Chapter 1

Introduction
“Science contributes to our culture in many ways, as a creative intellectual
activity in its own right, as a light which has served to illuminate man’s place
in the universe and as the source of understanding of man’s own
nature”(John F. Kennedy (1917–63).
MICROBIOLOGY is a specialized area of biology (Gr. bios-life+ logos-to study) that
concerns with the study of microbes ordinarily too small to be seen without magnification.
Microorganisms are microscopic (Gr. mikros-small+ scopein-to see) and independently living
cells that, like humans, live in communities. Microbiology: is the science that deals
with tiny organism or microbes (micro-organisms), that cannot be seen by
the necked eye and which need special techniques that are required to
isolate and growth them.
Micro - means very small, anything so small that it must be viewed with a
microscope. Bio- means “living organisms” and logy-means “the study of
“. Therefore microbiology is the study of small living organism i.e.
microscopic organisms. Hence: Microorganisms are organisms that cannot
be seen with naked eye. Microorganisms include a large and diverse group of
microscopic organisms that exist as single cell or cell clusters Microorganism includes:
Bacteria
Viruses
Protozoa
Unicellular fungi
Multicellular fugi
Unicellular algae and others
Ricktessia
Mycoplasma
Nematodes
viroides
Microorganisms are present everywhere on earth, which includes humans, animals, plants and
other living creatures, soil, water and atmosphere. Microorganisms are relevant to all of our lives
in a multitude of ways. Sometimes, the influence of microorganisms on human life is beneficial,
whereas at other times, it is detrimental. For example, microorganisms are required for the
production of bread, cheese, yogurt, alcohol, wine, beer, antibiotics (e.g., penicillin,
streptomycin, chloramphenicol), vaccines, vitamins, enzymes. Many products of microbes
contribute to public health as aids to nutrition, other products are used to interrupt the spread of
disease, and still others hold promise for improving the quality of life in the years ahead.

Microbes are also an important and essential component of an ecosystem. Molds and bacteria
play key roles in the cycling of important nutrients in plant nutrition particularly those of carbon,
nitrogen and sulphur. Bacteria referred to as nitrogen fixers live in the soil where they convert
vast quantities of nitrogen in air into a form that plants can use. Microorganisms also play major
roles in energy production. Natural gas (methane) is a product of bacterial activity, arising from
the metabolism of methanogenic bacteria. Microoragnisms are also being used to clean up
pollution caused by human activities, a process called bioremediation (the introduction of
microbes to restore stability to disturbed or polluted environments). Bacteria and fungi
have been used to consume spilled oil, solvents, pesticides and other environmentally toxic
substances. Microorganisms have also harmed humans and disrupted societies over the
millennia. Microbial diseases undoubtedly played a major role in historical events, it was in the
year 1347 when plague or ‘black death’ struck Europe and within 4 years killed 25 million
people, that is, one third of the population.

HISTORICAL DEVELOPMENTS IN MICROBIOLOGY

The beginnings
The study of microorganisms, or microbiology began when the first microscopes were developed
in 1665 by the English scientist, Robert Hooke who viewed many small objects and structures
using a simple lens that magnified approximately 30 times. His specimens included the eye of a
fly, a bee stinger, and the shell of a protozoan. Hooke also examined thin slices of cork, which
was the bark of a particular type of oak tree. He found that cork was made of tiny boxes that
Hooke referred to as ‘cells’. He published his work in a book Micrographie which contained a
miscellany of his thoughts on chemistry as well as a description of the microscope and its uses.
Hooke in 1665 described the fruiting structures of molds. Thus, Robert Hooke was the first
person to describe microorganisms.

MICROFOCUS 1.1
Antony van Leeuwenhoek (pronounced Layu-wen- hoek) was born on October 24, 1632 in
Delft, Holland (now Netherlands). In 1674, he made first observation of microoraganisms and
was the first person to observe and accurately describe and measure bacteria and protozoa,
termed by him, as animalcules” which he thought were tiny animals. In 1677, he became the
first person to describe spermatozoa and was one of the earliest to describe red blood
corpuscles. In 1680, he was elected a fellow of the Royal Society of London, and with Isaac
Newton and Robert Boyle, he became one of the first famous men of his time. He died on
August 30, 1723 at the age of 90. Because of his extraordinary contribution to microbiology, he
is considered as the father of bacteriology and protozoology

Antony van Leeuwenhoek (1632-1723)

Unicellular life was first described just a few years after Hooke recorded his observations of the
microscopic world. Antony van Leeuwenhoek (Microfocus 1.1) was a Dutch merchant who
polished grains of sand into lenses which were able to magnify 300 times and added a simple
focus mechanism. With his microscope, van Leeuwenhoek viewed rain and pond water,
infusions made from peppercorns, and scrapings from his teeth in the year 1674 and termed the
tiny microorganisms as ‘animalcules’. In 1676, van Leeuwenhoek sent his drawings to the
Royal Society of London. This has special significance to microbiology because it contained his
first detailed description of the microorganism.

