content

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  Protein is the major component of cell material. Because nitrogen is a part of protein, it is
possible to measure a bacterial population (or cell crop) in terms of bacterial Nitrogen.
 Bacteria have an average of 14 percent nitrogen per dry weight, but this number can vary
due to cultural differences or differences among species.
 This technique measures growth. First, harvest the cells and rinse them with water. Then
you can do a quantitative chemical analysis to determine the nitrogen content.
Disadvantages of Determination of Nitrogen Content Method
 The process of determining bacterial nitrogen is laborious. It can only be done on samples
that are free from all other sources.
 The method can only be used for highly concentrated populations. This and other reasons
make this method a preferred choice for research.
b. Determination of the Dry Weight of Cells
 To determine the population size, there are also methods that can measure changes in cell
mass. The determination of microbial dry mass is one approach. This method is the best
for measuring the mass of cells.
 This method involves the collection of cells in liquid medium. They are then dried in an
oven and weighed.
Advantages
 It’s a very simple process.
 This technique is especially useful for measuring the growth rate of filamentous
mushrooms.
 However, dry weight measurement is a reliable and accurate way to measure growth in
many organisms. It is often used in research.
Disadvantages
 It is not recommended to use it with dense suspensions. Cells must be free from any
extraneous matter.
 It can be time-consuming and sensitive. It is possible to centrifuge hundreds of milliliters
to obtain sufficient bacteria culture because the bacteria is so small.
 The amount of living material within cells may not always be indicated by dry weight. For
example, the intracellular reserve material poly-α-hydroxybutyrate can accumulate in
Azotobacter beijerinckii at the end of the log phase of growth and during the stationary

49
 phase and finally can comprise up to 74 percent of the dry weight of the cells; thus, the dry
weight may continue to increase without corresponding cell growth.

Indirect Methods of Cell Mass Measurement

The Indirect Method of Cell Mass Measurement is accomplished by measuring the turbidity of a
cell.
a. Turbidimetric Methods
 Spectrophotometry is a more sensitive and rapid method to measure cell mass.
 For turbidimetric measurements on cell mass, a spectrophotometer (or colorimeter) can be
used.
 The fact that microbial cells scatter light is the basis of spectropotometry. The number of
microbial cells within a population is roughly constant. Therefore, scattering light is
directly proportional and indirectly related to cell numbers.
 Increases in concentration lead to greater turbidity and less light being transmitted through
the medium.
 A spectrophotometer can measure the extent of light scattering, i.e. the decrease in
transmitted sunlight. This is known as the absorbance (optical density) of the medium.
 Absorbance is linearly related with cell concentration at absorbance levels below about
0.5. This value must be exceeded by the sample. If it exceeds that, it should first be diluted
and then absorbance measured.
 As long as there is enough turbidity to be able to measure the population, it can easily be
measured.

Advantages of Turbidimetric Methods

 Turbidimetry can be used to quickly and easily track growth.

Disadvantages of Turbidimetric Methods

 The culture must be dense enough that it registers some turbidity.
 It is possible that it may not be possible for cultures to be measured in cultures that have
been grown in darkened media or contain suspended material.
 Turbidity can also be caused by dead cells as well as living ones.

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b. Measurement of a Specific Chemical Change Produced on a Constituent of the Medium
 This method can be used to estimate cell mass by taking a species that makes an organic
acid from glucose fermentation.
 It is assumed that acid production under certain conditions and for a set time period is
proportional to the size of the bacterial population.
 The measurement of acid or other end products is an indirect method of measuring growth.
It is only applicable in very special cases.

Cell Activity Measurement

a. The Membrane Filtration Procedure
 Another common plating method traps bacteria in water samples using a membrane filter.
 After the filter has been placed on an agar media or a pad soaked in liquid media, it is
incubated until each colony forms.
 The colony count is a measure of the number and type of microorganisms present in the
sample. Selective media can also be used to identify specific microorganisms. This method
is particularly useful for analyzing water purity.
 These filters are known to have a uniform porosity that is small enough to trap
microorganisms.
 This method is especially useful in determining the amount of bacteria present in large
samples that have a small number of viable cells.
 The membrane with trapped bacteria is placed on a special plate that has a pad containing
the medium.
 To make it easier to identify certain kinds of organisms, special media and dyes are
available. The organisms form colonies during incubation and appear on the membrane’s
surface.

b. MPN (Most probable number) test

 This method involves making many copies of several dilutions in a culture and adding them
to tubes that contain a suitable liquid growth medium.
 Each tube is checked after incubation to see if there has been any growth.

