MICROBIOLOGY DMLT 1 SEMESTER 1 LECTURE NOTES

CHAPTER ONE: INTRODUCTION

1.1 Introduction to Medical Microbiology
Definition

 This is the study of living microscopic organisms in size that can not be
seen by naked eyes but with use of a microscope examples include
bacteria, viruses, protozoa, fungi and algae
 A term comes from two Greek words (i.e. Micro meaning small and bio
meaning life).
 The diameter is the smallest body that can be clearly dissolved and
seen with the naked eyes and is about 100μm but a few
microorganisms are small than this and therefore a microscope is
necessary for their observation.
 The light microscope under optimum conditions can dissolve bodies
down to 0.2μm and this includes all microorganisms except viruses, most
of which are smaller than this. Electron microscope is used to observe
viruses and can dissolve even the smallest virus of 0.01μm in diameter.
1.2 Branches of Microbiology
 Bacteriology: The study of bacteria and a specialist is called a
bacteriologist
 Mycology: The study of fungi. A specialist is called a mycologist
 Protozoology: The study of protozoa.
 Phycology (or algology): The study of algae.
 Parasitology: The study of parasites.
 Immunology: The study of the immune system. an Immunologist

 Virology: the study of viruses. The specialist is called a virologist

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Branches of Applied Microbiology
 Medical microbiology: The study of the pathogenic microbes and the role
of microbes in human illness. Includes the study of microbial pathogenesis
and epidemiology and is related to the study of disease pathology and
immunology.
 Pharmaceutical microbiology: The study of microorganisms that are related
to the production of antibiotics, enzymes, vitamins,vaccines, and other
pharmaceutical products and that cause pharmaceutical contamination
and spoil.
 Industrial microbiology: The exploitation of microbes for use in industrial
processes. Examples include industrial fermentation and waste water
treatment. Closely linked to the biotechnology industry. This field also
includes brewing, an important application of microbiology.
 Food microbiology and Dairy microbiology: The study of microorganisms
causing food spoilage and foodborne illness. Using microorganisms to
produce foods, for example by fermentation.
 Agricultural microbiology: The study of agriculturally relevant
microorganisms. This field can be further classified into the following:
 Veterinary microbiology: The study of the role in microbes in veterinary
medicine or animal taxonomy.
 Environmental microbiology: The study of the function and diversity of
microbes in their natural environments.
 Epidemiology: The study of the incidence, spread, and control of disease.

1.3 Microbiology and Medicine

The application of microbiology has given medicine its greatest success in

diagnosis, prevention and cure of diseases.

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Microorganisms and Disease

Only a small proportion of microorganisms that are bound in nature are

disease producing (pathogenic for man) therefore;

Pathogens are organisms that are capable of causing a disease. Most of

them are free living in the soil and water.

Some free-living microorganisms obtain energy from light or oxidation of

organic matter and they are termed as saprophytes

Commensals constitute the normal flora of a healthy body and they live on
the skin, in the mucous membrane of the upper respiratory tract, intestines,
and vagina and obtain their nourishment from the secretion of food residues.
Normally the commensals do not invade the blood and tissues.

Since they are generally harmless but under certain conditions as when the
body‟s defence mechanism is impaired they invade the tissues and cause
diseases thus acting as opportunistic pathogens.

True pathogens are the parasitic microorganisms that are adapted to

overcoming the normal defences of the body and invading the tissues. They
grow in the tissues, their production of toxins damage the tissues and cause
the manifestations of a disease.

The process of microbial invasion in the body is called infection. In addition,

the microbial diseases are often called the microbial infections.

Those infective diseases that are readily communicable from a person to a

person are called contagious or infectious diseases e.g. Cholera, plague,
measles, meningitis and dysentery e.t.c. Tetanus is an infective disease but not
contagious.

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To prove that a disease is caused by a particular microorganism, Koch‟s
postulates are applied; for any organism to be responsible for a particular
disease, it must be;

 Recovered in a pure form from the experimental animal.

 A pure culture must produce the same disease as in the experimental
animal.
 The organism must be isolated artificially.
 The organism must not be found in the healthy host.
 Serum of an infected person should contain titre antibodies, which are
specifically protective.

Immunity and Immunisation

Today, attenuated vaccines are used to protect against diseases like TB, Polio,
Measles and yellow fever.

Killed vaccines are used against diseases like Whopping cough, Influenza,
Typhoid while toxoids are infective in immunizing agents like Diphtheria and
Tetanus.

Antitoxins: they are immunoglobulins rised against a toxin in other animal and
administered to generate artificial, passive immunity, has immediate but short
term effect example rabies and tetanus antitoxins.

Toxoids: are toxins produced by organisms and are detoxified by treatment

with formaldehyde and used for the production of immunity in the receipient
e.g diphtheria, tetanus vaccines.

Epidemiology and Prevention of Infection

Many species of pathogens are derived exclusively or mainly from ill patients
as their sources but many others grow in and are disseminated from healthy

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persons known as carriers in whom they cause only limited sub-clinical
infections.

Some infections are called Zoonosis where they have their sources in animals
which are the natural hosts of the pathogen eg Rabies in dogs, bubonic
plague in rats, brucellosis in cattle, goats and pigs. Such infections from
external sources are termed as exogenous infections while Endogenous
infections are due to opportunistic invasions of the tissues by the commensals
in the body e.g. pneumonic infections of the lungs with pneumococci
resident in the throat.

1.4 History of microbiology

Modern microbiology History

Robert Hooke

The science of microbiology started with inventing of the microscope. The

English scientist Robert Hooke was credited being the first person to use the
microscope for academic study that was in the early 1660s. In 1665, Hooke
published his landmark book „Micrographia‟ that described the microscopic
world for the first time.

 Hooke studied plant sections in a particular cork and drew what he saw
which a matrix of tiny cylindrical-like structures was called cells. Later
researchers saw such structures in all types of living organisms and Hooke‟s
naming remained. Today, it is considered a foundation stone in the
understanding of microbiology.
 Robert Hooke is also remembered as the first person to report seeing
microbes under the microscope. In addition to the cellular structure of
plants, he also saw fungi, which he drew. He used a crude common
microscope, however because his lens was of poor quality, he was
apparently unable to see the bacteria.

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Edward Jenner

Small pox was one of the greatest problems of mankind. Thousands of years it
swept through human population killing 40% of its victims and living many of
the survivors scared for life, their face covered with a deep red pit. The
ancient Chinese recognized that whoever recovered from the small pox were
immune to small pox.

 Some unknown person in China perhaps noticing that even people who
only developed a few scabies were as immune as those whose bodies
were covered with scars.
 He took materials from a dried scaby and scratched it into uninfected
persons in attempt to immunize them. It worked and the process was
repeated by others, with the technique eventually reaching India. From
India, it traveled by several routes to Europe and in the 17 th century, it
came into common usage.
 The only problem was that the scabby from the victim contained a fully
virulent virus capable of producing the clinical disease. Thus, when one
person might have only a mild case and become immune, they shed the
virulent virus and were capable of starting their own epidemic. This was a
situation when Edward Jenner entered the picture. Through a series of
events, Jenner was led to the discovery of immunization and eventually to
the elimination of the small pox virus from the Earth.
 As a young man, he had lived in the country and had been told by a
milkmaid that he never had to worry about getting small pox because he
had had cowpox-a mild chronic disease of cows that milkmaids contract
on their hands as a rash.
 Later after Jenner became a physician and took up country practice, he
remembered the milkmaid‟s story. By 1796, he became convinced that the
story was true so he inoculated an 8 year old boy with cowpox and 8
weeks later, he inoculated the small boy with pus from a smallpox lesion.

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 The boy showed no effect and Jenner repeated the experiment as the
word of his result spread. Others began to test it and by 1803, it was
established as a medical procedure in England. It was from this risky
beginning that the science of immunization developed.
 While the scientists of the 18th and 19th centuries studied plant and animal
structures under the microscope, the real science of microbiology only
began in the later half of the 19th century. When high magnification
microscopes of good optical quality became more widely available, the
most notable person was Ferdinand .J. Cohn who in 1875 effectively found
the science of bacteriology. His main contribution was the classification of
bacteria and he coined (founded) the term Bacillus.
 Meanwhile in continental Europe where others like Antonie Van Leeuwen
Hook, in Netherlands, he was using microscopes to look at animals and
plant tissues. He examined a drop of rainwater that contained tiny
creatures that he called Animalicules and these were infant bacteria and
therefore he became the first person to study and see bacteria in 1632 -
1723.
 He was also born before his time although not the first person to discover
the microscope or a magnifying lens. He was also a Dutch merchant and
sometimes in delight Holland. He used a magnifying lens to view the quality
weave of the merchandise he purchased.

 He traveled to England in 1668 to view English cloth and therefore he saw

drawings of magnification of the cloth much greater. He became the first
microbiologist to use a microscope for viewing microorganisms.

Louis Pasteur

He was probably the greatest microbiologist of the 19 th century and around

1857, he developed the germ theory of diseases i.e. the idea that diseases
may be caused by microscopic organisms not visible by naked eyes. (That

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every disease to occur, there must be a causative agent) This was a
significant break in medicine that ultimately improved the health of everyone
on the planet.

 He was also able to prove that life itself did not spontaneously come
into being. Through a series of experiments, he successfully showed that
life could only be generated from existing life.
 Pasteur also showed that the fermentation process used in baking and
brewing was caused by microorganisms. Because of his work, he went
home and developed the process of sterilizing milk and this was named
after him.
 He is also remembered for the development of vaccines most notably
for rabies and anthrax.
 In addition, he identified and eliminated the disease in silk worms. He
was also interested in the idea of Panspermia that was promoted by
Lord Kelvin.

Robert Koch

In the late 1870s, a German country bacteriologist, physician became

interested in anthrax, a common disease of both the farmers and their
animals in rural practice.

Using a microscope, Koch saw a larger bacterium in the blood of an anthrax

victim.

He used his home as a laboratory and developed a basic microbiological

technique. Koch tested out the anthrax bacterium and purified i.e. he then
inoculated the purified bacterium into a healthy animal and produced the
classical clinical disease.

When he examined the blood of the inoculated animal, he was able to

isolate the same bacterium. He repeated the isolation, infection and disease

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cycle until he was sure that he had found the agent of anthrax, thus, he was
the first person to cultivate anthrax bacteria outside the body using blood
serum at body temperature building Pasteur‟s germ theory.

Koch also attracted many other bright scientists and together they
developed the basic techniques of (microorganisms) microbiology
laboratories we still use today. These include:

 The sterile culture techniques, pure culture techniques, the use of agar and
gelatin to produce a solid surface.
 The Gram staining and other staining techniques (or procedures).
In addition, Koch discovered the causative agent of cholera and TB. These
studies in combination to those of Pasteur established the germ theory of
disease. His procedure of defining the agent of any disease called Koch‟s
postulates, which consists of the following four steps established in 1884. These
are:

 The agent must be present in every case of disease i.e. isolate the
suspected agent for a disease victim.
 The agent must be isolated and cultured in vitro i.e. grow the agent in a
pure culture.
 The disease must be pre-produced when a pure culture of the agent is
inoculated into suspected host i.e. affects a healthy host and show that
the organism produces the classical clinical disease.
 The agent must be recoverable from the experimentary infected host i.e.
isolate the same organism for the new victim.

Joseph Lister

1827 to 1912, he introduced antiseptics in surgery by spraying carbonic acid.

On surgical instruments, wounds and dressings, he reduced surgical infections

due to bacterial infection and death.

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He instituted the use of chemicals and microbial for sanitation of objects that
come into contact with surgical wounds.

Alexander Flemming

In the early 19th century, he discovered penicillin, an antibiotic and the use of
chemotherapy.

Francesco Red

In 1688, Francesco who was an Italian physician refused the idea of

spontaneous generation by showing that rotting meat carefully kept from flies
will not spontaneously produce maggots.

Theodor Schwann

In 1836, Theodor Schwann helped to develop the cell theory of living

organisms. It states that all living organisms are composed of one or more cells
and that cell is basic function out of living organism.

Ernest Ruska

In 1931, he constructed the first electronic microscope.

Walter Hasse

He became interested in new science of microbiology joined Koch‟s

laboratory in 1881. He was the first man to study many aspects of bacterial
public health and bacterial metabolism.

1.5 Importance of Microorganisms to Man

 Many microbes are responsible for numerous beneficial processes such as

industrial fermentation (e.g. the production of alcohol, vinegar and dairy
products)

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 Antibiotic production and as vehicles for cloning in more complex
organisms such as plants. production of biotechnologically important
enzymes such as Taq polymerase, reporter genes for use in other genetic
systems and novel molecular biology techniques such as the yeast two-
hybrid system.

 Bacteria can be used for the industrial production of amino acids.

Corynebacterium glutamicum is one of the most important bacterial
species with an annual production of more than two million tons of amino
acids, mainly L-glutamate and L-lysine.

 Microorganisms are used for the biotechnological production of

biopolymers with tailored properties suitable for high-value medical
application such as tissue engineering and drug delivery.

 Microorganisms are beneficial for microbial biodegradation or

bioremediation of domestic, agricultural and industrial wastes and
subsurface pollution in soils, sediments and marine environments.

 There are also various claims concerning the contributions to human and
animal health by consuming probiotics (bacteria potentially beneficial to
the digestive system) and/or prebiotics (substances consumed to promote
the growth of probiotic microorganisms).

 Recent research has suggested that microorganisms could be useful in the

treatment of cancer. Various strains of non-pathogenic clostridia can
infiltrate and replicate within solid tumors.

 Clostridial vectors can be safely administered and their potential to deliver

therapeutic proteins has been demonstrated in a variety of preclinical
models.

 Microorganisms are disease causative agents for man.

 Some organisms like normal flora prevent colonization of pathogens.

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1.6 Normal Flora of Human Body

 They are microorganisms that reside on the surface and in deep layers of
skin, in the saliva and oral mucosa, in the conjunctiva, and in the
gastrointestinal tracts.

 They include bacteria, fungi, and archaea. Some of these organisms

perform tasks that are useful for the human host. However, the majority
have no known beneficial or harmful effect. Those that are expected to be
present, and that under normal circumstances do not cause disease, but
instead participate in maintaining health, are deemed members of the
normal flora.

 The bacteria of the normal flora consist of symbiots, commensals and

opportunists

Bacterial flora

 Populations of microbes such as bacteria and yeasts inhabit the skin and
mucosa. Their role forms part of normal, healthy human physiology,
however if microbe numbers grow beyond their typical ranges (often due
to a compromised immune system) or if microbes populate atypical areas
of the body (such as through poor hygiene or injury), disease can result.

 Many of the bacteria in the digestive tract, collectively referred to as the

gut flora e.g. Escherichia coli are able to break down certain nutrients
such as carbohydrates that humans otherwise could not digest. The
majority of these commensal bacteria are anaerobes. Normal flora
bacteria can act as opportunistic pathogens at times of lowered immunity.

 Actinomyces live in the mouth, where they are part of a sticky substance
called plaque. If this is not removed by brushing, it hardens into calculus
(also called tartar). The same bacteria also secrete acids that dissolve
tooth enamel, causing tooth decay.

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 The vaginal microflora consist mostly of various lactobacillus species such
as Lactobacillus acidophilus, L. iners , L. crispatus. Other lactobacilli found
in the vagina are L. delbruekii and L. gasseri. Disturbance of the vaginal
flora can lead to bacterial vaginosis.

Skin flora

 Actinobacteria (51.8%), Proteobacteria (16.5%), and Bacteroidetes (6.3%)

Conjunctival flora

 A small number of bacteria are normally present in the conjunctiva.

Staphylococcus epidermidis and certain coryneforms such as
Propionibacterium acnes are dominant. Staphylococcus aureus,
streptococci, Haemophilus species and Neisseria species sometimes occur.

 Some pathogens able to infect the conjunctiva, such as Neisseria

gonorrhoeae and Chlamydia trachomatis are thought to have special
processes allowing them to attach to the conjunctival epithelium.

Commensals

 These are organisms of the normal bacterial flora of the body. They live on
the skin and mucous membranes of the upper respiratory tract, intestines
and the vagina. They are mostly either beneficial or harmful to their hosts
and can protect by competing with potential pathogens.

 Normal floras actually have a beneficial effect by preventing colonization

of body sites by other potential pathogens e.g. in the skin (staphylococcus
epidermidis), mouth (staphylococcus aureus), vagina (lactobacilli), nose
and nasopharynx (Neisseria species) and intestines.

 Resident normal floras are normal floras which live/reside in the body of the
host for a long time e.g. Escherichia coli in the adult intestinal tract.

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 Transient normal floras are normal floras not of medical significance that
live on the skin and do not survive for a long time e.g. staphylococcus
epidermidis.

Parasitism

 A parasitic relationship is one in which one member of the association

benefits while the other is harmed.

 Parasitic symbioses take many forms, from endoparasites that live within
the host's body to ectoparasites that live on its surface. In addition,
parasites may be necrotrophic, which is to say they kill their host, or
biotrophic, meaning they rely on their host's surviving.

Opportunistism

These are organisms that can if a suitable opportunity arises become

pathogenic and cause disease. Such an opportunity may arise following;

 Transfer of a commensal from its usual habitat to another part of the body
where it can establish itself and cause a disease e.g. Escherichia coli in the
intestinal tract if it enters the urinary tract, it causes urinary tract infection
(UTI).
 Weakening of the person‟s natural immunity due to poor health, diseased
condition, malnutrition e.t.c for example staphylococcus aureus is a normal
flora in the nose and can become a pathogen and cause pneumonia in a
child with measles and influenza.
 Opportunistic organisms are often the cause of nosocomial infections
which are accidentally acquired by patients during a hospital stay due to
their defence mechanisms being weakened.
Colonization: This is the establishment of a stable population of bacteria or
pathogenic bacteria in the host‟s tissue and normally requires adhesion to the
mucosal cell surfaces.

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Carriers: Human carrier is a person who is infected by a pathogen but
experiences no diseases from it. Such a person can spread the pathogen for
a long time and be a source of infection to others without realizing it. The
carrier state is particularly important in transmission of typhoid.

Bacteria host relationship

Symbiosis

 Is often long-term interaction between different biological species. Which

previously had been used of people living together in community) to
describe the mutualistic relationship in lichens. Defined it as "the living
together of unlike organisms.

 Symbionts are organisms that usually benefit the person who is infected
e.g. the enteric bacteria that form part of the normal flora in the intestines
assist in the synthesis of vitamin K and some of the vitamins of the B
complex.

 Some symbiotic relationships are obligate, meaning that both symbionts

entirely depend on each other for survival. For example, many lichens
consist of fungal and photosynthetic symbionts that cannot live on their
own. Others are facultative, meaning that they can but do not have to live
with the other organism.

 Symbiotic relationships include those associations in which one organism

lives on another (ectosymbiosis, such as mistletoe), or where one partner
lives inside the other (endosymbiosis, such as lactobacilli and other
bacteria in humans or zooxanthelles in corals).

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Mutualism

 Mutualism is any relationship between individuals of different species

where both individuals derive a benefit.

 Mutualistic relationships may be either obligate for both species, obligate

for one but facultative for the other, or facultative for both. Many biologists
restrict the definition of symbiosis to close mutualist relationships.

 A large percentage of herbivores have mutualistic gut fauna that help

them digest plant matter, which is more difficult to digest than animal prey.

Commensalism

 Commensalism describes a relationship between two living organisms

where one benefits and the other is not significantly harmed or helped. It is
derived from the English word commensal used of human social
interaction.

 There are two other types of association: mutualism (where both organisms
benefit) and parasitism (one organism benefits and the other one is
harmed).

Pathogens: are a part of human body flora which have a bility to cause
disease.

Transmission of microorganisms

Endogenous transmission: when body microorganism cause disease.

Occurance of disease as result of organisms from human body

Exogenous transmission: transmission of microorganisms from the external

environment.

Sources of microorganisms: soil, water, air (aerosols), dust, vegetation.

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Routes of organism transmission

These may be different from natural infections; infections may be caused by

microorganisms entering the body through the skin, eyes, mouth and the
respiratory tract.

(i) Skin and Eyes (penetration)

Organisms may enter through the unbroken skin but it is more usual for them
to enter through cuts and scratches. Most of which may not be visible by
naked eyes.

The organisms are picked up from the hands on a bench or equipment that
has been accidentally contaminated by small usually unnoticed drops, spills
and splashes. They may be transferred to the face or eyes by the fingers.

(ii) Body contact by direct through sexual intercourse with infected individual
e.g hepatitis, HIV. Indirect contact with the contaminated objects with
infectous organisms

(iii) Through the mouth (ingestion)

Microorganisms may be ingested during the mouth pipetting either by direct

aspiration or from the ends of the pipette, which has been touched by the
contaminated fingers.

Direct fingers to mouth infection are also possible by eating food in the
laboratory. Food may become contaminated from the benches or fingers or
by contact with infected materials e.g. in refrigerators. Cigarettes or pipettes,
pens that are handled or placed on the laboratory benches, May also
transfer organisms to the mouth.

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(iv) Through respiratory tract (inhalation)

Most common laboratory procedures, microorganisms release aerosols i.e.

infected air droplets in the atmosphere. Small droplets evaporate and leave
behind droplet nuclei consisting of bacteria or viruses, which are too light to
settle, they are moved around the room or even the whole building and
through the small air current and ventilation system. If inhaled, big particles will
be captured in the nossal passage, small particles, however, will not be
trapped and may be inhaled right away into the lungs where they may start
an infection.

(v) Congenital or transplacental: mother to child transmission e.g HIV, syphilis.

(vi) Iatrogenic means: through blood transfusion or use of surgical instrument

with freshly collected blood.

(b) Outside the laboratory

Microorganisms from the laboratory may enter the bodies of the public by the
same Routes as those affecting laboratory workers but the sources are
different.

In general, the public may become infected as a result of an escape of

microorganisms during transportation of infectious specimens to and between
laboratories e.g. from healthy centres to hospitals.

Another source of infection can occur if the public is exposed to infectious

materials from the laboratory due to failure of laboratories to decontaminate
such materials.

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Some organisms are more hazardous to handle and more likely to infect
laboratory workers than others. In the programme formulated by WHO, there
are four groups of these risks.

CHAPTER TWO: CLASIFICATION OF BACTERIA

2.1 Taxonomic ranks and definitions

Bacteria are classified and identified to distinguish one organism from another
and to grow similar organisms by criteria of interest to a microbiologist or other
scientists. The most important level of this type of classification is the species‟
level.

Taxonomy

This is the science of classification, identification, naming, describing

organisms and nomenclature.

Taxonomic ranks

 These are categories used in the classification of living organisms.

Organism was grouped into two broad categories, the plantae and
animalia. In 1866 the third kingdom was established call protista which
would include all the single-celled organisms and those multicellular forms
not developing complex tissues.

 In 1957, Fungi as a kingdom was established and 1969 kingdom monera

was also named which included prokaryotics.

 In 1978 a new taxonomic rank called domain above the kingdom in the
taxonomic hierarchy was named. The domain of life accepted by most
biologists includes the eukarya, bacteria and archaea.

 Taxonomic rank in biological classification, rank is the level (the relative

position) in a taxonomic hierarchy.

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 Examples of taxonomic ranks are species, genus, family, and class .The
International Code of Zoological Nomenclature defines rank as:

 The level, for nomenclatural purposes, of a taxon in a taxonomic hierarchy

(e.g. all families are for nomenclatural purposes at the same rank, which
lies between superfamily and subfamily.
 Today, nomenclature is regulated by the nomenclature codes, which
allow names divided into an indefinite number of ranks. There are seven
main taxonomic ranks: kingdom, phylum or division, class, order, family,
genus, species. In addition, the domain (proposed by Carl Woese) is now
widely used as one of the fundamental ranks, although it is not mentioned
in any of the nomenclature codes.

Main taxonomic ranks

Latin English

Region domain

Regnum kingdom

Phylum Division phylum (in zoology) division (in botany)

Classis class

Ordo order

Familia family

Genus genus

Species species

 A taxon is usually assigned a rank when it is given its formal name.

 The basic rank is that of species. The next most important rank is that of
genus: when an organism is given a species name it is assigned to a genus,
and the genus name is part of the species name.

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 The third-most important rank, although it was not used by Linnaeus, is that
of family.

Classification

 This is the orderly arrangement of bacteria into groups. There is nothing

inherently about scientific classification and different groups of scientists
may classify some organisms differently e.g. clinical microbiologists are
interested in the serotypes, antimicrobial resistance.

Identification

This is the practical use of classification criteria to distinguish certain organisms

from others, to verify the authority of a strain or a particular reaction or to
isolate and identify the organisms that cause a disease.

Nomenclature

This is the means by which the characteristics of species are defined and
communicated among the microbiologists. A species‟ name should mean the
same thing to all microbiologists. Yet some definitions vary in different
countries or microbiologic speciality groups e.g. of organisms include;
Clostridium perfringenes in the USA is called Clostridium Welchi in England.

Biological classification or scientific classification in biology

Is a method to group and categorize organisms by biological type, such as

genus or species. Biological classification is part of scientific taxonomy
(Bergey‟s manual of systematic bacteriology).

Modern biological classification has its root in the work of Carolus Linnaeus,
who grouped species according to shared physical characteristics. These

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groupings have since been revised to improve consistency with the Darwinian
principle of common descent. Molecular phylogenetics, which uses DNA
sequences as data, has driven many recent revisions and is likely to continue
to do so. Biological classification belongs to the science of biological
systematics.

Species (the most specific category)

 A bacterial species is a distinct organism with certain characteristic

features or a group of organisms that resemble one another closely in
the most important features of their organization.

 Species is the basic and most important taxonomic group especially for
bacteria systematic. It is a distinct group of strains that have certain
distinguishing features and bear a close resemblance to one another in
the more essential feature of organization.

Sub species: These are variants within the species.

A strain: This is made up the descendants of a single isolation in pure culture

and the strain of a species is designated as the type strain.

Non-Taxonomic ranks

These are ranks below sub species such as biovars, serovars, phagovars,
pathovars which are often used to indicate groups of strains that can be
distinguished by some special characters such as antigenic makeup, biotype
strains having special biochemical properties; serotype strains have distinctive
antigenic properties

2.2 Bacterial Classification

 Bacteria are a large domain of single-cell, prokaryote microorganisms.

Typically a few micrometres in length.

Tsongo Lawrence Msc (MUK) 22

 They have a wide range of shapes, ranging from spheres to rods and
spirals.

 The definitive identification scheme for bacteria was first presented in 1984
in Bergey’s Manual of Systematic Bacteriology.

 In this scheme, bacteria were classified on the basis of many

characteristics. By their morphology (cell shape), staining reactions
example gram stain, Ziel neelsen stain, growth requirements and
biochemical characteristics.

 Bacteria can also be classified by their DNA composition using specialized

techniques.
 They are found in various forms, can occur in singles, pairs, clusters, chains
and also have different shapes e.g. cocci, rods (branching forms, spiral
shaped e.t.c.)

2.2.1 Bacterial Morphology

Bacteria cell morphology (shape) is classified into two typical examples

 coccus (spherical)
 bacillus (rod-like)

Coccus

They are divided into 3 groups;

(a) Cocci in clusters. The bacterial cells remain attached to each other when
dividing forming cells of cluster organism‟s example Staphylococci species.
Bacteria characterized by cells arranged in tetrad clusters (four cells in a
square formation) or large, often irregular, clusters.

Tsongo Lawrence Msc (MUK) 23

(b) Cocci in chains. The bacterial cells remain attached to each other forming
cells of chains when dividing example Streptococci species

(c) Cocci in pairs/diplococci. When dividing, the bacterial cell remain

attached to one another forming pairs of cells example Neisseria species

Note that, several species of bacteria are able to change their form
especially after growth on artificial media, they can appear as singles.
Organisms which show variation in shape, size or different forms are described
as Pleomorphic.

Characteristics of cocci

 Cocci are round, oval, Spherical bacteria.

 They measure about 0.5-1.0 μm in diameter.
 When multiplying, cocci may form pairs, singles, chains, clusters or irregular
forms.
 Cocci in pairs are called diplococci example Meningococci and
Gonococci.
 Cocci in chains are called Streptococci example Streptococcus
pyogenes.
 Cocci in irregular groups or clusters are called Staphylococcus example
Staphylococcus aureus.

Tsongo Lawrence Msc (MUK) 24

  Bacteria in the Sarcina genus typically form a cuboidal arrangement of
eight cells

Staphylococcus bacteria.

Tsongo Lawrence Msc (MUK) 25

. (ii) Rods (Bacilli)

Bacillus

Bacillus subtilis, Gram

stained

They are into different rods;

Tsongo Lawrence Msc (MUK) 26

(a) Straight rods example Escherichia
(b) Club-shaped rods example Corynebacterium
(c) Branching rods example Actinomyces
(d) Comma forms example Vibrios
(e) Spore forming rods example Clostridium and Bacillus
Characteristics of rods
 Rods are stick-like bacteria with rounded, tapered, square or swollen ends.
 They measure 1-10μm in length by 0.3-1.0μm in width.
 The short rods with rounded ends are often called coccobacilli.
 When multiplying, the bacterial rods usually remain attached to one
another but separate. However, they may form chains e.g. Streptobacilli
species, form branching chains e.g. Lactobacilli, form mass together
example mycobacterium leprae, remain attached at many angles
resembling Chinese letters example corynebacterium diphtheriae.
 Vibrios are small slightly curved rods (comma shaped) measuring 3-4μm by
0.5μm in width. They are motile with single flagellum at one end and show
rapid darting motility example Vibrio cholerae.

Spirillum

Spirillum is bacterium with a cell body that twists like a spiral. It is the third
distinct bacterial cell shape type besides coccus and bacillus cells.

Spirillum

Tsongo Lawrence Msc (MUK) 27

Characteristics

 Spirillum is a genus of Gram-negative bacteria (family Spirillaceae). Spirilla

are motile with proups of flagella at both ends.
 Spirochaetes are flexible, coiled, and motile and progress by rapid body
movement; they are small, coiled, rigid and measure 3-4μm in length.
 Spiral forms example are Spirochaetes (singular spirochaetes) Treponema
pallidum, Spirilla (singular spirillum)

CHAPTER THREE: ANATOMY OF A BACTERIA CELL

3.1 Bacterial Structure

Bacteria form a large group of pathogenic, saprophytic and free-living

microorganisms.

They are unicellular that produce by binary fission. Their cell type is divided
into two, Prokaryotic and Eukaryotic groups.