Biology of the 1700s was a body of knowledge without a focus. It consisted of observations of
plant and animal life and the attempts by scientists to place the organisms in logical order. The
dominant figure of the era was Carolus Linnaeus (1707–1778), a Swedish botanist who brought
all the plant and animal forms together under one Binomial nomenclature (naming of an
organism by two names—the genus and species) system of classification scheme. His book,
Systema naturae, was first published in 1735. Discovery of the microscopic world raised some
interesting queries and eventually led scientists to question some of the long-held beliefs. At that
time in history, the scientific community used a theory known as ‘spontaneous generation’ (the
doctrine that holds that lifeless objects give rise to living organisms) to explain the apparently
magical origins of life. The theory proposed that simple life forms arose spontaneously from
non-living materials and had its basis in the findings of Aristotle in the fourth century BC.
Although most people accepted spontaneous generation, the theory did have some strong
opponents. Among the first to dispute the theory of spontaneous generation was the Italian
scientist, Francesco Redi (1626–1697). He reasoned that flies had reproductive organs while
observing van Leeuwenhoek’s drawings. He suggested that flies land on pieces of exposed meat
and lay their eggs, which then hatch to maggots. This would explain the ‘spontaneous’
appearance of maggots. In the 1670s, Redi performed a series of tests in which he covered jars of
meat with fine lace, thereby preventing the entry of flies. The meat would not produce maggots
as it was protected and Redi temporarily put to rest the notion of spontaneous generation.
SCOPE AND HISTORICAL DEVELOPMENTS IN MICROBIOLOGY
Edward Jenner (1749–1823)
Although Redi’s work became widely known, the doctrine of spontaneous generation was too
firmly entrenched to be abandoned. In 1748, British clergyman, John Needham (1713–81) put
forth the notion that in flasks of mutton gravy, microorganisms arise by spontaneous generation.
He even boiled several flasks of gravy and sealed the flasks with corks as Redi had sealed his
jars. Still, the microorganisms appeared.
Italian scientist Abbe Lazzaro Spallanzani (1729–99) criticized Needham’s work. In 1767,
Spallanzani boiled meat and vegetable broths for long period of time and then sealed the necks
by melting the glass. As control experiments, he left some flasks open to the air, stoppered some
loosely with corks, and boiled some briefly, as Needham had done. After two days, he found the
control flasks swarming with organisms, but the sealed flasks had no organisms. Needham
countered that Spallanzani had destroyed the ”vital force” of life with excessive amounts of heat.
While the spontaneous generation was being debated, some of the scientists were concerned
about the transmission of the disease. In 1546, Italian scientist Girolamo Fracastoro held the
concept that “contagion is an infection that passes from one thing to another”. He recognized
three forms of passage, namely contact, lifeless objects, and air (Table 1.8). This notion received
little credibility that microorganisms were the substance of contagion. The German Athanasius
Kircher was paid little attention when he reported “microscopic worms” in the 1600s in the
blood of plague victims. Christian Fabricius was also neglected when he suggested in 1700s
that fungi might be the cause of rust and smut diseases in plants. Edward Jenner (Microfocus
1.2) was accorded honours in 1798 when he discovered immunization for smallpox, despite the
fact that he could not explain the cause of the disease. In 1847, Hungarian physician, Ignaz
Semmelweis reported that blood poisoning agent was transmitted to maternity patients by
physicians fresh from performing autopsies in the mortuary. Semmelweis showed that hand
washing in chlorine water could stop the spread of disease. His call for disinfection practices
were however largely unheeded because it implied that physicians were at fault.
The Classical Golden Age of Microbiology (1854–1914)
The science of microbiology blossomed during a period of about 60 years referred to as the
Golden Era of Microbiology. The period began in 1857 with the work of Louis Pasteur and
continued into the twentieth century until the advent of World War I. During this period,
numerous branches of microbiology were laid for the maturing process that has led to modern
microbiology. Louis Pasteur was the first to report the role of microorganisms in fermentation
in 1848, he achieved distinction in organic chemistry for his discovery that tartaric acid, a four
carbon organic compound, forms two different types of crystals. Pasteur successfully separated
the crystals while looking through the microscope. In 1854, at the age of 32, he was appointed
Professor of Chemistry at the University of Lille in northern France. Pasteur in 1857 unraveled
the mystery of sour wines. In a classic series of experiments, Pasteur clarified the role of yeasts
in fermentation of fruits and grains resulting in the production of alcohol.
He also found that bacteria were responsible for spoilage of wine. He firmly disproved the
spontaneous generation doctrine by his Swan-Neck Flask experiment. He proposed germ
theory of disease and discovered the existence of life in the absence of free oxygen (anaerobic
growth). He showed that mild heating could be used to kill microorganisms in broth
(pasteurization). Pasteur suggested methods to control pebrine disease in silkworm, isolated the
causative agent of cholera (Vibrio cholerae) and rabies (Lyssa) virus and also developed anti
rabies and anthrax (Bacillus anthracis) vaccines. Although Pasteur failed to relate a specific
organism to a specific disease, his work stimulated others to investigate the nature of
microorganisms and to ponder their association with disease.
German botanist, Ferdinand Cohn (1828–98), discovered that bacteria multiply by dividing into
two cells. He also observed that certain bacteria form an extremely resistant structure called
endospore in the cell.

Cohn described the entire life cycle of Bacillus (vegetative cell → endospore → vegetative cell).
He is credited with the use of cotton plugs for closing flasks and tubes to prevent the
contamination of sterile culture media. In 1866, Cohn studied the filamentous sulphur-oxidizing
bacterium Beggiatoa mirabilis and was the first to identify the small granules present in the cell
that are of sulphur, produced from the oxidation of H2S.
The definite proof of the germ theory of disease was offered by Robert Koch from East Russia,
now part of Germany. Koch’s primary interest was anthrax, a deadly blood disease in cattle and
sheep. In 1875, he injected mice with the blood of diseased sheep and cattle.
He then performed meticulous autopsies and noted that the same symptoms appeared regularly.
He isolated a few rod shaped bacilli from a mouse’s blood by placing the bacilli in the sterile
aqueous humor from an ox’s eye. The symptoms of anthrax appeared within hours. Koch
autopsied the animals and found their blood swarming with bacilli. He re-isolated the bacilli in
sterile aqueous humor. Koch’s procedures came to be known as Koch’s postulates (Fig. 1.2).
The four postulates are:
• The suspected microorganism must always be found in diseased but never in healthy
individuals.
• The microorganism must be isolated in pure culture (one free of all other types of microbes) on
a nutrient medium.
• The same disease must result when the isolated microorganism is inoculated into a healthy
host.
• The same organism must be re-isolated from the experimentally infected host.

