BACTERIOLOGY 1

BMA 1

Mrs Patience Ewane

PhD Fellow
ESS-UCAC
 Plan

1. Introduction to Microbiology
2. The Microbial World
3. Structure of bacteria
4. Classification of bacteria
5. Cultivation of bacteria
6. Bacterial nutrition
7. Bacterial growth
8. Bacterial genetics
9. Sterilization and disinfection
Learning Objective

• At the end of the lesson, the student

should be able to:

1. Identify the structure of bacterial cell

2. Do simple and differential staining

methods

3. Describe the essential nutrients required

for bacterial growth

4. Describe the mechanisms of genetic

variation in bacterial cell
1.1 INTRODUCTION TO
MICROBIOLOGY

Microbiology is defined as the study or

organisms and agents that are too small to
be seen with the naked eye that is the study
of microorganisms. Objects less than 1
millimetre in diameter cannot be seen
clearly and must be examined with a
microscope thus MCB is concerned with
organisms this small and even smaller.
However, some eukaryotic cells like bread
mould and filamentous algae are studied by
microbiologist, yet they are visible to the
naked eye.
Microorganisms include acellular entities
like viruses, prokaryotic and eukaryotic
cells. They are diverse and their
classification has always been a challenge
for taxonomists. Their early descriptions as
either plants or animals were too simple.
For instance some microbes are motile like
animals but also have cell walls and are
photosynthetic like plants. Procaryotic
cells (Greek pro- before, and karyon- nut
or kernel); organisms with a primordial
nucleus, have a much simpler morphology
and lack a true membrane-delimited
nucleus. Eucaryotic cells (Greek, eu- true
and karyon- nut or kernel) have a
membrane enclosed nucleus, they are more
complex morphologically and are usually
larger than procaryotes.

Cellular microorganisms are found in all

three domains of life: Bacteria, Archaea,
Eucarya. Organisms are divided into five
kingdoms namely: Monera, Protista,
Fungi, Animalia and Plantae.
Microorganisms (except viruses which are
acellular) are classified in the first three
kingdoms i.e Monera, Protista, Fungi.

Microbiology is defined not only by the

size of its subjects but techniques it uses to
study them. The development of
microbiology (MCB) as a scientific
discipline has depended on the availability
of the microscope and the ability to isolate
and grow pure cultures of microorganisms.
The development of these techniques is in
part due to the disproving of the Theory of
Spontaneous Generation and Koch’s
Postulate establishing that microrganisms
can cause disease.

MCB is a large discipline that will

continue to grow to impact human welfare,
with significance in science and industry.

Subdivisions of Microbiology

Bacteriology deals with bacteria.

Mycology deals with fungi. Virology deals
with viruses.
Five kingdom classification of organisms
 Five kingdom classification of organisms

Revision
1 Describe the field of microbiology in
terms of the size of its subject material
and the nature of its techniques
2 describe and contrast procaryotic and
eukaryotic cells
3 describe and contrast the five kingdom
classification system with the three
domain system. Why do you think
viruses are not included in either
system?
History of Microbiology

Mankind has always been affected by

diseases which were originally believed to
be visitations by the gods and meant to
punish evil doers. However, even before
microorganisms were seen, some
investigators suspected their existence and
responsibility for disease. Hippocratus
(between 450-380 B.C), father of
medicine, observed that ill health resulted
due to changes in air, winds, water,
climate, food, nature of soil and habits of
people.

Varro (117-26 BC) said that disease was

caused by animated particles invisible to
naked eye but which were carried in the air
through the mouth and nose into the body.

The Roman philosopher Lucretius (about

98-55 B.C) suggested that disease was
caused by invisible living creatures.

Girolamo Fracastorius (1500 A.C.)

proposed that the agents of communicable
disease were living germs, that could be
transmitted by direct contact with humans
and animals, and indirectly by objects ; but
no proof because of lacking experimental
evidence.

The earliest microscopic observations

appear to have been made between 1625
and 1630 A.C on bees and weevils by the
Italian Francesco Stelluti, using a
microscope probably supplied by Galileo.

In 1665, the first drawing of a

microorganism was published by Robert
Hooke in a publication titled
Micrographia.

Antony Van Leeuwenhoek (1632-1723

A.C.), was the first person to publish
extensive and accurate observations of
microorganisms. He observed
“animalcules” using simple microscope
composed of double convex glass lenses
held between two silver plates. He was the
first who properly described the different
shapes of bacteria. His microscope s could
magnify around 50 to 300 times and he
may have illuminated his liquid specimens
by placing them between two pieces of
glass and shining light through them at 45
angle to the specimen plane. This would
have provided a form of dark –field
illumination in which the organisms
appeared as bright objects against a dark
background and made bacteria clearly
visible.
Antony Van Leeuwenhoek’s Microscope
Circa
 Animacules

Although Leeuwenhoek was not concerned

about the origin of microorganism; many
other scientists were searching for an
explanation for spontaneous appearance of
living things from decaying meat,
stagnating ponds, fermenting grains and
infected wounds.

On the bases of this observation, two major

theories were formulated.

1. Theory of Abiogenesis

2. Theory of Biogenesis

 Theory of Abiogenesis deals with the

theory of spontaneous generation; stating
that living things originated from non-
living things. Aristotle (384-322 BC) was
the founder of the theory spontaneous
generation.

He observed spontaneous existence of

fishes from dried ponds, when the pond
was filled with rain.
Francesco Redi (1626-1697): He is the
scientist who first tried to set an
experiment to disprove spontaneous
generation.

 He put meat in a bottle and covered it

with a gauze.
 He observed that the flies laid eggs
from which the maggots developed.
 He said maggots did not developed
from meat but from flies egg.
 Spontaneous generation theory
 Theory of Biogenesis states that life
comes from pre-existing life. Louis Pasteur
(1822-1895 GC) was the scientist who
disproved the theory of abiogenesis. He
designed a large curved flask (Pasteur
goose neck flask) and placed a sterile
growth broth medium. Air freely moved
through the tube; but dust particles were
trapped in the curved portion of flask.
Microbial growth in the broth was not
seen. Therefore Pasteur proved that micro-
organisms entered to substrates through the
air and micro-organisms did not evolve
spontaneously.

Major contributions of Louis Pasteur

1. Microbial theory of fermentation

2. Principles and practice of sterilization

and pasteurization
3. Control of diseases of silk worms

4. Development of vaccines against

anthrax and rabies.

5. Discovery of streptococci

The germ theory of disease

The complete establishment of the germ

theory of disease depended on the work of
a German scientist, Robert Koch (1843-
1910).

Major achievements of Robert Koch

1. Discovery and use of solid medium in
bacteriology

2. Discovery of causative agents of

tuberculosis and cholera.

3. Koch’s phenomenon

4. Koch’s postulates

Koch’s postulates: proof of germ theory

of disease

A microorganism can be accepted as a

causative agent of an infectious disease
only if the following conditions are
satisfied.

1. The micro-organism should be found in

every case of the disease and under
conditions which explain the pathological
changes and clinical features.

2. It should be possible to isolate the

causative agent in pure culture from the
lesion.

3. When such pure culture is inoculated

into appropriate laboratory animal, the
lesion of the disease should be reproduced.

4. It should be possible to re-isolate the

bacterium in pure culture from the lesion
produced in the experimental animal.

5. Nowadays additional postulate is

mentioned i.e. Specific antibody to the
bacterium should be detectable in the
serum during the course of the disease.
It has not been possible to fulfil every one
of Koch’s postulates, but by adhering to
them as closely as possible, serious errors
have been prevented.

Exceptions to Koch’s postulates

1. Many healthy people carry pathogens

but do not exhibit symptoms of the disease.

2. Some microbes are very difficult or

impossible to grow in vitro (in the
laboratory) in artificial media. E.g.
Treponema pallidum

3. Many species are species specific. E.g.

Brucella abortus cause abortion in animals
but no report in humans.
4. Certain diseases develop only when an
opportunistic pathogen invades
immunocompromised host.
2. THE MICROBIAL WORLD:
TAXONOMIC CLASSIFICATION OF
ORGANISMS

TAXONOMY is the science of organismal

classification. Classification is the
assignment of organisms (species) into an
organised scheme of naming. Idealy these
schemes are based on evolutionary
relationships (i.e the more similar the
name, the closer the evolutionary
relationships). Thus, classification is
concerned

with:-
1. The establishment of criteria for
identifying organisms & assignment to
groups (what belongs where)

2. The arrangement of organisms into

groups of organism (e.g. At what level of
diversity should a single species be split
into two or more species?).

3. Consideration of how evolution resulted

in the formation these groups.

TAXON:-

„ A group or category of related organisms.

Two key characteristics of taxa are:

-Members of lower level taxa (e.g.

Species) are more similar to each other
than are members of higher level taxa (e.g.
Kingdom or domain).

-Member of specific taxa are more similar

to each other than any are to members of
different specific taxa found at the same
hierarchical level (e.g. Humans are more
similar to apes, i.e. comparison between
species, than either is similar to, for
example, Escherichia coli). Thus once you
know that two individuals are member of
the same taxon, you can inter certain
similarities between the two organisms.