Prokaryotic group
a) They are microorganisms with very simple cell structure that have no
nuclear membrane thus no definite nucleus e.g. bacteria such as
rickettsiae, Chlamydia, mycoplasm.
b) Prokaryotes multiply by simple dividing known as binary fission.
c) The cell contains simple enzyme systems that lack mitochondria.
d) The genetic material is not organized into chromosomes inside a nuclear
membrane but lies as a single piece of double stranded DNA inside the
cytoplasm.

Eukaryotic group

a) They are microorganisms with a complex cell structure similar to that of

higher organisms example Slime moulds, protozoa, & algae

Tsongo Lawrence Msc (MUK) 28

b) They have a nuclear membrane, which forms a defined nucleus.
c) The genetic material is differentiated into chromosomes which are
contained in a nuclear membrane to form a definite nucleus.
d) They multiply by a process known as mitosis
e) The cell is able to make its own energy because it possesses complex
enzyme systems, mitochondria and other organelles.

3.2 Typical shape and arrangement of bacterial cell

 A prokaryotic cell has five essential structural components: a nucleoid

(DNA), ribosomes, cell membrane, cell wall, and some sort of surface layer,
which may or may not be an inherent part of the wall.

Structurally, there are three architectural regions:

 Appendages (attachments to the cell surface) in the form of flagella and

pili (or fimbriae);
 A cell envelope consisting of a capsule, cell wall and plasma membrane;
 A cytoplasmic region that contains the cell chromosome (DNA) and
ribosomes and various sorts of inclusions.

Tsongo Lawrence Msc (MUK) 29

Cell envelope
 The cell envelope is a descriptive term for the several layers of material
that envelope or encloses the protoplasm of the cell.
 The cell protoplasm (cytoplasm) is surrounded by the plasma membrane, a
cell wall and a capsule.
Cell Wall

The cell walls of bacteria deserve special attention for several reasons:

 They are an essential structure for viability

 They are composed of unique components found nowhere else in nature.
 They are one of the most important sites for attack by antibiotics.
 They provide ligands for adherence and receptor sites for drugs or viruses.
 They cause symptoms of disease in animals.
 They provide for immunological distinction and immunological variation
among strains of bacteria.

Tsongo Lawrence Msc (MUK) 30

 The cell wall is an essential structure that protects the cell protoplast (the
region bound by and including the membrane) from mechanical damage
and from osmotic rupture or lysis.
 They are porous and therefore permeable to salt molecules
 They maintain the bacterial shape
 The cell wall is made of porous, rigid material that has high tensile
strength. Such a material is murein. Bacterial murein is a unique type of
peptidoglycan.
 Peptidoglycan is a polymer of sugars (a glycan) cross-linked by short
chains of amino acids (peptide).
 All bacterial peptidoglycans contain N-acetylmuramic acid, which is
the definitive component of murein.

In Gram-positive Bacteria (those that retain the purple crystal violet dye when
subjected to the Gram-staining procedure), the cell wall is thick (15-80
nanometers), consisting of several layers of peptidoglycan.

Running perpendicular to the peptidoglycan sheets are a group of molecules

called teichoic acids which are unique to the Gram-positive cell wall.

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Structure of the Gram-positive bacterial cell wall. The wall is relatively thick
and consists of many layers of peptidoglycan interspersed with teichoic acids
that run perpendicular to the peptidoglycan sheets.

In the Gram-negative Bacteria (which do not retain the crystal violet in the
Gram-stain procedure) the cell wall is relatively thin (10 nanometers) and is
composed of a single layer of peptidoglycan surrounded by a membranous
structure called the outer membrane.

The outer membrane of Gram-negative bacteria invariably contains a

unique component, lipopolysaccharide (LPS or endotoxin), which is toxic to
animals and is usually considered as part of the cell wall.

The wall is relatively thin and contains much less peptidoglycan than the
Gram-positive wall. Also, teichoic acids are absent. However, the Gram
negative cell wall consists of an outer membrane that is outside of the
peptidoglycan layer a unique group of lipoprotein molecules.

Tsongo Lawrence Msc (MUK) 32

The Outer Membrane of Gram-negative Bacteria

 Of special interest as a component of the Gram-negative cell wall is the

outer membrane.
 A discrete bilayered structure on the outside of the peptidoglycan sheet.
For the bacterium, the outer membrane is first and foremost a permeability
barrier, but primarily due to its lipopolysaccharide content, it possesses
many interesting and important characteristics of Gram-negative bacteria.
 The outer membrane superficially resembles the plasma membrane
except the outer face contains a unique type of Lipopolysaccharide
referred to by medical microbiologists as endotoxin because of its toxic
effects in animals.

Tthe outer membrane, cell wall, and plasma membrane of a Gram-negative

bacterium Lipopolysaccharide (LPS or endotoxin) is located on the outer face
of the outer membrane.

Correlation of the Grams stain with cell wall properties of Bacteria

Property Gram-positive Gram-negative

Thickness of wall thick (20-80 nm) thin (10 nm)

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Number of layers 1 2
Peptidoglycan (murein) content >50% 10-20%
Teichoic acids in wall Present Absent
Lipid and lipoprotein content 0-3% 58%
Protein content 0 9%
Lipopolysaccharide content 0 13%
Sensitivity to Penicillin G Yes no (1)
Sensitivity to lysozyme Yes no (2)

Cell Wall-less Forms

 A few bacteria are able to live or exist without a cell wall. The
mycoplasmas are a group of bacteria that lack a cell wall.

Capsules

 This is a polysaccharide layer outside of the cell wall polymer called a

capsule.
 A true capsule is a discrete detectable layer of polysaccharides deposited
outside the cell wall. A less discrete structure or matrix which embeds the
cells is a called a slime layer or a biofilm.
 A type of capsule found in bacteria called a glycocalyx or microcapsule is
a very thin layer of tangled polysaccharide fibers on the cell surface.

Tsongo Lawrence Msc (MUK) 34

Bacterial capsules outlined by India ink viewed by light microscopy.
Types of capsule
This is a true capsule, a discrete layer of polysaccharide surrounding the cells.
Aslime layer or biofilm, embedded more randomly in a polysaccharide matrix.
Polysaccharide films that may inevitably be present on the surfaces of
bacterial cells, but cannot be detected visually, are called glycocalyx.
Capsules are generally composed of polysaccharides; rarely do they contain
amino sugars or peptides.

Colonies of Bacillus anthracis. The slimy or mucoid appearance of a bacterial

colony is usually evidence of capsule production.

Functions of Capsules, Glycocalyx, Slime Layers and Biofilms

Function Example
Adherence to surface, tissue or substrate Streptococcus mutans - dental
in nature plaque
Resistance to engulfment by phagocytic Streptococcus pneumoniae -
cells lobar pneumonia
Resistance to killing and digestion by Bacillus anthracis - cutaneous
phagocytic cells anthrax
Resistance to attack by antibodies and Pseudomonas biofilm in cystic
drugs fibrosis patients
Protection against drying Azotobacter vinelandii - soil

Tsongo Lawrence Msc (MUK) 35

Reserve of nutrients carbohydrates for Streptococcus mutans - dental
subsequent metabolism plaque

The Cytoplasmic Membrane

 The cytoplasmic membrane of bacterial cells is a delicate and plastic

structure that completely encloses the cell cytoplasm (or protoplasm).
 The bacterial membrane is composed of 40 percent phospholipid and 60
percent protein. The phospholipids are amphoteric molecules, meaning
they have a water-soluble hydrophilic region (the glycerol "head")
attached to two insoluble hydrophobic fatty acid "tails".

Functions of the Cytoplasmic Membrane

Permeability Barrier

 This regulates the passage of substances into and out of the cell.
 The bacterial membrane freely allows passage of water and a few small
uncharged molecules

Transport of Solutes

 The presence of transport systems in the membranes allows the bacteria to

accumulate solutes and chemical precursors of cell material inside their
cytoplasm at concentrations which greatly exceed the concentrations in
the environment.
 Bacteria have a variety of types of transport systems which can be used
alternatively in various environmental situations.

 The most important transport systems are called active transport.

 Membranes may contain other enzymes involved in many metabolic

processes such as cell wall synthesis, septum formation, membrane
synthesis, DNA replication, CO2 fixation and ammonia oxidation

Tsongo Lawrence Msc (MUK) 36

 . Synthesis of membrane lipids (including lipopolysaccharide in Gram-
negative cells)
 Coordination of DNA replication and segregation with septum formation
and cell division
 Location of specialized enzyme systems, such as for CO2 fixation,
nitrogen fixation
 Synthesis of murein (cell wall peptidoglycan)

The Cytoplasm

o The cytoplasm of bacterial cells consists of an aqueous solution of three

groups of molecules such as proteins (enzymes), DNA, mRNA and tRNA;

o The cytoplasmic constituents of bacterial cells include the procaryotic

chromosome (nucleoid), ribosomes, and several hundred proteins and
enzymes.

Ribosomes
 The distinct granular appearance of the cytoplasm is due to the presence
and distribution of ribosomes.
 The ribosomes are smaller than cytoplasmic ribosomes of eucaryotes.
Procaryotic ribosomes are 70S in size, being composed of 30S and 50S
subunits. The 80S ribosomes of eucaryotes are made up of 40S and 60S
subunits.
 Ribosomes are involved in the process of translation (protein synthesis),
Characteristics of typical bacterial cell structures

Predominant chemical
Structure Function(s)
composition
Flagella Swimming movement
Protein
Pili
Mediates DNA transfer during
Sex pilus Protein
conjugation

Tsongo Lawrence Msc (MUK) 37

 Attachment to surfaces;
Common pili or
protection against Protein
fimbriae
phagotrophic engulfment
Attachment to surfaces;
Capsules protection against phagocytic
(includes "slime engulfment, occasionally killing Usually polysaccharide;
layers" and or digestion; reserve of nutrients occasionally polypeptide
glycocalyx) or protection against
desiccation
Cell wall
Prevents osmotic lysis of cell Peptidoglycan (murein)
Gram-positive
protoplast and confers rigidity complexed with teichoic
bacteria
and shape on cells acids
Peptidoglycan prevents osmotic
Peptidoglycan (murein)
lysis and confers rigidity and
surrounded by
Gram-negative shape; outer membrane is
phospholipid protein-
bacteria permeability barrier; associated
lipopolysaccharide "outer
LPS and proteins have various
membrane"
functions
Permeability barrier; transport of
Plasma solutes; energy generation;
Phospholipid and protein
membrane location of numerous enzyme
systems
Sites of translation (protein
Ribosomes RNA and protein
synthesis)
Highly variable;
Often reserves of nutrients;
Inclusions carbohydrate, lipid,
additional specialized functions
protein or inorganic
Chromosome Genetic material of cell DNA
Extrachromosomal genetic
Plasmid DNA
material

Tsongo Lawrence Msc (MUK) 38

 Appendages

Flagella

 Flagella are filamentous protein structures attached to the cell surface that
provide the swimming movement for most motile bacteria.
 Procaryotic flagella are much thinner than eucaryotic flagella; the
diameter of a procaryotic flagellum is about 20 nanometers
 The flagellar filament is rotated by a motor apparatus in the plasma
membrane allowing the cell to swim in fluid environments.
 The flagella apparatus consists of several distinct proteins: a system of rings
imbedded in the cell envelope (the basal body), a hook-like structure near
the cell surface, and the flagellar filament.

Typical Arrangement of Bacterial Flagella

Tsongo Lawrence Msc (MUK) 39

Examples of bacterial flagella arrangement schemes
A-Monotrichous; one flagella at one end of the bacteria e.g. in vibrio
cholerae
B-Lophotrichous; very many flagella at one end of the bacteria e.g.
Bartonella species
C-Amphitrichous; many or one flagella at both ends of bacteria e.g. spirillum
species
D-Peritrichous; many flagella on all the bacteria body e.g. Escherichia coli
and salmonella species
Detecting Bacterial Motility
 Since motility is a primary criterion for the diagnosis and identification of
bacteria, several techniques have been developed to demonstrate
bacterial motility, directly or indirectly.

1. Flagellar stains outline flagella and show their pattern of distribution. If a

bacterium possesses flagella, it is presumed to be motile.

Tsongo Lawrence Msc (MUK) 40

Staining techniques such as Leifson's method utilize dyes and other
components that precipitate along the protein filament and hence increase
its effective diameter.

2. Motility test medium demonstrates if cells can swim in a semisolid medium

such as O.75% agar is inoculated with the bacteria in a straight-line stab with
a needle. After incubation, if turbidity (cloudiness) due to bacterial growth
can be observed away from the line of the stab, it is evidence that the
bacteria were able to swim through the medium.

Bacterial cultures grown in motility test medium, the tube on left is a non
motile organism; the tube on right is a motile organism. Motility test medium is
a semi-soft medium that is inoculated with a straight needle. If the
bacteria are motile, they will swim away from the line of inoculation in order to

Tsongo Lawrence Msc (MUK) 41

find nutrients, causing turbidity or cloudiness throughout the medium. If they
are non motile, they will only grow along the line of inoculation.

3. Direct microscopic observation of living bacteria in a wet mount. One must

look for transient movement of swimming bacteria. True motility is confirmed
by observing a bacterium swim from one side of the microscope field to the
other side.

Fimbriae and Pili

 Fimbriae and pili are interchangeable terms used to designate short, hair-
like structures on the surfaces of procaryotic cells. Like flagella, they are
composed of protein.

 Fimbriae are shorter and stiffer than flagella, and slightly smaller in
diameter.

 Fimbriae are very common in Gram-negative bacteria, but occur in some

archaea and Gram-positive bacteria as well.

 They are most often involved in adherence of bacteria to surfaces,

substrates and other cells or tissues in nature.

Tsongo Lawrence Msc (MUK) 42

 In E. coli, a specialized type of pilus, the F or sex pilus, apparently stabilizes
mating bacteria during the process of conjugation, but the function of the
smaller, more numerous common pili is quite different.
Common pili (often called fimbriae) are usually involved in specific
adherence (attachment) of procaryotes to surfaces in nature.

 They are major determinants of bacterial virulence because they allow

pathogens to attach to (colonize) tissues and/or to resist attack by
phagocytic white blood cells.

 For example, pathogenic Neisseria gonorrhoeae adheres specifically to

the human cervical or urethral epithelium by means of its fimbriae;
enterotoxigenic strains of E. coli adhere to the mucosal epithelium of the
intestine by means of specific fimbriae; the M-protein and associated
fimbriae of Streptococcus pyogenes are involved in adherence and to
resistance to engulfment by phagocytes.

Fimbriae (common pili) and flagella on the surface of bacterial cells

Endospores

 Are dormant cell formed by a few groups of Bacteria as intracellular

structures, but ultimately they are released as free endospores.
 Biologically, endospores are a fascinating type of cell.

Tsongo Lawrence Msc (MUK) 43

 They are highly resistant to environmental stresses such as high
temperature (some endospores can be boiled for hours and retain their
viability), irradiation, strong acids, disinfectants, etc.
 They resist heating by autoclaving at 21˚C for 15 minutes or in a hot air
oven at 160-180˚C for 1 hour and can withstand dehydration, cold and the
action of disinfectants
 Endospores are formed by vegetative cells in response to environmental
signals that indicate a limiting factor for vegetative growth, such as
exhaustion of an essential nutrient.
 They germinate and become vegetative cells when the environmental
stress is relieved. Hence, endospore-formation is a mechanism of survival
rather than a mechanism of reproduction.
 When conditions of vegetative growth are not favourable especially when
carbon and nitrogen become unavailable, some bacteria are able to
survive by forming resistant endospores. Spore formation involves a change
in enzyme activity and morphology.

 Some spores may be positioned at the end of the bacterium called

Terminal spores or can be subterminal positioned near to one end or
centrally positioned.

 Shape: It can be circular, oval and centre.

 Size: It can be large, longer than the width of the cell and cause bulging of
the cell wall.

 Can be small and fit within the width of the cell.

 A spore is unable to multiply but when conditions for vegetative growth
return, it‟s able to produce a bacterial cell which is capable of
reproducing.

Tsongo Lawrence Msc (MUK) 44

Early and late stages of endospore formation

BACTERIAL GENETICS definitions

Mutations

 Mutations are heritable changes in genotype that can occur

spontaneously or be induced by chemical or physical treatments.
 The process of mutation is called mutagenesis and the agent inducing
mutations is called mutagen. Mutation rates of individual genes in
bacteria range from 10-2 to 10-10 per bacterium per division.

Gene transfers among bacteria done in three means

Transformation

Tsongo Lawrence Msc (MUK) 45

Transformation involves the uptake of free or naked DNA released by donor to
the recipient.

It is the gene transfer resulting from the uptake by a recipient cell of naked
DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria,
and Pneumococcus) can take up DNA from the environment and the DNA
that is taken up can be incorporated into the recipient's chromosome.

Conjugation

Bacterial conjugation is the transfer of DNA from a living donor bacterium to a

recipient bacterium.

Plasmids are small autonomously replicating circular pieces of double-

stranded circular DNA.

Transduction

 This involves the transfer of DNA between cells by bacteriophage.

 Most bacteriophage carry their genetic formation (the phage genome) as

length of double stranded

 DNA coiled up inside the protein coat. Other phages are known in which
the phage genome consists of single stranded DNA or RNA. But as far as
known, transducing phages all contain double stranded DNA.

 Bacteriophages are viruses that parasitize bacteria and use their

machinery for their own replication. During the process of replication inside
the host bacteria, the bacterial chromosome or plasmid is packaged into
the bacteriophage capsid.

Vectors

Tsongo Lawrence Msc (MUK) 46

 A vector is a replicating DNA molecule that can be used to transfer DNA
molecule or segments to a suitable host where it can replicate. Examples
of vectors include plasmids, phages, cosmids

Plasmids

 These are small circular extra chromosomal genetic elements that may
encode a variety of supplementary genetic information including the
information for self transfer for other cells by conjugation. The genes of
plasmids encode additional characteristics that improve the fitness of the
bacteria such as antibiotic resistance or production of colicins.

 OR Plasmids are extra chromosomal elements found inside a bacterium.

These are not essential for the survival of the bacterium but they confer
certain extra advantages to the cell.

CHAPTER FOUR: PATHOGENICCITY AND VIRULENCE

4. 1 Bacterial Pathogenicity
A pathogen is a microorganism that is able to cause disease in a human,
plant, animal or insect.
Pathogenicity is the ability to produce disease in a host organism or it is the
degree of pathogen to enter the body, multiply and cause a disease. I.e. the
ability to produce toxins
Virulence refers to the degree of pathogenicity of the microbe or it is the
ability of the pathogen to cause a disease
Aetiology It is the occurrence and existence of micro organisms
Infection this is the invasion of the body by a pathogen
Disease It is the impairment of the body or unwell being of the body

Tsongo Lawrence Msc (MUK) 47

Pathogenesis It is the process of disease development with its pathogenic
mechanism

4.2 Mechanisms of Bacterial Pathogenicity

1. Invasiveness is the ability to invade tissues. It encompasses mechanisms for
colonization (adherence and initial multiplication), production of extracellular
substances which facilitate invasion (invasins) and ability to bypass or
overcome host defense mechanisms.
2. Toxigenesis is the ability to produce toxins. Bacteria may produce two types
of toxins called exotoxins and endotoxins.
Exotoxins are released from bacterial cells and may act at tissue sites
removed from the site of bacterial growth.
Endotoxins are cell-associated substance. Bacterial toxins, both soluble and
cell-associated, may be transported by blood and lymph and cause
cytotoxic effects at tissue sites remote from the original point of invasion or
growth. Some bacterial toxins may also act at the site of colonization and
play a role in invasion.
Colonization
 The establishment of the pathogen at the appropriate body tissues.
Pathogens usually colonize host tissues that are in contact with the external
environment.

 Organisms that infect body tissues have usually developed tissue

adherence mechanisms and some ability to overcome or withstand the
constant pressure of the host defenses at the surface.

Bacterial Adherence to Mucosal Surfaces

 Bacterial adherence or attachment to body cells or tissue surface requires
the participation of two factors: a receptor and a ligand.
 The receptors are specific carbohydrate or peptide residues on the cell
surface.

Tsongo Lawrence Msc (MUK) 48

 The bacterial ligand, called an adhesin, is typically a macromolecular
component of the bacterial cell surface which interacts with the host cell
receptor.
ADHERENCE FACTOR DESCRIPTION

A surface structure or macromolecule that binds a bacterium

Adhesin
to a specific surface

A complementary macromolecular binding site on a

Receptor
(eukaryotic) surface that binds specific adhesins or ligands

Lectin Any protein that binds to a carbohydrate

A surface molecule that exhibits specific binding to a receptor

Ligand
molecule on another surface

The mucopolysaccharide layer of glucosaminoglycans

Mucous
covering animal cell mucosal surfaces

Filamentous proteins on the surface of bacterial cells that may

Fimbriae
behave as adhesins for specific adherence

Common pili Same as fimbriae

A specialized pilus that binds mating procaryotes together for

Sex pilus
the purpose of DNA transfer

Fimbriae in Enterobacteriaceae which bind specifically to

Type 1 fimbriae
mannose terminated glycoproteins on eucaryotic cell surfaces

Pili in certain Gram-positive and Gram-negative bacteria. In

Type 4 pili Pseudomonas, thought to play a role in adherence and biofilm
formation

Tsongo Lawrence Msc (MUK) 49

 Proteins that form the outermost cell envelope component of a
broad spectrum of bacteria, enabling them to adhere to host
S-layer cell membranes and environmental surfaces in order to
colonize.

A layer of exopolysaccharide fibers on the surface of bacterial

Glycocalyx cells which may be involved in adherence to a surface.
Sometimes a general term for a capsule.

A detectable layer of polysaccharide (rarely polypeptide) on

Capsule the surface of a bacterial cell which may mediate specific or
nonspecific attachment

A distinct cell wall component of the outer membrane of

Lipopolysaccharide
Gram-negative bacteria with the potential structural diversity to
(LPS)
mediate specific adherence. Probably functions as an adhesin

Teichoic acids and

Cell wall components of Gram-positive bacteria that may be
lipoteichoic acids
involved in nonspecific or specific adherence
(LTA)

Specific Adherence of Bacteria to Cell and Tissue Surfaces

o Tissue tropism: particular bacteria are known to have an apparent

preference for certain tissues over others, e.g. S. mutans is abundant in
dental plaque but does not occur on epithelial surfaces of the tongue;
the reverse is true for S. salivarius which is attached in high numbers to
epithelial cells of the tongue but is absent in dental plaque.

o Species specificity: certain pathogenic bacteria infect only certain

species of animals, e.g. N. gonorrhoeae infections are limited to humans;
Enteropathogenic E. coli K-88 infections are limited to pigs; E. coli CFA I

Tsongo Lawrence Msc (MUK) 50

 and CFA II infect humans; Group A streptococcal infections occur only in
humans.

o Genetic specificity within a species: certain strains or races within a

species are genetically immune to a pathogen

Invasion

 The invasion of a host by a pathogen is called infection. It may be aided

by the production of bacterial extracellular substances which act against
the host by breaking down primary or secondary defenses of the body.
These substances are called invasins.

 Most invasins are proteins (enzymes) that act locally to damage host cells
and or have the immediate effect of facilitating the growth and spread of
the pathogen.

 The damage to the host as a result of this invasive activity may become
part of the pathology of an infectious disease.

 Invasins usually act at a short range and may not actually kill cells as part
of their range of activity; exotoxins are often cytotoxic and may act at
remote sites

 Also, exotoxins typically are more specific and more potent in their activity
than invasins.

 Even so, some classic exotoxins (e.g. diphtheria toxin, anthrax toxin) may
play some role in colonization or invasion in the early stages of an infection,
and some invasins (e.g. staphylococcal leukocidin) have a relatively
specific cytopathic effect.

Enzymes that are involved as Spreading factors of the bacteria

Hyaluronidase it is produced by streptococci. Staphylococci and clostridia.
The enzyme attacks the interstitial cement ("ground substance") of connective
tissue by depolymerizing hyaluronic acid.

Tsongo Lawrence Msc (MUK) 51

 Collagenase is produced by Clostridium histolyticum and Clostridium
perfringens. It breaks down collagen, the framework of muscles, which
facilitates gas gangrene due to these organisms.

 Neuraminidase is produced by intestinal pathogens such as Vibrio

cholerae and Shigella dysenteriae. It degrades neuraminic acid (also
called sialic acid), an intercellular cement of the epithelial cells of the
intestinal mucosa.

 Streptokinase and staphylokinase are produced by streptococci and

staphylococci, respectively. Kinase enzymes convert inactive
plasminogen to plasmin which digests fibrin and prevents clotting of the
blood. The relative absence of fibrin in spreading bacterial lesions allows
more rapid diffusion of the infectious bacteria.

Enzymes that Cause Hemolysis and/or Leucolysis

 These enzymes usually act on the animal cell membrane forming a pore
that results in cell lysis, or by enzymatic attack on phospholipids, which
destabilizes the membrane.

 They may be referred to as lecithinases or phospholipases, and if they lyse

red blood cells they are sometimes called hemolysins.

 Leukocidins, produced by staphylococci and streptolysin produced by

streptococci specifically lyse phagocytes and their granules.

 Phospholipases, produced by Clostridium perfringens (i.e., alpha toxin),

hydrolyze phospholipids in cell membranes by removal of polar head
groups.

 Lecithinases, also produced by Clostridium perfringens, destroy lecithin

(phosphatidylcholine) in cell membranes.

 Hemolysins, produced by staphylococci (i.e., alpha toxin), streptococci

(i.e., streptolysin) and various clostridia, may be channel-forming proteins

Tsongo Lawrence Msc (MUK) 52

 or phospholipases or lecithinases that destroy red blood cells and other
cells (i.e., phagocytes) by lysis.

Staphylococcal coagulase

 Coagulase, formed by Staphylococcus aureus, is a cell-associated and

diffusible enzyme that converts fibrinogen to fibrin which causes clotting.

 Coagulase activity associated with pathogenic S. aureus and never

associated with nonpathogenic S. epidermidis,

 Or a staphylococcal lesion encased in fibrin (e.g. a boil or pimple) could

make the bacterial cells resistant to phagocytes or tissue bactericides or
even drugs which might be unable to diffuse to their bacterial target.

Some extracellular bacterial enzymes that are considered Invasins

Invasin Bacteria Involved Activity
Streptococci,
Hyaluronidase staphylococci and Degrades hyaluronic of connective tissue
clostridia
Collagenase Clostridium species Dissolves collagen framework of muscles
Neuraminidas Vibrio cholerae and
Degrades neuraminic acid of intestinal mucosa
e Shigella dysenteriae
Staphylococcus Converts fibrinogen to fibrin which causes
Coagulase
aureus clotting
Staphylococci and Converts plasminogen to plasmin which digests
Kinases
streptococci fibrin
Staphylococcus Disrupts neutrophil membranes and causes
Leukocidin
aureus discharge of lysosomal granules
Repels phagocytes and disrupts phagocyte
Streptococcus
Streptolysin membrane and causes discharge of lysosomal
pyogenes
granules
Streptococci,
Phospholipases or lecithinases that destroy red
Hemolysins staphylococci and
blood cells (and other cells) by lysis
clostridia
Clostridium
Lecithinases Destroy lecithin in cell membranes
perfringens
Phospholipas Clostridium
Destroy phospholipids in cell membrane
es perfringens

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 One component (EF) is an adenylate cyclase
Anthrax EF Bacillus anthracis which causes increased levels of intracellular
cyclic AMP
One toxin component is an adenylate cyclase
Pertussis AC Bordetella pertussis that acts locally producing an increase in
intracellular cyclic AMP

Avoiding Contact with Phagocytes

Bacteria can avoid the attention of phagocytes in a number of ways.

o Invade or remain confined in regions inaccessible to phagocytes. Certain

internal tissues (e.g. the lumen of glands) and surface tissues (e.g. the
skin) are not patrolled by phagocytes.

o Avoid provoking an overwhelming inflammatory response. Some

pathogens induce minimal or no inflammation required to focus the
phagocytic defenses.

o Inhibit phagocyte chemotaxis. E.g. Streptococcal streptolysin (which also

kills phagocytes) suppresses neutrophil chemotaxis, even in very low
concentrations.

o Hide the antigenic surface of the bacterial cell. Some pathogens can
cover the surface of the bacterial cell with a component which is seen as
"self" by the host phagocytes and immune system. Phagocytes cannot
recognize bacteria upon contact and the possibility of opsonization by
antibodies to enhance phagocytosis is minimized.

Inhibition of Phagocytic Engulfment

 Some bacteria employ strategies to avoid engulfment (ingestion) if

phagocytes do make contact with them.

 Resistance to phagocytic ingestion is usually due to a component of the

bacterial cell wall, or fimbriae, or a capsule enclosing the bacterial wall.

 Examples of antiphagocytic substances on the bacterial surface include:

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 Polysaccharide capsules of S. pneumoniae, Haemophilus influenzae,
Treponema pallidum and Klebsiella pneumoniae

 M protein and fimbriae of Group A streptococci

 Surface slime (polysaccharide) produced by Pseudomonas aeruginosa

Antigen associated with LPS of E. coli

 K antigen of E. coli or the analogous VI antigen of Salmonella typhi

Toxigenesis

This is the production of toxins by bacteria. There are two types of bacterial
toxins. The endotoxinc and exotoxins. Exotoxins are secreted outside the
microbial cells when organism is still alive.

Toxoids
 Are detoxified toxins which retain their antigenicity and their immunizing
capacity? The formation of toxoids can be accelerated by treating toxins
with a variety of reagents including formalin, iodine, pepsin, ascorbic acid,

ketones, etc. The mixture is maintained at 37 o at pH range 6 to 9 for

several weeks. The resulting toxoids can be used for artificial immunization
against diseases caused by pathogens where the primary determinant of
bacterial virulence is toxin production.

 Toxoids are the immunizing agents against diphtheria and tetanus that are
part of the DPT vaccine.

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Properties of toxins

Exotoxins Endotoxins

 Secreted by cells and found in  Integral parts of the microbial

high concentrations in liquid cells and are liberated upon
media. their disintegration.
 They are polypeptides with  They are polysaccharide with
molecular weight of 10,000- molecular weight <10,000
900,000.  Relatively heat-stable and
 They are relatively thermo-labile withstands temperatures of 60˚C
(destroyed at 60˚C). for several hours.
 They are highly antigenic i.e.  They induce production of
stimulates the production of antibodies.
antitoxins.  Weakly toxic and fatal to
 They are highly toxic and fatal to laboratory animals in 100 of
laboratory animals. microorganisms.
 They do not produce fever in the  They produce high fever in the
host. host.
 Examples include; Clostridium  Examples include; Streptococcus
tetani is a neurotoxin producer, species.
which affects the cells of the
central nervous system. Others
include; Clostridium species,
corynebacterium diphtheriae, E.
coli, Shigella, Vibrio Cholerae.
The toxins produced by enteric
pathogens are known as
Enterotoxins.