Fig. 1.2: The diagrammatic representation of the Koch’s criteria for proving that a specific
microorganism causes a
specific disease, i.e., the Koch’s postulates.
Koch chanced to observe in 1880 that a slice of potato contained small masses of bacteria,which
he termed colonies. Colonies contained millions of just one kind of bacteria. Koch concluded
that bacteria could grow and multiply on solid surfaces, and he added gelatin to his broth to
prepare a solid culture medium. He then inoculated bacteria to the surface and set the medium
aside to incubate. When colonies of the same bacterium grew together, a pure culture (an
accumulation of one type of microorganism formed by the growth of colonies of the
organism) formed. Koch could now inoculate laboratory animals with a pure culture of bacteria
and be certain that only one species of bacterium was involved. His work also proved that
bacteria, not toxins in the broth were the cause of the disease. Gelatin was replaced with agar as
a solidifying agent in the culture media as suggested by Fannie Eilshemius Hesse, wife of
Walter Hesse, an assistant in the Koch’s lab. Petri dish was also invented about this time by
Julius Petri, one of Koch’s assistants. In 1881, Koch demonstrated his pure culture techniques
in the International Medical Congress. Koch’s proof of the germ theory was presented in 1876.
Within two years, Pasteur had verified the proof and gone a step further. He reported that
bacteria were temperature-sensitive because chickens did not acquire anthrax at their normal
body temperature of 420C but did so when the animals were cooled down to 370C. He also
recovered anthrax spores from the soil and pointed out that cattle were probably infected during
grazing. One of Pasteur’s more remarkable discoveries was made in 1880 when a group of
inoculated chickens failed to develop chicken cholera. He had been working on ways to enfeeble
bacteria using heat, different growth media, passages among animals, and virtually anything he
thought might weaken them. Finally, he had developed two cultures whose ability to because
disease was reduced. The trick was to suspend the bacteria in a mildly acidic medium and allow
the culture to remain undisturbed for a long period of time. When it was inoculated to chickens
and later followed by a dose of lethal cholera bacilli; the animals did not become sick. This
principle is the basis for the use of many vaccines for immunity. Pasteur applied the principle to
anthrax in 1881 and found he could protect sheep against the disease. Koch isolated the tubercle
bacillus, the cause of tuberculosis. In 1884, Koch’s associate George Gafky, cultivated the
typhoid bacillus, and that same year another coworker, Friederich Loeffler, isolated the
diphtheria bacillus. In later years, Koch’s coworker, Emil von Behring, successfully treated
diphtheria by injecting antitoxin, a blood product (preparation of antibodies) obtained from
animals given injections of the toxin. For his work, von Behring was awarded the first Nobel
Prize in Physiology or Medicine. In 1885; Pasteur reached the zenith of his carrier when he
successfully immunized young Joseph Meister against the dreaded disease rabies. Although he
never saw the agent of rabies, Pasteur was able to cultivate it in the brains of animals and inject
the boy with bits of the tissue. The experiment was a triumph for Pasteur because it fulfilled his
dream of applying the principles of science to practical problems.

Why we study microbiology?

Some microorganisms are beneficial and others are harmful. Microbes
directly benefit or harm human beings. Some of these economic
importances of microorganisms are:
1. Microbes and
Health
Microbes cause diseases both in plants and animals. Hence microbiology
helps us to understand the fundamental nature of those microbes which
causes diseases. This will help to devise methods to prevent microbes from
causing diseases and to treat diseases through correct identification of the
causative agent.
Most of antimicrobial drugs are produced by microbes. Example,
Streptomycin is produced from Streptomycetes, Penicillin from fungus called
Penicillium, Bactracin from bacteria called Bacillus etc application of
microbiology have given medicine great success in the diagnosis,
prevention and cure of diseases:
 For clinical infection
 Epidemiology
 Use of aseptic techniques
 Base of immunization
 Differential role of antimicrobials
 Microbiology provides the ‘why’ for health care practice
 To enable to carry out microbiological research related to health
 Procedures of health care are based up on principles from
microbiology
2. Agriculture and food production
Micrograms play key role in nutrient recycling like nitrogen and
carbon thereby increases soil fertility which boosts production and
productivity.
Microorganisms also involved in food production and processing such
as making of
Yogurt, cheese, and wine bear, bread, etc
3. Pharmaceutical industries
4. Beverage and alcohol industries
5. Biotechnologies
Evolution of Microorganisms
Argument about the origin of living things grew with discovery of microbes
by the help of Leeuwen Hoeck’s microscope. Concerning the creation of
microorganism, there was two schools of thought: the spontaneous
generation theory and the biogenesis theory.
Spontaneous Generation Theory
The supporter of spontaneous generation (abiogenists) believe that
living things could develop from non- living matter (from inanimate
matter) without the need for a living progenitor to give them life.
The theory of spontaneous generation, which held that living organism,
could rise spontaneously from non-living matter. This was a long held
commonly believed principle. People had observed, for example, meat
becomes putrid with time and that the appearance of maggots coincides
with purification.
Examples of some Spontaneous Generation theory supporters
Aristotle (383-322BC)
Believed that life comes from decaying matter
Example: Decayed meat Maggot
Mud (dust) + water Fish
Dirty shirt + flour kept in pot in dark place Rat
Concluded that living organisms arise from air, water, dust and other
non- living matter
The Biogenesis theory
Opponents of Spontaneous Generation theory supported the theory of
biogenesis, which stated that all organisms arise only from other pre-living
organisms.
Francesco Radi (1626-1697AC) showed that flies do not spring forth
spontaneously from the rotting meat.
Meat boiled and sealed---------------------No flies
Meat boiled and unsealed-----------------Maggot
Lazzaro Spalanzanni (1729-1799 AC)
He w as one of the first to provide evidence that
microorganisms do not arise spontaneously in 1769.
He boiled the broth for 1 hr and sealed and found no microorganisms
grew in it.
However the abiogenesis supporters claimed that air is vital for
 spontaneous generation of microbes but Spalanzanni excluded air and
that is why he couldn’t get microbes.
Franz Schulze (1815-1873)
Passed air through strong sulfuric acid solution into the boiled broth
and found no microbes were able to grow.
However, the abiogenesis supporters claimed that sulfuric acid altered
the environment which is necessary for spontaneous formation of
microbes.
Louis Pasteur (1822-1895)
A chemistry professor in French
Modified Spalanzanni’s experiment
 The germ theory of infectious disease
The theory was conceived after the work of L. Pasteur by Robert Koch, a
German general practitioner who discovered that bacteria cause disease,
including tuberculosis in 1832.
Robert Koch (1843 - 1910)
Koch developed diagnostic techniques used for modern microbiology.
He introduced agar as a setting agent for bacteriological media, although
the discovery is attributed to Fran Hesse from observations made in her
kitchen. Koch applied a set of criteria for conclusively demonstrating the
etiology (specific cause) of an infectious disease collectively known as
Koch’s postulates. He also contributed to the development of pure culture.
The Germ Theory
 Also called kotch’s postulate
 Formulated by Robert Koch, a German scientist
 He established that germs were the causes of diseases not the end
product of diseases
 He discovered bacilli in the blood of cattle that had died of anthrax
 He grew these bacilli in the culture and examined under microscope.
Then injected in to health animals. The infected animals developed
symptoms of anthrax. He then re- isolated similar bacilli.
These experiments lead to the formulation of Koch’s postulates.
1. A specific disease is caused by a specific organism. A specific
organism can always be found in association with a given disease
2. The organism should be isolated and grown in the lab into a pure
culture.
3. When the artificially cultured organism inoculated into health but
susceptible animal it should produce symptoms of the same diseases
4. The organism should be re-isolated from artificially infected animal
and grown into pure culture in the lab.
 Drawbacks (Limitations) of Kotch’s postulate
1. Not every microorganism associated with
diseases. ex. Normal flora do not cause
diseases
2. Many health people carry pathogens but do not exhibit symptoms
of the disease (carriers)
3. Certain disease develops only when an opportunistic pathogen
invades a weakened host. Opportunistic m i c r o o r g a n i s m s c a n
c a u s e diseases only in immune compromised individuals.
4. Not all diseases are caused by microorganisms.
Eg. Diabetes, Asthma, hypertension etc
5. Some microbes are very difficult or impossible to grow in the
 laboratory in artificial media, such as most viruses and some bacteria
ex. Treponema pallidivm and M.leprae are uncultivable organisms
Contribution of Kotch’s postulate
1. Emphasizes the importance of lab cultivation of microorganism in
artificial growth media
2. Showed that a specific microbe has specific activity
3. Contributed to the development of pure culture