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  The assumption is that the last tube of the series that shows growth was inoculated using
between 1-10 cells and the next tube with between 11-100 cells.
 If there is no growth, the tube is presumed to not have received any cells.
 This allows you to estimate the number of cells present in your original sample.
 When a select medium is available that supports the growth of a particular type of microbe,
MPN values are used most often.
 It is used to assess microbial densities in water samples.

Table below shows a summary of methods for measuring microbial growth

Method Some Applications Manner in which Growth Is

Expressed
Microscopic count Enumeration of bacteria in Number of cells per ml
vaccines and cultures
Electronic enumeration Same as for microscopic count Same as for microscopic count
Plate count Enumeration of bacteria in Colony-forming units per ml
milk, water, foods, soil,
cultures, etc.
Membrane filter Same as plate count Same as plate count
Turbidimetric measurement Microbiological assay, Optical density (absorbance)
estimation of cell crop in broth,
cultures, or aqueous
suspensions
Nitrogen determination Measurement of cell crop from Mg nitrogen per ml
heavy culture suspensions to be
used for research in metabolism
Dry weight determination Same as for nitrogen Mg dry weight of cells per ml
determination
Measurements of biochemical
Milli-equivalents of acid per ml
activity e.., acid production by Microbiological assays
or per culture
cultures

52
 CONTROL OF MICROBIAL GROWTH

I. Definition and General Terms

General considerations for effective control

A. Sterilization: the process of killing (or removing) all microorganisms on an object or in a

material (e.g. liquid media).

B. Disinfection: the process of reducing the numbers of or inhibiting the growth of

microorganisms, especially pathogens, to the point where they no longer pose a threat of
disease.

C. Disinfectant: a chemical agent used to destroy microorganisms on inanimate objects such as

dishes, tables, and floors. Disinfectants are not safe for living tissues.

D. Antiseptic: a chemical agent that can be administered safely to external body surfaces or
mucous membranes to decrease microbial numbers. Antiseptics cannot be taken internally.

I. Chemical agents that prevent or inhibit microbial growth

1. Cidal agents: cause irreversible damage or death the target microbe independent of the
host.

2. Static agent: Static drugs inhibit growth or reproduction are dependent on the immune
system of the human host for elimination of the pathogens from the body. A bacteriostatic
compound inhibits growth and reproduction of microbes, but does not kill them. That is
why a graphical representation of the cell number does not decrease after the bacteriostatic
compound added, but remains constant over time.

II. Definitions Physical Methods

Heat is an economical and simple way to destroy microbes, heat

methods work by denaturing proteins.

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 A. Thermal death point (TDP): is the lowest temperature at which all bacteria in a liquid culture
are killed within 10 minutes.

B. Thermal death time (TDT): The time required to kill all bacteria in a liquid culture at a given
temperature.

C. Decimal reduction time (DRT):The time required to kill 90% of the ba cteria in a liquid
culture at a given temperature.

III. Factors Influencing Disinfectant Activity

A. Disinfectant activity
1. the number of microorganisms,

2. Types of microbes (some are more resistant than others, e.g., gram-positive bacteria are
generally more sensitive to antibiotics than are gram-negative ones)
3. physiology of organisms (growing organisms are more susceptible than dormant ones)

4. environment (pH. presence or absence of organic matter)

5. Mode action of the agent used. How does the agent kill or inhibit the microbe.

6. Temperature (increased temperatures generally enhance disinfectant activity).

a. Most antimicrobial agents exert their effect by damaging either the plasma membrane
or proteins or nucleic acids

7. Some desirable qualities of a good germicidal agent include

a. The agent most be rapid in action (even at low concentrations)
b. The agent should be soluble in water or alcohol and have long-term stability
c. The agent should be broad spectrum without being toxic to humans and animals tissues
d. The agent should be able to penetration of inanimate surfaces to sustain a cumulative
and persistent action
e. The agent should be resistance to becoming inactivated by organic matter
f. The agent should have the qualities of being non-corrosive or have non-staining
properties
g. The agent should be able to sanitize and deodorize
h. The agent should be affordable and readily available.

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IV. Control Methods

The factors one must take into account for a good germicidal agent to work :
1) The nature of the material being treated
2) the degree of contamination
3) The length of exposure time needed
4) The strength of the germicidal agent require upon the target microbe.

