MCB 201: GENERAL MICROBIOLOGY (DR G. C.

IHEANACHO SECTION)
Shapes and arrangement of bacteria. Identification of bacteria –Overview of cultural characteristics, Gram
staining, biochemical tests. Nutritional categories and biochemical activities of microorganism. Microbial
Nutrition, cultivation and growth;
Batch and continuous growth cultures; phases of growth in batch culture. Environmental factors influencing
growth: temperature, oxygen, pH, moisture, salinity, etc.

Antimicrobial agents: antibiotics, static and cidal agents, sterilization, disinfection, sanitization, antiseptic.
Methods of controlling microorganisms; physical, chemical and radiation techniques, Major groups of
disinfectants:

SHAPES AND ARRANGEMENTS OF BACTERIA

Bacteria has two common shapes: a. cocci (plural coccus) b. Rods (bacillus)
a. Cocci are roughly spherical in shape. They can exist individually but may also come in other
arrangements like:
i. Diplococcic – cocci divides and remains in pairs
ii. They may also exist in long chains of cocci which divide and remain attached to each
other, eg. Streptococcus, Enterococcus
iii. They can exist in clusters of cocci, eg. Micrococcus
b. Rods. This is often reffered to as bacillus (plural bacilli)
c. Bacteria can also be unusually shaped. The Actinomycetes form longhyphae that may branch
to produce a network called mycelium
d. Bacteria may also form rods twisted into spirals or helices which are called spirila if rigid or
spirochetes, if flexible.
e. Some bacteria are pleomorphic – showing variable shapes.
f. Some bacteria could also be flat or square shaped
IDENTIFICATION OF BACTERIA
Identification of bacteria (preliminary or conclusive) can be based on a number of factors namely:
a. Microscopic examination of specimen
b. Growth study and biochemical characteristics of isolates
c. Immunologic tests that detects antibodies and antigens
d. Bacteriophage typing
e. Molecular methods
NB. A bacteriophage is a virus that uses a bacteria as its host. Bacteriophage typing is a technique
which identifies bacteria based on the type of phage they are susceptible to.
Microorganisms are typically identified by growth pattern and biochemical activities/characteristics.
Bacteria growth are usually recognized as colonies on solid media or turbidity on liquid media. The
time of growth of the bacteria is also necessary for identification.

After observation of growth characteristics of pure cultures of bacteria, are noted, specific
biochemical tests can be carried out, eg carbohydrate fermentation.
BIOCHEMICAL TESTS

Source: MICROBIOLOGY (fourth edition) by Prescott, Harley and Klein

STAINING OF SPECIMEN
Living microorganisms can be examined directly under the microscope. However, there is need to
fix and stain microorganisms to enhance visibility of specific features and also preserve them for
future studies.
Fixation is the process of preserving the external and internal features of the microorganisms in a
stationary position. There are two types of fixation:
a. Heat fixation – this involves gentle flame heating of an air dried bacteria film. This is
usually employed to fix over all cell morphology.
b. Chemical fixation – involves the use of chemicals like ethanol, acetic acid, etc. it usually
permeate cells and inactivate cellular proteins and lipids.
DYES
They are of two types – Basic dyes and Acid dyes
BASIC DYES – Examples include: methylene blue, basic fuchsin, crystal violet, safranin, malachite
green. These are positively charged group (cataionic) and bond to negatively charged moleculeslike
nucleic acids and proteins.
ACID DYES – Examples include: eosin, rose Bengal, acid fuchsin. These are negatively charged
and bond to positive structures.

STAINING TECHNIQUES
1. SIMPLE STAIN. This type of stain involves a single staining agent and often employed to
determine size, shape and arrangement of bacteria. Examples of dyes used are crystal violet,
methylene blue and cabolfuchsin.
2. DIFFERNTIAL STAIN.
a. Gram stain. This type of stain divides bacteria into two groups based on their staining
properties. It was developed in 1884 by Christian Gram and divides bacteria into Gram
positive and Gram negative.
b. Acid fast stain. This differential staining technique is used especially for the genus
Mycobacterium
NUTRUITIONAL CLASSES/CATEGORIES
Two sources of energy are available to microorganisms
a. Light energy trapped during photosynthesis
b. Energy gotten from breakdown of organic and inorganic compounds
Phototrophs use light energy from the sun as energy source while chemotrophs get energy by
oxidizing organic or inorganic chemical compounds. Electron/hydrogen sources are either from
inorganic substances (lithotrophs) or organic substances (organotrophs)

MICROBIAL NUTRUITION AND CULTIVATION

Nutrients are necessary for microbial growth. They are defined as substances used in biosynthesis
and energy generation.