The Development of Pure culture

Pure culture is also called axenic culture, a culture which contains one and
only one species of microorganism.
Why pure culture is needed?
Pure culture is required to study the morphological, physiological,
nutritional, genetic characteristic, environmental requirements etc of a
given spp.
How pure couture is obtained? Two important procedures
1. Isolation: Separation of the a microbe of concern from a mixed
population
Microorganisms in nature exist in a mixed population. For isolation several
methods can be used: serial dilution, selective media, enrichment media,
etc.
2. Culturing (cultivation of Microorganisms): is the growing of the
isolated microbe on a specific media into large number
Types of culture media
Culture media can be classified based on a number of criteria such as
physical states, functional properties, chemical composition etc.
A. physical States
i. Liquid media (broth):
These media do not contain any solidifying agent used for the propagation of
large numbers of organisms, fermentation studies, and various other tests.
Examples: nutrient broth, citrate broth, glucose broth, litmus milk, etc.
ii. Solid media
Contain solidifying agent such agar, silica gel or gelatin. Nutrient agars,
blood agar, and Sabouraud’s agar are examples of solid media that are
used for developing surface colony growth of bacteria and molds. The
development of colonies on the surface of a medium is essential when
 trying to isolate organisms from mixed cultures.
Gelatin as solidifying
agent
Koch used gelatin as a solidifying agent to grow Bacillus anthracis.
However gelatin has a number of drawbacks:

0
A). It melts at 25 C. Hence, difficult to grow microbes that need
0
growth temperature above 25 C
B). Gelatin is a protein and as a result some microbes used as a
source of protein.

Agar as a solidifying
agent Produced from sea
wood called Gellidium
spp.
Agar has a number of advantages:
0
a). has melting point above 100 C,
0
b). Solidify at 45 C and hence ideal for most laboratory purpose,
C). It is totally inert with no nutritive value and indigestible by
microorganisms
d). highly transparent which is important to allow clear observation
of microbial colony.
A good solidifying agent is one that is not utilized by microorganisms,
does not inhibit microbial growth, and does not liquefy at room
temperature.
Agar and silica gel do not liquefy at room temperature and are utilized
by very few organisms. Gelatin, on the other hand, is hydrolyzed by
quite a few organisms and liquefies at room temperature.
iii. Semisolid media
Fall in between liquid and solid media. Although they are similar to
solid media in that they contain solidifying agents such as agar and
gelatin, they are more jellylike due to lower percentages of these
solidifiers. These media are particularly useful in determining
whether certain bacteria are motile or not.
B. Culture media classification based on chemical compositions
i. Synthetic media: type of media in which all the constituents are
chemically defined.
Eg. Nutrient agar
Used to study specific nutritional requirements of microorganisms.
E.g. E .coli can grow on minimal media since it can synthesize the remaining
nutrients
ii. Complex media: a type of media in which there is one or more
nutrients that is chemically undefined.
Usually extracted from biological materials e.g. Beef extract,
peptone ( casein)
Extract, yeast extract
Complex media satisfy the growth of most microorganisms.
iii. Natural media: Naturally available and used as growth media without
modification.
E.g. milk
C. Culture media classification based their functional properties
i. Simple media
 Allow the growth of all microorganisms. E.g. nutrient agar
ii. Differential media
 Used to differentiate a specific microorganism from a given mixed
population.
E.g. Blood Agar: serve as a substrate for some microbes such as S.
pyogenes which lysis blood.
Eosin methylene blue (EMB) agar differentiate lactose fermentors
from non lactose fermentors of enteric bacteria
iii. Selective media
Allow the growth of some bacteria and inhibit others
E.g. Bismuth sulfite agar inhibits all gram positive bacteria and gram
negative bacteria except salmonella
iv. Selective-differential media
Differentiate and select a specific microorganism from a given
mixed population e.g. MacCkoneky’s agar
Selectively grow gram negative bacteria and inhibit gram
positive bacteria
Differentiate lactose fermentors from non lactose fermentors
v. Enrichment medium
Unlike the above types of media, this medium may involve chemical,
physiological, nutritional and environmental factors.
Is used to enrich the required microbe
Examples:
Suppose you want to grow thermopiles, then incubate the culture
 0
at high temperature (> 40 C)
Suppose you want to grow only nitrogen fixing bacteria, then you have to
grow the mixed population on a medium which does not contain any
nitrogen source. Nitrogen fixing bacteria can get nitrogen from the
atmosphere. Suppose a sample is contaminated with fungi, bacteria, algae
and protozoa, if penicillin is added to the medium bacteria will be eliminated,
if polyene added except bacteria all are eliminated. Why?
vi. Transport medium: used to transport samples (urine, swab,
sputum, blood etc.) to laboratory. It helps the organism to survive until
used for diagnosis especially the pathogens
Examples:
 Glycerol saline- transport media used for stool especially for
dysentery bacilli test
 Bile peptone used to transport V.cholerae
 Cary- Blair medium- for faeces specimen (especially which contains
salmonella, Shigella campylobacter or vibrio species
vii. Anaerobic media: used to grow anaerobic microorganisms
Brewer’s Anaerobic Agar This solid medium is an excellent medium
for culturing anaerobic bacteria in Petri dishes
GasPak anaerobic jar ( anaerobic jar); Generates hydrogen that
removes oxygen from the media
viii. Mycobacterium culture media: Used to grow acid fact bacteria.
E.g. Lowenstein Johnson media (LJ) which contain egg as a solidifying
agent and middle brook which is a broth.
Methods and Practice of Decontamination
Control of microorganisms can be achieved by a variety of chemical and
physical methods. Sterilization is generally achieved by using physical
means such as heat, radiation and filtration. Agents which destroy
bacteria are said to be bactericidal. Chemical methods, whilst effective
at disinfection, are generally not reliable for achieving total sterility.
Agents which inhibit the growth and reproduction of bacteria without
bringing about their total destruction are described as bacteriostatic.
Sterilization: destruction or removal of all forms of microbial life
including spores. Heating is the most common method used for killing
microbes.