NOTE that taxa are dynamic, changing as

our knowledge of organism and
evolutionary relationships change.
BINOMIAL NOMENCLATURE

- Organisms are named using binomial

nomenclature (viruses are exceptions)

- Binomial nomenclature employs the

names of the two level taxa, genus and
species, to name a species. Binomial
nomenclature includes:

i. Genus comes before species (e.g.,

Escherichia coli)

ii. Genus name is always capitalized (e.g.,

Escherichia)
iii. Species name is never capitalized (e.g.,
coli)

iv. Both names are always either italicized

or underlined (e.g Escherichia coli)

v. The genus name may be used alone, but

not the species name (i.e saying or writing
“Escherichia “alone is legitimate while
saying or writing “coli” is not)

Classification hierarchy

Domain:

Kingdom:

Phylum:

Class:

Order:
Family:

Genus:

Species:

A Strain

a) A strain in some ways is equivalent to a

breed or subspecies among plants or
animal. Strain is the level below the
species

b) Two members of the same strain are

more similar to each other than either is to
an individual that is a member of a
different strain, even if all three organisms
are members of the same species

Bacterial species
- A bacterial species is defined by the
similarities found among its members.
Properties such as biochemical reactions,
chemical composition, cellular structures,
genetic characteristics, and immunological
features are used in defining a bacterial
species. Identifying a species and
determining its limits presents the most
challenging aspects of biological
classification for any type of organism.

- A formal means of distinguishing

bacterial species is by employing a
dichotomous key to guide the selection of
test used to efficiently determine those
bacterial properties most relevant to
bacterial identification

The five kingdom system

The five kingdom system was first

proposed in 1969 and is showing its age

The five kingdoms include:

i. Plantae (the plants)

ii. Fungi (the fungi)

iii. Animalia (the animals)

iv. Protista (the unicellular eukaryotes)

v. Monera (the prokaryotes)

Kingdom of Monera

Three categories:
- Eubacteria

Are our common, everyday bacteria, some

of which are disease – causing; also the
taxon from which mitochondria originated.

- Cyanobacteria

Are photosynthetic eubacteria, the taxon

from which chloroplast originated

- Archaeobacteria

Are distinctive in their adaptation to

extreme environments (e.g., very hot, salty,
or acidic) though not all archaeobacteria
live in extreme environments.

These distinctions are more phenotypic

than they are evolutionary (i.e., a
cyanobacteria is a eubacteria, and neither is
an archaebacteria).

Kingdom Protista

Protista like Monera consist mostly of

unicellular organisms. Distinctively,
however, the members of Kingdom
Protista are all eukaryotic while the
members of kingdom Monera are all
prokaryotic. Some members of Protista are
multicellular, however Kingdom Protista
represents a grab bag, essentially the place
where the species are classified when they
are not classified as either fungi, animals or
plants.
Kingdom Fungi

Unlike Protista, the eukaryotic fungi are

typically non – aquatic species. They
traditionally are nutrients absorbers plus
have additional distinctive features. They
do exist unicellular fungi, which we call
yeast

DOMAIN

The domain is a taxonomic category that,

depending on point of view, is either above
the level of kingdom or supersedes the
kingdom.

The domain system contains three

members

¾ Eukaryotes (domain Eukarya)

¾ Eubacteria (domain Bacteria)

¾ Archaebacteria (domain Archaea)

EUKARYOTIC CELL

Eu- true Karyote- nucleus

The eukaryotic cell has a true membrane

bound nucleus, usually containing multiple
chromosomes, a mitotic apparatus, a well-
defined endoplasmic reticulum and
mitochondria.

PROKARYOTIC CELL

Pro- primitive Karyote- nucleus

The prokaryotic cell possesses naked DNA

without associated basic proteins, divides
amitotically by binary fission and bounded
by a semi rigid cell wall.
Table 1.1. The distinguishing features
between

Features Eukaryotic Prokaryoti

cell c cell
Size 10µm 1µm
Nuclear Present Absent
membrane
Chromosome Multiple Single
Nucleolus Present Absent
Histones Present Absent
Sexual Present Absent
reproduction
Cytoplasmic 80 70
ribosomes
Mitochondria Present Absent
Endoplasmic Present Absent
recticulum
Lysosome Present Absent
Microfilament Present Absent
s and tubules
Site of Mitochondr Cell
oxidative ia membrane
phosphorylati
on
Site of Chloroplast Cell
photosynthesi membrane
s
Peptidoglycan Absent Present
Cell Proteins phospholipi
membranes and sterols ds

Bacterial Cell: General properties

• Typical prokaryotic cell

• Contain both DNA and RNA

• Most grow in artificial media

• Replicate by binary fission

• Almost all contain rigid cell wall

• Sensitive to antimicrobial agent

3. STRUCTURE OF BACTERIA

Bacterial structure is considered at three

levels.

1. Cell envelop proper: Cell wall and cell

membrane.

2. Cellular element enclosed within the cell

envelope: Mesosomes, ribosomes, nuclear
apparatus, polyamines and cytoplasmic
granules.

3. Cellular element external to the cell

envelope: Flagellum, Pilus and
Glycocalyx.
1. Cell envelop proper

A. Cell wall

Multi layered structure and constitutes

about 20% of the bacterial dry weight.
Average thickness is 0.15-0.5 μm. Young
and rapidly growing bacteria have thin cell
wall but old and slowly dividing bacteria
has thick cell wall. It is composed of N-
acetyl Muramic acid and N-acetyl
Glucosamine back bones cross linked with
peptide chain and pentaglycine bridge.
Fig. 1.1 Ultrastructure of Bacteria

Components of cell wall of Gram

negative bacteria

1. Peptidoglycan

2. Lipoprotein

3. Phospholipid

4. Lipopolysaccharide
Components of cell wall of Gram
positive bacteria

1. Peptidoglycan

2. Teichoic acid
Fig. 1.2 Cell wall of Gram Positive &
Gram Negative Bacteria

Functions of the bacteria cell wall

1. Provides shape to the bacterium

2. Gives rigidity to the organism

3. Protects from environment

4. Provides staining characteristics to the

bacterium
5. Contains receptor sites for
phages/complements

6. Site of action of antibody

7. Contains toxic components to host

Bacteria with defective cell walls

Bacteria without cell wall can be induced

by growth in the presence of antibiotics
and a hypertonic environment to prevent
lysis. They are of three types:

1. Protoplasts: Derived from Gram-

positive bacteria and totally lacking cell
walls; unstable and osmotically fragile;
produced artificially by lysozyme and
hypertonic medium: require hypertonic
conditions for maintenance.

2. Spheroplast: Derived from Gram-

negative bacteria; retain some residual but
non-functional cell wall material;
osmotically fragile; produced by growth
with penicillin and must be maintained in
hypertonic medium.

3. L- forms: Cell wall-deficient forms of

bacteria usually produced in the laboratory
but sometimes spontaneously formed in the
body of patients treated with penicillin;
more stable than protoplasts or
spheroplasts, they can replicate in ordinary
media.
B. Cell membrane

Also named as cell membrane or

cytoplasmic membrane. It is a delicate
trilaminar unit membrane. It accounts for
30% of the dry weight of bacterial cell. It is
composed of 60% protein, 20-30% lipids
and 10-20% carbohydrate.

Function of cell membrane

1. Regulates the transport of nutrients and

waste products into and out of the cell.

2. Synthesis of cell wall components

3. Assists DNA replication

4. Secrets proteins

5. Carries on electron transport system

6. Captures energy in the form of ATP
Cellular element enclosed within the cell
envelop

A. Mesosomes

Convoluted invagination of cytoplasmic

membrane often at sites of septum
formation. It is involved in DNA
segregation during cell division and
respiratory enzyme activity.
B. Ribosomes

Cytoplasmic particles which are the sites of

protein synthesis. It is composed of RNA
(70%) and proteins (30%) and constitutes
90% of the RNA and 40% of the total
protein.

The ribosome monomer is 70s with two

subunits, 30s and 50s.

C. Polyamines

They are of three types

. Putrescin

. Spermidine

.Spermine
It is found in association with bacterial
DNA, ribosomes and cell membrane.

Function of polyamines

1. Antimutagenic.

2. Prevent dissociation of 70s ribosome

into subunits.

3. Increase resistance of protoplast lysis.

D. Cytoplasmic granules: represent

accumulated food reserves.

Nature of granules

. Glycogen

. Poly-beta hydroxy butyrate

. Babes-Ernst (Volutin granules)

E. Nuclear apparatus
Well defined nucleus and nuclear
membrane, discrete chromosome and
mitotic apparatus are not present in
bacteria; so nuclear region of bacteria is
named as nuclear body, nuclear
apparatus and nucleoid.

Bacterial genome consists of single

molecule of double stranded DNA
arranged in a circular form. Besides
nuclear apparatus, bacteria may have extra
chromosomal genetic material named as
plasmids.

Plasmids do not play any role in the

normal function of the bacterial cell but
may confer certain additional properties.
(E.g. Virulence, drug resistance) which
may facilitate survival and propagation of
the microorganism.