About 95% of microbial cell dry weight is composed of few major elements (macroelements):
carbon, oxygen, hydrogen, nitrogen, sulphur, phosphorus, potassium, calcium, magnesium and iron.
The first six elements are components of protein, carbohydrates, lipids and nucleic acid while the
remaining four acts as cations playing diverse biochemical roles in the organism.

Microorganisms also require trace elements (microelements) like manganese, zinc, cobalt,
molybdenum, nickel and copper. Trace elements often play vital roles as part of enzymes and
cofactors.
The above elements can also be classified based on the quantities needed in media preparation. The
major elements (C,O,H,N,S,P) are required in grams per liter of culture media; the minor elements
(K,Ca,Mg,Fe) are required in milligram quantities while the trace elements are required in
micrograms.

Carbon, hydrogen and oxygen needs are often satisfied together. Molecules that serve as carbon
source often contribute both carbon and nitrogen source. Carbon is the chief component of the
skeleton of most organic compounds.

Nitrogen is needed for the synthesis of amino acids, purines, pyrimidines, some lipids and
carbohydrates as well as enzyme cofactors. Phosphorus is a constituent of nucleic acids,
phospholipids and nucleotides like ATP. Sulphur is required for amino acid synthesis and some
carbohydrate.

The nutritional requirements of microorganisms differ considerably among different species. A

Prototroph is a microorganism that requires the same nutrients as most naturally occurring
members of its species. Conversely, an Auxotroph is a mutated microorganism that has lost its
ability to synthesize an essential nutrient and needs to obtain it from the environment.

GROWTH FACTORS AND MICROBIAL GROWTH

Growth factors are organic compounds needed because they are essential cell components or
precursors of such components and cannot be synthesized by the organisms. They are employed in in
microbiological assays. There are 3 major classes of growth factors:
a. Amino acids b. purines and pyrimidines c. vitamins
Amino acids are for protein synthesis; purines and pyrimidines are for nucleic acid synthesis while
vitamins are part of enzyme cofactors.

Naturally, bacteria grow as colonies on surfaces. Thus understanding colony development is

essential for the microbiologist.

Most rapid microbial growth occur at the edge of the colony. Growth is much slower at the middle
of the colony. The difference in growth is due to gradient in nutrient, oxygen and toxic contents
between this two regions. The center of the colony is much thicker than the edge and as such oxygen
and nutrients do not diffuse easily into the center as it would into the edges. Also toxic metabolic
products will not be easily removed from the center, hence growth is slowed down. This results in
cells from the edges growing at maximum rates while those at the center dies.
BATCH AND CONTINUOUS GROWTH CULTURES:
a. Batch Culture:
In a batch culture, microorganisms are grown in a closed system with a fixed volume of nutrient-rich media.
The growth process begins with an inoculum of microorganisms, and the culture is allowed to grow without
the addition of fresh media or removal of waste. As the culture progresses, the microorganisms consume
nutrients and produce waste, leading to changes in environmental conditions such as nutrient depletion and
accumulation of toxins. Eventually, the growth slows down and enters a stationary phase.
b. Continuous Culture:
In contrast to batch culture, a continuous culture is a steady-state system where fresh media is continuously
supplied, and waste is simultaneously removed. This allows constant growth conditions to be maintained and
provides a continuous supply of biomass. Continuous cultures are usually operated in bioreactors and are
commonly used for industrial production, as they can sustain high cell densities and optimize product yield.

Figure showing Microbial Growth Curve in a Closed System. The four phases of the growth curve are identified on the
curve and discussed below

Phases of Growth in Batch Culture:

During the growth of microorganisms in a batch culture, several distinct phases can be observed:
1. Lag Phase: This is the initial phase after inoculation, where microorganisms adapt to the new environment
and prepare for growth. There is no substantial increase in cell number during this phase, but metabolic
activity is high as the cells synthesize essential molecules and enzymes.

2. Logarithmic (Exponential) Phase: In this phase, the microorganisms experience rapid growth, and the
population size increases exponentially. The cells divide at their maximum potential rate, and the growth rate
is constant under favorable conditions. This phase is characterized by an abundant supply of nutrients and
absence of toxic byproducts.
3. Stationary Phase: At a certain point, the growth rate slows down, and the population enters the stationary
phase. This occurs due to nutrient depletion, accumulation of waste products, and environmental limitations.
The number of viable cells remains relatively constant, as the rate of cell division equals the rate of cell death.
Metabolic activity decreases, and secondary metabolites may be produced.