Sterilization techniques
 1. Heat
One of the oldest forms of antimicrobial treatment is that of heating, and
in most cases this remains the preferred means of sterilization, provided
that it does not cause damage to the material in question. The benefits of
boiling drinking water have been known at least since the 4th century BC,
when Aristotle is said to have advised Alexander the Great to order his
troops to take this precaution. This of course was many centuries before
the existence of microorganisms had been demonstrated or perhaps even
suspected.
a) Moist heat ex.
autoclave
Moist heat kills microorganisms by coagulating and denaturing their
enzymes and structural proteins.
0
Autoclaving is done at 121 c 151b pressure for 15 minutes

E.g Media, solutions, dressing equipotent.

b) Dry heat
Kill microbes by oxidation of intracellular compounds ex. heating at
0
160 c for 2hrs
Direct flame
Incineration: burning to ashes e.g. Paper cups, contaminated dressings,
animal carcasses, bags.
Hot air oxidation Empty glass ware, metal, instrument, needles, glass
syringes. A schematic view of a steam- jacketed autoclave. The entering
steam displaces the air to a port in the bottom of the chamber and saturates
the environment with steam. The pressure rises to 15pascal and the
temperature rises to 121°C. Sterilization is achieved in approximately 15
minutes.
2. Filtration
Separations bacteria from suspending liquid e.g. Enzymes, vaccines
serum, antibiotics and sugar used to sterilize heat sensitive material. They
are available in a variety of pore sizes, according to the specific application
3. irradiation such as UV rays
used to sterilize pharmaceutical and dental supplies

4. Gas vapor sterilization ethylene oxide

a) damage protein of bacteria
eg. Plastic goods, polythene tubes, vaccines etc.
 b) Formaldehyde gas is disinfectant but
carcinogenic
c) H2O2 vapor
Disinfection
The process of destruction of vegetative forms of pathogenic
microorganisms but not necessary spores.
Are harmful to human tissue
Used to decontaminate inanimate object
Examples :Phenols: - damage and denature cell membrane of bacteria
Used to control surgical infections in operating room
Control odor in sewage
Irritate the skin and hence not commonly used
Halogens: I2 and Cl2
I2 (iodine) can be used as disinfectant or antiseptic
kills various microbes (bacteria, fungi, some viruses)
available as tincture i.e. in solution in a aqueous alcohol
Cl2: disinfectant, germicide (HOCl is formed when react
with water)
Different types of chlorine compounds used as disinfectant
Alcohols: widely used to clean skin prior to immunization to
vein puncture Ö disorganize the lipid structure in
membrane and denature protein Effective against
most MOs except spores and some viruses.
Eg.70% ethanol and 70% is propanol used for surface and skin
disinfection
Detergents (surfactants)
Are surface active agents E.g. Omo, Soap
Soap has little value as an antiseptic but is does have an important
function in the mechanical removal of microbes through scrubbing.
Antiseptics: are substances which either kill microbes or inhibit their
growth.
Asepsis: a technique that is used in preventing infections from
gaining cases to uninfected tissue.
Cleaning: is a removing process which may remove many Mos. It is a
necessary pre- requisite before sterilization and disinfection.
Germicide: chemical or physical agent that destroys most organisms
but not spores
Sporocide: chemicals that kills spores and vegetative cells as well
 Fungicide: chemicals that kill fungi
Fungistat: chemicals that stop the growth of fungi

Bacterial Staining Techniques

Stains are generally salts in which one of the ions is colored and the other is
colorless
Stains are chemicals containing chromophores, groups that impart color
e.g. methylene blue (MB); when dissociated into positively and
negatively charge, the methylene ion is colorful and the chloride ion is
color less
Types of stains (based on their charges)
1. Basic stain
The cation (+ve charge) is positively charged
React with negatively charged materials such as the cytoplasm
of bacteria e.g. crystal violet (CV), safranin, MB, basic fuchsine,
2. Acidic stain
Have negatively charged chromophores
Used to stain the background of the cell and leave the cell
transparent e.g. Nigrosine, Congo red
3. Neutral stains
 Neither the positive charge not the negative charge has
chromophore
 E.g. Indian Ink
Staining types (function)
1. Simple satins
Non specific stain
React with all microbes in identical fashion
Consists of only one dye
Used to increase contrast so that morphology, size, etc can be
determined easily
Example MB, CV, Safranin