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Examples of toxins, anthrax toxin, cholera enterotoxin, E.coli toxin, shiga toxin,
botulinum toxin, tetanus toxin, diphtheria toxins and staphylococcus
enterotoxins

Endotoxins
they are part of the outer cell wall of bacteria. They are associated with
Gram-negative bacteria as constituents of the outer membrane of the cell
wall. Until disintegration from the bacteria i.e Endotoxins are secreted within
the microbial cells when the organism is dead; results from autolysis, external
lysis, and phagocytic digestion of bacterial cells.

The biological activity of endotoxin is associated with the Lipopolysaccharide

(LPS).

Characteristics of Bacterial toxins

PROPERTY ENDOTOXIN EXOTOXIN
Lipopolysaccharide Protein (mw = 50-
CHEMICAL NATURE
(mw = 10kDa) 1000kDa)
RELATIONSHIP TO Extracellular,
Part of outer membrane
CELL diffusible
DENATURED BY
No Usually
BOILING
ANTIGENIC Yes Yes
FORM TOXOID No Yes
Relatively high (1
POTENCY Relatively low (>100ug)
ug)
SPECIFICITY Low degree High degree
ENZYMATIC ACTIVITY No Usually
PYROGENICITY Yes Occasionally

Other factors for pathogenicity include;

 Transmission route: for a pathogen to cause a disease, it must enter the

body by route which will enable it to reach a site where it can establish

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 itself and multiply e.g. an organism which cause gas gangrene must reach
deep tissues to find the anaerobic conditions necessary for its growth.
 Number of bacteria that invade. The bigger the number of organisms, the
faster the pathogenicity.
 Status of health of the person infected. The lower the immunity of the
patient, the easier the organism to spread into the host‟s tissues.

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 CHAPTER FIVE: STAINS AND STAINING METHODS

5.1 Introduction to Staining Methods

Uses of examination of stained preparations

 It enables the morphology, relative sizing and arrangement of

microorganisms to be seen clearly.
 Assists in detection of cells especially the pus cells.
 It also enables the bacteria to be differentiated example gram positive
from the gram-negative organisms.
Stains are chemically salts prepared from dyes.

Classification of Stains

They are classified into two;

 Natural Stains (dyes)

These are found mainly in histology and they include;

a) Haematoxylin: This is extracted from American tree called haematoxylon

campenchionum.
b) Orcein and Litmus: These are extracted from linchen.
c) Carmine: Extracted from female insect called coccus cacti.
 Synthetic Dyes
These are referred to as aniline dyes, which are derivatives of nitrobenzene.

Types of stains

All stains whether natural or synthetic are divided into 3 groups;

i. Acid stains
The colouring substance is contained in acid radical and the basic part of the
stain is colourless.

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Acid stains are not very often used in microbiology except they are used to
provide a background staining example Eosin stain, Nigrosin stain and acid
fuchsin.

ii. Basic stains

This is where the colouring substance is contained in the basic radical and the
acid radical is colourless. The basic component is positively charged cation.

Most bacteriological stains are basic example crystal violet, basic fuchsin,
methylene blue, haematoxylin O. The nucleic acid of the bacterial cell
attracts the positively charged ions of the basic stains.

iii. Neutral stains

Both the acidic and the basic components are coloured. They are formed
from precipitates of aqueous mixtures of acid and basic stains.

It stains both nucleic acid and cytoplasm exapmle Giemsa stain, Field‟s stain,
Wright‟s stain, Leishman stain

Staining Terminologies

 Indirect staining: this is where organisms are stained with the help of a
mordant. Mordants are metallic substances, which act as a link between
organisms and the stains. It first combines with the stain to form a complex,
which then attaches itself firmly to the tissue to be stained example phenol
contained in the carbol fuchsin stain used in the ZN method, and iodine,
which is added after application of crystal violet in gram staining.
 Direct staining/Simple the organism is stained directly by immersing it in the
stain or the stain is added to the organism then washed and observed. The
staining takes place without the use of a mordant e.g. Methylene blue
stains.
 Progressive staining this is where elements of the tissue are stained evenly
in a definite sequence without need for any differentiation.

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 Regressive staining this is where elements in the tissue are first over stained
and then differentiated until or only when the tissue component to be
stained retains the stain.
 Vital staining this is the staining of living tissues or cells. When the tissue is
stained while still part of the organism, it is called intro vital staining. When
stained after removal from the organism, it is called supra vital staining.
 Negative staining the organism remains unstained against the stained
background. Acid stains like Nigrosin and India ink are the most examples.
 Metachromatic staining the organism or parts of the organism stain
different colours from that of the stain itself e.g. Albert stain.
 Accentuators these are substances added to increase the staining power
and sensitivity OR; they are chemicals added to the stain so as to speed
up its reaction or to make its reaction more intense or selective. It does not
combine with a stain e.g., potassium hydroxide (KOH) added in the
Loeffler‟s methylene blue stain.
 Impregnation this is obtained by precipitating the solutions of metallic salts
on the surface of organism e.g. silver impregnation method.
 Differentiation or Decolourisation In what is termed as regressive staining
where, all the elements of a preparation are stained
 Counter stain this is the stain which is meant to assist the primary stain in
such away that it gives contrast e.g. in ZN method, the main stain is carbol
fuchsin and the counter stain is methylene blue which gives contrast to
assist in microscopy.
5.2 Making of Smears
 Use a clean glass slide which is free from grease.
 Mark or label the glass slide with a glass writing diamond grease pencil.
 Make fairly heavy smears from liquid cultures and thin smears from cultures
of solid media.

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 Use only one smear per slide and keep the slides apart when staining on a
staining rack. This prevents the transfer of acid fast organisms into another
slide.
 Never use coplin or other staining jars for staining acid fast organisms to
prevent transfer of these organisms.
 Use aseptic precautions during the inoculation of culture tubes or plates.
Smears from liquid media
 Sterilise a loop of a Bunsen flame.
 Withdraw one loopful of culture and transfer to a clean slide, then spread
the sample with a loop to form a thick film of a liquid.
 Sterilize the wire loop and allow the smear to airdry, then fix over a Bunsen
burner flame three times and stain the smear.
Smears from solid media

 Use aseptic procedures.

 Place one drop of distilled water on a clean slide.
 With a loop or straight wire, transfer to the slide a small portion of the broth
to be examined and emulsify. Mix in a drop of water or saline until a thin
homogenous smear is produced.
 Allow the smear to dry then fix and stain.
Hanging drop preparation

This method is used to show the motility of certain organisms.

Principle

When suspended in a fluid and examined microscopically many bacteria

are seen to be motile i.e. they move from one position to another.

A motile organism will actively change its position relative to other

organisms present. Therefore, this true motility should not be confused with

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 Brownian which is a vibration caused by a molecular bombardment or
convectional currents.

Procedure

 A clean glass microscope slide and a 22 mmsq coverslip are used.

 Make a ring of plasticine or Vaseline about 2cm in diameter at the centre
of the slide. Alternatively, use a well slide with a depression in the centre.
 Transfer a loopful of culture at the centre of the slide.
 Gently press the ring of Vaseline or plasticine onto the coverslip ensuring
that the drop of culture is in the centre of the circle and does not come in
contact with the slide.
 With a quick movement invert the slide so that the coverslip is upmost and
the drop is left hanging.
 Examine the hanging drop by microscopy under ×10, ×40 objectives to
investigate the motility.

Cover slip Drop of culture

Vaseline

Microscope slide
Making of wireloops

 Wireloops are made of platinum or nichrome wires.

 Loops are usually circles of approximately 1.5mm and 3mm in diameter of
a standard wire gauge and 26 or 27 thickness.
 Wind the wire aroud a metal rod of approximate diameter.
 With a pair of scissors, cut one arm of the wire at the junction.
 Bend back the loop to centre it and insert into a metal wire holder.

Loop

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Wire
9.3 Different Methods of Staining

Gram Staining
Purpose

It is a staining reaction, which differentiates or divides bacteria into two

categories depending on whether they can be decolorized with acetone
alcohol, acetone spirit or aniline oil after staining with one of the basic stains
i.e. methyl violet, crystal violet and gentian violet and applying a mordent
lugol‟s iodine, Those that resist decolourisation and remain blue or violet in
colour are termed as gram-positive organisms. Those that are decolorized and
take up the red colour of the counter stain such as Neutral red, dilute carbol
fuchsin and safranine are termed as the gram-negative organisms.
Principle
 Gram-positive organisms have acidic protoplasm of PH=2.0 therefore, they
have higher affinity for the basic dyes. While the gram-negative organisms
have less acidic protoplasm of PH=5.0 hence less affinity for the basic dyes.
 The gram-positive organisms have tiny pores on the cell wall, which is
composed of murein layer of peptidoglycans, teichoic acids hence less
diffusion of the dye through the cell wall. Whereas the gram-negative
organisms have larger pores, less peptidoglycans only lipopolysaccharides
and lipoproteins and faster diffusion through the cell wall.
 Gram-positive organisms have magnesium ribonucleate, which combines
with the basic dye to form a complex that does not diffuse out of the cell
wall whereas the gram-negative organisms do not have the magnesium
ribonucleate
 Examples of gram positive and gram negative organisms include;
Gram positives
 Staphylococcus species,Streptococcus species,Corynebacterium
species,Clostridium species,Listeria species,Bacillus species, Yeasts
Gram negatives

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 Coliforms,Vibrio, species,Haemophilus Spirochaetes, Niesseria

Reagents for Gram Stain (explain preparation of reagents in details)

Basic stains
 Methyl violet
 Gentian violet
 Crystal violet

Mordant
 Lugol‟s iodine
Decolouriser
 Acetone spirit
 Acetone alcohol
Counter stain
 Neutral red
 Dilute carbol fuchsin
 Safranine
Preparation of reagents
To prepare 1 litre of 0.5% Gentian violet;
Gentian violet…………………………………………………..5.0g
Distilled water………………………………………………….1000mls
Method
 Weigh the gentian violet powder in the cornical flask and dissolve in
distilled water.
 Stir using a glass rod until the powder has dissolved.
 Filter and transfer in a reagent bottle.
 Label the stain clearly with the date of preparation, the type of the stain,
the nature of the stain whether it is in use of in stock, the name of the
person who has prepared e.t.c.

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Lugol’s Iodine
To prepare 1 litre of Lugol‟s iodine;
Iodine………………………………………………………………10g
Potassium iodide……………………………………………………20g
Distilled water……………………………………………………....1000mls
Method

 Weigh iodine and potassium iodide powders separately.

 Dissolve the potassium iodide in about 100mls of distilled water, add iodine
and dissolve. Stir until the crystals have completely dissolved.
 Make up the volume to 1000mls with distilled water.
 Filter the solution and transfer to a brown bottle since iodine fades on
direct sunlight.
 Label systematically.
 For daily use, transfer a small portion to a brown dropper bottle and label.
Acetone spirit or alcohol
They come commercially prepared however, they are flammables.
To prepare, mix equal amounts of acetone and alcohol or acetone and spirit
e.g. methylated spirit.
Dilute carbol fuchsin

To prepare, dilute the strong carbol fuchsin 1 in 20 (1 volume of carbol fuchsin

in 19 volumes of distilled water), mix by inversion and label.

Method of Staining

 Prepare a smear on a clean glass slide and label. Allow the smear to air
dry at room temperature.
 Fix the smear by passing it (smear uppermost) over the flame three times or
in methanol for 2 minutes.

Tsongo Lawrence Msc (MUK) 66

 Place the slide on a staining rack over a sink or basin. Cover the smear with
0.5% gentian violet and leave for 30 seconds to stain.
 Wash the smear in a thin stream of clean water.
 Then cover the smear with Lugol‟s iodine stain for 30 seconds, wash with
clean water and decolourize with acetone spirit slowly, drop at a time and
stop as soon as no more blue colour comes off the smear.
 Wash immediately with clean water and avoid over decolourisation of the
smear.
 Cover the smear with 0.5% neutral red and allow staining for 30 seconds. If
using dilute carbol fuchsin, leave to stain for 1-2 minutes.
 Wash the smear in a thin stream of water to remove the excess stain.
 Wipe the back of the slide clean and allow to air dry.
 Examine the smear first with 40× objective to check the staining and the
distribution of bacterial cells then examine using 100 × objectives.
Results

 Gram-positive Bacteria appear dark purple.

 Yeast cells also dark purple.
 Gram-negative bacteria appear pale to dark red.
 Nuclei of pus cells red.
 Epithelial cells appear pale red.
Reporting of gram smears
It should include the following information;
 The number of bacteria present example (+++), (++) and (+) reported in
pluses.
 The gram reaction of the bacteria whether gram positive or gram negative
organisms.
 The morphology of the bacteria whether rods, cocci, diplococci, or
coccobacilli.
 Also, whether the organisms are intracellular.

Tsongo Lawrence Msc (MUK) 67

 The presence of yeast cells, number of pus cells and epithelial cells if any

Variation in gram reactions

Occasionally, gram-positive organisms loose their ability to retain the basic
dyes and stained gram negatively for the following reasons.
a) Cell wall damage due to antibiotic therapy or excessive heating when
fixing the smears
b) Over decolourisation of the smears.
c) Use of an iodine solution, which is too old that, appears yellow instead of
brown, which cannot act as an effective mordant.
d) Preparing of the smear from an old culture. Occasionally gram-negative
organisms may not be fully decolorized and appear gram positive if the
smear is too thick.
Quality control
 Failure to demonstrate bacteria by a gram stain technique can be due to
poorly prepared reagents.
 Regularly check the gram staining technique, paying attention to careful
decolourisation.
 Use smears prepared from;
1) Urethral swab for Neisseria gonorrhoeae.
2) High vaginal swab for candida albicans.

In an under-decolorized smear, gram-positive organisms (Neisseria

gonorrhoeae) appear blue.
While in an over-decolourised smear, the gram-positive organisms (candida
albicans) appear red.

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Ziehl Neelsen (Zn) Staining Method

This is a method that is used to distinguish bacteria species according to their

acid fastness. Example tubercle bacilli, mycobacterium leprae from the non-
acid fast

Principle

 Acid-fast bacilli are relatively impermeable and persistant with simple stains
e.g. crystal violet, gentian violet and methyl violet. But when stained with
strong dyes such as basic carbol fuchsin with 5% phenol and an
application of heat, they subsequently resist the decolourisation by strong
acids e.g. 3% acid alcohol or 20-25% sulphuric acid.

 The acid fast bacilli have an exceptionally rich valid contents of lipids, fatty
acids, higher alcohols especially mycolic acid and acid fastness that are
attributed to this i.e. amino acids which have higher molecular weight so in
order to stain the bacterial protoplasm, the higher molecular weight
hydroxyl acids that contain the carboxy group must be dissolved by heat.

 Mycobacterium detains the red colour of strong carbol fuchsin after

decolourising with acid solution while other bacteria and cells do not
detain the red colour and they take up the colour of the counter stain.

 Ziehl neelsen is used to stain mycobacterium tuberculosis (TB) in most

specimens, mycobacterium ulcerans in the skin, mycobacterium bovis in
urine and aspirates, mycobacterium leprae in skin snips, Nocardia species

 Cryptosporidium oocysts have a thinner layer of mycolic acid in their cell

wall and only resist decolourisation in weak solutions or acid alcohols and
therefore they are mostly demonstrated using the modified ZN technique.

Tsongo Lawrence Msc (MUK) 69

AFBs mean acid-fast bacilli only when an acid is used as a decolouriser while
AAFBs means acid alcohol fast bacilli only when acid and alcohol is used as a
decolouriser.

Reagents for Ziehl Neelsen (explain the preparation in details)

a) Primary stain (basic stain)

Strong carbol fuchsin

b) Decolouriser
 1% or 3% acid alcohol where 1% for modified ZN
 20-25% or 5% sulphuric acid.
c) Counter stain
0.1-0.5% methylene blue or malachite green
d) 70% ethanol for fixing.

To prepare 1 litre of strong carbol fuchsin

Basic fuchsin powder………………………………………………10g

Ethanol or methanol (absolute)……………………………………..100mls
Phenol crystals………………………………………………………50g
Distilled water………………………………………………………900mls
 Dissolve the basic fuchsin in alcohol in a cornical flask and stir using a glass
rod.
 Dissolve the phenol crystals in distilled water in another cornical flask.
 Mix the two solutions.
 Filter the strong carbol fuchsin reagent into a regent bottle and label
clearly with date of preparation, the type of the reagent, the name of the
person who has prepared.
 For daily use, transfer the portion of the filtered reagent into a dropper
bottle.

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Note; if phenol has become solid, melt it by putting the reagent bottle in
warm water. Weigh the liquid phenol in a beaker and mix with water (50mls).

Safety precautions
Wear gloves because phenol crystals are corrosive to the skin.
To prepare a litre of 20% sulphuric acid
Concentrated sulphuric acid……………………………………………200mls
Distilled water………………………………………………………….800mls
Method
 Measure 800mls of distilled water and transfer to a heat resistant flask.
 Add 20mls of the acid slowly because the mixture becomes hot and may
boil.
 Always add acid to water but „NEVER‟ add water to acid.
 Leave the solution to cool to room temperature.
 Transfer to the reagent bottle and label with 20% sulphuric acid, date and
indicate that it is corrosive.
 For daily use, transfer a small portion in a dropper bottle and label.
To prepare 3% acid alcohol

3% v/v HCl solution in 70% v/v of ethanol. To make a litre of 3% acid alcohol;

Use RV/O =X OR

 Measure 679mls of absolute ethanol or methanol.

 Add 291mls of distilled water.
 Add 30 ml concentrated HCl
 Transfer the solution to the clean bottle and mix well
 For use, transfer the small amount of the reagent to the dropper bottle that
can be closed when not in use.
 Label the bottle and mark it flammable.
 Store at room temperature and the reagent is stable indefinitely.

Tsongo Lawrence Msc (MUK) 71

This is 3% v/v HCl solution in 70% v/v alcohol

 Ethanol ……………………………………………………679mls
 Distilled water…………………………………………….291mls
 HCl (concentrated) ……………………………………….30mls
Precaution
Ethanol and methanol are highly flammable therefore do not use them in
open flame.
Concentrated HCl is a corrosive chemical therefore handle with care in a well
ventilated room. Its vapour is dangerous to the skin.
For 1% v/v acid alcohol use RV/O = X
This is a 1% HCl solution in 70% v/v alcohol
Ethanol ……………………………………………………693mls
Distilled water…………………………………………….297mls
HCl (concentrated) ……………………………………….10mls
To prepare 1 litre of 0.5% methylene blue
Methylene blue powder……………………………………..5.0g
Distilled water………………………………………………1000mls
 Weigh the powder and dissolve in distilled water in a cornical flask.
 Stir until the powder has completely dissolved.
 Filter into a reagent bottle and label.
 For daily use, transfer a small portion into a dropper bottle and also
labeled.

To prepare 1 litre of 70% alcohol

Methanol/ethanol…………………………………………….700mls
Distilled water……………………………………………….300mls

Tsongo Lawrence Msc (MUK) 72

  Measure the alcohol into the measuring cylinder and transfer to the
bottle with screw caps.
 Measure the water and add to it.
 Label with 70% alcohol, date and mark “flammable”.
Quality control
Test all the new batch of reagents e.g. strong carbolfuchsin with a smear
prepared from a known AFB sputum sample and stain using ZN method.
If no AFBs are seen or the staining is poor, discard the strong carbolfuchsin
and prepare a new batch.
Types of Ziehl Neelsen staining methods
There are two ZN staining methods due to the difference in different bacterial
strains.
Ziehl Neelsen method I
This is used for mycobacterium tuberculosis and mycobacterium ulcerans
both of which are strongly acid fast where a 3% acid alcohol or 20-25%
sulphuric acid is used to decolourise the smear.
Ziehl Neelsen method II
It is used for mostly mycobacterium leprae and some protozoa Oocysts
example cryptosporidium, isospora and cyclospora where 1% acid alcohol or
5% H2SO4 is used to decolourise the smear.
Hot and Cold Ziehl Neelsen staining methods

 In ZN methods I and II above, heating of the strong carbolfuchsin is

recommended to ensure the best possible staining of the M. tuberculosis
and M. leprae. In addition, this is termed as the hot ZN method.
 In the cold ZN staining method, heating of the strong carbolfuchsin is not
required.
 In this method, the concentration of the basic fuchsin and phenol is
increased by a wetting agent, a chemical that lowers the surface tension

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 of lipid, which is added to ensure rapid penetration of the stain, and the
chemical is called teapol.
 However, comparison between the cold and hot methods has shown that
both M. leprae and M. tuberculosis stain less well by the cold method.
Ziehl Neelsen method I of staining mycobacterium tuberculosis

Requirement

 Reagents as prepared above

 New, grease-free, clean and dry glass slides.
 Bunsen burner flame.
 Diamond pencil.
 Clean water
 Cotton wool.
Sample required

Depending to the doctor‟s request, ZN staining can be performed on most

samples e.g. sputum, aspirates i.e. C.S.F, synovial fluid e.t.c, pus, biopsy, urine,
swab specimens e.t.c.

Procedure

 Label a clean, new, unscratched grease-free glass slide using a diamond

pencil (do not use a grease pencil).
 Prepare smears of the specimen on a slide and leave them to air dry.
 Fix the smears by covering with 1-2 drops of 70% ethanol/methanol for 2-3
minutes or alternatively fix gently over a Bunsen burner flame by passing
the smear for 3 times.
 Place the slide on the staining rack and flood it with filtered strong
carbolfuchsin.
 Wrap some cotton wool around the end of an applicator stick or the glass
rod. Wet it with alcohol and light in a flame.

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 Heat the slide from underneath until vapour rises from the stain.
 Keep the stain hot by heating at 5 minutes interval for 15 minutes i.e.
repeat the procedure for heating for 5 minutes interval. Do not allow the
stain to boil or dry.
 Cool the stain for 5 minutes and do not breathe in the vapour.
 Rinse the smear with a thin stream of water over a sink or basin.
 Decolourise the smear by covering with 3% acid alcohol or 20-25%
sulphuric acid for 2-5 minutes or until the smear is sufficiently decolourised
i.e. pale pink for sulphuric acid, colourless for 3% acid alcohol.
 Rinse the smear in clean water.
 Counter stain by covering the smear with 0.5% methylene blue or
malachite green for 1-2 minutes.
 Rinse the smear well in clean water.
 Wipe the back of the slide clean and allow to air dry at room temperature
i.e. place it in a draining rack (do not blot to dry).
 Examine the smear microscopically first with 40× to see the distribution of
materials and then systematically with 100× to look for AFBs.
Care should be taken not to touch the smear with the end of oil dispenser
because this can transfer AFBs from one preparation to another.
After examining a positive smear, the oil immersion objective should be wiped
or cleaned.
Results
AFBs will appear red straight or slightly curved rods occuring in singles or in
small groups.
Other organisms, cells and the background will appear blue or green
depending on the counter stain used.
Note; sputum which is too much mucoid, emulsify in 10% potassium hydroxide
or sodium hydroxide.
Reporting of the sputum smears

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If any definite red bacillus is seen, report the smear as positive and an
indication of the number of bacteria present in pluses.
 More than 10 AFBs seen per field report as (+++)
 1-10 AFBs seen per field report as (++)
 10-100 AFBs seen per 100 fields report as (+)

If no red bacilli seen after examination the whole area of the smear, report
the smear as “No AFBs seen” (Do not report as negative because organisms
may be present but not present in those fields examined). And 1-9 AFBs per
100 fields, report the exact number e.g

Sputum specimen report

 Appearance: Blood stained purulent/mucoid sputum

 ZN stain: AFBs positive (++) seen, pus cells (+++)
If yeast cells are seen, they are supposed to be reported.
Note: There is no blood stained purulent sputum with no pus cells.
 For sputum, specimens examined macroscopically taking special note of
the appearance e.g. muco-salivary, mucoid, purulent, muco-purulent,
blood stained. If sputum is salivary, ask for a repeat.
 If muco-purulent and difficult to make the smear, homogenize with 10%
KOH or NaOH.
 Rinse the wire loop in the 5% Lysol in the beaker. Withstand to remove the
traces of sputum.
 When making a smear, heat the wire loop until red hot using a Bunsen
burner flame or heat lamp.
Quality control
 At regular interval and always whether a new batch is started, two sputum
smears of known high and low positivity should be stained with the routine

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 smears to check that the carbolfuchsin dye staining technique and the
microscopic examination of the smears is satisfactory.
 Do not recycle the slides for ZN staining because AFBs may remain on the
glass slide and retain their staining properties after sterilization and cleaning
leading to false positive results.
 Include positive and negative smears during ZN staining.
 Store all the positive slides for a period of 3 months.
Ziehl Neelsen staining method II
 The requirements are the same as in method I except the decolourisers
used are 1% acid alcohol and 5% sulphuric acid.
 Decolorize the smears rapidly washing with 1% acid alcohol or 5%sulphuric
acid. Washing for 10 seconds is usually adequate for skin smears and
about 15 seconds for nassal smears.
 After decolourisation, the smear should appear pale pink or almost
colourless.
Results
 Mycobacterium leprae appears as red solid bacilli or beaded fragmented
granules/forms occuring in masses.
 Macrophages and background materials appear blue or green
depending on the counter stain used.
Reporting of M. leprae smears
If masses of the bacilli are seen, report their number as in pluses as in method I
Cold ZN method
 Make smears on a grease-free slide, airdry, fix in alcohol or heat and label.
 Stain the smear with equal volumes of teapol and strong carbolfuchsin.
 Leave to stand for 30 minutes for M. tuberculosis and 20minutes for M.
leprae and other protozoa oocysts.
 Wash with clean water.

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 Decolourise with 3%, 1% acid alcohol or 20-25%, 5% sulphuric acid until no
more colour comes out.
 Wash again with clean water
 Counter stain with methylene blue or malachite green.
Results
 More than 10 AFBs seen per field report as (+++)
 1-10 AFBs seen per field report as (++)
 10-100 AFBs seen per 100 fields report as (+)
Kinyoun modification (or cold Ziehl–Neelsen modification technique)
 Procedure for Kinyoun staining is similar to the Ziehl-Neelsen stain, but does

not involve heating the slides being stained.

 The Kinyoun staining method uses carbolfuchsin as a primary stain,
followed by decolonization with an acid-alcohol solution and methylene
blue as a counter stain.

 Kinyoun carbolfuschsin has a greater concentration of phenol and basic

fuchsin and does not require heating in order to stain properly.

 When viewed under a microscope, a Kinyoun stained slide will show acid-
fast organisms as red and nonacid-fast organisms as blue.

Modified Ziehl Neelsen

Purpose
This method is used to demonstrate cryptosporidium, Isospora beli and
Cyclospora oocysts in stool samples and eggs of Taenia species. They are of
significance if found in large numbers in diarrhoea stools.
The oocysts may occur in normal stool samples. They are small bodies that
may be confused with the yeast cells in wet preparations. However, they are
distinguished by their acid-fast properties.
Required

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Strong carbolfuchsin, Methanol, 5% H2SO4, 1% acid alcohol, 0.1% methylene
blue or malachite green
Sample
Fresh stool sample
Method
 Prepare a thin smear of diarrhoea faeces on a clean grease-free glass
slide and label.
 Air-dry the smear (solid faeces can be emulsified in the saline before a
smear is made).
 Fix the smear in methanol for 5 minutes and allow to air dry.
 Stain with strong carbolfuchsin without heat for 20 minutes to 30 minutes or
you may use heat method for better staining of the organisms and reduce
the time to 10 minutes.
 Decolourise with 1% acid alcohol until no more colour comes out of the
slide.
 Counter stain with methylene blue or malachite green for 3 minutes.
 Rinse with water and air dry.
 Examine microscopically using 40×, 100 × objectives.
Results
Small, round, oval, spherical, pink-red staining bodies seen measuring about
5µm in diameter against a blue or green background.
The oocysts may contain a single deeply stained dot and some oocysts may
stain pale pink.
Yeasts if present often stain dull red
Reporting
Report the appearance of the stool sample i.e. colour or form, method for
microscopic examination and the presence or absence of the oocysts e.g.
App: Black semi-formed stool
Modified ZN stain: Cryptosporidium oocysts positive (+).

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Quality control
Include a positive slide in the procedure for oocysts of cryptosporidium.
Use charts showing cryptosporidium oocysts morphology.
Auramine Fluorochrome Staining Method
Purpose
This is a staining technique for detection of AFBs in sputum, C.S.F and other
specimens by fluorescence microscopy.
Principle
When certain dyes are exposed to U.V light of short wavelength, they absorb
energy and emit rays of long wavelength. Such a dye is called a
fluorochrome and it is said to fluoresce.
When tissues or organisms are stained with such dyes and examined under
U.V light in an adopted microscope, they are seen as fluorescent objects.
Auramine can be used in this way as a useful stain for the detection of M.
tuberculosis.
Merits of the method
 No danger in transferring organisms from one slide to another because
40×, 25× objectives which are used do not touch the preparation unlike
the oil immersion preparations.
 Can examine large fluid at ago.
Demerits of the method
Uses expensive equipment
Reagents
 Auramine O stain (basic dye)
 1% acid or 70% alcohol (decolouriser)
 Potassium permanganate (counter stain)-KMnO4
To prepare 1 litre of Auramine O
Auramine O……………………………………………….3g
Phenol crystals……………………………………………30.0g

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Distilled water…………………………………………….1000mls
 Dissolve the phenol crystals in about 200mls of distilled water under gentle
heat if necessary.
 Add Auramine gently and shake vigorously to effect the solution.
 Make up to 1000mls with distilled water and mix well.
 Filter, label and store in a dark stopper bottle. The stain is stable for several
months.
 For use, transfer a small portion of the stain in a dropper bottle and label.
To prepare 1 litre of 1% acid alcohol
Concentrated HCl……………………………………..10mls
70% Ethanol……………………………………………990ml

To prepare a litre of KMnO4

KMnO4 (powder)………………………………………….1.0g
Distilled water……………………………………………..1000mls
 Weigh the powder and dissolve it in about a half of the water and mix well
to dissolve. Add the remaining water and mix well.
 Filter and transfer to a brown reagent bottle.
 Label and store in a dark at room temperature. The reagent is stable for
several months.
Procedure for staining
 Prepare a thin smear of the specimen. Allow to airdry and fix with gentle
heat
 Apply the Auramine stain for 20 minutes
 Rinse with clean water
 Decolourise with 1% acid alcohol for 2-3 minutes
 Rinse with clean water
 Counter-stain with 1% KMnO4 for 1 minute
 Wash with clean water

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 Clean the back of the slide and air dry
 Systematically examine the smears for AFBs under a fluorescence
microscope using 40 × objectives
 Protect the smears from direct sunlight to prevent fading of the
fluorescence

Results
AFBs appear as bright white yellow luminous rods against a dark background
Reporting
If fluorescence AFBs are seen, report the smear as;
AFBs positive in pluses {(+), (++), (+++)} which gives an indication of the
number of the bacilli present.
If there are no fluorescent rods, report the smear as „NO AFBs seen‟.
Quality control
At regular intervals and always when a new batch of the stains is started, two
sputum smears of known high and low positivity should be stained with the
routine smears to check that Auramine O stain and the staining technique are
satisfactory.
Metachromatic stains

Certain bacteria contain granules seen in their protoplasm. The presence and
nature of these granules are used in the identification of these organisms‟
example volutin or metachromatic granules where the organisms will take a
different colour from that of the granules e.g. Albert‟s stain.