3. Cellular element external to the cell

envelop

A. Glycocalyx (capsule and slime layer)

Capsule is gel firmly adherent to cell

envelop.

Slime is gel easily washed off from cell

envelope.

All bacteria have at least a thin slime layer.

Capsule is composed of polysaccharide

and protein (D-Glutamate of Bacillus
anthracis)
Features of capsule

1. Usually weakly antigenic.

2. Not necessary for viability.

3. Endows virulence.

4. Protects from phagocytosis.

5. Capsulated strains are invariably non-

motile.

6. Visualized by negative staining and

capsule staining.

7. Detected by quellung phenomenon.

B. Flagellum
It is the organ of locomotion in bacterial
cell and consists of three parts. These are

.The filament

. The hook

. The basal body

The basal body and hook are embedded in

the cell surface while the filament is free
on the surface e of bacterial cell.

Their presence in bacterial cell is detected

by

. Hanging drop preparation

. Swarming phenomenon on surface of

plate agar

. Motility media
. Special staining methods

. Silver impregnation methods

. Dark –field microscopy

. Electron microscopy

Size: 3-20μm in length and 0.01-0.013μm

in diameter.

It is composed of protein named as

flagellin.

The flagella antigen in motile bacterium is

named as H (Hauch) antigen.

Flagella arrangements

1. Atrichous: Bacteria with no flagellum.

2. Monotrichous: Bacteria with single

polar flagellum.
3. Lophotrichous: Bacteria with bunch of
flagella at one pole.

4. Amphitrichous: Bacteria with flagella

at both poles.

5. Peritrichous: Bacteria with flagella all

over their surface.

It is the organ of motility found in

periplasmic space of spirochetes.

C. Pili (fimbriae)

It is hair like structure composed of protein

(pilin)

Two types (Based on function)

. Common pili: The structure for

adherence to cell surface.
. Sex pili: The structure for transfer of
genetic material from the donor to the
recipient during the process of conjugation.

Table 1.2. Comparison between flagella

and pili

Character Flagella Pilli

Size thickness ++++ +
Origin Cell Cell wall
membrane
Organ of Yes No
locomotion
Organ of No Yes
adhesion
Required for No Yes
conjugation
D. Spores

Resting cells which are capable of surviving

under adverse environmental conditions like
heat, drying, freezing, action of toxic
chemicals and radiation. Bacterial spore is
smooth walled and oval or spherical in shape.

 It does not take up ordinary stains.

 It looks like areas of high refractivity
under light microscope.
 It is significant in spread of disease and
indicator of sterility of materials.

Spores are detected by

. Simple staining methods

. Special staining methods

Endospores develop within vegetative
bacterial cells of genera like: Bacillus,
Clostridium (rods) and Sporisarcina (cocci).

Arrangements of spores

1. No bulging of cell wall

. Oval central

. Oval sub terminal

. Spherical central

2. Bulging of cell wall

. Oval sub terminal

. Oval terminal

. Spherical terminal

. Free spore

Endospores may be centrally located, close

to one end (subterminal) or definitely terminal.
Sometimes it may be so swollen that it swells
the sporangium.

Different spore types

1.4. Classification of bacteria

Bacterial classification depends on the

following characteristics.

1. Morphology and arrangement

2. Staining

3. Cultural characteristics

4. Biochemical reactions

5. Antigenic structure

6. Base composition of bacterial DNA

Morphology and staining of bacteria are the

commonly used characteristics to classify
bacteria.
1. Morphology of bacteria

When bacteria are visualized under a light

microscope, the following morphology are
seen.

1. Cocci (singular coccus): Round or oval

bacteria measuring about 0.5-1.0μm in
diameter. They are found in single, pairs
diplococci, chains or clusters.

Diplococci arise when cocci divide and remain

together forming pairs. Long chains result
when cells adhere after repeated divisions in
one plane as seen in the genera Streptococcus,
Enterococcus and Lactococcus.
Staphylococcus divides in random planes to
generate irregular grapelike clumps. Division
in two or 3 planes can produce symmetrical
clusters of cocci. Members of the genus
Micrococcus often divide in 2 planes to form
square groups of four cells called tetrads. In
genus Sarcina, cocci divide in 3 planes
forming cubical packets of 8 cells.

Bacilli (singular bacillus): Stick-like, rod-

like bacteria with rounded, tapered, square or
swollen ends; with a size measuring 1-10μm
in length by 0.3-1.0μm in width. Example:
 Bacillus megaterium Coccobacilli are short
and round thus resemble cocci. Coccobacilli
(singular coccobacillus): Short rods.

Spiral: Spiral shaped bacteria with regular or

irregular distance between twisting. Vibrios
most closely resemble rods as they are
comma-shaped. Spiral-shaped procaryotes can
either be classified as spirilla which usually
have tufts of flagella at one or both ends of the
cell or spirochetes which are flexible and have
a unique, internal flagellar arrangement

E.g. Spirilla and spirochetes

2. Staining of bacteria

Bacterial staining is the process of colouring

of colourless bacterial structural components
using stains (dyes).The principle of staining is
to identify microorganisms selectively by
using dyes, fluorescence and radioisotope
emission. Staining reactions are made possible
because of the physical phenomena of
capillary osmosis, solubility, adsorption, and
absorption of stains or dyes by cells of
microorganisms.

Individual variation in the cell wall

constituents among different groups of
bacteria will consequently produce variations
in colours during microscopic examination.
Nucleus is acidic in character and hence, it has
greater affinity for basic dyes. Whereas,
cytoplasm is basic in character and has greater
affinity for acidic dyes.

There are many types of affinity explaining

this attraction force:

1. hydrophobic bonding

2. reagent-cell interaction

3. reagent-reagent interaction

4. ionic bonding

5. hydrogen bonding

6. covalent bonding

Why are stains not taken up by every

microorganism?
Factors controlling selectivity of microbial
cells are:

1. number and affinity of binding sites

2. rate of reagent uptake

3. rate of reaction

4.rate of reagent loss (differentiation or

regressive staining)

Properties of dyes

Why dyes colour microbial cells?

Because dyes absorb radiation energy in

visible region of electromagnetic spectrum i.e.,
light (wave length 400-650). And absorption is
anything outside this range is colourless. E.g.,
acid fuschin absorbs blue green and transmits
red.
General methods of staining

1. Direct staining

Is the process by which microorganisms are

stained with simple dyes. E.g., methylene
blue

2. Indirect staining – is the process which

needs a mordant. A mordant is the substance
which, when taken up by the microbial cells
helps make dye in return, serving as a link or
bridge to make the staining recline possible. It
combines with a dye to form a coloured
“lake”, which in turn combines with the
microbial cell to form a “ cell-mordant-dye
complex”.

It is an integral part of the staining reaction

itself, without which no staining could
possibly occur. E.g., iodine. A mordant may
be applied before the stain or it may be
included as part of the staining technique, or it
may be added to the dye solution itself.

An accentuator, on the other hand is not

essential to the chemical union of the
microbial cells and the dye. It does not
participate in the staining reaction, but merely
accelerates or hastens the speed of the staining
reaction by increasing the staining power and
selectivity of the dye.

Progressive staining
- Is the process whereby microbial cells are
stained in a definite sequence, in order that a
satisfactory differential coloration of the cell
may be achieved at the end of the correct time
with the staining solution.

Regressive staining

- With this technique, the microbial cell is first

over stained to obliterate the cellular desires,
and the excess stain is removed or decolorized
from unwanted part.

Differentiation (decolourization)

- Is the selective removal of excess stain from

the tissue from microbial cells during
regressive staining in order that a specific
substance may be stained differentially from
the surrounding cell. Differentiation is usually
controlled visually by examination under the
microscope

Uses

1. To observe the morphology, size, and

arrangement of bacteria.

2. To differentiate one group of bacteria from

the other group.

Biological stains are dyes used to stain

microorganisms. Types of microbiological
stains

. Basic stains
. Acidic stains

. Neutral stains

NB: This classification is not based on PH of

stains. Basic stains are stains in which the
colouring substance is contained in the base
part of the stain. The acidic part is colourless.
Eg. Acidic stains are stains in which the
colouring substance is contained in the acidic
part of the stain. The base part is colourless. It
is not commonly used in microbiology
laboratory. Eg. Eosin stain

Neutral stains are stains in which the acidic

and basic components of stain are coloured.
Neutral dyes stain both nucleic acid and
cytoplasm. Eg. Giemsa stain

Types of staining methods

1. Simple staining method

2. Differential staining method

3. Special staining method

1. Simple staining method

It is type of staining method in which only a

single dye is used. Usually used to
demonstrate bacterial morphology and
arrangement. Two kinds of simple stains

1. Positive staining: The bacteria or its parts

are stained by the dye.

Eg. Carbol fuchsin stain, Methylene blue

stain, Crystal violet stain

Procedure:

. Make a smear and label it.

. Allow the smear to dry in air.

. Fix the smear over a flame.

.Apply a few drops of positive simple stain

like 1% methylene blue,

1% carbolfuchsin or

1% gentian violet for 1 minute.