2. Differential stain
Is specific
More than one (two or more) dyes used
e.g. Gram’s stain, spore stain, capsule stain, flagella stain, AFB stain
The Gram’s Stain
In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally
stumbled on a method which still forms the basis for the identification of
bacteria.
While examining lung tissue from patients who had died of pneumonia, he
discovered that certain stains were preferentially taken up and retained by
bacterial cells. Over the course of the next few years, Gram developed a
staining procedure which divided almost all bacteria into two large groups -
the Gram stain
Four stains used (CV, Alcohol, MB, I2/kI, safranin )
Procedure for Gram's Staining
After the smear has been prepared, dried, heat-fixed, and cooled off,
proceed as follows: Prepare a smear and fix it. Fixing can b done heat
1. Place slide on staining rack and cover specimen with crystal violet.
Let stand for 1 minute.
2. Wash briefly in tap water and shake off excess.
3. Cover specimen with iodine solution and let stand for 1 minute.
4. Wash with water and shake off excess.
Tilt slide at 45° angle and decolorize with the acetone-alcohol
solution until the purple color stops running (maximum for 20
seconds). Wash immediately with water and shake off excess.
6. Cover specimen with safranine and let stand for 1 minute.
7. Wash with water, shake off excess, and gently blot dry. The smear
is now ready to be read. (Use oil immersion lens.)
Principle of Gram's Stain
The crystal violet stain is the primary stain, which stains everything in the
smear blue. The Gram's iodine acts as a mordant that causes the crystal
violet to penetrate and adhere to the gram-positive organisms. The
acetone-alcohol mixture acts as the decolorizer that washes the stain
away from everything in the smear except the gram-positive organisms.
The safranine is the counter-stain that stains everything in the smear that
has been decolorized: pus cells, mucus, gram-negative organisms. The
gram-negative organisms will stain a much deeper pink than the pus cells,
and mucus will stain even lighter pink than the pus cells.
 CHAPTER 2

Methods to Study Microorganisms

Principles of Microscopy

Depending upon the principle on which magnification is based, microscopes

are classified in to two categories; light (optical) and electron microscope.

Light microscopy, in which magnification is obtained by a system of optical

lenses using light waves, includes:

1 Bright-field microscopy
2 Dark-field microscopy
3 Fluorescence microscopy
4 Phase-contrast microscopy
Bright-field microscope is the most widely used instrument for routine
microscopic work while the other types of microscopy are used for special
purposes or research investigations.

1. Bright-Field Microscopy

In bright-field microscopy, the microscopic filed (the area observed) is

brightly lighted and the microorganisms appear dark because they absorb
some of the light.

Ordinarily, microorganisms do not absorb much light, but staining them with
a dye greatly increases their light-absorbing ability, resulting in greater
contrast and color differentiation.

Generally microscopes of this type produce a useful magnification of about X

1, 000 to X 2,000. At magnifications greater than X2, 000 the image
becomes fuzzy.

2. Dark-field microscopy

In this case, a dark background is established, and only the object is

illuminated. A special condenser scatters the light and causes the light to hit
the object from different directions. Some light is reflected from the object
in to the lens, and the object is seen clearly, but since there is no direct
background light, the surrounding area appears dark.

This microscopy is important in the diagnoses of certain diseases when small

live organisms near the limit of resolution of the microscope must be
observed. For example, the syphilis spirochete, Treponema palladium may
be seen in scrapings taken from a skin lesion. Since stain is not used, the
organisms may be seen moving about with a characteristic rotary motion.

3. Phase-contrast microscopy

This allows organisms to be seen alive and without staining. One can easily
see the internal details which cannot be easily visualized with bright-field
microscopy. Bacterial granules, fine structure of protozoa and fungi may be
studied.

The phase-contrast microscope contains a series of special filters and

diaphragms that split the light beam and throw the rays slightly out of phase.
The separated light then passes around microscopic objects as well as
through them, and small differences in the densities of the objects show up
as different degrees of brightness and contrast.

4. Fluorescent Microscopy

The process consists of coating a microbe with fluorescent dye such as

flourescein and illuminating it with ultraviolet light. As the electrons in the
dye are excited, they move to high energy levels, then quickly drop back
giving off the excess energy as visible light. The object appears to fluoresce.
The most important application of this microscopy is in the fluorescent
antibody technique (FAT).

Resolving Power of Microscope

Resolving power is the ability to distinguish two adjacent points as distinct

and separate. Mere increase in size (greater magnification) without the
ability to distinguish structural details (greater resolution) is not beneficial.
To state it differently, the largest magnification produced by a microscope
may not be the most useful because the image obtained may be unclear or
fuzzy.

Resolving power varies for each objective and is calculated as follows:

R.P. = ۸ ۸ = wave length of light (The wave length of visible light is

550nm) 2xNA NA= Numerical
aperture of the lenses

Numerical aperture for low power objective is 0.25, for the high power
objective is 0.65 and for oil immersion objective is 1.25.

The R.P. for the low-power objective is calculated as R.P. =550nm=1.1μm

2x0.25

As the resolving power of this objective is 1.1μm, objects smaller than this
figure cannot be seen clearly or two objectives placed close to each other at
a distant smaller than 1.1μm may not be seen as two separate objects.
The simplest optical microscope is the magnifying glass and is good to about
ten times (10X) magnification. The compound microscope has two systems
of lenses for greater magnification, 1) the ocular or eyepiece lens that one
looks into and 2) the objective lens, or the lens closest to the object.

Eyepiece Lens: the lens at the top that you look through. They are usually
10X or 15X power.

Tube: Connects the eyepiece to the objective lenses

Arm: Supports the tube and connects it to the base

Base: The bottom of the microscope, used for support

Illuminator: A steady light source used in place of a mirror. If your

microscope has a mirror, it is used to reflect light from an external light
source up through the bottom of the stage.
Stage: The flat platform where you place your slides. Stage clips hold the
slides in place. If your microscope has a mechanical stage, you will be able
to move the slide around by turning two knobs. One moves it left and right,
the other moves it up and down.

Revolving Nosepiece or Turret: This is the part that holds two or more
objective lenses and can be rotated to easily change power.

Objective Lenses: Usually you will find 3 or 4 objective lenses on a

microscope. They almost always consist of 4X, 10X, 40X and 100X powers.
When coupled with a 10X (most common) eyepiece lens, we get total
magnifications of 40X (4X times 10X), 100X, 400X and 1000X.