Albert’s method of metachromatic staining

Reagents
Solution I (Albert‟s stain)…………………………………..1 llitre
Toluidine blue………………………………………………1.5g

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Malachite green…………………………………………….2.0g
Absolute ethanol……………………………………………20mls
Glacial acetic acid………………………………………….10mls
Distilled water……………………………………………...970mls
 Weigh toluidine blue and malachite green and transfer to a right bottle.
 Add glacial acetic acid.
 Put 300mls of water. Mix well to dissolve the stains.
 Add ethanol and remaining water then mix well.
 Filter, label and store in the dark at room temperature.
 The stain is stable for several months.
 For use, put small amounts of the stain into a dropper and label.
Solution II (Albert’s iodine)
To make 1500mls;
Potassium iodide……………………………………………15g
Iodine……………………………………………………….10g
Distilled water………………………………………………1500mls
 Weigh the potassium iodide and transfer to a clean brown bottle.
 Add about 400mls of distilled water and mix well until the potassium iodide
is fully dissolved.
 Make up to 1500mls with distilled water and mix well.
 Label the bottle and store in the dark at room temperature.
 Prepare a fresh reagent if the colour fades.
Procedure

 Make a smear from 18-24 hour culture from a loeffler‟s serum agar.
 Dry and fix with gentle heat.
 Add solution I for 3-5 minutes, wash with clean water and blot carefully.
 Apply solution II for 1 minute and wash with clean water.
 Wipe at the back of the slide clean and air dry.

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 Examine the smear microscopically with 40× to check the staining and
distribution of the materials.
 Then with 100× objective to look for bacteria staining metachromatic
granules.
Results
Metachromatic granules appear bluish black. Bacteria cells appear green
while other objects or structures appear pale green.
The staining methods are routinely used to demonstrate corynebacterium
diphtheriae.
Pugh’s stain
It is used for staining of corynebacterium diphtheriae.
Reagents
Toluidine blue……………………………………………1g
Ethyl alcohol (Absolute)…………………………………20mls
Glacial acetic acid……………………………………….50mls
Distilled water…………………………………………..950mls
Combine the glacial acetic acid with distilled water. Add toluidine blue dye
dissolved in alcohol, filter and label.
Procedure
 Prepare a smear from an 18-24 hour loeffler‟s serum culture agar of the test
organism. Allow to dry and fix with gentle heat.
 Apply the stain for 2-3 minutes.
 Wash with water, blot carefully and dry with gentle heat.
Results
Granules stain reddish purple and the remainders of the organisms stain light
blue.
Capsule Stains
In ordinary staining methods example gram stain are often unstained and are
clear around the stained organisms.

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In order to stain the capsules, use direct/positive or indirect/negative
techniques.
Crystal violet capsule stain
Reagents
1% crystal violet
20% copper sulphate
Method
 Prepare a thin smear, air dry without fixing in heat.
 Apply 1% crystal violet for 2 minutes (no heat). Heat disturbs the capsule
shape.
 Wash with 20% copper sulphate
 Blot carefully/air dry and examine under 40× objective.
Results
Capsules are pale violet; bacterial cells appear deep violet or deep purple.
Negative Staining
Principle
This is a technique where the capsules of the organisms remain unstained over
a stained dark background. Negative staining is commonly used for
demonstrating fungal capsules example Crytococcus neoformans, bacterial
capsules example Klebsiella Speciesp and bacterial spore.
There are mainly two methods of negative staining;
a. Nigrosin methylene blue
Solution 1

Nigrosin ……………………………………………..50-100g
Distilled water……………………………………….1000mls
Formalin……………………………………………..0.5-1ml
 Dissolve the nigrosin in warm distilled water and add formalin as a
preservative.
Solution 2

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Loeffler’s methylene blue
Saturated solution of methylene blue in alcohol…………………………300mls
Potassium hydroxide 0.01% in distilled water…………………………..1000mls
Procedure of staining
 Put a loopful of culture on the slide.
 Add a loopful of freshly filtered solution. Mix the two and allow drying then
fixing with gentle heat.
 Rinse rapidly with clean water and blot carefully to dry.
Results
Capsule is unstained and the background will be dark grey because of the
nigrosin.
Organisms demonstrated are; streptococcus pneumoniae, bacillus anthracis,
clostridium welchii, Cryptococcus noeformans, klebsiella species.
India Ink
This is a technique where a capsule of an organism remains unstained against
a dark background used commonly for demonstrating fungal capsules
example Cryptococcus neoformans, bacterial capsules example klebsiella
species and bacterial spores.
Preparation of the India Ink
 2% Chromium Mercury
 Concentrated India Ink solution. Dissolve 2g of Chromium Mercury in100
mls of distilled water to make 2%.
 Add 2 drops of concentrated India Ink to the solution of 2% to make a
working India Ink solution
 Store in a brown bottle
 Filter and label with date, name of reagent, e.t.c and store for use
 India ink is stable for long time
 Quality control the reagent by using a known specimen with a capsulated
organism

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 The capsule will clearly appear unstained against a dark background of
the India ink.
Staining Procedure
 Put a drop of bacterial/fungal suspension on a clean slide.
 Add a drop of India ink and mix gently.
 Put and press a coverslip lightly onto to remove excess liquid and air to
give a thin layer of the preparation.
 Examine under 10× and 40× objectives.
Results
Bacteria and fungi appear refractile, capsule clearly unstained against a dark
background.

Stained bacteria cell

Unstained capsule of an organism

Spore Stains
These are stains that stain spores more intensely than other parts of the cell.
Principle
Spores have cell walls impermeable to stains however; they can be stained
by heating preparation.
The spore wall stained resists decolourisation by alcohol treatment/any other
decolouriser.
Types of Spore Stains
a) Fuchsin methylene blue spore stain
b) Malachite green spore stain
1) Fuchsin methylene blue spore stain
Reagents:
ZN carbol fuchsin staining solution
3% ferric chrolide aqueous solution

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 5% sodium sulphate as decolouriser
0.3% methylene blue as a counter stain

Procedure
 Make a thin smear, air dry and fix with gentle heat.
 Apply strong carbol fuchsin staining solution for 2-5 minutes.
 Heat the back of the slide until steam rises.
 Cool and wash in clean water.
 Decolourise with 3% ferric chrloride for 1-2 minutes.
 Wash with clean water.
 Counter stain with 0.3% methylene blue for 1 minute.
 Wash with clean water, air dry and examine using 10 × objectives.
Results
Spores will be bright red. In vegetative will be blue.
2) Malachite Green Spore Stain
Reagents
0.5% malachite green
0.5% safranine counter stain
Procedure
 Make a thin smear on a slide, air dry and fix in gentle heat.
 Flood the slide with warm malachite green for 5 minutes.
 Wash with water.
 Counter stain with safranine for 30 seconds.
 Wash air dry and examine using 40 × objectives.
Results
Spores will be green; other parts of the organism will be red.
Simple Stains
These are stains, which are used to show the morphology of organisms, and
sometimes they are used also as counter stains to show the background.

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Methylene Blue

It‟s routinely used to demonstrate gonococci also used as a counter stain.

Preparation
Saturated solution of methylene blue in alcohol 1.5g/100mls of
alcohol……………50mls
Distilled water……………………………………………………………………….950mls
Procedure
 Make a smear, air dry and fix.
 Apply the stain for 30 seconds.
 Wash with water, air dry and examine under 100×
Results
Bacteria will appear blue
Malachite Green
Used as a counter stain for ZN method.
Preparation
Malachite green (powder)…………………………………………..1g
Distilled water………………………………………………………1000mls
 Dissolve the powder in distilled water, filter and label.
Procedure
 Make a thin smear and fix.
 Apply the stain for 30 seconds, wash, air dry and examine using 100 ×
objectives.
Results
Bacteria appear green.
Loeffler’s Alkaline Methylene Blue

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These are basic dyes routinely used for demonstrating of the morphology of
organisms in smears. It stains more intensely than neutral stains and show some
degree of chromatic stains i.e. may acquire other colors apart from blue.
Preparation
Methylene blue (1.5g/95%) alcohol………………………………………..30mls
0.01%KOH in distilled water……………………………………………..1000mls
 Measure the KOH solution then mix with water and add methylene blue.
Filter and label.
Procedure
 Make a thin smear and fix.
 Apply the stain for 30 seconds, wash, air dry and examine using 100 ×
objectives.
Results
Bacterial cells appear blue.
Polychromed Methylene Blue

This is made by allowing some of the loeffler‟s methylene blue to ripen slowly.
The stain is kept in bottle, which are half filled and shaken at intervals to mix
the contents thoroughly.
The slow oxidation of the methylene blue forms a violet compound which
gives the stain it is polychrome properties. The ripening takes 12 or more
months to complete. It‟s used to demonstrate anthrax organisms.
 Make a thin smear and fix.
 Apply the stain for 30 seconds, wash, air dry and examine using 100 ×
objectives.
Results
Real polychromatic staining stains the organisms blue and other parts show
other colours e.g. pink

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5.4 General Methods Used In Identification of Micro-Organisms in the
Laboratory
Direct Examination
 Macroscopically by their appearance, Microscopically by their motility,
morphology and staining reactions example Gram stain, ZN, India Ink
Serological methods
By use of Antibody and antigen reaction, Culture On plates containing
nutrients required for microbial growth
 Identification of organisms by either bio-chemical reactions based on the
enzymes, By using sensitivity patterns, Animal inoculation,
 DNA composition

CHAPTER SIX: REQUIREMENTS FOR BACTERIA GROWTH

This is divided into two major categories;

(i) Nutritional Requirements
(ii) Physical Requirements

6.1 Nutritional Requirements

The growth of bacteria depends on adequate supply of stable nutrients and

the specific nutrient requirement vary according to the natural environmental
adaptation of different species. Some species are able to grow under a wide
range of conditions but others especially more strict organisms e.g.
Gonococci is very strict and restricted in their requirements are; Carbon,
Hydrogen, Oxygen, Nitrogen, Sulphur, Phosphorus. Other elements such as
Iron, Sodium, Manganese, and Magnesium are required in small amounts.

Nitrogen and Carbon can be supplied in water that is essential for any
growth.

(a) Carbon source

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The majority of the bacteria including all the parasitic species require organic
nutrients such as Carbohydrates, amino acids, peptides and lipids to serve as
a source of Carbon and energy. Organisms that use CO2 as their carbons
source and synthesize other substances they need are Autotrophs. While
Heterotrophs are organisms that require glucose or another organic carbon
source from which they obtain energy and intermediates for synthetic
processes.

(b) Nitrogen source

The main inorganic source of Nitrogen used in biosynthesis by pathogenic

bacteria is ammonia usually in form of ammonium salts (NH 4+). It can be
provided directly in the environment or indirectly by diamination of organic
nitrogenous nutrients such as amino acids or by reduction of nitrites.

(c) Inorganic salts

Bacteria requires a supply of inorganic salts for growth particularly phosphates
and sulphates among the anions and sodium, potassium magnesium, iron,
manganese, calcium among the cations.
(d) Organic compounds

Certain organic compounds such as amino acids, nucleotides,

monosaccharides, lipids and co-enzymes must be either synthesized by the
organism or provided as nutrients in the environment. Microorganisms vary
widely in their biosynthetic capacity and this variation is reflected in their
nutrient requirement example some bacteria can synthesize all their amino
acids from carbon as energy source together with ammonium and sulphate
ions. Other bacteria are unable to synthesize any of their amino acids and
these therefore, become their essential nutrients that must be supplied in their
growth media.

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If a co-enzyme or an essential part of a co-enzyme is required as a nutrient, its
required in small catalytic amount and sometimes known as a growth factor
or microbial vitamin.

10.2 Physical Requirements

Bacterial growth can be extremely rapid if a cell can double its size and
divide within 30 minutes. It must therefore, be capable of synthesizing its
weight of the cell material within this period.

(i) Oxygen

Some bacteria strictly grow in the presence of oxygen. These bacteria are
called strict or obligate aerobes e.g. Pseudomonas aeruginosa.

Some grow strictly anaerobically i.e. in absence of oxygen but they grow in
the presence of nitrogen and these are called strict or obligate anaerobes
e.g. Clostridium species.

Majority grows in the presence or absence of oxygen and they are called
facultative anaerobes example Staph. Aureus, E. coli, Klebsiella, Salmonella
species Shigella species, Proteus species

Some bacteria require reduced amount of oxygen but increased amount of

carbondioxide and these are called Micro-aerophiles or Carboxyphiles e.g.
Niesseria Gonorrhoea, Brucella abortus.

(ii) Temperature

Influence on growth;

For each species, there is a definite range of temperature for growth. Within
that range there is an optimum range of temperature for best growth. For
most bacteria especially the ones living as commensals or pathogens in
humans or other warm-blooded animals, the temperature range is between

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20-40˚C with 37˚C being optimum. Bacteria that grow within this range are
called Mesophiles e.g. Salmonella, E. coli, Klebsiella.

Few bacteria especially the saprophytes require temperatures of less than

20˚C. Some may require -2˚C. Such bacteria are called Psychrophiles. In
addition, are responsible for spoilage of refrigerated foods and they are less of
medical importance e.g. Listeria monocytogenes with optimum growth
temperature of +4˚C.

Another group is the non-parasitic bacteria called thermophiles, which grow

at high temperatures between 55˚C-80˚C. These organisms are important as
the cause of spoilage in under-processed canned foods e.g. Bacilli
stearothermophilus.

(iii) Moisture

It‟s estimated that 4/5 by which of a bacterial cell consists of water as in case
of other microorganisms. Moisture is absolutely necessary for growth.

Drying in air injures many microbes and different species vary widely in their
ability to survive when dried under natural conditions thus with Gonococcus,
Treponema pallidum appear to dry quickly whereas with Tubercle bacilli (TB),
staph. aureus and the small pox virus may survive drying especially spores of
Bacillus anthracis which can survive for 60 years when dry an a plate.

(iv) Hydrogen concentration (PH)

A suitable environment PH is an essential factor in microbial metabolism and

growth. The majority of commensals and pathogenic bacteria grow best at a
neutral or slightly alkaline reaction.

Some bacteria however, grow in the presence of a considerable degree of

acidity and these are termed as Acidophilic e.g. Lactobacilli species. Others

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are very sensitive to acids but tolerant to alkali e.g. Vibrio cholerae. Bacteria
that grow in neutral ph are termed as neutrophilic.

(v) Light and other radiations

Darkness provides favourable conditions for growth and viability. Ultraviolet

rays are rapidly bacteriocidal e.g. direct sunlight or radiation from mercury
lamp even diffuse daylight as it enters a room through a window glass
significantly shortens the survival of microorganisms. Bacteria that require light
for growth are called photoautrophs

Bacteria are also killed by ionizing radiation.

(vi) Osmotic pressure

Relative to the cells of the higher organisms, bacteria are tolerant to change
in the osmotic pressure of their environment and can grow in media with
widely valid contents in salts, sugars, and other solutes. This is partly a
reflection of mechanical strength of the cell wall. For most species, the
maximum concentration of NaCl permitting growth is between 5-10%. Those
that can grow in a higher salt concentration up to saturation are known as
Halophilic (Osmophiles). These are saprophytes whose importance lies in
causing spoilage of food preserved in salt and sugar. They are not pathogens.

Sudden exposure of bacteria to solutions of high salt concentrations e.g. 2-

25% NaCl may cause plasmolysis (temporally shrinkage of cytoplasmic
membrane).

Sudden transfer from a concentration to a weak solution may cause swelling

or busting of the cell.

(vii) Mechanical and sonic stress

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Although a bacterial cell wall has considerable strength and elasticity, its
possible to rupture and kill them by exposure to mechanical stresses.

A bacterial suspension may be largely disintergrated by subjection to a very

vigorous shaking with glass beads or ultrasonic vibrations. These measures are
used in isolating a large molecular component of the cell.

6. 3 Bacterial Growth Phases

When an innoculum of cells from a pure culture of bacteria is introduced into
a suitable nutrient media and incubated under appropriate conditions,
almost all the cells have the potential to growth at a very rapid rate. This
growth by the cells is normally associated with multiplication.

Their multiplication is by simple binary fission and growth under favourable

conditions occurs very rapidly in about every 30 minutes.
The time taken for one cell to divide into two is known as the generation time.

Example;

Time (Minutes) Generation Number Of Cells

0 0 1

30 1 2

60 2 4

90 3 8

120 4 16

150 5 32

When a comparatively small number of organisms are taken in a culture and

inoculated into fresh growth media, the number of cells inoculated may
multiply a million times or more during growth. If the number of cells present at

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different times after inoculation is measured and the number is plotted in
relation to time, the resultant growth is referred to as Batch growth curve.

Two types of growth curves can be drawn according to the number of cell
counts that is used.

(i) Total count

This is based on the number of cells present irrespective of whether they are
living or not. In microbiology, an organism is considered living or viable if it‟s
capable of continued multiplication. If its not, it‟s called dead or non-viable.

(ii) Viable count

This measures only those cells, which are capable of growing.

A Typical Growth Curve

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Lag Phase

In this period, there is no appreciable multiplication of cells although they may

increase in size or show marked metabolic activities.

This phase can be looked often as presenting the time taken by the organism
to adopt itself in the growth medium. The cells in an inoculum may be so
depleted, enzymes, metabolic intermediates or other factors that are
sometimes required for these materials or cells to build up to their optimum
levels- may be reduced.

Alternatively, if the composition of the new medium differs significantly from

that in which the inoculum was growing, entirely the new enzymes may be
required to be synthesized.

Bacteria will start adjusting to the environment and starts to divide, while
others are slow at adjusting. This results into a condition that though there will
be increase in the number of cells, the resultant number will not be a multiple
of the original.

Log/Exponential Phase

In this phase, the cells divide at a constant rate because of growth, there is a
linear relationship between time and number of cells.

The nutrients, enzymes are available for growth and therefore cells adapt to
the new environment and begin to reproduce.

If the number of bacterial cells on the culture media increase 10 times in a

given period of time, they will increase 100 folds twice that time and so fourth.

The actual rate of growth is directly related to generation time (time between
divisions of the bacterium under particular environmental conditions).

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Some cells begin to die at this stage due to their toxins, which are their
products, but still the number of bacteria multiplying will be greater than those
dying.

Stationery Phase

In the due course, the rate of multiplication decreases until it ceases

altogether and cells pass into a stationery phase. It is theoretically possible to
reach a stable number of cells by the establishment of equilibrium between
the rate of growth and death.

However, the common situation is one in whish neither growth nor death
occurs where the number of cells dying equals the number of cells multiplying.
Exhaustion of essential nutrients in the media, accumulation of waste products
and presence of toxins in the media most commonly cause the stationery
growth.

Decline/Death Phase

After a period in the stationery phase, the cells in the culture medium begin to
die. They become incapable of growth when transferred into a fresh medium.

The causes of this death or loss of viability are many. The most determinant
being the nature of the fact that causes the cells of the bacteria to cease of
growth that sets on the stationery phase i.e. in this phase the number of
bacteria dying is greater than the number of cells multiplying.

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CHAPTER SEVEN: CULTURE MEDIA

7.2 Culture media

They are nutrients used for culturing bacteria or substances that contain
nutrients for the growth and multiplication of bacteria.

 Culturing is the growing of bacteria artificially on media which contain

nutrients that favor their growth.
 A good culture media must contain the right amount of required nutrients
and correct osmotic pressure and PH.
Like any other living organism, bacteria require water, carbohydrates,
proteins, minerals for their growth or survival.

Inoculum

These are micro organisms used in inoculation on the culture plate.

Incubation

This is the process of maintaining a bacterial culture at a particular

temperature for a set length of time in order to measure the bacterial growth.

Plating

This is inoculating or seeding of microorganisms into a culture media plate, for

their growth.

Bacterial culture media

There are various reasons why bacteria have to be grown (cultured) in the
laboratory on artificial culture media. One of the most important reasons
being its utility in diagnosing infectious diseases.

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 Isolating a bacterium from sites in body normally known to be sterile is an
indication of its role in the disease process.
 Indeed, isolating an organism from the clinical specimen is the first step in
proving its role as an etiologic agent.
 Culturing bacteria is also the initial step in studying its morphology and its
identification. Bacteria have to be cultured in order to obtain antigens
from developing serological assays or vaccines.
 Certain genetic studies and manipulations of the cells also need that
bacteria be cultured in vitro.
 Culturing bacteria also provide a reliable way estimating their numbers
(viable count). Culturing on solid media is another convenient way of
separating bacteria in mixtures.

Bacteria infecting humans (commensals or pathogens) are

chemoorganoheterotrophs. When culturing bacteria, it is very important to
provide similar environmental and nutritional conditions that exist in its natural
habitat. Hence, an artificial culture medium must provide all the nutritional
components that a bacterium gets in its natural habitat.

7.3 Ingredients of culture media or nutritional requirements of culture media

Some of the ingredients of culture media include water, agar, peptone,

casein hydrolysate, meat extract, yeast extract and malt extract. While tap
water is suitable for culture media, it must not be used if it contains high
amount of minerals. In such situations, distilled or dematerialized water should
be used

(i) Water it is an important solvent for nutrients. All organisms require water
for their growth. It must be free from inhibiting chemicals e.g. lead and
copper which are toxic for growth of bacteria and therefore use
distilled or de-ionized water.

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(ii) Carbohydrates they are in the form of simple or complex sugars. They
are a required source of carbon, nitrogen and energy.
Other chemical substances are necessary such as sulphur, phosphorus
and small traces of metal salts and certain vitamins or vitamin like
substances called essential metabolites.
Also required for differentiating bacteria e.g. lactose fermentors are
indicated by change of colour of the indicator and usually
accompanied by production of gas.

(iii) Peptone is a byproduct of protein (plant or animal) digestion. Proteins

are often obtained from heart muscle, casein, and fibrin or soya flour
and are digested using proteolytic enzymes such as pepsin, trypsin or
papain. The final product contains peptones, proteoses and amino
acids besides a variety of inorganic salts including phosphates,
potassium and magnesium. Casein hydrolysate is obtained from
hydrolysis of milk protein casein using HCl or trypsin.

All forms of peptones are not coagulated by heat. It provides nitrogen,

mineral salts, vitamins and some provide carbohydrates. Peptone is used in
varying amounts depending on different types of requirements. In some high
amounts peptone is used in blood culture, in high tryptophan amount for
indole test media and tryptophan with high peptone for blood culture.

Qualities of a good peptone

 It should have the ability to support the growth of bacteria.

 It should not have fermentable sugars because they may affect
biochemical tests by either giving false positives.
 It should have low content of contaminating bacteria.
 It should have low content of copper ions.

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 It should have a neutral PH where most organisms grow.
 It must be soluble in water.
 The powder should be light and dry.
(iv) Agar: it is a gelling agent and it is prepared from sea weeds. It consists of
long chain polysaccharide units.
It has lower temperatures of 32-37°C hence it can be used to add heat labile
nutrient. It is used to solidify the culture media as it has a high gelling strength.
It provides calcium and organic ions. It should not contain impurities like
organic salts, protein-like materials, traces of long fatty chain acids.

There are two common types of agar

a) Japanese agar
b) New Zealand agar

Gelatin
Gelatin is a protein derived from the collagen of the skin, hide and bone. It
forms a gel at a concentration of (12-15%) in a nutrient broth.

Qualities of a good agar

 It should not add any food value to the media i.e. not support growth but
only help in solidification.
 It should not have large particles that may need filtering.
 It should melt at 98°C and set at 42°C.
 It should yield a gel at a concentration of 1-2% (that is weight per volume
depending on the make).
 It should be liquefied by very few marine organisms.
 It should be free from toxicity and microorganisms.
(v) Mineral salts it is required for cell growth, this includes sulphur from
sulphate, phosphorus from phosphates, magnesium, NaCl and Ca 2+ are
required in small quantities (traces).

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(vi) Meat extracts Meat extract is obtained by hot water extraction of lean
beef and then concentrated by evaporation. Meat extract contains
gelatin, albumoses, peptrones, proteoses, amino acids, creatinine,
purines, and accessory growth factors. These provide amino acids.
Malt extract is prepared by extracting soluble materials from sprouted
barley in water at 55oC and concentrated by evaporation. It contains
maltose, starch, dextrin, glucose and small amounts of protein and
protein breakdown products and growth factors and Vitamin. Meat
extract is incorporated in very many culture media.

(vii) Yeast extracts Yeast extract is prepared from washed cells of bakers‟
yeast and contains wide range of amino acids, growth factors and inorganic
salts. It is used in many culture media as a growth stimulant example XLD

(ix) Blood it should be collected aseptically and must be rendered none

coagulating by defibrination or addition of citrate or oxalate. Frozen or lysed
blood should not be used. Use horse, sheep and rabbit blood.

(x) Serum it can be obtained commercially. It should be stored at 3-5°C.

(xi) Blood products this can be in form of;

 Horse serum
 Haemolysed horse blood
 Oxalated human blood
 Defibrinated horse blood or sheep.
It is mainly horse and sheep blood mainly used. Defibrinated blood is good for
fastidious organisms like haemophilus because of the release of V-factors.
Haemolysed or lysed blood should be used for special purpose e.g. growing
corynebacterium, clostridium species and haemophilus species.
In sensitivity testing lysed horse blood is added to the media to improve the
reaction.

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(xii) Bile, bile salts and derivatives Bile is a product of liver and its composition
varies according to its animal sources. It contains bile pigments, bile acids
either in free or coagulates forms, fatty acids, cholesterol, inorganic salts,
mucin, lecithin, sulphates, gluconic acids and urea. Bile salts are sodium salt
with a conjugated bile acids.

(xii) Environmental factors Apart from these nutritional requirements, certain

other conditions before they will grow satisfactorily in artificial culture media
e.g. gaseous, temperature, PH, moisture are required.

Types of culture media

They are three types of culture media i.e.

 Liquid/broth culture media

 Solid culture media
 Semi solid culture media

7.4 Classification and Preparation of culture media

Bacterial culture media can be classified in at least three ways; Based on

consistency, based on nutritional component and based on its functional use.
Classification based on consistency
Culture media are liquid, semi-solid or solid. Liquid media are sometimes
referred as “broths” (e.g nutrient broth).
Liquid media

 They are available for use in test-tubes, bottles or flasks. In liquid medium,
bacteria grow uniformly producing general turbidity.
 Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus)
are known to grow as a thin film called „surface pellicle‟ on the surface of

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 undisturbed broth. Bacillus anthracis is known to produce stalactite growth
on ghee containing broth.
 Sometimes the initial turbidity may be followed by clearing due to autolysis,
which is seen in penumococci. Long chains of Streptococci when grown in
liquid media tend to entangle and settle to the bottom forming granular
deposits but with a clear medium.
 Culturing bacteria in liquid media has some drawbacks. Properties of
bacteria are not visible in liquid media and presence of more than one
type of bacteria can not be detected. Liquid media tend to be used
when a large number of bacteria have to be grown.
 Culture media are suitable to grow bacteria when the numbers in the
inoculum is suspected to be low.
 Inoculating in the liquid medium also helps to dilute any inhibitors of
bacterial growth. This is the practical approach in blood cultures.

 Culturing in liquid medium can be used to obtain viable count (dilution

methods).

Solid media
Liquid medium can be rendered solid by the addition of certain solidifying
agents. Agar agar (simply called agar) is the most commonly used solidifying
agent. The word "agar" comes from the Malay word agar agar (meaning
jelly).

 It is also known as kanten, China grass, or Japanese isinglass.

 Agar is chiefly used as an ingredient in desserts throughout Japan.
 It is an unbranched polysaccharide obtained from the cell membranes of
some species of red algae such as the genera Gelidium and Gracilaria, or
seaweed (Sphaerococcus euchema). Commercially it is derived primarily
from Gelidium amansii.

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 Agar is composed of two long-chain polysaccharides (70% agarose and
30% agarapectin).
 It melts at 95oC (sol) and solidifies at 42oC (gel), doesn‟t contribute any
nutritive property,
 It is not hydrolysed by most bacteria and is usually free from growth
promoting or growth retarding substances. However, it may be a source of
calcium & organic ions.
 Most commonly, it is used at concentration of 1-3% to make a solid agar
medium.

 New Zealand agar has more gelling capacity than the Japanese agar.
Agar is available as fibres (shreds) or as powders.

For preparing agar in Petri plates, 3% agar (by weight) is added to the broth
and autoclaved, when the medium is at ~50oC, it is poured on to sterile Petri
plates and allowed to set. For preparing agar containing media in test-tubes,
the culture medium is mixed with 3% agar and heated with stirring to melt. This
ensures that all the tubes get equal amounts of agar. These tubes can then
be sterilized by autoclaving.

Semi-solid media
reducing the amount of agar to 0.2-0.5% renders a medium semi-solid.

 Such media are fairly soft and are useful in demonstrating bacterial motility
and separating motile from non-motile strains (U-tube and Cragie‟s tube).
 Certain transport media such as Stuart‟s and Amie‟s media are semi-solid
in consistency. Hugh & Leifson‟s oxidation fermentation test medium as well
as mannitol motility medium is also semi-solid.

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Biphasic media
sometimes, a culture system comprises of both liquid and solid medium in the
same bottle.

 This is known as biphasic medium (Castaneda system for blood culture).

 The inoculum is added to the liquid medium and when subcultures are to
be made, the bottle is simply tilted to allow the liquid to flow over the solid
medium.
 This obviates the need for frequent opening of the culture bottle to
subculture

Other solidifying agents

Besides agar, egg yolk and serum too can be used to solidify culture media.

 While serum and egg yolk are normally liquid, they can be rendered solid
by coagulation using heat.
 Serum containing medium such as Loeffler‟s serum slope and egg
containing media such as Lowenstein Jensen medium and Dorset egg
medium are solidified as well as disinfected by a process of inspissation.

Classification based on nutritional component

Media can be classified as simple, complex and synthetic (or defined). While
most of the nutritional components are constant across various media, some
bacteria need extra nutrients.