. Wash off the stain with water.

. Air-dry and examine under the oil immersion

objective.

2. Negative staining: The dye stains the

background and the bacteria remain unstained.
Eg. Indian ink stain, Negrosin stain

2. Differential staining method

Multiple stains are used in differential staining

method to distinguish different cell structures
and/or cell types. Eg. Gram stain and Ziehl
Neelson stain
A. Gram staining method

Developed by Christian Gram. Most bacteria

are differentiated by their Gram reaction due
to

differences in their cell wall structure.

Gram-positive bacteria are bacteria that stain

purple with crystal violet after decolourizing
with acetone-alcohol.

Gram-negative bacteria are bacteria that

stain pink with the counter stain (safranin)
after losing the primary stain (crystal violet)
when

treated with acetone-alcohol.

Required reagents:
. Gram’s Iodine

. Acetone-Alcohol

. Safranin

Procedure:

1. Prepare the smear from the culture or from

the specimen.

2. Allow the smear to air-dry completely.

3. Rapidly pass the slide (smear upper most)

three times through the flame.

4. Cover the fixed smear with crystal violet for

1 minute and wash with distilled water.

5. Tip off the water and cover the smear with

Gram’s iodine for 1 minute.

6. Wash off the iodine with clean water.

7. Decolorize rapidly with acetone-alcohol for
30 seconds.

8. Wash off the acetone-alcohol with clean

water.

9. Cover the smear with safranin for 1 minute.

10. Wash off the stain wipe the back of the

slide. Let the smear to air-dry.

11. Examine the smear with oil immersion

objective to look for bacteria.

Interpretation:

. Gram-positive bacterium ……………Purple

. Gram-negative bacterium …………..Pink

B. Ziehl-Neelson staining method

Developed by Paul Ehrlichin 1882, and

modified by Ziehl and Neelson

Ziehl-Neelson stain (Acid-fast stain) is used

for staining Mycobacteria which are hardly
stained by Gram staining method.
Once the Mycobacteria are stained with
primary stain it cannot be decolorized with
acid, so named as acid-fast bacteria.

Reagents required:

. Carbol-fuchsin

. Acid-Alcohol

. Methylene blue/Malachite green

Procedure for Ziehl-Neelson staining method

1. Prepare the smear from the primary

specimen and fix it by passing through the
flame and label clearly

2. Place fixed slide on a staining rack and

cover each slide with concentrated carbol
fuchsin solution.
3. Heat the slide from underneath with spirit
lamp until vapour rises (do not boil it) and
wait for 3-5 minutes.

4. Wash off the stain with clean water.

5. Cover the smear with 3% acid-alcohol

solution until all colour is removed (two
minutes).

6. Wash off the stain and cover the slide with

1% methylene blue for one minute.

7. Wash off the stain with clean water and let

it air-dry.

8. Examine the smear under the oil immersion

objective to look for acid fast bacilli.

Interpretation:

Acid fast bacilli…………..Red

Back ground………………Blue

Reporting system

0 AFB/100 field …………………No AFB

seen

1-2 AFB/ 300 field……………….. Scanty

1-10 AFB/100 field………………1+

11-100AFB/100 field.……………2+

1-10 AFB/field…… ……………3+

>10 AFB/field……………… ….4+

NB: AFB means number of acid fast bacilli

seen.
3. Special stains

a. Spore staining method: Malachite green

Procedure:

1. Prepare smear of the spore-forming bacteria

and fix in flame.

2. Cover the smear with 5% malachite green

solution and heat over steaming water bath for
2-3 minutes.

3. Wash with clean water.

4. Apply 1% safranin for 30 seconds.

5. Wash with clean water.

6. Dry and examine under the oil immersion

objective.

b. Capsule staining method: Welch method

Procedure:

1. Prepare smear of capsulated bacteria.

2. Allow smear to air-dry; do not fix the
smear.

3. Cover the smear with 1% aqueous crystal

violet for 1 minute over steaming water bath.

4. Wash with 20% copper sulfate solution. Do

not use water.

5. Dry and examine under the oil immersion

objective.

1.5 BACTERIAL NUTRITION

Bacteria, like all cells, require nutrients for the
maintenance of their metabolism and for cell
division. Bacterial structural components and
the macromolecules for their metabolism are
synthesized from the elements. These are
called macroelements or macronutrients
because they are required in relatively large
amounts. The most important elements of
bacteria are carbon, hydrogen, oxygen,
sulphur, phosphorus and nitrogen which
constitute carbohydrates, lipids, proteins and
nucleic acids. The other four macroelements
exist in the cell as cations and play a variety of
roles. E.g K+ is required for enzyme activity,
Ca+ contributes to the heat resistance of
endospores, Mg2+ serves as a cofactor for
many enzymes, Fe2+ is part of cytochromes
and a cofactor for enzymes. In addition are
micronutrients or trace elements such a
manganese, zinc, cobalt, molybdenum, nickel
and copper. There are only two sources of
energy available to organisms: light and the
energy derived from oxidizing organic and
inorganic molecules.

Hydrogen and oxygen: Obtained from water.

Essential for the growth and maintenance of
cell.

Nitrogen: Constitutes 10% of dry weight of

bacterial cell. Obtained from organic
molecules like proteins and inorganic
molecules like ammonium salts and nitrates.

NB: Main source of nitrogen is ammonia, in

the form of ammonium salt.
Carbon sources

Organisms require a source of carbon for the

synthesis of numerous organic compounds that
comprise protoplast. Depending on their
requirements, bacteria can be classified as:

1. Autotrophs: Free-living, non-parasitic

bacteria which use carbon dioxide as carbon
source.

The energy needed for their metabolism can be

obtained from:

 Sun light-Photoautotrophs
 Inorganic compounds by oxidation-
Chemoautotrophs
2. Heterotrophs: Parasitic bacteria require
more complex organic compounds as their
source of carbon and energy. Human
pathogenic bacteria are heterotrophs.

The principal source of carbon is carbohydrate

which is degraded either by oxidation, in the
presence of oxygen, or by fermentation, in the
absence of oxygen, to provide energy in the
form of ATP.

Energy sources

Phototrophs: use light as their energy source

Chemotrophs: obtain energy from the

oxidation of compounds either organic or
inorganic

Electron source
  Lithotrophs: i.e rock-eaters use reduced
inorganic substances as their electron
source
 Organotrophs: extract electrons from
reduced organic compounds

Nutritional types

Photolithotrophic autotrophs: use light

energy and have CO2 as their carbon source.
Photosynthetic protists and cyanobacteria
employ water as their electron donor and
release oxygen. Others such as purple and
green sulphur bacteria cannot oxidize water
but extract electrons from inorganic donors
like hydrogen, hydrogen sulphide and
elemental sulphur. Example purple and green
sulphur bacteria
Chemoorganotrophic heterotrophs
(chemoheterotrophs,
chemoorganoheterotrophs or just
heterotrophs): use organic compounds as
sources of energy, hydrogen, electrons and
carbon. Frequently the same organic nutrient
will satisfy all these requirements. Essentially
all pathogenic microorganisms are
chemoheterotrophs. Example most non-
photosynthetic microbes including pathogens,
fungi and archaea

 Photoorganotrophic heterotrophs
(photoorganoheterotrophs) are common
inhabitants of polluted lakes and streams. They
can also grow as photoautotrophs with
molecular hydrogen as an electron donor.
Example purple nonsulfur bacteria, green
nonsulfur bacteria.

 Chemolithotrophic autotrophs
(chemolithoautotrophs) oxidize reduced
inorganic compounds such as iron, nitrogen of
sulphur molecules to derive both energy and
electrons for biosynthesis. Sulphur oxidizing
bacteria (below), nitrifying bacteria, iron
oxidizing bacteria

Purple sulfur bacteria

 Chemolithoheterotrophs (mixotrophs)
use reduced inorganic molecules as their
 energy and electron source, but derive their
carbon from inorganic sources. They greatly
contribute to chemical transformation of
elements (conversion of ammonia to nitrate or
sulphur to sulphate) that continually occur in
the ecosystem. Example some sulphur
oxidizing bacteria like Beggiatoa seen below.

 Prototrophs: Wild-type bacteria with normal

growth requirements.
 Auxotrophs: Mutant bacteria, which require
an additional growth factor not needed by the
parental or wild type strain.

Growth factors

Growth factors are organic compounds that are

required by microorganisms in small amounts
which the cell cannot synthesize from other
carbon source. These are:

 Amino acids: needed for protein synthesis

 Purines and pyrimidines: needed for nucleic
acid synthesis
 Vitamins: make up all or part of enzyme
cofactors and needed in only small amounts.
Some microorganisms require many vitamins
for growth e.g Enterococcus faecalis needs 8
vitamins.
Other growth factors are heme from
haemoglobin and cytochrome required by
Haemophilus influenza while some
Mycoplasma need cholesterol.

On the other hand, some microorganisms are

able to synthesize large quantities of vitamins
that can be used to manufacture these
compounds for human use. Example riboflavin
(Clostridium, Candida, Ashbya,
Eremothecium), coenzyme A
(Brevibacterium), Vit B12 (Streptomyces,
Propionibacterium, Pseudomonas) β carotene
(Dunaliella), Vit D (Saccharomyces).