The shortest lens is the lowest power; the longest one is the lens with the
greatest power. The high power objective lenses are retractable (i.e. 40XR).
This means that if they hit a slide, the end of the lens will push in (spring
loaded) thereby protecting the lens and the slide.

Condenser Lens: The purpose of the condenser lens is to focus the light
onto the specimen. Condenser lenses are most useful at the highest powers
(400X and above). Microscopes with in stage condenser lenses render a
sharper image than those with no lens (at 400X).

Diaphragm or Iris: Many microscopes have a rotating disk under the

stage. This diaphragm has different sized holes and is used to vary the
intensity and size of the cone of light that is projected upward into the slide.

ELECTRON MICROSCOPE

An electron microscope is a type of microscope that uses an electron

beam to illuminate a specimen and produce a magnified image. An electron
microscope (EM) has greater resolving power than a light microscope and
can reveal the structure of smaller objects because electrons have
wavelengths about 100,000 times shorter than visible light photons. They
can achieve better than 50 pm resolution and magnifications of up to about
10,000,000x whereas ordinary, non-confocal light microscopes are limited by
diffraction to about 200 nm resolution and useful magnifications below
2000x.
The electron microscope uses electrostatic and electromagnetic lenses to
control the electron beam and focus it to form an image. These electron
optical lenses are analogous to the glass lenses of a light optical microscope.

Electron microscopes are used to investigate the ultra structure of a wide

range of biological and inorganic specimens including microorganisms, cells,
large molecules, biopsy samples, metals, and crystals. Industrially, the
electron microscope is often used for quality control and failure analysis.
Modern electron microscopes produce electron micrographs, using
specialized digital cameras or frame grabbers to capture the image.

Transmission Electron Microscope (TEM)

The original form of electron microscope, the transmission electron

microscope (TEM) uses a high voltage electron beam to create an image. The
electron beam is produced by an electron gun, commonly fitted with a
tungsten filament cathode as the electron source. The electron beam is
accelerated by an anode focused by electrostatic and electromagnetic
lenses, and transmitted through the specimen that is in part transparent to
electrons and in part scatters them out of the beam.

When it emerges from the specimen, the electron beam carries information
about the structure of the specimen that is magnified by the objective lens
system of the microscope. The spatial variation in this information (the
"image") may be viewed by projecting the magnified electron image onto a
fluorescent viewing screen coated with a phosphor or scintillator material
such as zinc sulfide.

Alternatively, the image can be photographically recorded by exposing a

photographic film or plate directly to the electron beam, or a high-resolution
phosphor may be coupled by means of a lens optical system or a fibre optic
light-guide to the sensor of a CCD (charge-coupled device) camera. The
image detected by the CCD may be displayed on a monitor or computer.

Resolution of the TEM is limited primarily by spherical aberration, but a new

generation of aberration correctors have been able to partially overcome
spherical aberration to increase resolution. Hardware correction of spherical
aberration for the high-resolution transmission electron microscopy (HRTEM)
has allowed the production of images with resolution below 0.5 angstrom (50
picometres) and magnifications above 50 million times. [9] The ability to
determine the positions of atoms within materials has made the HRTEM an
important tool for nano-technologies research and development.

An important mode of TEM utilization is electron diffraction. The advantages

of electron diffraction over X-ray crystallography are that the specimen need
not be a single crystal or even a polycrystalline powder, and also that the
Fourier transform reconstruction of the object's magnified structure occurs
physically and thus avoids the need for solving the phase problem faced by
the X-ray crystallographers after obtaining their X-ray diffraction patterns of
a single crystal or polycrystalline powder. The major disadvantage of the
transmission electron microscope is the need for extremely thin sections of
the specimens, typically about 100 nanometers. Biological specimens
typically require be chemically fixing, dehydrating and embedding in a
polymer resin to stabilize them sufficiently to allow ultrathin sectioning.

Scanning Electron Microscope (SEM)

Unlike the TEM, where electrons of the high voltage beam carry the image of
the specimen, the electron beam of the scanning electron microscope (SEM)
[11]
does not at any time carry a complete image of the specimen. The SEM
produces images by probing the specimen with a focused electron beam that
is scanned across a rectangular area of the specimen (raster scanning).
When the electron beam interacts with the specimen, it loses energy by a
variety of mechanisms. The lost energy is converted into alternative forms
such as heat, emission of low-energy secondary electrons and high-energy
backscattered electrons, light emission (cathode) or X-ray emission, which
provide signals carrying information about the properties of the specimen
surface, such as its topography and composition. The image displayed by an
SEM maps the varying intensity of any of these signals into the image in a
position corresponding to the position of the beam on the specimen when
the signal was generated. In the SEM image of an ant shown at right, the
image was constructed from signals produced by a secondary electron
detector, the normal or conventional imaging mode in most SEMs.

Generally, the image resolution of an SEM is about an order of magnitude

poorer than that of a TEM. However, because the SEM image relies on
surface processes rather than transmission, it is able to image bulk samples
up to many centimetres in size and (depending on instrument design and
settings) has a great depth of field, and so can produce images that are good
representations of the three-dimensional shape of the sample. Another
advantage of SEM is its variety called environmental scanning electron
microscope (ESEM) can produce images of sufficient quality and resolution
with the samples being wet or contained in low vacuum or gas. This greatly
facilitates imaging biological samples that are unstable in the high vacuum
of conventional electron microscopes.

Limitations of Electron Microscope

Electron microscopes are expensive to build and maintain, but the capital
and running costs of co focal light microscope systems now overlaps with
those of basic electron microscopes. Microscopes designed to achieve high
resolutions must be housed in stable buildings (sometimes underground)
with special services such as magnetic field cancelling systems.

The samples largely have to be viewed in vacuum, as the molecules that

make up air would scatter the electrons. One exception is the environmental
scanning electron microscope, which allows hydrated samples to be viewed
in a low-pressure (up to 20 Torr or 2.7 kPa) and/or wet environment.

Scanning electron microscopes operating in conventional high-vacuum mode

usually image conductive specimens; therefore non-conductive materials
require conductive coating (gold/palladium alloy, carbon, osmium, etc.) Low-
voltage mode of modern microscopes makes possible observation of non-
conductive specimens without coating. Non-conductive materials can be
imaged also by a variable pressure (or environmental) scanning electron
microscope.