 Those bacteria that are able to grow with minimal requirements are said to
non-fastidious and those that require extra nutrients are said to be
fastidious.
 Simple media such as peptone water, nutrient agar can support most non-
fastidious bacteria.

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 Complex media such as blood agar have ingredients whose exact
components are difficult to estimate.
 Synthetic or defined media such as Davis & Mingioli medium are specially
prepared media for research purposes where the composition of every
component is well known.
Classification based on functional use or application
These include basal media, enriched media, enrichment media, selective,
indicator/differential media, transport media and holding media.

Basal Media

These are simple media e.g. nutrient agar and nutrient broth that support the
growth of microorganisms with less nutrient requirements, are basically simple
media that supports most non-fastidious bacteria. Peptone water, nutrient
broth and nutrient agar considered basal medium
Addition of extra nutrients in the form of blood, serum, egg yolk to basal
medium makes them enriched media.

Uuses

 They form a basis for the preparation of enriched media.

 They are used for maintaining stock cultures for certain strains of bacteria.
 They are used for sub culturing microorganisms from differential and
selective media for other biochemical and serological tests example
nutrient agar. It is a nutrient broth which has been solidified by addition of
agar Examples of gelling agents include;
Japanese agar to yield 2% concentration, Newsealen agar to yield 1.2%
concentrations

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Enriched media

These are media that are enriched with whole blood, lysed blood, serum
extracts, peptones or vitamins to support the growth of pathogens that
require additional nutrients or growth stimulants.

Uses

 They support the growth of fastidious organisms example Haemophilus

species, Neisseria species, Streptococcus species example of such media
include; tryptone, soya media, chocolate agar, blood agar and Loeffler‟s
serum slope.
Blood Agar

 It is a widely used media that act both as enriched and differential media
by showing haemolysis (β and α haemolysis).
 It is prepared by adding sterile blood to molten sterile nutrient agar that
has been put to 50°C to give 5-10% to final concentration.
However, the concentration of blood may be varied to suit the special
purposes.
 A fairly thick layer of media is prepared to avoid drying.
 10% blood agar (BA) does not give clear haemolysis and hence
production of double layer blood agar.

Preparation of blood agar plate

There are two types of BA plates to be prepared;

 Single layer BA plate

 Double or layered BA plate

Single Layered Blood Agar Plate

Provided are;

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 Molten sterile nutrient agar set at 50°C
 Sheep or rabbit or horse or goat‟s blood i.e. human blood should not to be
used because it may contain substances inhibitory to the growth of some
pathogens.
 Petri dishes.
Method
 Choose the correct BA concentration which should be either 5-10% final
concentration of BA plate.
 Use the formula RV = X where;
O R= required concentration of the BA to be
prepared.
V= required volume of BA or final volume of BA to
be prepared.
O= original concentration of blood (100%)
X= number of mls of blood to be added to the
molten sterile nutrient agar or Columbia agar
base.
Example
 You are provided with 20mls of molten Columbia agar base
 5mls of sheep blood
 With the materials given prepare blood agar (BA) showing your working
clearly.
Solution
Title: Preparation of blood agar (BA) plate
Given: 20mls Columbia agar base, 5mls of sheep blood, Petri dishes.
Required: To prepare a BA plate
Method
 Choice of BA concentration (concentration is usually 5-10%)
 Use of formula; RV = X where R = 5%

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 O
V = 20mls
O = 100%
X =?
X = 5 × 20
100
X = 1mls
Note: Include units i.e. a standard volume of media in the plate is 20mls

Preparation of Blood Agar Plate

 From the 20 ml of the nutrient agar base, pipette away 1ml of the agar
and replace it with 1ml of BA.
 Aseptically remove 1ml from the 20ml e.g. 20ml - 1ml = 19 ml + 1ml of
blood.
 Mix gently and avoid forming air bubbles.

Note: allow the blood to warm at room temperature before being added to
the nutrient agar.
 Dispense aseptically 20ml amount in a sterile Petri dish depending to the
size.
 Allow the plate to set for few minutes like 30 minutes
 Label the plate on the back with the date of preparation, type of media,
give it a batch number and quality control and then store for use.

Layered blood agar

Uses
 A thin layer of blood enables haemolytic reactions to be seen more
clearly.
 Reduces the amount of blood agar to be needed.

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Preparation
Method
 Prepare a thin layer of nutrient agar base in a plate by pouring about 7-
8ml of sterile agar in a plate to give a thickness of 0.5mm i.e. pipette 8ml
and release 8ml in the centre of the plate and allow to set.
 Choose a BA concentration as in a single layer example 5-10%
 Still use the formula RV = X
O
Calculate: RV = X
O where; R = 5%
V = 20-8 = 12 ml
O = 100%
X = 5 × 12
100
X = 0.6ml

 Pipette 0.6ml away from the 12ml of nutrient agar and replace it with 0.6ml
of blood from what is given.
 Mix gently then pour the same amount of the Blood Agar as that of the
nutrient agar on the plate (i.e. 8ml in this case) or to maintain a standard
plate, pour the whole 12 ml of blood agar and allow to set.
 Perform quality control on layered blood agar and store for use.

Chocolate agar

 Known as heated blood agar or lysed blood agar. The procedure is similar
to that of blood agar preparation except that the blood is added while
the molten blood agar base is still hot at 60 – 70 oC

 This lyses the blood cells and releases their contents into the medium. This
process turns the medium brown, hence the name. This medium is
especially useful in growing Hemophilus and Neisseria.

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 Serum for medium can be obtained from animal blood but must be filtered
through membrane or seitz filter before use.

Selective media

 They are designed to inhibit unwanted commensal or contaminating

bacteria and help to recover pathogen from a mixture of bacteria.

 Selective media are agar based; any agar media can be made selective
by addition of certain inhibitory agents that don‟t affect the pathogen.

 OR These are media which contain substances that inhibit/prevent or

slowdown the growth of microorganisms rather than pathogens of which
the media are meant or intended example DCA (Deoxychollate citrate
agar) which contain bile salts, sodium deoxychollate that inhibit organisms‟
growth except salmonella and shigella, Xylose Lysine Deoxycholate Agar
(XLD)

 Various approaches to make a medium selective include addition of

antibiotics, dyes, chemicals, alteration of pH or a combination of these.
 Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin,
Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover
S.aureus contain 10% NaCl. Potassium tellurite medium used to recover
C.diphtheriae contains 0.04% Potassium tellurite. McConkey‟s Agar used
for Enterobacteriaceae members contains Bile salt that inhibits most gram
positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover
P.aeruginosa contains cetrimide. Crystal Violet Blood Agar used to recover
S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium
used to recover M.tuberculosis is made selective by incorporating
Malachite green. Wilson & Blair‟s Agar for recovering S.typhi is rendered
selective by the addition of dye Brilliant green.

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 Selective media such as TCBS Agar and Monsur‟s Tellurite Taurocholate
Gelatin Agar used for isolating V. cholerae from fecal specimens have
elevated pH (8.5-5.6), which inhibits most other bacteria.

DCA (Deoxychollate citrate agar)

Principle
It is a selective and differential media used to isolate salmonella and shigella.
By adding extra bile salts it can isolate Yersinia.
Sodium deoxychollate inhibits unwanted organisms, neutral red and lactose
helps in the differentiation of the lactose fermentors from the non-lactose
fermentors.
Formula

Lab-lemco powder………………………………………..5.0g
Peptone……………………………………………………5.0g
Lactose…………………………………………………….10g
Sodium thiosulphate…………………………………….....5.4g
Sodium citrate……………………………………………..8.5g
Sodium deoxychollate…………………………………….5.0g
Neutral red………………………………………………...0.02g
Ferric citrate……………………………………………….1g
Agar……………………………………………………….12g
Distilled water……………………………………………..1 litre
PH of 7.3±0.2

Xylose Lysine Deoxycholate (XLD) Agar

Principle

It is a selective growth medium used in isolation of salmonella and shigella

species in clinical specimens. It has a PH of 7.4 leaving it with a bright pink or

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red appearance due to the indicator phenol red. Sugar fermentation lowers
the PH and the phenol red indicator registers this by changing to yellow.
Salmonella ferments the sugar xylose to produce acid while shigella colonies
cannot do this and therefore, remain red.
After exhausting the xylose supply, the salmonella colonies will decarboxylate
lysine increasing the PH once again to alkaline thus mimicking the red shigella
colonies.
Salmonella metabolises thiosulphate to produce hydrogen sulphide which
leads to the formation of colonies with black centres and allows them to be
differentiated from similarly coloured shigella colonies.
Formula
Yeast extract powder……………………………………………………...3.0g
L-lysine HCL……………………………………………………………..5.0g
Xylose…………………………………………………………………….3.8g
Lactose……………………………………………………………………7.5g
Sucrose……………………………………………………………………7.5g
Sodium deoxychollate…………………………………………………….1g
NaCl………………………………………………………………………5.0g
Sodium thiosulphate……………………………………………………...6.8g
Ferric ammonium citrate…………………………………………………0.8g
Phenol red………………………………………………………………...0.08g
Agar agar…………………………………………………………………12.5g

Enrichment media

They are liquid media that also serves to inhibit commensals in the clinical
specimen. Selenite F broth, tetrathionate broth and alkaline peptone water
are used to recover pathogens from fecal specimens or These are fluid or
liquid selective media that increases the number of pathogens by containing

Tsongo Lawrence Msc (MUK) 116

enrichment or substances that discourage the multiplication of unwanted
bacteria example selenite F, selenite U and alkaline peptone water

Selenite F broth

It is an enrichment media used for isolation of salmonella and shigella. The

selenite in the media inhibits coliform bacilli while permitting salmonella to
grow in faeces and urine before sub culturing on XLD agar.

Ingredients

Sodium hydrogen selenite (NaHSeO3)…………………………………4g

Peptone…………………………………………………………………5g
Disodium hydrogen phosphate (Na2HPO4. 12H2O)…………………..9.5g
Sodium dihydrogen phosphate (NaH2PO4. 2H2O)……………………0.5g
Distilled water………………………………………………………….1 litre

Preparation

 Dissolve the ingredients in sterile water and heat to 75-80°C for

complete dissolution.
 Boil for 10 minutes.
Note: Selenium salts ingredients are toxic, also includes volatile
derivatives e.g. hydrogen selenite and when inhaled it can induce
abnormalities in future.
 Adjust the PH to 7.1 and dispense the solution in 5ml amount in a screw
capped universal bottle.
 The selectivity of the medium diminishes if excessive amount of organic
material are inoculated.
 When inoculating food stuffs in order to isolate salmonella, pre-
enrichment broth culture medium is preferred.

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Differential/Indicator media
Certain media are designed in such a way that different bacteria can be
recognized on the basis of their colony colour.

These are media which contain indicators, dyes, metabolic substrates so that
those bacteria that utilize them appear as differently coloured colonies. Such
media are called differential media or indicator media example TCBS agar
which contain bromothymol blue to act as an indicator to differentiate
sucrose fermenting from the non-sucrose fermenting vibrios and MacConkey
which contains neutral red as an indicator to differentiate lactose fermentors
from the non-lactose fermentors.

When a particular carbohydrate is incorporated into a medium and a mixture

of bacteria inoculated on it, only that bacterium that can ferment it produces
acid. This change in pH is detected by using a pH indicator incorporated in
the medium and the bacterium that can ferment the sugar appears in a
different colour. This approach is used in MacConkey‟s agar, CLED agar, TCBS
agar, XLD agar, CVBA (Crystal violet blood agar)

MacConkey‟s agar is the most commonly used media to culture and identify
gram negative bacilli (especially enterobacteriaceae members). It contains
bile salts (selective agent), lactose (sugar), peptone and neutral red (pH
indicator), agar and water.

Those bacteria that can ferment lactose produce pink coloured colonies
where non-lactose fermenting colonies produce colourless colonies. Similarly,
Vibrio cholerae produces yellow coloured colonies on sucrose containing
TCBS medium.
Reduction of potassium tellurite to metallic tellurium by Corynebacterium
diphtheriae results in production of black coloured colonies on PT agar.

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Production of H2S by Salmonella typhi results in production of black coloured
colonies on Wilson & Blair‟s medium. Enterococcus fecalis produces black
coloured colonies on bile esculin agar due to reduction of esculin to esculetin.
Detection of hemolysis on blood agar can be considered as an indicator
property of Blood agar. BA differentiates haemolytic organisms.

MacConkey
It is a differential media with low selectivity and used for growth of
enterobacteria.
The presence of lactose and neutral red differentiates lactose fermentors from
the non lactose fermenting organisms.
The bile salt it contains inhibits the growth of non intestinal bacteria. However,
the concentration of sodium tanocholate can be adjusted to suit less torelant
organisms and omission of NaCl in the media prevents proteus from swarming

Formula for MacConkey

Peptone………………………………………………..20g/l
Lactose………………………………..………………..10g/l
Bilesalts………………………………………..……….5g/l
Neutral red…………………………………………….0.075g/l
Agar…………………………………………………...12g/l
Distilled water…………………………………………1000ml
PH normally……………………………………...........7.4 ± 0.2
 Preparation follows manufacturer‟s instructions.
 Store at 2-8°C, preferably in plastic bag to avoid loss of moisture.
 It has a shelf life of about 4 weeks (life span).

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Cystine Lactose Electrolyte Deficient (CLED)
CLED medium is a valuable non inhibitory growth medium used in isolation
and differentiation of urinary organisms. Being electrolyte deficient, it prevents
the swarming of proteus species.
Lactose fermentors produce yellow colonies on CLED agar and the non
lactose fermentors appear blue.
The medium has a PH of 7.3
Formula
Peptone………………………………………………4g/l
Lab-lemco powder…………………………………..3g/l
Tryptone……………………………………………..4g/l
Lactose………………………………………………10g/l
L-cystine……………………………………………..128mg/l
Bromothymol blue……………………………………20mg/l
Agar…………………………………………………..15g/l
Thiosulphate citrate bile sucrose agar (TCBS)
It is a selective growth medium used in the isolation of Vibrio cholerae species.
Principle
The high concentration of thiosulphate and citrate and the strong alkalinity of
the medium largely inhibit the growth of enterobacteriaceae.
Ox-bile and cholate suppress primarily Enterococci. Any coliform bacteria
which grow may not metabolise sucrose only a few sucrose positive proteus
strain can grow to form yellow vibroid like colonies.
The mixed indicator thymol blue-bromothymol blue changes its colour to
yellow when acid is formed even in this strongly alkaline medium.
Formula
Peptone………………………………………………5.0g
Yeast extract…………………………………………5.0g
Sodium citrate……………………………………….10.0g

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Sodium thiosulphate…………………………………10.0g
Ox-bile……………………………………………….5.0g
Sodium cholate………………………………………3.0g
Sucrose………………………………………………20g
Iron III citrate………………………………………..1.0g
NaCl………………………………………………….10g
Thymol blue………………………………………….0.04
Bromothymol blue…………………………………...0.04
Agar agar…………………………………………….14g
Preparation
 Follow the manufacturer‟s instructions.
 Do not autoclave
 Boil to heat
 PH 8.6±0.2
The plates are clear and green-blue.

Transport media

Clinical specimens must be transported to the laboratory immediately after

collection to prevent overgrowth of contaminating organisms or commensals.

This can be achieved by using transport media. Such media prevent drying
(desiccation) of specimen, maintain the pathogen to commensal ratio and
inhibit overgrowth of unwanted bacteria. Some of these media (Stuart‟s &
Amie‟s) are semi-solid in consistency. Addition of charcoal serves to neutralize
inhibitory factors.

Cary Blair medium and Venkatraman Ramakrishnan medium are used to

transport feces from suspected cholera patients. Sach‟s buffered glycerol
saline is used to transport feces from patients suspected to be suffering from

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bacillary dysentery. Pike‟s medium is used to transport streptococci from
throat specimens.

These are mostly semisolid media used to protect pathogens from drying or
being out grown by commensals due to delay since the patient from whom
the sample is taken is far from the laboratory,

Examples of transport media include; the cary-blair transport media which is

mainly used for enteric pathogens like Vibrio cholerae: Thioglycolate broth for
strict anaerobes. Stuart transport medium - a non-nutrient soft agar gel
containing a reducing agent to prevent oxidation, and charcoal to neutralize
certain bacterial inhibitors- for gonococci, and buffered glycerol saline for
enteric bacilli.

Venkat-Ramakrishnan (VR) medium for vibro cholerae. Amies transport

media with or without charcoal for ensuring the viability of Neisseria species
and it is a modification of Stuart‟s transport media. Alkaline peptone water PH
8.6 for Vibrio cholerae transportation.

Stuart’s Transport Media

It is a semi solid media used to maintain the viability of gonococci during their
transport and the distilled water used for preparing the media should be free
from chloride therefore, water should have passed through anion exchange
resin.

Formula
Sodium thioglycochollate…………………………………………….1g
Sodium glycerophosphate……………………………………………10g
Calcium chloride……………………………………………………...0.1g
Agar…………………………………………………………………..6g
Methylene blue………………………………………………………4ml

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Deionised water……………………………………………………...1 litre

Preparation of Stuart’s transport media

 The solids are dissolved in de-ionized water
 The PH is adjusted to 7.3 – 7.4 and add methelene blue solution
 Distribute the media in small bijour bottles
 Autoclave at 121 oC for 15 minutes, and then tighten the caps
immediately
 When the media has cooled, it should be colourless.
Anaerobic media
Anaerobic bacteria need special media for growth because they need low
oxygen content, reduced oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin
and vitamin K. Such media may also have to be reduced by physical or
chemical means. Boiling the medium serves to expel any dissolved oxygen.
Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05%
cysteine or red hot iron filings can render a medium reduced.
Robertson cooked meat that is commonly used to grow Clostridium spps,
medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient
broth.
Before use the medium must be boiled in water bath to expel any dissolved
oxygen and then sealed with sterile liquid paraffin. Thioglycollate broth
contains sodium thioglycollate, glucose, cystine, yeast extract and casein
hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential
indicator that is incorporated in the medium. Under reduced condition,
methylene blue is colourless.
Preparation and storage
Care must be taken to adjust the pH of the medium before autoclaving.
Various pH indicators that are in use include phenol red, neutral red,

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bromothymol blue, bromocresol purple. Dehydrated media are commercially
available and must be reconstituted as per manufacturers‟ recommendation.
Most culture media are sterililized by autoclaving. Certain media that contain
heat labile components like glucose, antibiotics, urea, serum, blood are not
autoclaved.
These components are filtered and may be added separately after the
medium is autoclaved. Certain highly selective media such as Wilson and
Blair‟s medium and TCBS agar need not be sterilized. It is imperative that a
representation from each lot be tested for performance and contamination
before use.
Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks.
Certain liquid media in screw capped bottles or tubes or cotton plugged can
be held at room temperature for weeks.
Broth culture media
They are fluid or liquid culture media used for many purposes e.g. a) Selenite F
broth media, peptone water.
a) Peptone water
Uses
 Used for culturing microorganisms.
 Can be used to enrich microorganisms.
 It forms a basis for carbohydrate fermentation.
 Used as a media for Vibrio culture.
 Used for indole test production.
Formula
Peptone………………………………………………….10g
Sodium chloride…………………………………………1g
Distilled water……………………………………………1 litre
Preparation
 Dissolve the ingredients in one litre of distilled water.

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 Mix well
 Put in small bijour bottles.
 Autoclave at 121°C for 15 minutes.
 Label, store in a sterile place for use.
b) Nutrient broth
 Used for preparation of other culture media
 It should be of best quality;
They are 3 types of nutrient broth
(i) Meat infusion broth which consists of watery extracts of lean meat and
to which paptone has been added.
(ii) Meat extracts broth. It is a mixture of commercial peptone and meat
extract.
(iii) Digest broth which consists of watery extract of lean meat that has
been digested by proteolytic enzyme. It is economically good for
fastidious organisms. However, cultures tend to dry out rapidly in this
broth and the proteolytic enzymes used for digesting of trypsin are
maintained at optimum temperature of 37°C (and PH of enzyme
activity).
c) Brain heart infusion broth
 It is used for blood culture and contains sodium polyanthol sulphonate.
 It acts as a good anticoagulant for blood.
 It is a non inhibitory media.
d) Liquoid broth media
It is used for blood culture and it contains; liquid called polyanthol sodium
sulphonate which is obtained from kodakor or kock. It acts as a good
anticoagulant and it is non-inhibitory.

Formula

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Liquido solution 5% in 0.85% sodium chloride in water…………………….10mls
Nutrient broth…………………………………………………………………1 litre
Preparation
 Mix the ingredients and adjust the PH to 7.6
 Distribute in bijour bottles and autoclave at 121°C for 15 minutes.
e) Cooked meat medium broth

It is a nutritious broth medium originally known as Robertson‟s heart medium.

 It is used as an enrichment medium for both anaerobic and non-

anaerobic organisms.
 It is used for maintenance of stock culture for anaerobes.
 It enhances the fast growth of organisms.
 Helps in differentiation of species of bacteria by reddening of meat as
protelitic activity (blackening).
Formula
Heart muscle…………………………………………………….454g
Peptone…………………………………………………………10g
Lab-lemco powder………………………………………………10g
Sodium chloride ………………………………………………..5.0g
Glucose…………………………………………………………2.0g
Preparation
 Use small test tubes and put in them 1 gram of cooked meat medium
granules.
 Add 10mls of distilled water.
 Mix and sterilize at 121°C for 15 minutes.
 Allow to set when cool.
 Tighten the caps.
PH is 7.0-7.4
Uses of common liquid media

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 Used for demonstration of carbohydrate fermentation.
 Used for enrichment of few microorganisms example selenite F, selenite U,
alkaline peptone water.
 Used for demonstration of gas production by using Durham‟s tube.
 Used for demonstration of motility.
 Used for indole production.
 Used for cultivation of organisms for agglutination test.
 Used for emulsification of organisms for sensitivity testing.
Preparation of carbohydrate sugars

Fermentation is an anaerobic process by which carbohydrates are broken

down to produce acid or acid and gas. Some organisms have the ability to
ferment certain carbohydrates which is used in their identification.

Formula

Peptone, Sodium chloride, Meat extract, an indicator Phenol red/Andrade‟s

indicator.
Carbohydrate is added in the final concentration of 0.5-1.0% water.
 The media is then dispersed in tubes or bottles containing small Durham‟s
tubes which must be inverted and completely filled with the medium.
 The indicator in the medium will reveal the production of acid and the
inverted Durham‟s tube will trap any bubbles of gas that may be formed.

Durham’s tube
completely filled with
medium Durham’s tube
showing bubble of
gas

Carbohydrate
media

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11.5 Identification Media

Tripple Sugar Iron (TSI) AGAR

Purpose

It is a composite media that is for identification of enterobacteria by either

sugar fermentation or hydrogen sulphide production.
Principle
It determines the ability of an organism to attack specific carbohydrates in
the basal media and, also to determine if hydrogen sulphide is produced. The
media is composed of three sugars; lactose, sucrose and dextrose (glucose).
The alkaline in the slant and acid in the butt indicates fermentation of only
glucose. Acid in the butt and slant indicates the fermentation of the three
sugars. Alkaline in the butt and slant indicates no fermentation of any sugar
but only peptone is utilized.
Formula
Lab-lemco powder………………………………………….3g
Yeast extract………………………………………………..3g
Peptone……………………………………………………..20g
Sodium chloride…………………………………………….5g
Lactose………………………………………………………10g
Sucrose………………………………………………………10g
Glucose/dextrose…………………………………….………1g
Ferric citrate………………………………………………….0.3g
Sodium thiosulphate…………………………………………0.3g
Phenol red in plenty or any amount
Distilled water………………………………………………..1 litre
PH ±2
Preparation
 Follow the manufacturer‟s instructions.

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 Aliquot the media in tubes and autoclave at 121°C for 15 minutes.
 The tubes containing media should be sloped after autoclaving and the
length of the slopes should be the same as that of the butt.
The two reaction chambers are created and incubation will also be under
two conditions both aerobic and anaerobic conditions.
When inoculating, the wire should not reach at the bottom since this may
lead to the pulling of the media upwards resulting into space which may be
mistaken as displacement by gas thus giving false positive results of gas
production.

Interpretation of TSI agar

 Bacteria that ferment any of the three sugars in the medium will produce
byproducts.

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 These byproducts are usually acids, which will change the color of the red
pH-sensitive dye (phenol red) to a yellow color.
 Position of the color change distinguishes the acid production associated
with glucose fermentation from the acidic byproducts of lactose or sucrose
fermentation. Many bacteria that can ferment sugars in the anaerobic
butt of the tube are enterobacteria.
 Some bacteria utilize thiosulfate anion as a terminal electron acceptor,
reducing it to sulfide. If this occurs, the newly-formed hydrogen sulfide (H2S)
reacts with ferrous sulfate in the medium to form ferrous sulfide, which is
visible as a black precipitate. Examples of sulfide-producing bacteria
include Salmonella, Proteus, Citrobacter and Edwardsiella species. The
blackening of the medium is almost always observed in the butt (bottom)
of the medium.
 All lactose fermenters result in yellow slant/yellow butt (acid/acid reaction),
whereas non-lactose fermenters may result in pink/yellow or yellow/yellow
(if sucrose is fermented). Blackening of the butt due to H 2S production may
mask the acid reaction (yellow) in the butt. Salmonella enterica serovar
Typhi may result in blackening of the medium at the interface of butt and
slant.
 Under anaerobic conditions (as occur toward the bottom of the tube)
some bacteria use H+ as an electron acceptor and reduce it to hydrogen
gas. This is not very soluble and may accumulate as bubbles along the
inoculation track, between the agar and the glass, or in the fluid which
accumulates at the bottom of the slant. Hydrogen production may lift the
agar from the butt of the tube or fracture the agar. Carbon dioxide, if
produced, may not show as bubbles because it is far more soluble in the
medium.

Slant Butt Colour Utilisation

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 Alkaline Acid Red/yellow Only glucose is fermented and
peptone
Acid Acid Yellow/yellow Glucose, lactose and sucrose are
fermented.
Alkaline Alkaline Red/red No fermentation of sugars and
only peptone is utilized.

Method of Innoculating

 The medium is innoculated by streaking the slant in Zig zag manner and
stabbing the butt with pure culture of the test organism.
 Cotton wool plug is best suitable for capping the TSI tubes and this ensures
that sufficient atmospheric oxygen is readily available for the metabolism
of peptone at the surface of the slant.
 The tubes are incubated for 18-24 hours and results read and recorded.
 Since the indicator used is phenol red, yellow in the media indicates acid
reaction and red indicates an alkaline reaction. The test also shows
whether the organism is capable of producing hydrogen sulphide by
blackening of the medium.
 It also shows whether the organism produces gas or not which is indicated
by the presence of gas bubbles in the butt.

Typical reactions of some selected Bacteria

BACTERIUM SLANT BUTT H2S COMMENTS

Shigella Causes food infection
R Y
dysenteriae dysentery
Salmonella
R YG + Causes food poisoning
typhimurium
Salmonella typhi R Y + Causes typhoid fever
Aerobacter Similar to Klebsiella,but
Y YG
aerogenes nonrespiratory
Escherichia coli Y YG Most common of GIflora
one of "paracolon" group
Citrobacter freundii Y YG +
(non-pathogenic)

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 Causes GU infections, highly
Proteus vulgaris Y YG +
motile
Klebsiella R or Pneumonia in debilitated
Y
pneumoniae YG patients (nosocomial)
Pseudomonas GI inhabitant, causes wound,
R R
aeruginosa GU infections
Alcaligenes GI inhabitant, opportunistic
R R
faecalis pathogen of GU

Quality Control Organisms

Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853
Proteus vulgaris ATCC 13315
ATCC- American Type collection Culture
Kligler Iron Agar
Kligler Iron Agar is used for the identification of enterobacteria by the rapid
detection of lactose and glucose fermentation (with or without gas
production), as well as the production of hydrogen sulfide.
Principles
The fermentations of lactose and glucose, used to differentiate species of
enterobacteria, result in acidification which makes phenol red (pH indicator)
turn yellow.
Microorganisms not fermenting lactose (Salmonella or Shigella) initially
product a yellow slant due to the acidification resulting from glucose present
in small quantities. When the glucose has been exhausted in the aerobic
portion of the slant, the reaction becomes basic by oxidation of the acids
Produced, resulting in the phenomenon of a red color on the surface of the
medium. This color does not appear in depth in the butt, where the color
remains yellow.

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Bacteria fermenting lactose and glucose make the slant and the butt turn
yellow because of the production of large quantities of acid. This is sufficient
to maintain an acid pH on the surface.
Microorganisms which ferment neither of these two sugars do not change the
color of the medium.
The production of H2S is revealed in the base of the medium by the
appearance of black iron sulfide, due to the reduction of thiosulfate in the
presence of ferric citrate.
The production of gas (H2, CO2) resulting from sugar fermentations is shown
by the appearance of gas bubbles or by a fragmentation of the agar.
Preparation
Suspend 58.0 g of dehydrated medium (BK034) in 1 liter of distilled or
deionized water.
Slowly bring to boiling, stirring with constant agitation until complete
dissolution.
Dispense in tubes, Sterilize in an autoclave at 121°C for 15 minutes.
Incline the tubes so as to obtain a butt 3 cm in height and an oblique slant.
Instruction for use
Using a suspected colony taken from a selective isolation medium, inoculate
the butt by stabbing in the center and the inclined surface by closely spaced
streaks.
Pure cultures taken from the center of well isolated colonies must be used to
avoid cross reactions which will make identification impossible.
Incubate at 37°C for 24 hours with caps loosely screwed to favor gas
exchanges.