Revision questions

 Discuss the ways in which

microorganisms are classified based on
 their requirements for energy, carbon
and electrons
 Describe the nutritional requirements
for the major nutritional groups and
give some microbial examples of each
 What are growth factors?
 What are vitamins?
 How can humans put to use a microbe
with a specific growth factor
requirement?
 List the growth factors that
microorganisms produce industrially
 Why do you think amino acids, purines
and pyrimidines are often growth
factors while glucose is not?
6 BACTERIAL GROWTH

It is an orderly increase in all the cellular

components of an organism. It is an increment
in biomass (longer or larger cells) and it is
synchronous with bacterial cell reproduction
like budding or binary fission. It is usually not
convenient to investigate the growth and
reproduction of individual microorganisms
because of their small size. Therefore when
studying growth, the changes in the total
population number are followed.

Procaryotic cell cycle

The cell cycle is the complete sequence of

events extending from the formation of a new
cell through the next division. Most
procaryotes reproduce by binary fission
although some reproduce by budding,
fragmentation.

Steps of binary fission

1 a parent cell prepares for division by

enlarging its cell wall, cell membrane and
overall volume

2 the septum begins to grow inward as the

chromosomes move toward opposite ends of
the cell. Other cytoplasmic components are
distributed to the two developing cells

3 the septum is synthesized completely

through the cell centre and the cell membrane
patches itself so that there are two separate cell
chambers. At this point, the 2 daughter cells
are divided. Some species separate completely,
while others remain attached forming chains,
doublets or other arrangements

Two pathways function during the cell cycle:

One pathway replicates and partitions the

DNA into progeny cells

The other carries out cytokinesis (septum

formation and progeny).
The bacteria growth curve

Binary fission and other cell division processes

bring about an increase in the number of cells
in a population. The population growth is
studied by analyzing the growth curve of the
microbial culture. When bacteria are in liquid
medium, they are usually grown in batch
culture or closed system, i.e in closed vessel
with a single batch medium. Since no fresh
medium is provided during incubation,
nutrient concentrations decline and
concentrations of wastes increase. The growth
of microorganisms reproducing by binary
fission can be plotted as the logarithm of the
number of viable cells versus the incubation
time. The resulting curve has four distinct
phases.
1. Lag phase

Microorganisms are introduced into fresh

culture medium, no immediate increase in cell
number. The period of adaptation with active
macro molecular synthesis like DNA, RNA,
various enzymes and other structural
components. It is the preparation time for
reproduction; no increase in cell number. This
phase may be necessary because cells may be
old and depleted ATP, essential cofactors and
ribosomes that must be synthesized before
growth can begin. The medium may be
different from the one the microorganism was
growing previously hence it may need to
synthesize new enzymes to use different
nutrients. They may also be injured and
require time to recover. This phase varies in
length with the conditions of the
microorganism and the nature of the medium.
It may be long if the inoculum is from an old
culture or refrigerated. When a young,
vigorously growing exponential phase culture
is transferred to fresh medium of the same
composition, the lag phase will be short or
absent.

2. Exponential (log) phase

The period of active multiplication of cells.

Cell division precedes at a logarithmic rate
(maximal rate), and determined by the
medium, genetic potential and condition of the
culture. The rate of growth is constant with
cell divisions at regular intervals. The curve
rises smoothly rather than in jumps because
each individual divides at a slightly different
moment. The population is most uniform in
terms of chemical and physiological properties
during this phase thus this phase is usually
used in biochemical and physiological studies.

3. Maximal stationary phase

The period when the bacteria have achieved

their maximal cell density or yield.

There is no further increase in viable bacterial

cell number. The growth rate is exactly equal
to the death rate. Attained by bacteria at a
population level of around 109 cells per ml.
final population size depends on nutrient
availability and other factors as well of type of
microorganism. A bacterial population may
reach stationary growth when one of the
following conditions occur:

1. The required nutrients are exhausted.

Aerobic bacteria are limited by O2 depletion

2. Inhibitory end products are accumulated

(toxic waste)

3. Physical conditions do not permit a further

increase in population size

4 Senescence and death

For many years, the decline in viable cells

following stationary cells was described
simply as the ‘death phase’. It was assumed
that detrimental environmental changes like
nutrient deprivation and the build-up of toxic
wastes caused irreparable harm resulting in
loss of viability was often not accompanied by
a loss in total cell number, it was assumed that
cells died but did not lyse. It is thought that
these cells are temporarily unable to grow at
least under lab conditions a phenomenon
called viable but non-culturable (VBNC). It is
thought to be the result of a genetic response
triggered in starving stationary phase cells.
Once appropriate conditions are available,
VBNC cells resume growth. An alternative to
VBNC is programmed cell death. This predicts
that a fraction of the microbial population is
genetically programmed to commit suicide. In
this case non-culturable cells are death and the
nutrients they leak, enable the eventual growth
of those cells in the population that did not
initiate suicide. They sacrificed themselves for
the larger population.
4. Decline phase

The period at which the rate of death of

bacterial cells exceeds the rate of new cell
formation. There is drastic decline in viable
cells. Few organisms may persist for so long
time at this period at the expense of nutrients
released from dying micro-organisms.

During this phase, the bacterial population

continually evolves so that actively
reproducing cells are those best able to use the
nutrients released by their dying brethren and
best able to tolerate the accumulated toxins.
This dynamic process is marked by successive
waves of genetically distinct variants.
Quantitative measurement of bacterial
growth

Bacterial growth is measured by determining

number of bacteria.

The common measuring methods are

1. Viable plate count

The most common plating method of

estimating bacterial growth which involves
counting the number of bacterial colonies
grown on solid media after incubation of the
inoculated media for 18-24 hours. Two
methods used include the pour plate and
spread plate method. In both methods, a
diluted sample of the bacteria are dispersed
over solid agar surface. Each microorganism
develops into a distinct colony. The number of
colonies can be calculated from the number of
colonies formed and the sample dilution.
Example if 1ml of a 1x10-6 dilution yielded
150 colonies, the original sample contained
around 1.5 x 108 cells per ml. The count is
made more accurate by the use of a special
colony counter.

Another method first traps bacteria in aquatic

samples on a membrane filter. The filter is
then placed on an agar medium or on a pad
soaked with liquid media and incubated until
each cell forms a separate colony. A colony
count gives the number of microorganisms in
the filtered sample and special media can be
used to select specific microorganisms.
Procedure

• The sample is serially diluted.

• The suspension is inoculated on solid media

by surface spread technique i.e. the suspension
is spread

• The plate is incubated for 18-24 hrs to allow

the bacteria to grow and form colonies.

• The concentration of bacteria in the original

sample can be determined by counting the
visible colonies multiplied by the dilution
factor.

Number of colonies =Number of colonies Χ

dilution factor X Volume of sample

NB: The statistically significant plate count is

between 30 and 300 colonies.
Less than 30 colonies on a plate are not
accepted for statistical reasons.

Greater than 300 colonies on a plate are too

close to distinguish an individual colony
forming unit (too numerous to count).

Limitation of viable plate count: It selectively

in favour of a certain group of bacterial
population.
2. Direct count

It involves direct microscopic counting of

bacteria in the sample using counting chamber.
It is relatively quick and does not need the
sample to be incubated.

Procedure

 Serial dilution of the sample

 Fill known area and volume of the
counting chamber with the sample
 Total number of bacteria in the sample per
unit volume is equal to the No of bacteria
in the sample Χ the No. of squares Χ
dilution factor.
3. Turbidimetric method
It is the method of determination of bacterial
growth in liquid media. Bacterial growth
increases the turbidity of liquid to absorb light.
The turbidity of the suspension is determined
by spectrophotometer.
Factors influencing bacterial growth in vitro:

 Not all bacterial species grow under

identical environmental conditions.
 Each bacterial species has a specific
tolerance range for specific environmental
parameters.
 Outside the tolerance range environmental
conditions for a bacteria to reproduce, it
 may survive in dormant state or may lose
viability.
 Rates of bacterial growth are greatly
influenced by the following environmental
parameters.

. Nutrition

. Temperature

. Oxygen

. PH

. Salinity

. Pressure

. Light radiation
Conditions suitable for bacteria growth

1 Nutrition: The following nutrients must be

provided for optimal bacterial growth.

• Hydrogen donors and acceptors

• Carbon source

• Nitrogen source

• Minerals: sulfur and phosphorus, trace

elements

• Growth factors: amino acids, purines,

pyrimidines and vitamins.

2. Temperature

Temperature tolerance range: The minimum

and maximum temperature at which a micro-
organism can grow; is different in different
species of bacteria.
Optimal growth range of temperature: The
temperature at which the maximum growth
rate occurs; and results in the shortest
generation time of bacteria.