Major Methods for Microbial Control

V. Physical Agents
VI. Mechanical Agents
VII. Chemical Agents

V. Physical Agents

A. Moist heat
1. Methods include boiling, pasteurization, and autoclaving.

a. Very inexpensive and readily available; usually 1000C for 15 minutes kills many
vegetative cells and viruses are killed/inactivated within 10 minutes at 100 0C.
B. Pasteurization

2. Primarily used to decrease the number of pathogenic organisms in food without adversely
affecting the flavor;
a) Ultra pasteurization of milk is 1300C - 1400C for 1 - 2 seconds
b) flash pasteurization is 720C for 15 seconds
c) normal pasteurization at 630C for 30 minutes.

C. Autoclaving
a. Steam under pressure-the most effective moist heat method; usually 121.5 0C
at 15 psi for 15 minutes.

D. Dry heat
1. Direct flaming or incineration and hot air (1600C-1700C).
1600C for 1.5 to 2 hours or 1700C for 1 hour

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E. Freezing
1. Temperatures between 00 and 70C may inhibit the reproduction of certain
organisms or the production of toxins.

2. Rarely bactericidal

3. Not be an effective method of disinfection

a. Quick freezing is often used to store microorganisms for long periods of
time
b. Slow freezing causes severe damage to cellular constituents and may be
bactericidal.

F. Desiccation
Drying or freeze-drying can be used to inhibit growth (via inhibition of enzymes);
organisms remain viable.

G. Irradiation and its Effects

a. Dependents on wavelength and on intensity and duration of exposure.

b. Ionizing radiation (alpha, beta, gamma, and x-rays, cathode rays, high-energy
protons and neutrons) exhibits a high degree of penetration. It creates free radicals
in the medium, leading to the denaturation of proteins and nucleic acids. It can
result in mutations.
1) Used in food preservation processes
2) Viruses and spores are somewhat resistant.
3) Gram-negative bacteria are more sensitive to ionizing radiation than are
gram-positive organisms.

c. Ultraviolet radiation is a form of non-ionizing radiation.

1)Low degree of penetration.
a) Results in thymine dimers (cross-linkages) in DNA that interfere with
replication.

d. Microwaves
Microwaves usually do not kill directly organisms
1) Kills indirectly from heat generated in micro waved materials.
2) Visible light can cause oxidation of some light-sensitive materials.

56
VI. Mechanical Agents

A. Filtration
1. Mechanical means of removing microorganisms. The liquid or gas is passed through a filter
with pores small enough to prevent passage of microbes. This method can be used
for substances that are sensitive to heat.

a. "High Efficiency Particulate Air (HEPA) filters: removes at least 99.97% of airborne
particles 0.3 micrometers (µm) in diameter.
b. Ultra Low Particulate Air (ULPA) filter: remove from the air at least 99.999% of dust,
pollen, mold, bacteria and any airborne particles with a size of 120 nanometers or
larger.
B. Osmotic pressure
1. Extremely hypertonic conditions can cause plasmolysis (i.e., contraction of all the
cell membrane away from the cell wall).

VII. Chemical and gas

1. Referred to as cold sterilization
Halogens agents (chlorine, iodine), Quaternary ammonium compounds, Heavy metals (Ag, Cu,
Hg, Zn), Oxidizing agents, Aldehydes. Alcohols. Phenolics, Ethylene Oxide

2. Chlorhexidine is similar to hexachlorophene.

a) Chlorhexidine is often combined with detergents or alcohol as a disinfectant of
skin.
b) Effective disinfectant of most vegetative bacteria and enveloped viruses.
c) Used in surgical hand scrubs.

3. Quaternary ammonium compounds or quats are cationic detergents

a)Bactericidal against gram-positive bacteria, less effective against gram-negative
bacteria.

b)Quats are also fungicidal, amoebicidal, and are effective against enveloped viruses.
1) The permeability of the membrane is affected denaturing proteins

VIII. Method for the evaluation of the effectiveness of an antimicrobial agent

A. Evaluating the Potency of a Disinfectant
1. Disinfectant quickly kills microorganisms without causing damage to the
contaminated material
a) Potency is affected by

57
 1) Concentration of the agent
2) Length of exposure
3)Temperature
4) pH
5) Interfering organic matter

2. Kirby-Bauer disk diffusion method

a) Disk impregnated with chemical placed onto a bacterial media culture

b) Zone of clearing measured around disk and information used to determine effectiveness
of chemical compound

3. Use dilution test

a) A chemical that can be greatly diluted and still be effective gets a high rating for
effectiveness

b) Direct spray method

1) Used to test chemicals that are not water soluble.

c) Tissue toxicity text

1) Tests antiseptic quality through exposure of tissue culture systems to dilutions of
the agent.

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