Small, stable specimens such as carbon nanotubes, diatom frustules and

small mineral crystals (asbestos fibres, for example) require no special
treatment before being examined in the electron microscope. Samples of
hydrated materials, including almost all biological specimens have to be
prepared in various ways to stabilize them, reduce their thickness (ultrathin
sectioning) and increase their electron optical contrast (staining). These
processes may result in artifacts, but these can usually be identified by
comparing the results obtained by using radically different specimen
preparation methods. It is generally believed by scientists working in the
field that as results from various preparation techniques have been
compared and that there is no reason that they should all produce similar
artifacts, it is reasonable to believe that electron microscopy features
correspond with those of living cells. Since the 1980s, analysis of cryofixed,
vitrified specimens has also become increasingly used by scientists, further
confirming the validity of this technique.

The stereo or stereoscopic or dissecting microscope is an optical

microscope variant designed for low magnification observation of a sample,
typically using light reflected from the surface of an object rather than
transmitted through it. The instrument uses two separate optical paths with
two objectives and eyepieces to provide slightly different viewing angles to
the left and right eyes. This arrangement produces a three-
dimensional visualization of the sample being examined. [1] Stereomicroscopy
overlaps macro photography for recording and examining solid samples with
complex surface topography, where a three-dimensional view is needed for
analyzing the detail.
The stereo microscope is often used to study the surfaces of solid specimens
or to carry out close work such as dissection, microsurgery, watch, circuit
board manufacture or inspection, and fracture surfaces as in fracto-
graphy and forensic engineering. They are thus widely used
in manufacturing industry for manufacture, inspection and quality control.
The stereo microscope should not be confused with a compound
microscope equipped with double eyepieces and a binoviewer. In such a
microscope, both eyes see the same image, with the two eyepieces serving
to provide greater viewing comfort. However, the image in such a
microscope is no different from that obtained with a single monocular
eyepiece.
Stereo Microscope

Differences to Normal Optical Microscopes

Unlike a compound light microscope, illumination in a stereo microscope
most often uses reflected illumination rather than transmitted(diascopic)
illumination, that is, light reflected from the surface of an object rather than
light transmitted through an object. Use of reflected light from the object
allows examination of specimens that would be too thick or otherwise
opaque for compound microscopy. Some stereo microscopes are also
capable of transmitted light illumination as well, typically by having a bulb or
mirror beneath a transparent stage underneath the object, though unlike a
compound microscope, transmitted illumination is not focused through a
condenser in most systems.[2] Stereoscopes with specially-equipped
illuminators can be used for dark field microscopy, using either reflected or
transmitted light.[3]
Scientist using a stereo microscope outfitted with a digital imaging pick-up
and fibre-optic illumination

Great working distance and depth of field are important qualities for this type
of microscope. Both qualities are inversely correlated with resolution: the
higher the resolution (i.e. the greater the distance at which two adjacent
points can be distinguished as separate), the smaller the depth of field and
working distance. Some stereo microscopes can deliver a useful
magnification up to 100×, comparable to a 10× objective and 10× eyepiece
in a normal compound microscope, although the magnification is often much
lower. This is around one tenth the useful resolution of a normal compound
optical microscope.
The large working distance at low magnification is useful in examining large
solid objects such as fracture surfaces, especially using fibre-
opticillumination. Such samples can also be manipulated easily so as to
determine the points of interest. There are severe limitations on sample size
in scanning electron microscopy, as well as ease of manipulation in the
specimen chamber.

Magnification
There are two major types of magnification systems in stereo microscopes.
One is fixed magnification in which primary magnification is achieved by a
paired set of objective lenses with a set degree of magnification. The other is
zoom or pancraatic magnification, which are capable of a continuously
variable degree of magnification across a set range. Zoom systems can
achieve further magnification through the use of auxiliary objectives that
increase total magnification by a set factor. Also, total magnification in both
fixed and zoom systems can be varied by changing eyepieces.
Intermediate between fixed magnification and zoom magnification systems
is a system attributed to Galileo as the "Galilean optical system" ; here an
arrangement of fixed-focus convex lenses is used to provide a fixed
magnification, but with the crucial distinction that the same optical
components in the same spacing will, if physically inverted, result in a
different, though still fixed, magnification. This allows one set of lenses to
provide two different magnifications; two sets of lenses to provide four
magnifications on one turret; three sets of lenses provide six magnifications
and will still fit into one turret. Practical experience shows that
such Galilean optics systems are as useful as a considerably more expensive
zoom system, with the advantage of knowing the magnification in use as a
set value without having to read analogue scales. (In remote locations, the
robustness of the systems is also a non-trivial advantage.)

Illumination
Small specimens necessarily require intense illumination, especially at high
magnifications, and this is usually provided by a fibre-optic light source. Fiber
optics utilizes halogen lamps which provide high light output for a given
power input. The lamps are small enough to be fitted easily near the
microscope, although they often need cooling to ameliorate high
temperatures from the bulb. The fibre-optic stalk gives the operator much
freedom in choosing appropriate lighting conditions for the sample. The stalk
is encased in a sheath that is easy to move and manipulate to any desired
position. The stalk is normally unobtrusive when the lit end is near the
specimen, so usually does not interfere with the image in the microscope.
Examination of fracture surfaces frequently need oblique lighting so as to
highlight surface features during fractography, and fibre-optic lights are ideal
for this purpose. Several such light stalks can be used for the same
specimen, so increasing the illumination yet further.
More recent developments in the lighting for dissecting microscopes include
the use of high-power LEDs which are much more energy efficient than
halogens and are able to produce a spectrum of colors of light, making them
useful for fluorophore analysis of biological samples (impossible with a
halogen or mercury vapor light source).
Digital Display with Stereo Microscopes
Recently, video dual CCD camera pickups have been fitted to stereo
microscopes, allowing the images to be displayed on a high resolution LCD
monitor. Software converts the two images to an integrated anaglyph 3D
image, for viewing with plastic red/cyan glasses, or to the cross converged
process for clear glasses and somewhat better color accuracy. The results
are viewable by a group wearing the glasses. More typically, a camera
attached to one of the eyepieces will record conventional 2D images.