Based on different optimal growth temperature

requirement, bacteria are divided into:

. Psychrophilic bacteria:15-20° C; grow best at

low temperature range

. Mesophilic bacteria: 30-37°C; grow best at

middle temperature range

. Thermophilic bacteria: 50-60°C; grow best at

high temperature range

NB: Most human pathogens and many of the

normal flora of human bodies have an optimal
temperature of 37°C. Therefore they are
mesophilic bacteria.
3. Oxygen

Base on oxygen requirements and tolerance,

bacteria are divided as:

. Obligate aerobes

. Obligate anaerobes

. Facultative anaerobes

. Microaerophiles

• Obligate aerobic bacteria grow only when

free oxygen is available to support their
respiratory metabolism. They obtain ATP by
using oxygen as a final electron acceptor in
respiration.

• Obligate anaerobic bacteria grow in the

absence of oxygen; exposure to oxygen kills
anaerobes.
• Facultative anaerobic bacteria grow in the
presence or absence of oxygen. They obtain
ATP by fermentation or anaerobic respiration

• Microaerophilic bacteria grow best at

reduced oxygen tension; high oxygen tension
is toxic to them.

4. Hydrogen ion concentration

It is a measure of acidity and alkalinity. PH <7

is acidic

PH =7 is neutral

PH>7 is alkaline

• Neutrophilic bacteria grow best at near

neutral PH value.

• Acidicophilic bacteria prefer to grow at low

PH value (acidic medium).
• Alkalinophilic bacteria prefer to grow at high
PH value (alkaline medium).

• Most pathogenic bacteria grow best at PH of

6-8.

5. Salinity

Salt content of the medium affects bacterial

growth.

Halophilic bacteria grow best at high salt

concentration.

. Moderate halophiles require 3% salt

concentration.

. Extreme halophiles require 15% salt

concentration.

Most bacteria cannot tolerate high salt

concentration. High salt concentration disrupts
membrane transport systems and denatures
proteins of bacteria but halophiles have
adaptive mechanisms to tolerate high salt
concentration.

6. Pressure

Osmotic pressure: The pressure exerted on

bacterial cell surface as a result of difference
in solute concentration between the inside and
outside of a cell.

Osmotolerant bacteria can grow in solutions

with high solute concentration.

Osmophilic bacteria grow best at high

hydrostatic pressure.

Hydrostatic pressure: The pressure exerted

by the weight of a water column.
High hydrostatic pressures more than 200
atmosphere generally inactivates enzymes and
disrupts membrane transport process.

Barotolerant bacteria can grow at high

hydrostatic pressure. Barophilic bacteria grow
best at high hydrostatic pressure.

7. Light radiation

Photosynthetic bacteria require light in the

visible spectrum to carry out photosynthesis.

CULTIVATION OF BACTERIA ON
CULTURE MEDIA

Culture media

It is the media containing the required

nutrients for bacterial growth.
 Uses: Isolation and identification of micro-
organisms

. Performing antimicrobial sensitivity tests

Common ingredients of culture media

. Peptone

. Meat extract

. Yeast extract

. Mineral salts

. Carbohydrates

. Agar

. Water

Peptone: Hydrolyzed product of animal and

plant proteins: Free amino acids, peptides and
proteoses (large sized peptides).
It provides nitrogen; as well carbohydrates,
nucleic acid fractions, minerals and vitamins.

Meat extract: supply amino acids, vitamins

and mineral salts.

Yeast extract: It is bacterial growth

stimulants.

Mineral salts: these are: Sulfates as a source

of sulfur.

. Phosphates as a source of phosphorus.

. Sodium chloride

. Other elements

Carbohydrates: Simple and complex sugars

are a source of carbon and energy.

.Assist in the differentiation of bacteria.

Eg. Sucrose in TCBS agar differentiates vibrio
species.

Lactose in MacConkey agar differentiates

enterobacteria.

Agar: It is an inert polysaccharide of seaweed.

It is not metabolized by microorganism.

Property

. It has high gelling strength

. high melting temperature(90-95°C)

. low gelling temperature

. It forms firm gel at 1.5% W/V concentration.

. It forms semisolid gel at 0.4-0.5% W/V

concentration.

Uses:

. Solidify culture media

. May provide calcium and organic ions to
inoculated bacteria.

Water: Deionized or distilled water must be

used in the preparation of culture media.

Types of culture media

1. Basic /Simple / All purpose media

It is a media that supports the growth of micro-

organisms that do not require special nutrients.

Uses :

. To prepare enriched media

. To maintain stock cultures of control

bacterial strains

. To subculture pathogenic bacteria from

selective/differential medium prior to
performing biochemical or serological tests.
Eg. Nutrient Broth, Nutrient Agar

2. Enriched media

Media that are enriched with whole blood,

lyzed blood, serum, special extracts or
vitamins to support the growth of pathogenic
bacteria.

Eg. Blood Agar, Chocolate Agar

3. Enrichment media

Fluid media that increases the numbers of a

pathogen by containing enrichments and/or
substances that discourage the multiplication
of

unwanted bacteria. Eg. Selenite F broth media,

Alkaline peptone water

4. Selective media
Media which contain substances (Eg.
Antibiotics) that prevent or slow down the
growth of bacteria other than pathogens for
which the media are intended.

Eg. Modified Thayer –Martin Agar

Salmonella-Shigella (SS) agar

1. Differential media

Media to which indicator substances are added

to differentiate bacteria.

Eg. TCBS Agar differentiates sucrose

fermenting yellow colonies of Vibrio cholerae
to non-sucrose fermenting blue colonies of
other Vibrio species.
NB: Most differential media distinguish
between bacteria by an indicator which
changes colour when acid is produced
following carbohydrate fermentation.

2. Transport media

Media containing ingredients to prevent the

overgrowth of commensals and ensure the
survival of pathogenic bacteria when
specimens cannot be cultured soon after
collection.
e.g. Amies transport media, Stuart media,
Kelly-Blair media

Choice of culture media

The selection of culture media will depend on:

1. The major pathogens to be isolated, their

growth requirements and the features by which
they are recognized.

2. Whether the specimens being cultured are

from sterile sites or from sites having normal
microbial flora.

3. The cost, availability and stability of media.

4. The training and experience of laboratory

staff in preparing, using and controlling
culture media.
Forms of culture media
1. solid culture media

2. semisolid culture media

3. Fluid culture media

1. solid culture media

. Plate cultures in petri dishes

. stab/slope cultures in tubes and bottles

Uses: Description of bacterial colonies

• size : diameter in mm

• Out line : circular, entire, wavy, indented

• Elevation: flat, raised, low convex and dome

shaped.
• Transparency: transparent, opaque, and
translucent.

• Surface: smooth (mucoid) and shiny, rough

and dull.

• Color: colorless, white, pink, and pigmented

• changes in medium Eg. Hemolysis in Blood

Agar

Blackening of medium due to hydrogen sulfide

production.

2. Semisolid culture media

Uses:

. as an enrichment media

. as motility media
3. Fluid culture media

Bacterial growth in fluid media is shown by a

turbidity in the medium.

Uses :

. as an enrichment media

. as biochemical testing media

. as blood culture media

COMMON BIOCHEMICAL TESTS

Litmus milk reduction test:

Requirement:

Litmus milk medium

Wire loop

Bunsen burner

Test bacteria

Method:

. Inoculate 0.5 ml of sterile litmus milk

medium with the test bacteria.

. Incubate at 35-37 °c for up to 4 hrs.

. Observe for changes in colour every 30 min.

Results: Change in color of medium from pink
to white or pale is suggestive

of enterococci

CAMP test (Christie, Atkin, Munich

Paterson )

Principle: Streptococcus. agalactiae produce

protein named as camp factor, which interacts
with staphylococci βhemolysin on sheep red
blood cell.
Method:

. Streak Staphylococcus aureus isolate across

sheep blood agar plate.

. Inoculate the test bacteria at right angle to

staphylococci without touching it.

. Incubate over night at 35-37°c.

. Formation of an arrow-head shaped area of

hemolysis indicates interaction of camp factor
with staphylococci hemolysin.
Bacitracin test

Principle: Streptococcus pyogenes is sensitive

to bacitracin but other kinds of streptocci are
resistant to bacitracin.

Method:

. Streak a blood agar plate with the isolated

organism.

. Place bacitracin disc in the streaked area.

. Incubate the plate for 24 hours at 37°C.

. Examine the plate for a zone of no-growth

around the disc.

No-growth around the disc...… S. pyogenes

Growth around the disc …… Other

streptococci
Optochin test

Principle: S. pneumoniae is sensitive to

optochin disc unlike other alpha-hemolytic
streptococci.

Method:

. Streak a blood agar plate with the isolated

organism.

. Place optochin disc in the streaked area.

. Incubate the plate for 24 hours at 37°c.

. Examine the plate for a zone of no-growth
around the disc.

No-growth around the disc..…S. pneumoniae

Growth around the disc …other Alpha

hemolytic streptococci

Carbohydrate utilization test

Method:
. Prepare saline suspension of test bacteria

. Add 0.1ml of bacterial suspension into each

of four test tubes containing glucose, lactose,
maltose and sucrose carbohydrate discs.

. Incubate in a water bath at 37°c and examine

at 30 min intervals for 5 hrs for change in
color.