Results
Kligler's medium supplies four types of information:
(1) Glucose fermentation

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Red butt: glucose not fermented, yellow butt: glucose fermented
(2) Lactose fermentation
Red slant: lactose not fermented, yellow slant: lactose fermented
(3) Gas production
Production of gas bubbles in the butt of the tube,
(4) Formation of H2S
Formation of a black color between the butt and the slant or along the
inoculation stab

Typical composition
(Can be adjusted to obtain optimal performance)
For 1 liter of medium:
Tryptone.........................................................................................20.0 g
Yeast extracts ....................................................................................3.0 g
Meat extracts .....................................................................................3.0 g
Glucose............................................................................................1.0 g
Lactose ..........................................................................................10.0 g
Sodium chloride ...............................................................................5.0 g
Sodium thiosulfate ...........................................................................0.5 g
Ferric ammonium citrate ..................................................................0.5 g
Phenol red...................................................................................25.0 mg
Bacteriological agar .......................................................................15.0 g
PH of the ready-to-use medium at 25°C: 7.4 ± 0.2
Lysine Iron Agar
Lysine Iron Agar is used for the differentiation of microorganisms on the basis
of lysine decarboxylase and hydrogen sulfide production.
Principles
Enzymatic Digest of Gelatin provides carbon, nitrogen, and amino acids
required for good growth of a wide variety of organisms. Yeast Extract

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provides vitamins and cofactors required for growth, and additional sources
of nitrogen and carbon. Dextrose is an energy source. L-Lysine is the substrate
used to detect lysine decarboxylase and lysine deaminase enzymes. Ferric
Ammonium Citrate is an indicator of hydrogen sulfide production. Sodium
Thiosulfate is added as a source of inorganic sulfur. Bromcresol Purple, a pH
indicator, is yellow at or below pH 5.2 and purple at or above pH 6.8. Agar is
the solidifying agent.
Formula / Liter
Enzymatic Digest of Gelatin ...................................................... 5 g
Yeast Extract.............................................................................. 3 g
Dextrose ..................................................................................... 1 g
L-Lysine..................................................................................... 10 g
Ferric Ammonium Citrate ......................................................... 0.5 g
Sodium Thiosulfate.................................................................... 0.04 g
Bromcresol Purple ...................................................................... 0.02 g
Agar ........................................................................................... *13.5 g
Final pH: 6.7 ± 0.2 at 25°C
Formula may be adjusted and/or supplemented as required to meet
performance specifications.

Adjustment Of pH of the Media

It is essential that all media are adjusted to the correct PH. This is performed
using a PH meter. However, manual methods can be used example
Colorimetric method and lovibond comparator method. Colorimetric
method, an indicator is added to the medium and to a standard buffer
solution. The medium is adjusted until the colours are matched.
In Lovibond comparator, the sample of medium plus indicator (phenol red) is
matched against a permanent coloured glass standard.

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7.6 Quality Control of culture media

Physical Appearance

If the medium is stored for an excessively long time under adverse conditions
or has been improperly prepared, the following signs may develop and these
should be documented.

Presence of turbidity or a precipitate indicates that some constituent has

come out of the solution.
Colours darker than normal may indicate overcooking of sugar containing
media, incorrect pH or incorrect mixture of ingredients.
Colour lighter than normal may also indicate incorrect mixture of
ingredients or a wrong pH.
Prolonged storage of medium after pouring in plates causes its
dehydration and makes it unfit for use. Dehydration of the medium can
be reduced by preparing only required number of plates of media and
storing them by sealing plates in plastic bags.

Sterility testing

A few media are used without terminal sterilization, but these are exceptions;
most media must be sterile when they are inoculated. Each batch of medium,
whether prepared in the laboratory or received from a commercial source,
should be sampled for sterility.

This is best done by removing 1-5% of the batch and placing it in a

bacteriologic incubator at 35° C for 48 hours.
If contaminants appear in the medium as a result of inadequate sterilization, a
new lot should be obtained. Those containers that are used for sterility testing
should be discarded at the completion of the test, since they are unsuitable

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for inoculation because of the dehydration that occurs after up to 48 hours in
the incubator.
Growth
Determine the ability of the medium to support the growth of suspected
organisms by inoculating the medium with a typical stock culture isolate.
Performance of Plated Media
Samples of plates from each batch are selected for performance testing and
are inoculated with the appropriate stock cultures.
For each type of medium, at least two or three microorganisms having growth
characteristics with „positive‟ and „negative‟ results for the medium should be
used.
The size of inoculum and method of inoculating the test plates must be
standardized as closely as possible.
In general, control organisms should be selected from an actively growing
broth culture and a standard loopful of culture seeded directly onto the test
medium, which is then streaked so as to obtain isolated colonies.
After appropriate incubation, the results of the performance test are
recorded. The medium is released for use in the clinical laboratory only if the
results indicate satisfactory performance. In initiating a quality control
programme, one must establish some priorities, such as beginning by testing
those media that are most likely to demonstrate deficiencies. Top priority
should be given to blood agar, chocolate agar and Thayer Martin agar
media. Secondary priority should be accorded to selective enteric media
such as MacConkey agar, EMB, XLD and bile salt agars.

7.7 Storage of Culture Media

 Store the media in a cool dark place with bottles tightly capped.
 Shelf life can go up to years provided there is no change in the volume or
appearance of the media to suggest contamination.

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 Plate media should be stored at 2-8°C in sealed plastic bags to prevent
drying. Before use, the surfaces of the plates should be dry.
 Plates must not be exposed to sunlight.
 Unused plates should not be left on the bench for overnight to prevent
contamination and deterioration.
 Most slopes and fluid media can be stored at room temperature between
20-28°C.
 New batches of media should be dated and stored separately from the
earlier batches.
Preparation of Swabs (Charcoal Swabs)

 The swabs of absorbent cotton wool are boiled for five minutes in
phosphate buffer of 0.7 moles/litre at PH 7.4
 Shake off excess moisture and immerse in 1% water suspension of finaly
powdered charcoal BDH (British Drug Health activated charcoal).
 Shake off excess moisture and place swabs in test tubes.
 Plug the tube with cotton wool and sterilize in a hot air oven at 160°C for 90
minutes.
Note: Commercially prepared treated charcoal is available.

 Alkaline peptone water PH 8.6 for transportation of Vibrio cholerae.

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7.8 Methods of Specimen Inoculation

1) Stabbing method
It is a culture method made by inserting an inoculating needle or wire with the
inoculum down the centre of a solid media contained in a test tube mostly
used for setting biochemical tests.

2) Slanting culture method

Is a method for maintaining bacteria in which the inoculum is streaked on the
surface of the agar that has solidified in inclined glass tubes forming slopes or
slants?

3) Streaking method
This is the method of applying cultures to a solid medium where a loop of the
medium is dragged along the surface of the solid medium from where
colonies are at the point of the streak.

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 Surface streaking using a wire loop

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4) Pour plating method
This is where the test organism suspension is poured onto the media for
growth.
Principle
The bacteria are mixed evenly, distributed and separated throughout the
liquid. The method agar is then poured into an empty plate and allowed to
solidify. After inoculation discrete bacterial colonies can then be formed
growing both on the agar media and in agar then on the isolated colonies
can be a separately picked of the isolation plate and transfer to new sterile
medium.

5) Drop plating

Drops of the test organisms in suspension are put on culture media for growth.

6) Rolling or spreading method

This is where a loopful of the inoculum is rolled on a solid medium or broth

medium for their growth.

7.9 System of Bacterial Identification

1) Cultural characters
a) Colonial appearance on a solid media. Here we consider the size or
diameter of the colony, the outline of the colony or the nature of the
margins e.g. having entire margins.
The elevation of the colony e.g. convex colony, the translucent of the
colony either clear or opaque, shape of the colony either round or
irregular. Texture of the colony either smooth or dull is also described.

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 Ability or inability to grow on the media containing inhibitory substances
e.g. streptococcus pyogenes which is β-haemolytic does not grow on
MacConkey while staphylococcus aureus grows on it.
b) Change in the media α and β-haemolysis on a BA and lactose
fermentation on MacConkey.
c) Growth conditions of a particular organism. Here look for oxygen,
carbondioxide and temperature growth.
d) Pigmentation. Some bacteria have golden yellow appearance like
staphylococcus aureus or typical yellow like micrococcus.
e) Size. Small ranging from 1.5-2.0mm like in staphylococcus species. Pin-head
colonies ranging from 0.5-1mm e.g. streptococcus and Enterococcus
species. Large colonies ranging from 3-5mm.
f) Shape. Colonies are either round or irregular.
g) Lactose fermentation. Can be seen on MacConkey and CLED. Red-pink
and yellow colonies lactose fermentors on MacConkey and CLED while
white or grey non-lactose fermentors.
h) Haemolysis only on a BA. Some bacteria have the ability to produce
haemolysin. Clear zones around the colonies on a BA is a sign of β-
haemolysis e.g. streptococcus pyogenes.
Green zones around the colonies on a BA is a sign of α-haemolysis e.g.
streptococcus pneumoniae.
i) Margin. Can be in different forms;
Entire margin of colonies e.g. in staphylococcus and E. coli
Serrated or crenated margins of colonies as commonly seen in bacillus
Undulating margins as in pseudomonas species
j) Elevation of the bacterial colonies
Can be flat, raised, low convex, dome-shaped, umbonate, convex
papillate surface

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k) Swarming. Some bacteria have the ability to swarm due to being highly
motile e.g. proteus species. This can be reduced by;
 Increasing the concentration of agar-agar.
 Reducing the concentration of electrolytes.
 Grow the organism on MacConkey without salt.

2) Morphology and staining reactions of individual organisms

 Gram stain: this gives the gram reaction of an organism, the size, shape,
grouping and arrangement of endospores of the organism.
 ZN: sputum smears that have been stained using ZN technique will show
presence of AFBs which is a presumptive diagnosis of tubercle bacilli.
 Unstained wet preparation that is examined under dark ground
microscopy which reveals the morphology of delicate organisms such as
spirochaetes e.g. treponema pallidum causing syphilis.
3) Biochemical reactions This can differentiate those species that can not be
differentiated by morphology and cultural characters.
 Sugar fermentation test. Some organisms ferment sugars to acids and gas
or to acid without gas.
 End product detection test e.g. indole and hydrogen sulphide production.

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 Possession of certain enzyme activities e.g. oxidase, catalase, urease,
coagulase, gelatins, lactinase.
 Gas-liquid chromatograph for the analysis of metabolic end-products, e.g
fatty acids and alcohols produced by anaerobolic bacteria.
4) Molecular differentiation By gene characterization and gene spacing e.g.
polymerase chain reaction (PCR).
5) Antibiotic sensitivity tests. Organisms are tested for their ability to grow in
the presence of different concentrations of antimicrobial agents by agar or
broth dilution and disc diffusion test where zone sizes are measured, It helps as
a guide to therapy, Epidemiological makeup in tracing hospital crosses
infection, In identification of organisms e.g. bacitracin for streptococcus
pyogenes and optochin for streptococcus pneumoniae.
5) Antigenic characters

i. Antibody reaction. Species and types of bacteria can be identified by

specific antibody reactions or by polyclonal and monoclonal
antibodies e.g. shigella dysenteriae polyclonal (1-7), polyclonal 2 (8-
10), monoclonal antibodies.

ii. Counter immunoelectrophoresis (CIE). This is used for detection of

pneumococcal and certain antigens. Antigens and antibodies are put
in separate wells in agar and subjected to an electric current. An
antigen which is negatively charged moves towards the anode and
antibody which is positively charged moves towards the cathode.
Where the two meet, if the antigen is specific to the antibody a line of
precipitation is formed e.g. in streptococcus pneumoniae.

iii. Enzyme Linked Immunosorbent Assay (ELISA)

This can be used to look for antibodies produced against bacteria,
viruses or parasites and to also detect for antigens.

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6) Typing of bacteria

This is done for epidemiological purposes in tracing the sources and modes of
spread of infections in a community.
Methods of typing

They are mainly 2 methods; biotyping and serotyping methods. Special

biochemicals or serological tests may be used to distinguish particular
biotypes or serotypes that show variation in metabolic behaviour or antigenic
structure.

7) Animal pathogenicity and toxigenicity

Diagnosis of tuberculosis and other diseases used to be done by this method

but it is being replaced by culture method. Animal test for demonstrating
bacteria toxins have also been superseded or replaced by invitro tests.
However, animals are models for most disease investigations where suspected
specimen is innoculated either intraperitoneally or intradermally into the
animal. The animal is observed for clinical presentation of the disease.

8) Gene Probes

These are cloned fragments of DNA molecules that recognize

complementary sequences within microorganisms and binding is detected by
targeting the DNA with a radioactive reagent that can be developed to give
a coloured reaction.

7.10 Common Methods for isolation of pure culture

 Pure culture of microorganisms that form discrete colonies on solid media,

e.g., yeasts, mostbacteria, many other microfungi, and unicellular
microalgae, may be most commonly obtained by plating methods such
as streak plate method, pour plate method and spread plate method.

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Special Methods for Isolation on Pure Culture
1. Single Cell Isolation methods

 An individual cell of the required kind is picked out by this method from
the mixed culture and is permitted to grow. The following two methods are
in use.

(i) Capillary pipette method

 Several small drops of a suitably diluted culture medium are put on a

sterile glass-coverslip by a sterile pipette drawn to a capillary. One then
examines each drop under the microscope until one finds such a drop,
which contains only one microorganism. This drop is removed with a sterile
capillary pipette to fresh medium. The individual microorganism present in
the drop starts multiplying to yield a pure culture.

(ii) Micromanipulator method

 Micromanipulators have been built, which permit one to pick out a single
cell from a mixed culture. This instrument is used in conjunction with a
microscope to pick a single cell (particularly bacterial cell) from a
hanging drop preparation. The micro-manipulator has micrometer
adjustments by means of which its micropipette can be moved right and
left, forward, and backward, and up and down. A series of hanging drops
of a diluted culture are placed on a special sterile coverslip by a
micropipette.

2. Enrichment Culture Method

 Generally, it is used to isolate those microorganisms, which are present in
relatively small numbers or that have slow growth rates compared to the
other species present in the mixed culture. The enrichment culture strategy

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 provides a specially designed cultural environment by incorporating a
specific nutrient in the medium and by modifying the physical conditions
of the incubation. The medium of known composition and specific
condition of incubation favors the growth of desired microorganisms but, is
unsuitable for the growth of other types of microorganisms.

Proof of Purity of Cultures

 Assuming that one has isolated a pure culture, how does one establish
that it is pure? A pure culture is one in which the cells are all of one kind,
i.e., demonstrate "likeness". Hence, the proof of purity of cultures consists
of demonstrating the "likeness" of microorganisms in the culture. It is based
on certain criteria as follows:

 .The microorganisms look alike microscopically and stain in the same

fashion.

 When plated, all the colonies formed look alike.

 Streaks, stabs, etc. are uniform.

 Several isolated colonies perform identically, i.e., ferment the same

sugars, and so on.

Maintenance and Preservation of Pure Cultures

Subculturing, refrigeration, paraffin method, cryopreservation, and

lyophilization (freeze drying).

Refrigeration
Pure cultures can be successfully stored at 0-4°C either in refrigerators or in
cold-rooms. This method is applied for short duration (2-3 weeks for

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 bacteria and 3-4 months for fungi) because the metabolic activities of the
microorganisms are greatly slowed down but not stopped. Thus their
growth continues slowly, nutrients are utilized and waste products released
in medium. This results in, finally, the death of the microbes after sometime.
Paraffin Method
 This is a simple and most economical method of maintaining pure cultures
of bacteria and fungi. In this method, sterile liquid paraffin in poured over
the slant (slope) of culture and stored upright at room temperature. The
layer of paraffin ensures anaerobic conditions and prevents dehydration
of the medium. This condition helps microorganisms or pure culture to
remain in a dormant state and, therefore, the culture is preserved for
several years.
 Cryopreservation
Cryopreservation (i.e., freezing in liquid nitrogen at -196°C) helps survival of
pure cultures for long storage times. In this method, the microorganisms of
culture are rapidly frozen in liquid nitrogen at -196°C in the presence of
stabilizing agents such as glycerol that prevent the formation of ice
crystals and promote cell survival.
Lyophilization (Freeze-Drying)
 In this method, the culture is rapidly frozen at a very low temperature (-
70°C) and then dehydrated by vacuum. Under these conditions, the
microbial cells are dehydrated and their metabolic activities are stopped;
as a result, the microbes go into dormant state and retain viability for
years. Lyophilized or freeze-dried pure cultures and then sealed and
stored in the dark at 4°C in refrigerators. Freeze-drying method is the most
frequently used technique by culture collection centers.

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 CHAPTER EIGHT: ANAEROBIASIS

8.1 Culturing of Anaerobes

Anaerobic atmosphere is essential for the growth of strict anaerobes such as

clostridium species, bacteroids and anaerobic streptococci.
Anaerobic incubation also helps to differentiate pathogens and also isolate
facultative anaerobes from specimens containing Commensals e.g.
streptococcus pyogenes from throat swabs.
The haemolytic reaction of β-haemolytic streptococci is more pronounced
following anaerobic incubation.
There are several methods of obtaining anaerobic conditions (anaerobiasis)
growing microorganisms under anaerobic conditions example
1. Anaerobic jar with hydrogen from cylinders
2. Anaerobic jar with gas generating kits
3. Using sodium dithionate agar (in lids of culture plates)
4. By using reducing agents in culture media such as heated iron strip, sodium
thioglycolate or cysteine
5. Using meat granules in the medium to act as oxygen scavengers
maintaining the anaerobic status in the bottom of tube.
6. Using copper coated steal wool to remove oxygen.

Anaerobic jar using hydrogen from cylinders

Anaerobic conditions are obtained by removing most of the air from the
anaerobic jar and replacing it with hydrogen preferably hydrogen mixed with
nitrogen and carbondioxide.
In the presence if a catalyst the hydrogen reacts with the remaining oxygen
to form water.

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The commonest catalyst used is palladium. The catalyst is able to work if it is
dry and not inactivated by hydrogen sulphide or other products of bacterial
mechanism.
After use, the catalyst must be dried in a hot air oven and inspected for
damage to avoid any explosions brought about by ignition of the catalyst
powders linking into the jar.
The risk can be avoided by using low temperature catalysts that are special.

To check that anaerobiasis is obtained

This is done by use of chemical indicators preferably bacterial indicators. The
chemical indicators include; Lucas semi-solid thioglychollate methylene blue
which is long lasting and in the absence of oxygen, the indicator is colourless
and in the presence of oxygen it is blue.
Preparation of Lucas semi-solid indicator
 Mix 12 drops of 9% v/v thioglychollate acid with 5mls of a 2% v/v sodium
tetraborate solution in a bottle.
 Add 2 drops of 0.02% w/v phenol red solution; the colour should be pale
pink.
 Add 10mls of 0.01% w/v methylene blue solution and 10mls of previously
melted watery agar of 0.05% w/v.
 Mix well and distribute in small tubes.
 Heat in a container of boiling water until the indicator is colourless.
 Seal each tube. For use, open the tube and attach carefully to the side
arm of the anaerobic jar.
Biological Indicators

A suitable bacterial indicator is pseudomonas aeruginosa which is a strict

aerobe and when cultured in Simon‟s citrate agar, growth produces an
alkaline reaction which changes the colour of the media from green to blue.

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 In an anaerobic atmosphere, no growth occurs and therefore the media
remains green.

Method of Use

Inlet valve

Outlet valve

Catalyst Tubing attached to

a low pressure
source of hydrogen
Lucas semi
solid indicators

Control
Culture plates
culture
Wash bottle (to see the
flow of hydrogen

 Place the inoculated plates inverted in the anaerobic jar and replace the
lid in position to close the inlet valve.
 Connect the outlet valve to the vacuum pump and suck out the air until
the manometer indicates that there is a negative pressure in the jar of
about 30-40mmHg.
 Close the valve and discard the pump.
 Attach the inlet valve to a low pressure source of hydrogen or if available
to the supply containing hydrogen, nitrogen and carbondioxide.
 Open the inlet valve to allow gas to enter fast, the gas enters rapidly and
then slow as hydrogen combines most of water.
 This is followed by entry of more gas to replace the gases that have been
combined.

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 Close the inlet valve, disconnect the jar and place it in an incubator set at
37°C.
 After incubation and before opening the jar checks that the chemical
indicator is colourless.
 Open the jar by unscrewing of the valves by allowing the air to enter and
remove the lid.
 Examine the biological indicator pseudomonas citrate agar culture for
growth and change of colour of the media.
 If it is blue and growth has occurred then anaerobic conditions have been
obtained. Culture plates must be re-incubated anaerobically in a different
anaerobic jar. The catalyst of the fault jar must be checked and the jar
examined for leakage.

Care and Maintenance of Anaerobic jars

 Besides ensuring that the catalyst is dry and fully activated, the Lucas semi-
solid methylene blue indicator should be checked for any drying of the
medium.
 Anaerobic jar should be kept clean when not in use and stored in a dry
place.
 The washers of the rim of the jar should be inspected regularly for wear
including the outlet and inlet seals. If the anaerobic conditions are not
being obtained and the catalyst is not faulty, check the jar for leakage.

Anaerobic jar using gas generating kits

 In this system, the hydrogen required combines with free oxygen in the
anaerobic jar which is slowly released from chemicals (NaHO 3) contained
in a foil envelope.
 The chemicals are activated by the addition of water and a slow
temperature catalyst is used to bring about the reaction and the indicator
is enclosed to bring anaerobiasis and carbondioxide will be released.

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Anaerobic jar using gas generated from candles

 This is provided by candles placed in an anaerobic jar after being lit to

provide a 5-10% Carbon dioxide environment.

 An alternative 10% carbon dioxide atmosphere in a jar of 3 litre capacity

can be obtained by mixing 0.7g of Sodium bicarbonate with 1.7g tartaric
acid or with 2.4g citric acid. Remember to immediately before closing the
jar moisten the chemicals with water.

CHAPTER NINE: SPECIMEN EXAMINATION

9.1 Laboratory Reception of Microbiological Specimens

 If pathogens are to be isolated successfully, the type of specimen, its

collection, time and method of its dispatch to the laboratory must be
correct.

 Adequate information about the patient‟s condition and antimicrobial

treatment must also be sent with the specimen.

 The correct type of specimen to collect will depend on the pathogens to

be isolated e.g a cervical swab not a vaginal swab is required for the most
successful isolation of Neisseria gonorrhoeae from a woman or sputum not
saliva is essential for isolation of respiratory pathogens.

 Specimens such as urine and sputum are best collected soon after a
patient wakes up. This is the best time for collection because the organisms
will have had opportunity to multiply over several hours. Blood for culture is
best collected when the patient‟s body temperature begin to rise. The
time of collection for most other specimens will depend on the condition of
the patient and the time agreed between the medical, nursing and
laboratory staff for the delivery of specimens to the laboratory.

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 Every effort must be made to collect specimens for microbiological
investigation before antimicrobial treatment is started.

 The laboratory should issue written instructions (SOPs) to all those

responsible for collecting specimens including the staff of the wards,
outpatient clinics and health centres on how to collect the specimens.

 Use a collection technique that will ensure specimen containers only those
organisms from the site where it was collected.

 Strictly sterile (aseptic) procedure is essential when collecting from sites

that are normally sterile e.g. the collection of blood, CSF or effusions. This is
necessary to avoid contamination of these specimens and to protect the
patient.

 Avoid contaminating discharges or ulcer materials with the skin

commensals. The swabs used to collect the specimen must be sterile and
the absorbent cotton wool which the swabs are made must be free from
antibacterial substances.

 Collect bacteriological specimens in sterile, leakproof, dry containers free

from all traces of disinfectants. Containers must be clean but need not to
be sterile for the collection of faeces and sputum. To avoid breakages, the
containers made from autoclavable plastic should be used whenever
possible provided they are leakproof.

 Containers given to patients must be easy for them to use. Patients should
be instructed in aseptic collection of specimens and should be asked to
avoid contaminating outside of containers. If contamination occurs, the
outside of the containers must be wiped with a tissue or a cloth soaked in a
disinfectant before the specimen is sent to the laboratory.

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 Those responsible for collecting specimens should report any abnormal
features such as cloudiness in the specimen, any abnormal colouration or
the presence of pus, blood, mucous and parasites. If the specimen is clear,
it should be indicated.

 Each specimen must be clearly labeled with the date, time of collection,
the patient‟s name or identification number, ward or health centre, tests
required to be done, age, and sex. Slides with one end frosted should be
used for making smears so that a lead pencil can be used to label them.

 Each specimen must be accompanied with a request form, which is well

filled with the entire patient‟s information.

(i) The patient‟s name or identification number, age, sex, ward or health
centre.
(ii) Type of the specimen, date and time of its collection.
(iii) Investigations required e.g. Gram stain, ZN, Culture.
(iv) Clinical notes giving the patient‟s illness, suspected diagnosis and any
antimicrobial treatment that has been started at home and in hospital.
(v) Signature and the name of the requesting doctor or person.

 Those delivering, receiving and examining specimens with dangerous

pathogens must be informed. If a specimen is likely to contain a highly
infectious organism, such specimen should be labeled „High risk‟ and
whenever possible carrying a warning symbol or put on such specimens a
warning symbol such as a red dot, star or a triangle which is immediately
recognized as meaning that the specimen is dangerous and must be
handled with extra care.

Specimens that should be marked as „High risk‟ include;

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 Sputum likely to contain mycobacterium tuberculosis.
 Faecal specimens that may contain Vibrio cholerae or salmonella typhi.
 Fluids from ulcers that may contain anthrax bacilli or treponemes.
 Specimens from patients with suspected hepatitis, HIV or AIDS, viral
haemorrhagic fever and plague.
 Immediately after collection, high-risk specimens must be sealed inside
a plastic bag or in a container with tight fitting lid. The request form must
not be placed in the bag or in the container with the specimen.

 Keep register of all specimens received at the reception; record all the
details like names, identification number, wards or health centres of the
patients, type of specimen, investigation required, date and the time of
collection. Computers, counter books can be used for this purpose.

 Specimens for microbiological investigations should be delivered in the

laboratory as soon as possible.

 When a delay in delivery is unavoidable, a suitable chemical

preservative or culture transport media must be used. Thus prevents
organisms from drying due to enzyme reaction, change of PH or lack of
essential nutrients.

A transport medium is usually needed to preserve anaerobes

 Amies transport medium is mainly used on swab specimens especially for

Neisseria gonorrhoeae. It is a modification of Stuart‟s transport media.
Example of preservatives include; boric acid added to urine,
cetylpyridinium chloride-sodium chloride added to sputum for isolation of
T.B, cary-blair medium is used as a transport medium for faeces that may
contain salmonella, shigella, Vibrio species and campylobacter.
 Refrigeration at 4-10°C can help to preserve cells and reduce the
multiplication of commensals in unpreserved specimens. Specimens for

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 isolation of Haemophilus species, streptococcus pneumoniae and
Neisseria species however must be refrigerated because coldness kills
these pathogens.
 Smears collected by a ward staff or outpatient clinics must be placed in a
safe, dry box to protect from dust, ants and the laboratory should provide
wards with petridishes or other containers in which to place and transport
dry glass preparations.

Referral specimens
 Specimens for referral (dispatch) to microbiology laboratory must be kept
well and safely. If specimens are to be mailed, the regulations regarding
the sending of pathological specimens through the post should be
obtained from the post office and followed exactly.

 When transporting or dispatching microbiological specimens for referral;

 Keep register for the specimens referred or dispatched. Record the name,
number, ward, or health centre of the patient, type of specimen,
investigations required, date of referral and method of sending the
specimen e.g. emailing, hand-delivery and by post office.
 Ensure that specimen containers are free from cracks and cap the
leakproof seal around the container. Cap with adhesive tape to prevent
loosening and leakage in transit.
 Use sufficient packing material to protect a specimen especially if a
container is a glass tube or bottle. In this case, use a plastic container to
act as a secondary container. Place the packed container in a strong
protective box or tin and seal completely. If the specimen is a fluid, use
sufficient absorbent material to absorb any material in cases of any
breakage or leakage.
 Mark all specimens that may contain highly infectious organisms „High risk‟.

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 Dispatch smear slides in a plastic slide container or use a slide carrying box
or envelope.
 Label specimens that are referred by mail; fragile with care, pathological
specimen and address the package clearly.

 If the specimen is likely to deteriorate unless kept cool, transport it in an

insulated container such as a polysterene box or thermos flask containing
ice packs. The specimen must be sealed inside a waterproof bag or tin to
prevent the labeling being washed off as a result of water from the
icepacks.

Dispatch of microbiological results

 Results should be cross-checked, signed and stamped before dispatch by

senior technology laboratory personnel.

 Each individual result is placed and sealed in its individual envelope and
delivered by laboratory personnel to the respective doctors.

 Keep record of the results dispatched

 Results should be signed for by the receiving officer.

9.2 Examination of Microbiological Specimens

9.2.1 Sputum

Possible pathogens

 Mycobacterium tuberculosis, Mycoplasma pneumonia, Streptococcus

pneumonia, Streptococcus pyogenes, Staphylococcus aureus,
Haemophilus influenza, Klebsiella pneumonia, Pseudomonas aeruginosa,
Proteus species,Yersinia pestis, Fungi actinomyces

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 Blastomyces dermatitidis, Candida albicans, Nocardia asteroids,
Histoplasma capsulatum, Aspergillus fumigates, Cryptococcus neoformans,
Paragonimus westermani
Sputum becomes contaminates with small number of commensal organisms
from upper respiratory tract and mouth as it is being collected since it passes
through the pharynx and the mouth.
Commensals organisms
 Staphylococcus aureus, Staphylococcus epidermidis (albus),
Streptococcus viridians, Lactobacilli, Streptococcus pneumonia,
Enterococci, Micrococci, Diphtheroids, Yeasts like fungi, Neisseria,
Coliforms, Haemophilus influenza (non capsulated strains), Fusobacteria
Collection, transport and storage of sputum

(i) Give the patient a clean, dry, wide-naked, leakproof container and
request him or her to cough deeply to produce a sputum specimen.
(ii) Collect two sputum specimens;
 Collect on the spot called a spot sputum sample.
 The early morning called the early morning sputum sample.
(iii) The specimen must be sputum but not saliva. The early morning sample
should be collected soon when the patient wakes and before any
mouth wash. If the patient is a young child and it is not possible to
obtain sputum. Gastric wshimgs can be used for the isolation of M.
tuberculosis.
(iv) Label the specimen with all the necessary information.
(v) Deliver the sputum sample to the laboratory which should be
accompanied with a well filled laboratory request form within 2 hours of
collection. Or in case of delay in delivery, keep at 4°C. Refrigeration to
slowdown the multiplication of commensals.

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(vi) If pneumonia pathogens are suspected. Collect a purulent part of
sputum on a cotton wool swab, then insert in a container of Amies or
Cary-blair transport medium. Label the container using a lead pencil.
 For M. tuberculosis, use cetylpyridinium chloride-sodium chloride as a
transport medium.
 For referral sputum samples, refer to the notes back on packing and
transport of specimens.
Note; specimens for the isolation of Haemophilus species, streptococcus
pneumoniae and Neisseria must never be refrigerated.

Laboratory Examination of Sputum

Day 1
(a) Macroscopic eaxmination
Describe the appearance of the sputum (macroscopic examination). The
sputum specimen can either be;
 Purulent mostly pus and green-looking.
 Mucopurulent with pus and mucous sometimes green-looking.
 Mucoid mostly, mucous.
 Muco-salivary, mucous with small amounts of saliva.
 Bloody or blood-stained, containing blood.