4. Death Phase: Eventually, the number of viable cells starts to decline in the death phase. The rate of cell
death exceeds cell division due to the exhaustion of nutrients, accumulation of toxic metabolites, and other
adverse conditions. The decline may be gradual or rapid, depending on the severity of the environmental
factors. Understanding the different growth phases in batch culture is crucial for optimizing industrial
processes, predicting growth dynamics, and studying microbial physiology and behavior.

ENVIRONMENTAL FACTORS INFLUENCING GROWTH

Environmental factors play a crucial role in influencing the growth of microorganisms. Some key
environmental factors include:
Temperature: Microorganisms have specific temperature ranges in which they can thrive. The optimal
growth temperature varies among different types of microorganisms. For example, psychrophiles grow best at
low temperatures, while thermophiles thrive at high temperatures.
pH Level: Microorganisms exhibit different pH preferences. Some prefer acidic conditions (acidophiles),
while others prefer alkaline conditions (alkaliphiles). The pH level affects the activity of enzymes within
microorganisms, thus influencing their growth.
Moisture: Water availability is essential for microbial growth. Microorganisms require water for various
metabolic processes and reproduction. Moisture content in the environment affects the availability of water
for microbial activities.
Oxygen Availability: Microorganisms can be categorized into aerobic (require oxygen), anaerobic (do not
require oxygen), and facultative anaerobic (can grow with or without oxygen) based on their oxygen
requirements. Oxygen availability affects the growth and metabolism of microorganisms.
Nutrient Availability: Microorganisms require essential nutrients such as carbon, nitrogen, phosphorus, and
trace elements for growth. The availability and composition of these nutrients in the environment influence
microbial growth.
Light: Light availability affects the growth of photosynthetic microorganisms, such as algae and
cyanobacteria. These microorganisms utilize light energy for photosynthesis, which is crucial for their growth
and reproduction.
Salinity: Microorganisms exhibit varying tolerance to salt concentrations. Some microorganisms, known as
halophiles, thrive in high-salt environments, such as salt lakes or brine pools. Others, known as halotolerant or
halophilic, can tolerate moderate to high salt concentrations.
Environmental Competition: Microorganisms compete with each other for resources, including nutrients,
space, and sunlight. The presence and abundance of other microorganisms in the environment can influence
the growth and survival of a particular microbial population.
Environmental Toxins: Environmental toxins, such as heavy metals, pesticides, and pollutants, can inhibit or
even kill microorganisms. These substances may disrupt microbial metabolism, hinder nutrient uptake, or
damage cell structures, thus affecting their growth.
Understanding and manipulating these environmental factors are crucial for various fields, including
agriculture, food preservation, medical research, and environmental management. By controlling these
factors, scientists and professionals can optimize conditions for desired microorganisms or limit the growth of
harmful ones.

ANTIMICROBIAL AGENTS
ANTIMICROBIAL AGENTS: These are agents that kills microorganisms or inhibit their growth. The
activities of microbial agents is affected by these factors:

1. Population size. Because an equal fraction of a microbial population is killed during each interval, a larger
population requires a longer time to die than a smaller one. The same principle applies to chemical
antimicrobial agents.

2. Population composition. The effectiveness of an agent varies greatly with the nature of the organisms
being treated because microorganisms differ markedly in susceptibility. Bacterial endospores are much more
resistant to most antimicrobial agents than are vegetative forms, and younger cells are usually more readily
destroyed than mature organisms. Some species are able to withstand adverse conditions better than others.
Mycobacterium tuberculosis, which causes tuberculosis, is much more resistant to antimicrobial agents than
most other bacteria.

3. Concentration or intensity of an antimicrobial agent. Often, but not always, the more concentrated a
chemical agent or intense a physical agent, the more rapidly microorganisms are destroyed. However, agent
effectiveness usually is not directly related to concentration or intensity. Over a short range a small increase in
concentration leads to an exponential rise in effectiveness; beyond a certain point, increases may not raise the
killing rate much at all. Sometimes an agent is more effective at lower concentrations. For example, 70%
ethanol is more effective than 95% ethanol because its activity is enhanced by the presence of water.