Result: Change in color from red to yellow-

orange indicates carbohydrate utilization.

BILE SOLUBILITY TEST

This helps to differentiate S. pneumoniae,
which is soluble in bile and bile salts, from
Viridans streptococci which are insoluble.

Principle

A heavy inoculum of the test organism is

emulsified in physiological saline to give a
turbid suspension. The bile salt sodium
deoxycholate is then added. The test can also
be performed by adding the bile salt to a broth
culture of the organism. The bile salt dissolves
Streptococcus pneumoniae as shown by a
clearing of the turbidity within 10-15 minutes.
Viridans streptococci are not dissolved and
therefore there is no clearing of the turbidity.
Requirement

Sodium deoxycholate100g/l

Physiological saline (sodium chloride, 8.5g/l)

Method

• Emulsify several colonies of the test

organism in a tube containing 2ml of sterile
physiological saline, to give a turbid
suspension.

• Divide the organism suspension between two

tubes.

• To one tube, add 2 drops of the sodium

deoxycholate reagent and mix.
• To the other tube, add 2 drops of sterile
distilled water and mix.

• Leave both tubes for 10-15 minutes.

• Look for a clearing of turbidity in the tube

containing the sodium deoxycholate.

Results

Clearing of turbidity -------------- Probably

Streptococcus pneumoniae

No clearing of turbidity -----------organism is

probably

Not Streptococcus pneumoniae

There should be no clearing of turbidity in the
tube to which distilled water was added. If
there is, repeat the test.

Note: Some strains of S. pneumoniae are not

dissolved by bile salts, and very occasionally
some strains of viridans streptococci give a
positive tests.

Controls

Bile solubility positive control: Streptococcus

pneumoniae.

Bile solubility negative control: Streptococcus

faecalis.
CATALASE TEST

This test is used to differentiate those bacteria

that produce the enzyme catalase, such as
staphylococci, from non-catalase producing
bacteria such as streptococci.

Principle
Catalase acts as a catalyst in the breakdown of
hydrogen peroxide to oxygen and water. An
organism is tested for catalase production by
bringing it into contact with hydrogen
peroxide. Bubbles of oxygen are released if the
organism is a catalase producer. The culture
should not be more than 24 hours old.

Care must be taken if testing an organism

cultured on a medium containing blood
because catalase is present in red cells. If any
of the blood agar is removed with the colony, a
false positive reaction will occur. It is usually
recommended, therefore, that catalase testing
be performed from a blood free culture
medium such as nutrient agar.

Requirement
a. Hydrogen peroxide, 3% H2O2

Note: Shaking the reagent before use will help

to expel any dissolved oxygen. False positive
reactions may occur if the hydrogen peroxide
contains dissolved oxygen.

Method

• Pour 2-3ml of the hydrogen peroxide

solution into a test tube.

• Using a sterile wooden stick or a glass rod,

remove a good growth of the test organism and
immerse it in the hydrogen peroxide solution.

Note: A nichrome wire loop must not be used

because this may give a false positive reaction.

• Look for immediate bubbling.

Results
Active bubbling ----------------- Positive test

Catalase produced

No release of bubbles ---------- Negative test

No catalase produced

Note: if the organism has been cultured on an

agar slope, pour about 1ml of the hydrogen
peroxide solution over a good growth of the
organism, and look for the release of bubbles.
Caution: performing the test on a slide is not
recommended because of the risk of
contamination from active bubbling. If the
rapid slide technique is used, the hydrogen
peroxide solution should be added to the
organism suspension after placing the slide in
a Petri dish. The dish should then be covered
immediately, and the preparation observed for
bubbling through the lid.

Controls

Positive catalase control: Staphylococcus

species.

Negative catalase control: Streptococcus

species.
CITRATE UTILIZATION TEST

This test is one of several techniques used to

assist in the identification of enterobacteria.
The test is based on the ability of an organism
to use citrate as its only source of carbon and
ammonia as its only source of nitrogen.

Principle

The test organism is cultured in a medium

which contains sodium citrate, an ammonium
salt, and the indicator bromothymol blue.
Growth in the medium is shown by turbidity
and a change in colour of the indicator from
light green to blue, due to the alkaline reaction,
following citrate utilization.

Requirement

Koser’s citrate medium or Simmon’s citrate

Method

Using a sterile straight wire, inoculate 3-4ml

of sterile Koser’s citrate medium with a broth
culture of the test organism.

Note: Care must be taken not to contaminate

the medium with carbon particles, such as
from a frequently flamed wire.

Incubate the inoculated broth at 35 – 37°C for

up to 4 days, checking daily for growth

Results
Turbidity and blue colour ---------- Positive
test:

Citrate utilized

No growth -------------------------Negative test:

Citrate not utilized

Controls

Positive citrate control: Klebsiella pneumoniae

Negative citrate control: Escherichia coli.

COAGULASE TEST
This test is used to differentiate
Staphylococcus aureus which produces the
enzyme coagulase, from S. epidermidis and S.
saprophyticus which do not produce
coagulase.

Principle

Coagulase causes plasma to clot by converting

fibrinogen to fibrin.

Two types of coagulase are produced by most

strains of S. aureus:

Free coagulase which converts fibrinogen to

fibrin by activating a coagulase – reacting
factor present in plasma. Free coagulase is
detected by the appearance of a fibrin clot in
the tube test.
Bound coagulase (clumping factor) which
converts fibrinogen

Directly to fibrin without requiring a coagulase

– reacting factor. It can be detected by the
clumping of bacterial cells in the rapid slide
test. It is usually recommended that a tube test
should be performed on all negative slide tests.
A tube test must always be performed if the
result of the slide test is not clear, or when the
slide test is negative and the Staphylococcus
has been isolated from a serious infection.

Requirement

a. Undiluted human plasma (preferably

pooled) or rabbit plasma. The plasma should
be allowed to warm to room temperature
before being used.
Plasma from EDTA (ethylenediamine – tetra –
acetic acid) or citrate anticoagulated blood is
usually used.

Note: Occasionally citrate-utilizing organisms

such as Klebsiella can cause the clotting of
citrated plasma in the tube test. This can be
prevented by adding heparin to the citrated
plasma. It is also possible for human plasma to
contain inhibitory substances which can
interfere with coagulase testing. Adequate
controls must be included for both slide and
tube tests.

Method for slide test (to detect bound

coagulase) Place a drop of physiological saline
on each end of a slide, or on

two separate slides.

Emulsiy a colony of the test organism in each
of the drops to

make two thick suspensions.

Note: Colonies from a mannitol salt agar

culture are not

suitable for coagulase testing. The organism

must first be

cultured on nutrient agar or blood agar.

Add a drop of plasma to one of the

suspensions, and mix gently.

Look for clumping of the organisms within 10

seconds.

No plasma is added to the second suspension.

This is
used to differentiate any granular appearance
of the

organism form true coagulase clumping.

Results

Clamping within 10 secs ------------------------

Staphylococcus aureus

No clumping within 10 secs-----------No bound

coagulase produced.

Controls

Positive coagulase control: Staphylococcus

aureus.

Negative coagulase control: Escherichia coli

or

Staphylococcus epidermidis

Method for tube test (detect free coagulase)

Dilute the plasma 1 in 10 in physiological
saline (mix 0.2 ml of

plasma with 1.8ml of saline).

Take three small test tubes and label:

T = Test organism (18-24h broth culture)

Pos = Positive control (18-24h staph. Aureus

broth

culture)

Neg = Negative control (sterile broth)

A suitable broth is brain heart infusion

Pipette 0.5ml of the diluted plasma into each

tube.

Add 5 drops (about 0.1ml) of the test organism

culture to the

tube labelled ‘T’.

Add 5 drops of the Staphylococcus aureus
culture to the tube

labelled ‘Pos’.

Add 5 drops of sterile broth to the tube

labelled ‘Neg’.

After mixing gently, incubate the three tubes at

35-37°C.

Examine for clotting after 1 hour. If no

clotting has occurred,

examine at 30minute intervals for up to 6

hours.

When looking for clotting, gently tilt each

tube.

Most Staphylococcus aureus strains produce a

fibrin clot within 1
hour of incubation. There should be no fibrin
clot in the

negative control tube.

Results

Fibrin clot ----------------------------- S. aureus

No fibrin clot ------------------------- No free

coagulase produced

DEOXYRIBONUCLEASE (DNAse) TEST

This test is used to differentiate Staph. Aureus

which produces the
enzyme DNAse from other staphylococci
which do not produce

DNAse. It is particularly useful if plasma is

not available to peform a

coagulase test or when the results of a

coagulase test are difficult to interpret.

Principle

Deoxyribonuclease hydrolyzes
deoxyribonucleic acid (DNA).

The test organism is cultured on a medium

which contains DNA.

After overnight incubation, the colonies are

tested for DNAse

production by flooding the plate with a weak

hydrochloric acid
solution. The acid precipitates unhydrolyzed
DNA. DNAse producing

colonies are, therefore surrounded by clear

areas indicating DNA

hydrolysis.