If sputum is mostly saliva, report the specimen as unsuitable for examination

and request for another specimen.
(d) Microscopic examination of sputum
Perform Ziehl Neelsen (ZN) stained smear.
 Make a thin smear using a purulent part of the sputum on a grease-free
clean slide.
 If the sputum contains blood, lyse the blood clots by saponin solution.
 Allow the smear to air-dry in a safe place and fix with alcohol or over a
flame.

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 Stain the by ZN method for M. Tuberculosis.
 Examine using 100 × objectives to look for AFBs.

Auramine Fluorochrome method

Perform Auramine Fluorochrome technique on request and when facilities are
available.
 Examine using 100 × objectives to look for AFBs.

Do a Gram stain method

 Make a thin smear using a purulent part of sputum on a clean glass slide.
 Allow to air-dry in a safe place or fix using a flame.
 Stain using gram technique and examine the smear for pus cells and
bacteria under 100× objective.
 Look for gram positive and gram negative rods such as streptococcus
pneumoniae, Haemophilus influenza, and Klebsiella pneumoniae.
 However, small numbers of diplococci, cocci and rods may be seen in a
normal sputum because they form part of the normal microbial flora of the
upper respiratory tract.

Note: In most cases, the gram staining technique is done in case of a

negative ZN smear.

Saline preparation method

 Used in examination of sputum for Paragonimus westermani parasite
 Transfer a small amount of sputum on a slide.
 Add a drop of physiological saline.
 Mix and cover with a coverslip.
 Examine using 10×, 40× objectives to look for the Paragonimus eggs.

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 Morphologically, the P. westermani eggs are yellow-brown, oval in shape
and large. Measures 80-100×55µm. there is a flattened operculum (lid) at
one end of the egg and a thickening of the shell at the other end.
 Other parasites that may be seen in sputum are; Ascaris lumbricoides
larvae, Hookworm larvae and strongyloides larvae.
Potassium Hydrroxide (KOH) preparation method

 Used in examination of sputum for fungal infections

 Transfer a small amount of sputum on a glass slide and add 10 – 20% KOH
solution.
 Mix and cover the preparation with a coverslip.
 Examine the preparation for fungi using 10×, 40× objectives and look for
yeasts.
Crag and India ink
 For Cryptococcus neoformans, do a crag test or India ink wet preparation
on sputum.
 Liquefy the sputum with N-Acetyl-L-Cysteine (NALC) and NaOH which acts
as a decontaminant.
 Vortex for 2 seconds and incubates for 15 minutes.
 Centrifuge to get a deposit.
 Use a deposit to perform an India ink test and supernatant for the crag
test.
Culture of sputum
 Inoculate on Blood agar (BA), MacConkey, chocolate agar, Sabouraud
agar
 Incubate the BA plate aerobically and chocolate agar plate in a CO 2
enriched atmosphere at 35-37°C for 24-48 hours.
 Either first emulsify the purulent sputum in about 5mls of sterile normal saline
then inoculate the washed sputum.

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 TB cultures use Lowenstein Jensen medium. Decontaminate the sample
with NaOH. The organism grows after 5-6 weeks of incubation.

Sodium hypochlorite technique for concentration of AAFBs

 Chances of detecting AAFBs in sputum smears are significantly increased
when sputum is first treated with 5% v/v sodium hypochlorite followed by
centrifugation or overnight sedimentation.

 Sodium hypochlorite kills mycobacterium TB and it is also safer for the

laboratory staff.
 Sputum treated with sodium hypochlorite can not be used for culture.
Method

 Transfer 1-2mls of sputum to a screw cap universal bottle or other container

that can hold 20-30mls.
 Add an equal volume of 5% v/v sodium hypochlorite solution and mix well.
 Leave at room temperature for 10-15 minutes shaking at interval to
breakdown the mucus in the sputum.
 Add about 8mls of distilled water and mix well.
 Centrifuge at 3000g for 15 minutes.
 Using a Pasteur pipette, remove and discard the supernatant fluid.
 Mix the sediment and transfer a drop of a well mixed sediment to a clean,
scratch-free slide.
 Spread the sediment to make a thin preparation and allow to air dry.
 Heat fix the smear and stain using the ZN technique.
 Examine microscopically for AAFBs and report your findings.
Day 2 and onwards

 Examine and report the cultures

 Look for pathogenic bacterial colonies like streptococcus pneumoniae,
pseudomonas aeruginosa.

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 Set the antimicrobial sensitivity testing e.g. optochin disc which is sensitive
to streptococcus pneumoniae.
 Pneumococcal antigen can be detected in sputum by serological
diagnosis using co-agglutination method or counter immuno-
electrophoresis. However, the test is expensive.

On routine examination of sputum, Perform

 Appearance of the specimen (macroscopy)

 ZN stain
 Gram stain
 culture
Note;

Other investigations are additional and are done on request of the doctor.

Specimens should be examined under a safety cabinet if possible.

Pneumonia and bronchopneumonia should be cultured as soon as possible

because they don‟t survive well in the specimen.

9.2.2 Routine examination of urine

Possible Pathogens

 Staphyloccocus aureus, Haemolytic streptococci, Enterococci, Escherichia

coli, Proteus species, Pseudomonas aeruginosa, Klebsiella species,
Salmonella typhi and paratyphi, M. tuberculosis, Mycoplasma, Candida,
Chlamydia.
Salmonella typhi and paratyphi can be found in the urine of about 25% of
enteric fever from the third week of infection.

Parasites found in urine

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  Schistosoma haematobium causing urinary schistosomiasis (Bilharzia),
Trichomonas vaginalis as vagina contamination, Microfilaria such as
wunchereria bancrofti and onchocerca volvulus, Intestinal parasites can
also be found in urine but it indicates faecal contamination.

Commensals

 Urinary tract is normally sterile except the urethra which may contain a few
commensals such as; Acinetobacter species, Yeasts in female urethra,
Diphtheroids,

Collection and transportation of Urine specimen

Types of urine collection

i. Midstream urine
Use
For urine microscopy and culture to investigate bacterial infections of the
urinary tract.
Collection
 Give a patient a sterile, dry, wide-naked, leak-proof container and explain
the importance of collecting the specimen with as little contamination as
possible (clean-catch specimen).
 Instruct the female to clean the area around the urethra opening with
clean water, dry the area and collect the urine with the labia held apart.
 To collect a midstream urine sample, instruct the patient to pass a small
amount of urine into the toilet or latrine to ensure that the bacteria, cells or
parasites that have entered the urethra from the vagina can be flashed
away or out.
 Then collect about 20mls of urine in a bottle and pass the remaining urine
in the bladder into the toilet.

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 Label the container with the date, name, identification number of the
patient, time of collection and deliver the specimen with a request form to
the laboratory immediately.
 In case of delays in delivering and examination, the urine should be
refrigerated at 4°C or use boric acid as a preservative. 0.1g of boric acid
powder in 10mls of urine and mix well.
 Urine for culture must not be preserved with bactericidal chemicals such as
thymol, bleach, HCl, acetic acid and chloroform.
Changes that occur in the Urine when unpreserved at room temperature

 Any bacteria in the urine will multiply so that the bacterial count will be
unreliable.
 Ammonia will be released that will increase the PH of the specimen which
will result in destruction of cells and casts.
 The bacteria will also breakdown any glucose which may be present.
 If white cells, red cells and casts are present, they will begin to lyse
especially in a concentrated specimen.
 The concentration of the urine may be altered.
 If bilirubin is present, it will be oxidized to biliverdin which can not be
detected and urobilinogen will be oxidized to urobilin.
Why urine is preserved

 Bacteria remain viable without multiplying.

 White blood cells and casts are also well preserved.
 No interference in the measurement in urinary protein and glucose.
Random urine sample
Use
Used for qualitative chemical testing.
Collection

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 Random urine can be collected at any time of the day into a clean
universal container.
 About 15 – 20 ml of the urine sample should be collected
First morning sample
Use
It is used for the detection of mycobacterium infection of the urinary tract,
pregnancy testing and trichomonas vaginalis.
Collection
 To collect a first morning urine sample, provide a patient with a large clean
container and tell him to empty the bladder into the toilet before resting in
the night. And on arising in the morning, collect all the urine in the
container.
 About 15 – 20 ml of the urine sample should be collected

Terminal urine sample

Use
Used to demonstrate the ova of Schistosoma haematobium
Collection
 To collect a terminal urine sample, provide a patient with a clean
container and instruct him to pass most of it into the toilet and to collect
the last portion of the urine in the container.
 In infants and babies, a random urine sample collected as clean as
possible may be used for all types of urine investigations.

24-hour urine specimen

Use: Used for estimation of Urine volume and protein discharged by the
patient in 24-hour.

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All the urine passed by patient within the 24-hour time should be collected in
a wide mouth 4L container capacity.

16.2.3 Laboratory Examination of Urine

Macroscopic examination of urine
Color, Appearance and Odour
Color
Normal fresh sample of urine varies from pale to dark yellow.
The yellow color is also due to presence of urobilin, uroerythrin and
urochrome. These are the degradation product of urobilinogen when it is
exposed to air.
Pale urine: indicates
Diabetes Inspidus, Chronic renal failure, Very dilute urine,
Reddish urine; indicates Presence of blood in urine (haematuria and
haemoglobinuria), certain drugs and food pigments
Brownish yellow or green with yellow foam This is due to the presence bile
pigment associated with jaundice.
Brown black This is associated with poisoning i.e. lead, mercury, and arsenic,
Hemorrhages and tumors
Milky white It is due the presence of fat globules and
Appearance
 Normal urine is clear in appearance.
 Abnormal urine appears turbid, cloudy which indicates puscells (pyuria),
bacteria (Bacteriuria) following renal and urinary tract infection.
 Turbid can be due to non-pathological conditions due to precipitation of
mucin from urinary tract or precipitation of calcium phosphate. This
disappears with the addition of dilute acetic acid
Odour
 Freshly passed normal sample has the characteristic aromatic odour due
to volatile organic acid when the urine is allowed to stand urea which is

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 the main component of urine gets decomposed by bacterial action to
ammonia which results in the ammonical odour of urine.
 A foul smell indicates UTI, a fruity smell is associated with the presence of
ketone bodies in urine, and a mousy smell is related to phenyl ketoneuria
Appearances of urine in some diseases
URINE APPEARANCE POSSIBLE DISEASES
 Cloudy containing pus cells  bacterial urinary infection
 Red and cloudy containing red  Urinary schistosomiasis and
cells bacterial infection
 Brown and cloudy containing red  Black water fever and other
cells conditions that cause travascular
haemolysis.
 Yellow-brown containing bilirubin  Acute viral hepatitis and
obstructive jaundice
 Yellow-orange containing urobilin  Haemolysis and hepatocellular
or oxidized urobilinogen jaundice
 Milky-white containing chyle  Wunchereria bancrofti that causes
bancroftian filariasis
Note; The infection of the bladder is called cystitis. Urine is not a specimen for
Neisseria gonorrhea

Volume

Clinical significance

 The measurement of urine volume discharged by the patient is done only

for 24-hour specimen.
 The 24-hour urine discharge by a healthy adult is 1.5 liter, which varies with
fluid intake diet and physiological condition of the body.
 The urine discharged during the day is about 3-4 times night volume.

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 The children discharge about 1 liter of urine and infant‟s approximately
600-ml.
 Increase discharge of urine greater than 2000 ml/24 hours is called polyuria
and is due to the deficiency of ADH hormone.
 ADH is responsible for regulating the amount of water absorbed by the
body while urine is passing through tubules.
 When the urine discharged is less then 400 ml/24 hour is called oliguria. This
occurs in hot weather and diseases of kidney (pyelonephritis).
 Complete suppression of urine for 12 hour or less then 100 ml urine / 24 hour
is called anuria. It is observed in case of blood transfusion reaction,
cardiac failure, acute renal failure, surgical shock, and excessive diarrhoea
and vomiting.

Specific gravity

Clinical significance

 Specific gravity is a density of a substance as compared to water, which is

taken as 1.000.
 It depends upon the amount of solute present and also on the
temperature of the solution.
 It increases with increasing concentration of solute and decreases with
increasing temperature.
 The specific gravity of a normal sample ranges from 1.016 to 1.025
 However excessive water intake dilutes the urine and specific gravity is less
than 1.016 reduced perspiration and poor water intake with increase the
specific gravity above 1.025.
 If the kidney is functioning properly, the first morning specimen should have
specific gravity greater than 1.020.

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 Increased discharge of urine with decreased specific gravity is typical of
Diabetes Inspidus.
 High volume of urine discharge with increase specific gravity suggests
Glucoseuria associated with kidney failure.

There are three methods for determining the specific gravity.

Urinometer method, Refractometer method and urine strip method.

Proteinuria

 Proteinuria is a condition in which the urine contains too much protein.

Proteinuria is also known as albuminuria, due to the presence of one type
of protein, albumin, in the urine. It is often caused by dysfunctional kidneys,
high blood pressure, or diabetes and it is a sign of chronic disease or
damage to the kidneys.

Causes

There are three main mechanisms that cause proteinuria;

 Due to disease in glomerulus

 Because of increased quantity of proteins in serum (overflow proteinuria)
 Due to low reabsorption at proximal tubule

 Proteins, components of blood, are essential for various body processes.

Normally, the kidneys block the passage of protein, keeping it in the blood
for delivery to the body‟s tissues and organs. In proteinuria, however,
protein in the blood passes through the kidneys and into the urine. Higher
than normal blood pressure is a common cause of proteinuria.

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 The kidneys are damaged by the elevated blood pressure, which
eventually results in the passage of protein into the urine.
 Other causes of proteinuria include diabetes, kidney infection, or other
types of kidney inflammation.
 Nephrotic syndromes (i.e. intrinsic renal failure)
 Pre-eclampsia
 Eclampsia
 Toxic lesions of kidneys
 Diminished oncotic pressure. Symptoms of diminished oncotic pressure
may include ascites, edema and hydrothorax.
 Proteinuria is a serious medical condition if Left untreated, it may lead to
serious. Serious symptoms include difficulty in urinating (dysuria), inability to
urinate, difficulty breathing (dyspnea) or shortness of breath, lethargy or
altered mental status, or chest pain or pressure.
 Some people with proteinuria do not have symptoms. However, if you
have proteinuria, you may notice urine that appears frothy, or you may
experience swelling in the body (edema). Proteinuria is usually detected
during a simple urine analysis.

 Proteinuria can also be caused by certain biological agents, such as

bevacizumab (Avastin) used in cancer treatment, or by excessive fluid
intake (drinking in excess of 4 litres of water per day).

Measurement

 Proteinuria is diagnosed by a simple dipstick test, although it is possible

for the test to give a false negative reading, even with nephrotic if the
urine is dilute. False negatives may also occur if the protein in the urine is
composed mainly of globulins or Bence-Jones proteins because the
reagent on the test strips, bromphenol blue, is highly specific for albumin.

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 Traditionally, dipstick protein tests would be quantified by measuring the
total quantity of protein in a 24-hour urine collection test, and abnormal
globulins by specific requests for protein electrophoresis.
 Alternatively the concentration of protein in the urine may be compared
to the Creatinine level in a spot urine sample. This is termed the
protein/Creatinine ratio (PCR).

Glycosuria or glucosuria

 This is the excretion of glucose into the urine. Ordinarily, urine contains no
glucose because the kidneys are able to reclaim all of the filtered glucose
back into the bloodstream.
 Glycosuria is nearly always caused by elevated blood glucose levels, most
commonly due to untreated diabetes mellitus.
 Rarely, glycosuria is due to an intrinsic problem with glucose reabsorption
within the kidneys themselves, a condition termed renal glycosuria.
Glycosuria leads to excessive water loss into the urine with resultant
dehydration, a process called osmotic diuresis.

 Glucose in urine can be identified by Benedict's qualitative test.

 A urine dipstick can show a false-positive glucosuria if someone is taking
Pyridium medications that relieve symptoms of urinary tract infection

Microscopic examination of urine

Perform a wet (microscopy) examination

 On fresh uncentrifuged urine, mix uniformly and examine for bacteria, cells
and casts

 Place 0.05mls of well mixed fresh urine on a clean glass slide, cover with
the coverslip and examine the preparation using ×10, ×40 objective to look
for the following abnormal features;

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Bacteria

This can be in form of rods, cocci and can be motile or non-motile indicating
bacterial urinary infection. They indicate bacterial urinary infection.

Report in absolute numbers or pluses as the number per high power field.

Pus cells (White cells)

 They are dead or inflammatory white blood cells. These are identified by
their nucleated structures with uneven margins.
 They are bigger than RBC‟s and do not lyse with the addition of 2% acetic
acid.
 They are round 10-15µm in diameter larger than red blood cells.
 They contain cytoplasmic granules and lobed nucleus.
 They are associated with bacterial urinary infection and are often found in
clamps.
 They are usually reported as the number per HPF either in pluses or
absolute e.g. 20wbcs/HPF, or pus cells: 20-30/HPF, or pus cells: 25/HPF.
 The normal urine may contain few white cells of less than 5/HPF. However,
high number may be present in the female urine.

Red cells

 They are smaller and more refractile than pus cells.

 They have a definite outline and contain no granules but seen as

colourless.

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 When the urine is isotonic, they have a round appearance

 When it is hypertonic, they will appear smaller than normal and often
crenated.

When it is hypotonic, they will appear larger than normal (swelling) which can
be easily ruptured.

 Red cells are reported in absolute numbers or pluses.

 The red cells in urine (haematuria) may be found in urinary schistosomiasis,
bacterial infection, acute glomerulonephritis (inflammation of the
glomerulus of the kidney), sickle cell disease, leptospirosis, infective
endocarditis, in calculi (stones in the urinary tract) and haemorrhagic
conditions.
 The presence of abnormal numbers of red blood cells in the urine may be
due to; Glomerular disease, Tumors that erode any part of the urinary tract,
Kidney trauma, Renal infarcts, Acute tubular necrosis, Upper and lower
urinary tract infections, Nephrotoxins, Traumatic catheterization, Passage
of renal stones, Physical stress.
 Red cells in urine of women may be due to menstruation.

Yeast cells

 These are differentiated from the red cells by their oval shape. Some of
them usually show single budding.

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 Are also confirmed in a wet preparation by running a drop of dilute acetic
acid under the coverslip where red cells will be haemolysed by the acid
and yeast cells will not.
 Are seen in urine of women with vaginal candidiasis and occasionally in
specimens from diabetic patients.
 Yeast cells are reported in absolute numbers or pluses

Epithelial cells

 They are nucleated and vary in shape and size.

 These are larger then both RBC‟s and WBC‟s are nucleated with angular
sides.
 The presence of few squamous epithelial cells is considered to be normal
but increased sheding indicates renal degeneration. They usually indicate
inflammation of the urinary tract or vaginal contamination of the
specimen.
 They are driven from linings of the genital urinary system.
 They are 3 types and squamous epithelial cells are the largest of all.

Oval fat bodies

 These are another type of cells and epithelial cells that undergo fatty
degeneration and form oval fat bodies. The presence of fat globules

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 indicates a serious pathological condition like nephritic syndrome caused
by diabetes mellitus or toxicity of renal system

Casts

 They consist of solidified protein.

 They are cylindrical in shape because they are formed in the kidney
tubules.
They are several types;
Hyaline casts
These are colourless and empty and associated with damage to glomerular
filter membrane, chronic renal disease. A few may be seen following
excessive exercise or strainous exercise or during fever, dehydration, exposure
to heat and emotional stress.

Waxy casts
 They are hyaline casts that have remained in the kidney tubules for a
longtime.
 Are thicker and denser than hyaline casts.
 Are appearing indented or twisted and may be yellow in colour.
 They usually indicate tubular damage and can sometimes be seen in renal
failure.

Cellular casts
 They contain white cells or red cells.

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 Red cell casts appear orange-red and indicate haemorrhage into the
renal tubules or glomerular bleeding.
 White cell casts are found when there is inflammation of the kidney pelvis
or tubules. Indicates kidney mal-functioning. They areYellow-brown
pigmented casts may be seen in the urine of jaundiced patients.

Granular casts
 They are casts containing irregular sized granules originating from
degenerated cells and proteins.
 They are associated with renal damage.

Crystals

 These have a characteristic refractile appearance.

 Normal urine contains many chemicals from which crystals can form and
therefore, the finding of most crystals in urine has little importance.
 Crystals should be looked for in fresh urine if calculi (stones) in the urinary
tract are suspected.
 The most common crystals seen in acidic urine are urates, consisting of uric
acid, amorphous urates and sodium urate.
 They are the only normal crystals found in acidic urine that appear
coloured.

Tsongo Lawrence Msc (MUK) 178

 They are seen in a variety of shapes and identification is best made by
colour (yellow to reddish-brown).
 Increased levels of uric acid crystals are seen in leukaemia (cancer of the
blood).

Calcium oxalate crystals

 They are seen in acidic urine but can be seen in neutral and rarely in
alkaline urine.
 They are easily recognised as colourless but resemble envelopes, oval form
may also occur. The crystals are associated with diets and often seen
following large doses of ascorbic acid and also indicate calculi in urinary
tracts.

Tri-phosphate crystals
 They are found in alkaline urine.
 Are most easily identified because in their routine form, they appear as
colourless prisms.
 They are seen in large numbers in urine that has been left standing at room
temperature.

Other crystals which may be found in rare disorders include;

 Cystine crystals: are yellow, dark coloured and look like needles mast
together. Found in severe liver disease.
 Tyrosine crystals: are also yellow, dark coloured and look like needles mast
together and found in severe liver disease.
 Cholesterol crystals: are like rectangles with cut out corners and are found
in kidney disease (severe) when alphatic vessel has ruptured into the renal
pelvis.

Tsongo Lawrence Msc (MUK) 179

 Sulphonamides: Are found in urines of patients being treated with
sulphonamides.
When deposited in the urinary tract, they can cause haematuria.

Parasites in urine

Eggs of Schistosoma haematobium

 Recognised by their large size and spine at one end (Terminal spine)
 The urine will contain red cells and proteins. Centrifuge the urine to
concentrate the eggs. Then do a wet preparation and examine using 10×,
40× objectives.

Trichomonas vaginalis
 Found in the urine of the women with acute vaginitis.
 Are little larger than white cells and easily detected in fresh urine because
they are motile.
 They move by flagella and undulating membrane and occur in urine as a
contamination.

Wunchereria bancrofti
 The urine appears milky-white or reddish pink (chyle mixed with blood).
 They occur in urine when urogenital lymphatic vessel ruptures.

 The microfilaria is large 225-300×10µm.

 They are motile and sheathed and no nuclei are present in the tail when
stained.

Onchocerca volvulus
 Found in the urine of patients with onchocerciasis especially in heavy
infections.
 The larvae is large 280-330×7µm

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 They are unsheathed with slightly enlarged head end and tail which is
sharply pointed and contains no nuclei.

 The intestinal parasite found in urine occurs as contamination especially in

young girls when the eggs are washed off in external genitalia as the urine
is being passed e.g. Enterobius vermicularis.

Spermatozoa cells

 They are found in urine of men and recognised by their head and long
thread-like tail.

 They are also motile in fresh urine and may appear in normal condition.
However, if seen must be reported because on the other hand may
indicate a disease in males.

Perform a gram stain on urine deposit

 It is done on request of a doctor.

 It allows to determine or to decide whether there is a urinary infection or
whether the bacterial cells are contaminants.
 Smears are done when bacteria or pus cells are seen in the urine wet
preparation.
 Centrifuge about 10mls of urine, and then make a smear of the deposit on
the slide.
 Airdry and stain by gram stain method.
 Examine using 40×, 100× objectives to look for bacteria associated with
urinary infection.
Vaginal contamination is detected by mixed bacterial flora including positive
rods e.g. Lactobacilli.

If gram negative intracellular diplococci in pus cells are seen, ask for a
urethral swab specimen for examination e.g. Neisseria gonorrhoea.

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Perform a ZN on urine deposit

 This is done on doctor‟s request and it is done if renal TB is expected to look

for AFBs in urine specimen.
 Make a smear of the deposit from centrifuged early morning urine deposit.
 Airdry, fix and stain by ZN method.
 Examine the smear under 100 × objectives for AFBs or AAFBs.

Culture of urine

 Culture and sensitivity testing is required if the urine contains bacterial cells,
casts, proteins e.t.c.
 It is not necessary to culture urine which is microscopically and
biochemically normal except in pregnancy where asymptomatic
bacteraemia may occur.
 Culture media used are;
 Cystine lactose electrolyte deficient (CLED) agar
 Blood agar
 MacConkey agar
 Sabouraud agar for yeast cells
 Mix the urine well and inoculate a loopful of urine on the above media.
 Incubate the inoculated plates aerobically at 35-37°C for overnight.
 If salmonella typhi is suspected, inoculate a loopful of urine in selenite
broth; incubate the broth at 37°C overnight.
 If renal TB is suspected, culture the urine on acid Lowenstein Jensen slope
for isolation of AFBs but done on request.
 Allow the early morning urine to sediment overnight and use the deposit to
culture. The urine should be decontaminated for 10 minutes. Incubate the
slope aerobically at 37°C for 5-6 weeks.
Day two and onwards

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 The following day subculture on xylose lysine deoxycholate (XLD) agar, If
salmonella typhi is suspected and inoculated a loopful of urine in selenite
broth then incubate the plate aerobically at 37°C for 24 hours.

 Examine the culture plates for any bacterial growth and report the
findings.
 On BA and MacConkey plates look for colonies that will be E. coli, Proteus
species, Pseudomonas aeruginosa, Klebsiella species, staphylococcus
species, Enterococci
 On XLD look for salmonella colonies.
 On LJ slopes look for colonies that will be mycobacterium tuberculosis.
 Set antimicrobial sensitivity testing using sensitivity testing agar.
 For urine chemistry, perform the following tests; urine proteins, sugars,
bilirubin, urobilinogen, ketone bodies, refer to clinical chemistry.
 Examine the urine under dark-field microscope if Leptospirosis is suspected.

9.2.3 Cerebrospinal Fluid (C.S.F)

C.S.F. is a fluid filled by the lateral ventricle deep within each cerebral
hemisphere.

Functions of C.S.F

 To provide a fluid cushion to protect the brain and the spinal cord from
mechanical injury caused by any sudden movement of the body.
 To carry nutrients to the brain and the spinal cord and remove waste
substances.
 To maintain a constant pressure inside the head and around the spinal
cord.

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C.S.F is examined to diagnose the inflammation of the meninges and the
spinal canal caused by microorganisms leading to infection known as
meningitis.

Possible pathogens

Bacteria causing pyogenic bacterial meningitis include; Streptococcus

pneumonia, Staphylococcus aureus, Streptococcus agalactiae (Group B),
Listeria monocytogenes, Bacillus anthracis, Neisseria meningitides,
Haemophilus influenza (type B), Escherichia coli, Salmonella typhi, Proteus
species, Klebsiella species, Pseudomonas aeruginosa, Flavobacterium
meningo-septicum, Mycobacterium TB, Treponema pallidum, Leptospira
enterogens

Anaerobes such as; Bacteroids species may be found in C.S.F when there is
brain abscess.

Viruses, Enteroviruses especially echoviruses causing viral meningitis

Fungi, Cryptococcus neoformans that cause fungal meningitis or

cryptococcal meningitis.

Protozoa, Trypanosome species, Amoebae such as Naegleria fowleri and

acanthamoeba causing amoebic meningoencephalitis. Larva of dirofilaria
immitis and anglostrongylus cantonensis

Commensals

 The C.S.F has no normal microbial flora, any microorganisms found in C.S.F
is a pathogen.
Pathology
 Pathogens reach the meninges in the blood stream or occasionally by
spreading from the nearby sites such as the middle ear and the nossal

Tsongo Lawrence Msc (MUK) 184

 sinuses. And the inflammation of the membranes that cover the brain and
spinal cord is called meningitis.
 Meningitis of the new born is called neonatal meningitis and if it is
suspected, blood should be cultured.
 In developing countries meningitis epidemics are usually caused by
Neisseria meningitidis. Serotype A and B outbreaks are common in Africa
and South America, in the Middle East and Asia.
 The epidemic usually occurs frequently in hot dry seasons.
Collection of C.S.F

 C.S.F for laboratory investigations is usually obtained by lumber puncture

(Spinal tap) and by ventricular puncture from infants.
 It must be collected by an experienced health worker or medical officer
and must be performed under aseptic procedures. If possible, it should be
collected before antimicrobial treatment.
 The sample of C.S.F is taken between the 3rd and 4th or 4th and 5th vertebra.
C.S.F specimen containers

Three specimen containers are required for C.S.F collection

 In sterile bottle for collection of culture sample (Microbiological

examination).
 In sequestrene (EDTA) bottle for haematological test such as C.S.F cell
count and differential cell count.
 Fluoride bottle containing sodium fluoride oxalate anticoagulant for
estimation of chemistry tests such as sugars and proteins.
Materials required for C.S.F collection

 Skin disinfectant i.e. 70% alcohol

 Specimen container (sterile, fluoride and EDTA bottles)
 Sterile gauze and adhesive plaster

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 Sterile lumber puncture needles of gauge 22/
3.5 inch for adults, 23/2.5 inch for
children.
 Trans-isolate (TI) medium used as a transport medium
 Gloves, syringes and needles.
Procedure

 A sterile wide bore needle is inserted between the 4 th and 5th lumber
vertebra and C.S.F is allowed to drip into dry sterile containers under
aseptic conditions.
 Collection can also be done in one sterile bottle and aseptically transfer
aliquots (portions) of the sample into TI medium, fluoride and EDTA bottles.
Storage

Store the C.S.F at room temperature. In cases of delay in examination, the

sample is stored at 35-37°C until it is examined.

Transportation

 C.S.F should reach the laboratory immediately after collection as soon as

possible.
 For referral, C.S.F sample should be inoculated in trans-isolate medium and
a portion of C.S.F in a sterile bottle which should be transported and
packed under suitable conditions and sent immediately to the referral
laboratory (see the packing and transportation of samples).
 A delay in examination of C.S.F sample reduces the chances of isolating
pathogens, it reduces the glucose level due to glycolysis especially if not
collected in a fluoride bottle.
 If trypanosomes are present, they will not be found because they rapidly
loose their motility, therefore, a C.S.F sample should be considered as an
emergency specimen.
 For referral transportation, the sample should be transported at 37°C.