4. Duration of exposure. The longer a population is exposed to a microbicidal agent, the more organisms are
killed. To achieve sterilization, an exposure duration sufficient to reduce the probability of survival to 10 –6 or
less should be used.

5. Temperature. An increase in the temperature at which a chemical acts often enhances its activity.
Frequently a lower concentration of disinfectant or sterilizing agent can be used at a higher temperature.

6. Local environment. The population to be controlled is not isolated but surrounded by environmental
factors that may either offer protection or aid in its destruction. For example, because heat kills more readily
at an acid pH, acid foods and beverages such as fruits and tomatoes are easier to pasteurize than foods with
higher pHs like milk. A second important environmental factor is organic matter that can protect
microorganisms against heating and chemical disinfectants. Biofilms are a good example. The organic matter
in a surface biofilm will protect the biofilm’s microorganisms; furthermore, the biofilm and its microbes often
will be hard to remove. It may be necessary to clean an object before it is disinfected or sterilized. Syringes
and medical or dental equipment should be cleaned before sterilization because the presence of too much
organic matter could protect pathogens and increase the risk of infection. The same care must be taken when
pathogens are destroyed during the preparation of drinking water. When a city’s water supply has a high
content of organic material, more chlorine must be added to disinfect it.
Definition of terms
1. Antibiotics: a microbial product or its derivative that kills susceptible microorganisms or inhibits their
growth.
2. Chemotherapeutic agents: these are compounds used to destroy pathogens or inhibit their growth at
concentrations low enough to avoid doing damage to the host. They can either be Cidal or static
a. Cidal: these agents kills it target population. Some cidal agents could be static at low
concentrations
b. Static: these group of agents reversibly inhibit growth. If the agent is removed, the organism will
recover.
3. Sterilization: the process by which all living cells, viable spores, viruses and viroids are either
destroyed or removed from an object or habitat.
4. Disinfection: killing, inhibition or removal of microbes that may cause disease. It usually refers to
treatment of inanimate objects with chemicals.
5. Sanitization: reduction of microbial population on an inanimate object to levels judged safe by public
health standards
6. Antiseptic: chemical agents applied to tissue to prevent infection by killing or inhibiting pathogens.

METHODS OF CONTROLLING MICROORGANISMS: PHYSICAL AND CHEMICAL METHODS

PHYSICAL METHODS
Heat and other physical agents are normally used to control microbial growth and sterilize objects. Most
frequently employed physical agents are heat, low temperatures, filtration, and radiation.

Heat
Fire and boiling water have been used for sterilization and disinfection for centuries. Heating is still one of the
most popular ways to destroy microorganisms. Either moist or dry heat may be applied.
Moist heat readily kills viruses, bacteria, and fungi. Exposure to boiling water for 10 minutes is sufficient to
destroy vegetative cells and eucaryotic spores. Unfortunately the temperature of boiling water (100°C or
212°F) is not high enough to destroy bacterial endospores that may survive hours of boiling. Therefore
boiling can be used for disinfection of drinking water and objects not harmed by water, but boiling does not
sterilize. Many objects are best sterilized in the absence of water by dry heat sterilization. The items to be
sterilized are placed in an oven at 160 to 170°C for 2 to 3 hours. Microbial death apparently results from the
oxidation of cell constituents and denaturation of proteins. Although dry air heat is less effective than moist
heat— Clostridium botulinum spores are killed in 5 minutes at 121°C by moist heat but only after 2 hours at
160°C with dry heat—it has some definite advantages. Dry heat does not corrode glassware and metal
instruments as moist heat does, and it can be used to sterilize powders, oils, and similar items. Most
laboratories sterilize glass petri dishes and pipettes with dry heat. Despite these advantages, dry heat
sterilization is slow and not suitable for heatsensitive materials like many plastic and rubber items.

APPROXIMATE CONDITIONS FOR MOIST HEAT KILLING

Low Temperatures
Although our emphasis is on the destruction of microorganisms, often the most convenient control technique
is to inhibit their growth and reproduction by the use of either freezing or refrigeration. This approach is
particularly important in food microbiology (see p. 970). Freezing items at _20°C or lower stops microbial
growth because of the low temperature and the absence of liquid water.

Filtration
Filtration is an excellent way to reduce the microbial population in solutions of heat-sensitive material, and
sometimes it can be used to sterilize solutions. Rather than directly destroying contaminating microorganisms,
the filter simply removes them. There are two types of filters.