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 For very short distances, it should be transported at room temperature.
Laboratory Examination of C.S.F

Materials required

 Clean glass slide

 Coverslips
 Counting chambers (Fuchs-rosenthal)
Reagents

 Glucose oxidase reagent for C.S.F sugar estimation or

 Glucose strip reagent for C.S.F glucose estimation
 White blood cell diluting fluid e.g. Toluidine blue or isotonic methylene
blue. Sometimes Turk‟s solution is used.
 India ink or Nigrosin stain (As negative staining for capsulated organisms
especially the fungus).
 Gram stain reagent to differentiate gram positives and gram negative
organisms
 ZN staining reagent for AFBs
 Protein standards or salphosalicylic acid (SSA) for C.S.F protein estimation
 Pandy‟s reagent for protein globulins
 Culture media for culturing the specimen.
 Typing sera for typing bacteria.
 Antibiotic discs for setting sensitivity tests.
Methods of C.S.F examination

Description of C.S.F appearance

 Note the colour and clearity of the C.S.F.

 Normal C.S.F is clear, bright and colourless.

 Abnormal C.S.F may have the following abnormal appearances;

Cloudy and purulent

 C.S.F indicates meningitis caused by pyogenic bacteria such as Neisseria
meningitidis. Cloudy (+) may be seen in viral and protozoa meningitis.

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Blood in C.S.F

 Describing the C.S.F as blood stained or bloody, which is often a result of

traumatic lumber puncture, pathogenically causes of blood in C.S.F
include;

 Recent subarachnoid haemorrhage, Meningitis due to Naegleria fowleri

Note; Smears for pathogens and culture should be carried only on bloody
C.S.F. other tests such as cell counts, biochemical tests are not performed
because they are always in

Yellow coloured or xanthochromic C.S.F

This is seen after centrifuging due to breakdown of haemoglobin.
Indication
 It indicates earlier subarachnoid haemorrhage.
 Cerebro-tumour
 Spinal constriction
 Jaundice
Presence of a spider web clot
 Clotted C.S.F indicates an increase in fibrinogen.
 When there is spinal constriction.
 In pyogenic meningitis.
 In TB meningitis
 If the C.S.F is allowed to stand for long hours, a spider web clot may form
on the surface of the fluid. A few sterile glass bids are added to the fluid,
shaken to breakdown the clot.
Perform cell count
Total white blood cell count
Required

 Fuchs rosenthal ruled counting chamber

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 Diluting fluid i.e. Toluidine blue or isotonic methylene blue
Method

 Mix uncentrifuged C.S.F sample collected in EDTA bottle.

 Dilute the fluid 1 in 2 with the diluting fluid by mixing 1 drop of C.S.F with 1
drop of diluting fluid.
 Assemble the Fuchs rosenthal counting chamber.
 Using a capillary tube, fill the chamber with a well mixed diluted C.S.F
avoiding overflow into the channels of the chamber.
 Wait for 2 minutes for cells to settle while in moist condition.
 Focus the cells and chamber rulings under 10 × objectives, then 40 ×
objectives for proper identification of the white cells.
 Count under 10× objective for all the white cells in the five large squares
i.e. 4 at the corners and one at the centre.
Calculation factor and results

Dilution = 1 in 2. Dilution factor (DF) = 2

Depth of the counting chamber = 1/5 mm
Number of squares counted = 5
Let the number of cells counted be n
Total number of cells = number of cells counted × DF
In 1 mm3 of C.S.F depth of counting chamber × number of squares
counted

n×2 = n×2
5 × 0.2 1
Therefore; Total number of cells is n × 2
E.g. if the cells counted are 20
Total number of cells is 20 × 2 = 40 cells/mm3
If it is per litre, it will be n × 106

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Note; If the cell count is high so that it is difficult to count, make 1 in 5 dilution
or 1 in 10 dilution of the C.S.F, refill the chamber and count then multiply the
correct used dilution factor.
Counting chamber

1 2

3 4

Fuchs rosenthal counting chamber areas are 1, 2, 3, 4 and 5 which are for cell
count. Alternative method is using improved neubauer counting chamber.

Normal C.S.F contains up to 5 × 106 cells per litre or 5 cells/mm3

Perform a differential white blood cell count on C.S.F

 Make a thin smear of C.S.F from the deposit and stain with Giemsa,
Leishmann or modified field‟s stain.
 Examine and differentiate the polymorphonuclear cells from the
lymphocytes.
 When lymphocytes are predominant may indicate viral meningitis, TB and
protozoa meningitis.

Do a wet preparation

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This is done for motile amoebae e.g. Naegleria fowleri and trypanosomes. In
this case a fresh C.S.F sample is needed which is 15 minutes of collection so
that a wet preparation would show motile organisms.

Method

 A drop of C.S.F deposit is put on a clean glass slide and covered with a
coverslip.
 Examine using 40 × objectives and observe any motile organisms and
report the findings.
 Stain the smear from the deposit with field‟s stain/Giemsa to demonstrate
the presence of trypanosomes and other cellular forms e.g. morular or
mott cells (the immunoglobulin producing cells which are suggestive of the
infection with trypanosomes in the central nervous system).
 Morular cells contain IgM and are thought to be degenerate plasma cells.
 They are larger than most lymphocytes, stain dark red mauve and contain
vacuoles.
Identification of Naegleria fowleri in C.S.F

This can be identified by growth on E. coli and ability to become flagellates in

water.

Method

 Spread a pure, washed culture of E. coli on a plate of 1.5% non-nutrient

agar.
 When the surface of the plate is dry, inoculate it with 2 drops of C.S.F and
incubate in a moist atmosphere at 37°C for overnight and examine the
plate for growth of amoebae using a magnifying lens.
To show the ability of amoebae to become flagellates, transfer a portion of
the agar containing amoebae to a tube containing about 1ml of sterile
distilled water and incubate at 37°C for 4 hours.

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 Examine every half an hour for flagellated forms.
 Naegleria fowleri amoebae become flagellates usually within 2 hours.
Perform negative staining using India ink or Nigrosin
Purpose

It is used to demonstrate capsulated organisms particularly in fungal

meningitis when true pathogenic yeasts are present in C.S.F especially
Cryptococcus neoformans.

Principle of India ink

The stain does not stain the yeast-Cryptococcus neoformans. It therefore

appears as a refractile organism unstained against a dark background
provided by India ink.

Method

 Transfer a drop of C.S.F deposit from centrifuged sample to a glass slide.

 Add a drop of India ink and mix.
 Cover with a coverslip and remove the excess stain with a soft tissue or
cotton wool (do not make the preparation too thick otherwise the cells
and capsules will not be seen).
 Examine the preparation microscopically using 10×, 40× objectives and
look for oval or round cells some with the buds, irregular in size, refractile
and surrounded by a capsule showing unstained against a dark
background given by India ink.
Results

Stained bacterial cell

Dark background

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 Clear unstained capsule

Reporting

India ink preparation: Capsulated organisms seen (+++)

Or; No capsulated organisms seen in case of negative results.

Do a crag test

For Cryptococcus neoformans antibodies as one of serological tests (Crag-

Cryptococcal antigen)

Perform TPHA or VDRL/RPR

For Treponema pallidum in cases of neurosyphilis

Do biochemicals test the protein and glucose on C.S.F?

Use a sample collected in a fluoride bottle for chemistry tests.

Note; the specimen with blood is not suitable for protein, sugar and cell count.

On such specimens, make smears for bacteriological examination and culture

e.g. gram stain and report the presence of pus cells and organisms seen.

The routine biochemical testing of C.S.F to investigate meningitis includes;

 Glucose estimation
 Total protein estimation
 Pandy‟s testing for screening of raised globulins.
C.S.F glucose estimation

 If the C.S.F is not preserved with fluoride, the glucose must be estimated
within 20 minutes of collection to avoid false low results due to glycolysis.
 If the C.S.F is cloudy, centrifuge the fluid and use the supernatant for
chemistry test.

Tsongo Lawrence Msc (MUK) 193

 Use the colorimetric glucose oxidase method or the O-Toluidine method for
blood sugar to determine the C.S.F sugar.
 Alternatively, use a semi-quantitative method by using Benedict‟s
solution/reagent and a reagent strip test for measuring urine glucose can
be used. Results are read and interpreted as for urine chemistry.

 Normal concentration of C.S.F glucose levels is 2.5-4.0 mmol/l (45-72mg/dl).

 Low C.S.F glucose levels is found in pyogenic meningitis or glucose may
even be absent.
 Raised C.S.F glucose levels are found in hyperglycaemia and encephalitis
following damage to cerebral capillaries. In viral meningitis, the glucose
levels are usually normal.
C.S.F total protein estimation

 Proteins can be estimated from fluoride oxalate C.S.F sample from

unpreserved C.S.F specimen.
 Colorimetric and semi-qualitative methods are used to estimate C.S.F
proteins.
 Sulphosalicylic acid is not recommended because it precipitates albumin
with greater turbidity than globulin.
 Normal C.S.F total protein is 0.15-0.40g/l or 15-40mg/dl only traces of
globulin are found in normal C.S.F.
Use of reagent strip for measuring C.S.F protein

The reagent strip tests used to test for proteins in urine detects mainly albumin.
If such strips are used to screen increases in C.S.F proteins, Pandy‟s test must
also be performed to detect increases in C.S.F globulins.

Pandy’s test

Principle

Tsongo Lawrence Msc (MUK) 194

The test detects rises in C.S.F globulins which occur in meningitis.
Reagents
 Pandy‟s reagent (Phenol saturated solution)
Phenol crystals…………………………………………29g
 Distilled water…………………………………………30mls
Add the phenol crystals in water and transfer to a reagent bottle. Label and
mark corrosive. The reagent is stable at room temperature.

Method

 Pipette 1ml of Pandy‟s reagent into a small glass tube.

 Use a dropper pipette to deliver a large drop of supernatant fluid from a
centrifuged C.S.F specimen into the bottle, holding the tube at high level.
DO NOT MIX.
 Look for immediate cloudness around the drop of C.S.F indicating the
presence of excess globulin.
 Report the test results as; immediate cloudness-Pandy‟s test positive. Or
no cloudness-Pandy‟s test negative.
Note; after a few minutes, cloudness may disappear. An increase in total
protein with a positive Pandy‟s test occurs in all forms of meningitis.

Normal C.S.F is Pandy‟s test negative.

Perform a gram stain

 Centrifuge a portion of C.S.F in a sterile bottle.
 Make a smear of a deposit on a clean glass slide.
 Airdry, fix over a flame or in absolute methanol and stain by gram
technique.
 Examine the smear for pus cells and bacteria using 40×, 100× objectives
and look for gram positive and gram negative organisms which can be
intracellular diplococci (Meningococci).

Tsongo Lawrence Msc (MUK) 195

 Gram positive deep staining yeast cells probably Cryptococcus
neoformans, sometimes the large capsules that surround the cells can be
detected as unstained areas.
Reporting

India ink: No capsulated organisms

Gram stain: Gram positive yeast cells
Culture: results to follow.
Do a ZN staining method
 A C.S.F specimen which may contain AFBs may appear with a spider web
clot. AFBs if present will be trapped in this web clot.
 Extract the web clot from the C.S.F and put on a clean glass slide.
 Use a deposit from a sterile tube, airdry and fix in methanol or by gentle
heat.
 Stain using ZN technique for AFBs.
 Examine using 100 × objectives and observe for the presence of AFBs as
red rods.
Reporting

Report in pluses if AFBs are seen. If negative report No AFBs seen.

Culture of C.S.F
The C.S.F should be cultured as soon as after collection. If the C.S.F appears
cloudy (+) centrifuge in a sterile tube for 15-20 minutes and use the deposit for
inoculating the plate.

 Culture on chocolate (heated Blood agar media) MacConkey agar and

Sabouraud agar for yeast cells and also BA.
 Incubate the chocolate plate in carbondioxide enriched atmosphere and
the rest of the plates aerobically at 37°C for 12-24 hours.

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 Sabouraud agar plate should be incubated for up to 72 hours and
checking for growth every night of incubation.
Day two and onwards

Examine and report the culture plates for any growth.

 On chocolate agar, look for colonies that would be Neisseria meningitidis,

Strep. Pneumoniae and Haemophilus influenza.
 On BA and MacConkey agar, look for any gram positive and negative
bacterial colonies.
 On Sabouraud agar, look for colonial features of C. neoformans.
 Set the antimicrobial sensitivity testing on growth colonies.
OTHER EFFUSIONS
An effusion is a fluid which collects from a body cavity due to inflammatory
response and include;

 Synovial fluid from joints

 Pleural fluid from the pleural cavity
 Pericardial fluid from the pericardial sac,
 Asciatic fluid from abdominal cavity
 Hydrocele fluid usually from the sac surrounding the testis.
Normal appearance: yellow and clear.

Abnormal may appear: purulent, turbid

Laboratory diagnosis as in C.S.F examination

 Hydrocele fluid is usually due to wunchereria bancrofti microfilaria.

9.2.4 Urogenital Specimens

They include; Urethral swabs, Cervical swabs, Vaginal swabs, High vaginal
swabs (HVS), Fluid or pus from genital ulcers

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Collection

Urethral swabs

 Patients should not pass urine for 2 hours before the specimen is collected.
 Clean around the urethral opening using a swab moistened with sterile
normal saline.
 Gently massage the urethra from up downwards and collect a sample of
pus on a sterile cotton wool swab.
 Label the specimen systematically.
Cervical specimens

 Moisten a vaginal speculum with sterile warm water and insert into the
vagina.
 Clean the cervix using a swab moistened with sterile normal saline.
 Pass a sterile cotton wool into endocervical canal and gently rotate the
swab to obtain a specimen, and then label.
Vaginal specimen

 Collect a sample of vaginal discharge on a sterile cotton wool swab.

Transportation of the specimen

 Insert the swabs in a container of Amies transport media, break off the
swab stick to allow the bottle top to be replaced tightly.
 If trichomonas vaginalis or gardnerella vaginalis infection is suspected, do
not use Amies transport media. Immediately send the specimen as soon as
possible for examination or store at 37°C.
Other specimens e.g. for detection of Treponema pallidum;

 Wear protectives and clean around the ulcer using a swab moistened in
saline.
 Then collect the sample fluid on a coverslip and insert it on a slide.

Tsongo Lawrence Msc (MUK) 198

 Label and immediately deliver the preparation in the laboratory for
examination by dark field microscopy.
Laboratory Diagnosis

Perform macroscopic examination

 By reporting the appearance of vaginal discharge. In trichomonas
vaginalis, the discharge is yellow-green purulent with PH of over 4.5.

 In candida albicans, it is white with the PH below 4.5.

 In gardnerella vaginalis, the discharge is grey, offensive, and thin with PH of

over 4.5.

Microscopic examination
 Do a wet preparation in saline or KOH (which lyses epithelial cells) to
detect T. vaginalis (it should be examined as soon as possible after
specimen collection) pus cells and yeast cells (examined using 10×, 40×
objectives) and clue cells (bacilli on the epithelial cells) which occurs in
gardnerella vaginalis.
 KOH lyses epithelial cells so that T. vaginalis, yeast cells and pus cells can
be seen clearly in the fluid against a clear background. The preparation
must not be too thick.
 Make another wet preparation for dark field microscopy to detect
Treponema pallidum.
 Make smears and stain with Giemsa stain.
 Examine using 100 × objectives to identify the detailed morphology of T.
vaginalis.
Perform a gram stain

 Make smears of the specimen.

 Airdry and fix with absolute methanol.
 Stain by gram technique.

Tsongo Lawrence Msc (MUK) 199

 Examine using 40×, 100× objectives and look for bacteria, pus cells yeast
cells e.t.c.
 For gonococci (Neisseria gonorrhoea), look for pus cells containing gram
negative diplococci (intracellular) but if the pus cells have been
damaged, the organisms may be seen lying outside the pus cells
(extracellular).
 For candida species, look for large gram positive yeast cells that would be
candida albicans or other species.

Culture the specimen

 Culture on modified New York city medium (MNYC) for Neisseria
gonorrhoea, on BA, chocolate agar, MacConkey agar, cooked meat
media and Sabouraud medium for candida species (yeast cells) and
serum slopes.
 Inoculate the specimens on the plates above and incubate chocolate
agar, MNYC plate anaerobically. Other plates aerobically both at 35-37°C
for overnight. But Sabouraud agar up to 48 hours.
 Then the following day, examine the plates for any growth of organisms
and give a report.
 On the third day, examine the sensitivity testing and give a report.
Possible pathogens

 Neisseria gonorrhoea, Streptococcus pyogenes, Chlamydia trachomatis,

Staphylococcus aureus, Listeria monocytogenes, Clostridium perfringenes,
Escherichia coli, Proteus
Trichomonas vaginalis, Candida species, Enterococci, Anaerobic
streptococci, Haemophilus ducreyi, Treponema pallidum
Commensals

Urethral swabs

Tsongo Lawrence Msc (MUK) 200

 Diphtheroids, Acinetobacter, Enterobacteria, Skin commensals may be
present
Vaginal swabs

 Lactobacilli, Clostridium species, Gardnerella vaginalis, Yeast cells,

Bacteroids species, Fusobacteria, Macoplasm, Small numbers of
Diphtheroids
Cervical swabs

 It is sterile hence no normal flora

9.2.5 Pus Swabs

Collection

 Pus from wounds, abscesses, burns and sinuses special care should be
taken to avoid contaminating specimens with commensal organisms from
the skin.
 Collect the pus from abscess when it is incised and drain or after it has
ruptured naturally.
 A specimen from wounds should be collected before an antiseptic
dressing is applied. Fresh pus swabs are collected for examination and
strictly sterile cotton wool swabs are used to collect the pus samples from
an infected surgical wound.
 Label the specimens and transport to the laboratory immediately in a cold
box to avoid death of fastidious organisms.
Laboratory diagnosis

Macroscopic examination
 Pus samples are examined for their appearance. They can appear
purulent, blood stained and moist.
 Note the colour of the specimen which can be whitish, grayish or yellowish.
Microscopic examination

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Do a wet preparation in KOH if fungal infection is suspected

 Make a smear in KOH covered with a coverslip.

 Then wait for 5 minutes for the specimen to clear.
 Examine using 10×, 40× objectives and look for oval, round, single budding
cells which can be yeast cells.
Perform a gram stain

 Make an evenly spread smear of the specimen on a clean glass slide.

 Allow the smear to air-dry.
 Fix in absolute methanol.
 And then stain by gram technique.
 Examine the smear for gram positive and gram negative organisms using
40×, 100× objectives.
Carryout a ZN smear if TB is suspected

 Make a smear of a specimen

 Airdry, fix and stain by ZN technique.
 Examine using 100× objectives for AFBs.
Culture the specimen
 Inoculate the specimen on 70% Sheep blood agar, Chocolate agar,
Sabouraud agar, cooked meat media, Neomycin blood agar,
MacConkey and Nutrient agar.
 Use a sterile wire loop to inoculate the plates and incubate at 37°C for
overnight.
 Chocolate and Neomycin plates are incubated in 20% carbondioxide.
On day two, examine and report the cultures.

Possible pathogens

 Streptococcus Viridans, Staphylococcus aureus, Streptococcus

perfringenes and tetani, E. Coli, Bacteroids species, Mycobacterium

Tsongo Lawrence Msc (MUK) 202

 tuberculosis, Klebsiella species, Entamoeba histolytica amoebae (pus
aspirated from liver abscess), Pseudomonas aeruginosa, Proteus species,
Anaerobic cocci, Candida species, Cryptococcus neoformans,
Clostridium tetani and perfringenes
Commensals

 Those that have contaminated the specimens from the skin, clothing, soil
or from air if an open wound.

9.2.6 Ear, Nose and Throat Swabs

Collection

 Ear swabs are collected on a sterile cotton wool swab or aspirate a small
amount of discharge in a sterile container. Take care not to contaminate
the swab. Place it in a container of Amies transport medium
 Nose swab specimen, collect using a sterile cotton wool swab moistened
with sterile peptone water, gently swab the inside surface of the nose.
Replace the swab in its sterile container taking care not to contaminate
the specimen.

 For nasopharyngeal aspirates, gently pass a sterile catheter through one

nostril as far as nasopharynx. Attach a sterile syringe to the catheter and
aspirate a specimen of mucopus. Then put in a sterile container.
 Throat and mouth swab specimens. Using a good light and a handle of a
tongue depressor depress the tongue and swab the affected area using a
sterile cotton wool swab, curved at an angle of 45°. Taking care not to
contaminate the swab with saliva, return it to it‟s sterile container.
 Label all the swab specimens and transport them to the laboratory
immediately after collection.
Laboratory examination

Examine the specimen for appearance

Tsongo Lawrence Msc (MUK) 203

It is moist, pus-like, purulent e.t.c.

Microscopic examination
 Make a wet preparation in KOH if fungal infection is suspected. After 10
minutes when the preparation has cleared, examine using 10×, 40×
objectives and look for yeast cells.
 Perform a gram technique
 Make a smear (evenly spread) of the specimen on a slide, airdry and fix
in absolute methanol.
 Stain by gram technique.
 Examine the smear for pus cells, bacteria under 10 × objective and
vicenti‟s organisms in case of throat swabs.
 Vincenti‟s organisms are seen as gram negative spirochaetes e.g.
Borrellia vicenti.
 Do Albert‟s stain for corynebacterium diphtheriae.
 Make a smear on a clean slide, airdry and stain by Albert‟s technique.
Culture the specimen
 Use Chocolate agar, Blood agar, MacConkey and Sabouraud agar
plates.
 Incubate the Chocolate agar in carbon dioxide atmosphere and the rest
aerobically all at 35-37°C for overnight. Sabouraud agar for 48 hours.
Day two and onwards, report after examining the cultures for any bacterial
growth.

Possible pathogens

Ear swabs

 Staph. Aureus, Strep. Pyogenes, Strep. Pneumoniae, Pseudomonas

aeruginosa, E. coli,
Klebsiella species, Proteus species, Bacteroids, Haemophilus influenza

Tsongo Lawrence Msc (MUK) 204

Commensals

 The middle ear is normally sterile. Commensal organisms may be found in

the external ear such as;
 Strep. Viridians, Bacillus species, Corynebacterium species, Staph.
Epidermidis, E. coli
Other Coliforms inclusive
Nossal swabs

 Staph. aureus
 Strep. Pyogenes
 Niesseria meningitidis
 Haemophilus influenza
Commensals

 Strep. Pneumoniae, Strep. Viridians, Enterococci, Staph. Species,

Micrococci, Diphtheroids, Neisseria and Haemophilus species
Throat and mouth swabs

 Strep. Pyogenes, C. diphtheria, C. ulcerans, Candida albicans, Vincenti‟s

organisms, Bacteroids
Commensals

 Strep. Viridians, Strep. Pneumoniae, Staph. Epidermidis, Staph. Albus,

Micrococci
Lactobacilli, Diphtheroids, Fusobacteria, Coliforms, Bacteroids, Neisseria
pharyngitidis
H. influenza (non capsulated strains)

9.2.7 Eye Specimens

Collection

 This is done by experienced healthy worker.

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 Conjuctival scrapings are collected to detect Chlamydia trachomatis.
 In case of eye discharge, use a dry, sterile cotton wool swab to collect the
discharge and insert in the Amie‟s transport media and label.
Laboratory examination

Macroscopic
 Examine for appearance of the specimen.

Culture the specimen

 Culture on BA, MacConkey, Chocolate, Loeffler‟s serum slopes, Modified
New York medium (MNYC) for gonococcal conjunctivitis.
 Incubate the chocolate plate in the carbondioxide atmosphere at 37°C
and the rest of the plates aerobically at 37°C for overnight.
 Examine the specimen microscopically by performing a gram stain and
examine using the 100× objective to look for gram positive and gram
negative organisms, intracellular diplococci that could be Neisseria
gonorrhoeae.
Do a Giemsa stain for Chlamydia trachomatis
 Make a smear of a specimen, airdry and fix in methanol.
 Then stain by Giemsa technique.
 Examine the smear using 100× objective and look for epithelial cells that
contain inclusion bodies. Chlamydia inclusion bodies vary in size and
shape and often touch or surround the nucleus of the epithelial cells. They
stain blue mauve. If it is more mature, it may contain red mauve staining
element.
Report
Chlamydia inclusion bodies present or
No Chlamydia inclusion bodies seen.
Day two and onwards, examine and report any growth of cultures.
Possible pathogens

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 Staph. Aureus, Strep. Pneumoniae, Strep. Pyogenes, Neisseria
gonorrhoeae, H. influenza
P. aeruginosa, Chlamydia trachomatis, Enterobacteria, Moraxella lacunata
Commensals

 Strep. Viridans, Staph. Species, Diphtheroids, non pathogenic Neisseria

species, Moraxella species.

9.2.8 Stool Specimens

Possible pathogens

 Shigella species, Salmonella, Vibrio cholera, Campylobacter enteritis, E.

coli in case of children below 5 years. Above 5 years is a normal flora,
many parasites and viruses.
 In case of immuno compromised patients, Clostridium perfringenes type A
and C, Bacillus cereus (Toxin), Candida albicans, Yersinia enterocoritica.

 Gastrointestinal tract has many normal flora mostly gram positives and
negatives.

Collection of stool

 Give a patient a clean, dry, leakproof container, free from disinfectant

and ask the patient not to contaminate the faeces with urine.
Note; the container need not to sterile.

 Rectal swabs are collected by inserting cotton wool swabs into the rectum
for about 10 seconds and avoid contamination with the bacteria from the
anal skin.
 Label the specimen and send to the laboratory immediately.
Transportation

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 Insert the rectal swab in a container of cary-blair transport media where
salmonella, shigella, Vibrio survive very well up to 48 hours.
 If cholera is suspected, transfer about 1ml of the specimen into 10mls of
sterile alkaline peptone water of a PH 8.6.
 Label and send to the laboratory within 8 hours of collection.
If worms or tapeworm segments are present, transfer to a container of normal
saline and send to the laboratory.

Laboratory examination

i. Describe the appearance of the specimen.

ii. Examine the specimen microscopically in saline, eosin and iodine.
iii. Do motility and slide immobilization test if cholera is suspected.
 Examine an alkaline peptone water culture for vibrios with a rapid and
darting motility under a dark field microscope.
Culture
 If the specimen is formed or semi-formed, make a thick suspension of it in
about 1ml of sterile peptone water.
 Inoculate on xylose lysine deoxycholate agar (XLD) and selenite F broth.
Incubate both at 37°C for overnight.
 XLD is a selective media for isolation of salmonella and shigella and should
be incubated aerobically.
 Selenite F broth is an enrichment medium and selective broth for
salmonella.
 Inoculate the specimen on campylobacter selective media if the
organism is suspected.
 Inoculate the specimen in alkaline peptone water at PH 8.6 and on
Thiosulphate citrate bile salt (TCBS) and incubate at 37°C for 5-8 hours if
cholera is suspected.

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 MacConkey agar and salmonella shigella agar (SSA) if Yersinia
enterocoritica is suspected. Incubate aerobically at room temperature of
20-28°C up to 48 hours.
Day two and onwards, examine and report the growth cultures then set
antimicrobial sensitivity testing.

9.2.9 Skin Specimens

Collection

 Use a sterile, dry, cotton wool swab; collect a sample of discharge from
the infected tissue. If there is no discharge, use a swab moistened in a
sterile normal saline to collect the specimen.
 If the tissue is deeply ulcerated and necrotic, aspirate the sample of the
infected material from the blisters from the walls of the ulcer using a sterile
needle and syringe.
 Skin specimens of the ringworms, clean affected or infected area with 70%
ethanol, then collect the skin scales, crusts, pieces of nails or hair on a
clean piece of paper.
 Skin (snips) for M. leprae, collect from the earlobe because it survives well
in the coldest parts of the body from visible lesions.
 The backs of both forearms, the front of the thighs just above the knees
and front of the legs just below the knees are some of the sites.
 Clean the site with 70% ethanol; hold a fold of the skin tightly between the
thumb and forefinger until it becomes pale due to loss of blood.
 Using a sterile blade, make a small cut through the skin surface 5mm long
and 2-3mm deep. The smear should be bloodless. Then place on a new
clean glass slide.
 Airdry and fix in methanol or heat.
Laboratory diagnosis

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 Culture the specimen on BA, MacConkey, Sabouraud agar for fungal
infection, Lowenstein jenstein media for TB.
 Inoculate and incubate the BA and MacConkey at room temperature for
48 hours.
 Perform a gram stain technique, KOH preparation for ringworm fungi,
Giemsa or Wayson‟s stained smear for bubonic plague and examine to
look for the bipolar staining of Yersinia pestis.
 ZN method preferably a modified method is examined to look for AFBs of
M. leprae.
 Polychrome methylene blue is done if cutaneous anthrax is suspected.
 Make a wet preparation for dark microscopy to detect motile Treponemes
if yaws and pinta are suspected.
 Day two and onwards, examine and report the culture and set
antimicrobial sensitivity testing.

Possible pathogens

 Mycobacterium leprae, Mycobacterium ulcerans, Treponema species,

Bacillus anthracis
Corynebacterium ulcerans, Staphylococcus aureus, Strep. Pyogenes, E.
coli, Proteus
Yersinia pestis, P. aeruginosa, Vincenti‟s organisms, Fungi e.g. ringworm
fungi, candida species, Parasites e.g. leishmania species, onchocerca
volvulus, drancunculus medinensis
Commensals

 Staph. Species, Micrococci, Anaerobic Streptococci, Strep. Viridians,

Enterococci
Diphtheroids, E. coli and other Coliforms

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 REFERENCES

1. Aycliffe, G. A. J., Collins, B. J. & Taylor, L. J. (2000). Hospital acquired

infections: Principles and prevention. Arnold.
2. John, DT & Petri, WA (2006). Markell and Voge's Medical Parasitology:
Saunders
3. Virella , G. (2007) Medical Immunology. Informa Healthcare Publishers.
4. Brooks, GF, Carroll, KC, Butel, JS, et al (2010). Jawetz, Melnick, & Adelberg's
Medical Microbiology: McGraw-Hill Medical
5. Monica Cheesbrough (1991 ) Medical Laboratory Manual for Tropical
Countries Vol 1: Tropical Health Tech
6. Monica Cheesbrough ( 1991) Medical Laboratory Manual for Tropical
Countries Vol 2: Tropical Health Tech
7. James D. Watson, Micheal Gilman, Jan Witkowski and Mark Zoller,
Recombinant DNA, 2nd Edition, Scientific American Books, New York 1992
8. Practical biochemistry-principles and techniques, 4 th Edition, by Keith
Wilson and John Walker, Cambridge University press, 1994.
9. Biochemical Techniques- theory and practice, by John F. Robyt and
Bernard J. White, 1987.

10. Molecular Cell Biology, 4th editionHarvey Lodish, Arnold Berk, S Lawrence
Zipursky, Paul Matsudaira, David Baltimore, and James Darnell.New York:
W. H. Freeman; 2000

11. Molecular biology of the cell – by Alberts; 2001

12. Chemistry, Cell Biology and Genetics: Volume One by Robert Brooker,
Robert J. Brooker in Books

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