Depth filters consist of fibrous or granular materials

that have been bonded into a thick layer filled with twisting channels of small diameter. The solution
containing microorganisms is sucked through this layer under vacuum, and microbial cells are removed by
physical screening or entrapment and also by adsorption to the surface of the filter material. Depth filters are
made of diatomaceous earth (Berkefield filters), unglazed porcelain (Chamberlain filters), asbestos, or other
similar materials.

Membrane filters have replaced depth filters for many purposes. These circular filters are porous
membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate,
polyvinylidene fluoride, or other synthetic materials. Although a wide variety of pore sizes are available,
membranes with pores about 0.2 _m in diameter are used to remove most vegetative cells, but not viruses,
from solutions ranging in volume
from 1 ml to many liters.

Radiation
Ultraviolet (UV) radiation around 260 nm is quite lethal but does not penetrate glass, dirt films, water, and
other substances very effectively. Because of this disadvantage,UV radiation is used as a sterilizing agent
only in a few specific situations. UV lamps are sometimes placed on the ceilings of rooms or in biological
safety cabinets to sterilize the air and any exposed surfaces. Because UV radiation burns the skin and
damages eyes, people working in such areas must be certain the UV lamps are off when the areas are in use.
Commercial UV units are available for water treatment. Pathogens and other microorganisms are destroyed
when a thin layer of water is passed under the lamps.

Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. It will destroy bacterial
endospores and vegetative cells, both procaryotic and eucaryotic; however, ionizing radiation is not always as
effective against viruses.

The Use of Chemical Agents in Control

Although objects are sometimes disinfected with physical agents, chemicals are more often employed in
disinfection and antisepsis.

Phenolics
Phenol was the first widely used antiseptic and disinfectant. In 1867 Joseph Lister employed it to reduce the
risk of infection. Phenolics: Phenolic disinfectants, such as ortho-phenylphenol, have broad-spectrum
antimicrobial activity. They disrupt cell membranes, denature proteins, and deactivate enzymes. Phenolics are
commonly used in healthcare settings, laboratories, and as general-purpose disinfectants.

Alcohols
Alcohols are among the most widely used disinfectants and antiseptics. They are bactericidal and fungicidal
but not sporicidal; some lipid-containing viruses are also destroyed. The two most popular alcohol germicides
are ethanol and isopropanol, usually used in about 70 to 80% concentration. They act by denaturing proteins
and possibly by dissolving membrane lipids. A 10 to 15 minute soaking is sufficient to disinfect thermometers
and small instruments.

Halogens
A halogen is any of the five elements (fluorine, chlorine, bromine, iodine, and astatine) in group VIIA of the
periodic table. They exist as diatomic molecules in the free-state and form salt like compounds with sodium
and most other metals. The halogens iodine and chlorine are important antimicrobial agents. Iodine is used as
a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins. At higher concentrations,
it may even kill some spores.

Heavy Metals
For many years the ions of heavy metals such as mercury, silver, arsenic, zinc, and copper were used as
germicides. More recently these have been superseded by other less toxic and more effective germicides
(many heavy metals are more bacteriostatic than bactericidal). Heavy metals combine with proteins, often
with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins.

Quaternary Ammonium Compounds (Quats):

Quats are cationic surfactants that disrupt the cell membranes of microorganisms, leading to their destruction.
They are effective against a wide range of bacteria, fungi, and some viruses. Quats are commonly used in
household cleaners, surface disinfectants, and some hand sanitizers.

Detergents are organic molecules that serve as wetting agents and emulsifiers because they have both polar
hydrophilic and nonpolar hydrophobic ends. Due to their amphipathic nature detergents solubilize otherwise
insoluble residues and are very effective cleansing agents.

Aldehydes
Formaldehyde and glutaraldehyde are examples of aldehyde disinfectants. They have excellent antimicrobial
activity and can inactivate bacteria, viruses, and spores. These disinfectants work by cross-linking proteins
and nucleic acids, rendering them non-functional. Aldehyde disinfectants are commonly used for high-level
disinfection and sterilization in healthcare setting

Sterilizing Gases
Many heat-sensitive items such as disposable plastic petri dishes and syringes, heart-lung machine
components, sutures, and catheters are now sterilized with ethylene oxide gas Ethylene oxide (EtO) is both
microbicidal and sporicidal and kills by combining with cell proteins. It is a particularly effective sterilizing
agent because it rapidly penetrates packing materials, even plastic wraps