Requirement

a. DNAse agar plate

Up to six organisms may be tested on the same

plate.

b. Hydrochloric acid, 1 mol/l

Method

Divide a DNAse plate into the required

number of strips by
marking the underside of the plate. Using a
sterile loop or swab, spot – inoculate the test
and control

organisms. Make sure each test area is clearly

labeled.

Incubate the plate at 36-37°C overnight.

Cover the surface of the plate with 1mol/l

hydrochloric acid

solution. Tip off the excess acid.

58. Look for clearing around the colonies

within 5minutes of

adding the acid.

Results

Clearing around the colonies -----------------

DNAse positive strain.
No clearing around to colonies -------------
DNAse negative strain.

Controls

Positive DNAse control: Staphylococcus

aureus.

Negative DNAse control: Staphylococcus

epidermidis.
HYDROGEN SULPHIDE (H2S)
PRODUCTION

The detection of hydrogen sulphide gas (H2S)

is used mainly to

assist in the identification of enterobacteria

and occasionally to

differentiate other bacteria such as Bacteroides

and Bruceila

species. H2S is produced when sulphur –

containing amino

acids are decomposed.

Use of Kligler iron agar (KIA) to detect H2S

This medium is suitable for detecting H2S

production by
enterobacteria. H2S is detected by the ferric
citrate contained in

the medium.

Inoculate the test organism into KIA and

incubate it at

appropriate temperature over night.

Observe blacking of the medium

Lead acetate paper test to detect H2S

When a sensitive technique for detecting H2S

production is required,

the lead acetate paper test is recommended.

Inoculate a tube or bottle of sterile peptone

water or nutrient

broth with the test organism.

Insert a lead acetate paper strip in the neck of
the bottle or tube

above the medium, and stopper well.

Incubate the inoculated medium at 35-37°C,

and examine daily

for a blackening of the lower part of the strip.

Results

Blackening ----------------------------- Positive

test H2S produced

No blackening ------------------------- Negative

test No H2S

produced.

Controls

Positive hydrogen sulphide control: Proteus

vulgaris
Negative hydrogen sulphide control: Shigella
species

INDOLE TEST

Testing for indole production is important in

the identification of enterobacteria. Most
strains of Escherichia coli, Proteus vulgaris,
Providencia rettgeri, Morganella morgani,
and providencia species break down the amino
acid tryptophan with the release of indole.
Principle

The test organism is cultured in a medium

which contains tryptophan. Indole production
is detected by Kovac’s or Ehrlich’s reagent
which contains 4(P)-
dimethylaminobenzaldehyde. This reacts with
the indole to produce a red coloured
compound. In the

following method the use of the combined

motility indole urea (MIU) medium is
described. A Kovac’s ragent paper strip is
inserted in the neck of the tube, and indole
production is indicated by a reddening of the
strip. Indole is a volatile substance (easily
vaporized). The tube must be well stoppered
during incubation. The indole test can also be
performed by culturing the organism in
tryptone water or peptone water containing
tryptophan, and detecting indole production by
adding Kovac’s or Ehrlich’s reagent to an 18-
24h culture.

Requirement

Motility indole urea (MIU) medium

MIU medium indicates whether an organism is

motile or non-motile, indole positive or
negative, and urease positive or negative.

Method

Using a sterile straight wire, inoculate 5ml of

sterile MIU medium with a smooth colony of
the test organism. Place an indole paper strip
in the neck of the MIU tube above the
medium, and stopper the tube, incubate at 35-
37°C overnight.

Examine for indole production by looking for

a reddening of the lower part.

Results

Reddening of strip -----------------------------

Positive test

Indole produced

No red colour ----------------------------------

Negative test

No Indole produced

Note: If the reaction is weak, confirm the

result by adding 1ml of

Kovac’s regent to the culture. Examine for a

red colouring of the
surface layer within 10 minutes.

Controls

Positive indole control: Escherichia coli

Negative indole control: Enterobacter

aerogenes.
Motility Test
This is shown by a spreading turbidity from
the stab line or a turbidity throughout the
medium (compare with an uninoculated tube).

Urease production

This is shown by a red-pink colour in the

medium.
NITRATE REDUCTION TEST

This test is used to differentiate members of

the Enterobacteriaceae that produce the
enzyme nitrate reductase, from Gram negative

bacteria that do not produce the enzyme.

The test is also helpful in differentiating
Mycobacterium species as explained.

Principle

A heavy inoculum of the test organism is

incubated in a broth containing nitrate. After 4
hours, the broth is tested for the reduction of
nitrate to nitrite by adding sulphanilic acid
reagent. If nitrite is present, the acid reagent is
diazotizex and forms a pink-red compound
with alpha-naphthylamine. When nitrite is not
detected it is necessary to test whether the
organism has reduced the nitrate beyond
nitrite. This is done indirectly by checking
whether the broth still contains nitrate. Zinc
dust is added which will convert any nitrate to
nitrate. If no nitrite is detected when the zinc
dust is added, it can be assumed that all the
nitrate has been reduced beyond nitrite to
nitrogen gas or ammonia by a nitrate reducing
organism.

Requirement

a. Nitrate broth

b. Sulphanilic acid reagent

c. Alphanaphthylamine reagent

d. Zinc dust

Method

Inoculate 0.6 ml of sterile nitrate broth with a

heavy growth of the test organism.

Incubate at 35-37°C for 4 hours.

Add 1 drop of sulphanilic acid reagent and 1

drop of
alphanaphthylamine reagent.

Shake to mix and look for a red colour.

Results

Red colour ----------------------------- Positive

test

Nitrate reduced

If no red colour is produced, add a very small

amount (knife point) of

Zinc dust powder. Look again for a red colour

and interpret as follows:

Red colour ----------------------------- Negative

test

No reduction of nitrate
No red colour ------------------------- Positive
test

Nitrate reduced

Controls

Positive nitrate reduction control: Escherichia

coli.

Negative nitrate reduction control:

Pseudomonas aeruginosa.

OXIDASE TEST (Cytochrome Oxidase)

The oxidase test is used to assist in the

identification of Pseudomonas, Neisseria,
Vibrio, and Pasteurella species, all of which
produce oxidase enzymes.

Principle

A piece of filter paper is soaked with a few

drops of oxidase reagent.

A colony of the test organism is then smeared

on the filter paper. If the organism is oxidase -
producing, the phenylenedialamine in the

reagent will be oxidized to a deep purple

colour. Occasionally the test is performed by
flooding the culture plate with oxidase reagent
but this technique is not recommended for
routine use because the reagent rapidly kills
bacteria. It can be useful, however, when
attempting to isolate Niesseria gonorrhoeae
colonies from mixed cultures in the absence of
a selective medium. The oxidase positive
colonies must be removed and subcultured
within 30 seconds of flooding the plate.

Important: Acidity inhibits oxidase enzyme

activity. The oxidase test must not be
performed, therefore, on colonies that produce

fermentation on carbohydrate – containing

media, such as sucrose fermenting Vibrio
cholerae colonies on TCBS medium, Sub-
inoculation on nutrient agar is required before
the oxidase test can be performed reliably.
Non – fermenting colonies, however, can be
tested.

Colonies tested from a medium that contains

nitrate may give unreliable oxidase test results.
Requirement

− Oxidase reagent

Freshly prepared

This is a 10g/l solution of tetramethyl –p-

phenylenediamine

dihydrochloride.

Note: Oxidase reagent is easily oxidized.

When oxidized, it is blue in colour and must
not be used.

Method

Place a piece of filter paper in a clean petri

dish and add 2 or 3 drops of freshly prepared
oxidase reagent.
Using a piece of stick or glass rod (not an
oxidized wire loop), remove a colony of the
test organism, and smear it on the filter paper.

Look for the development of a blue – purple

colour within a few seconds.

Results

Blue-purple colour
------------------------------------- Positive test

(within 10 seconds) Oxidase produced

No blue – purple colour

------------------------------- Negative test

(within 10 seconds) No oxidase produced

Note: ignore any blue – purple colour that

develops after 10 seconds.
Controls

Positive oxidase control: Pseudomonas

aeruginosa.

Negative oxidase control: Escherichia coli.

OXIDATION – FERMENTATION (O-F)
TEST

This test is used to differentiate those

organisms that oxidize. Carbohydrates
(aerobic utilization) Such as Pseudomonas
aeruginosa, from those organisms that ferment
carbohydrates (anaerobic utilization) such as
members of the Enterobacteriaceae.

Principle

The test organism is inoculated into two tubes

of a tryptone or peptone agar medium
containing glucose (or other carbohydrate) and
the indicator bromothymol blue. The
inoculated medium in one tube is sealed with a
layer of liquid paraffin to exclude oxygen.
Fermentative organisms utilize the
carbohydrate in both the open and sealed tubes
and the colour of the medium changes from
green to yellow. Oxidative organisms,
however, are able to use the carbohydrate only
in the open tube. There is no carbohydrate
utilization in the sealed tube (medium remains
green).

Although most genera of aerobic bacteria are

either carbohydrate oxidizers or fermenters,
the production of acid may be slow and
therefore cultures are usually incubated for 7-
14 days.

Requirement

a. Oxidation fermentation (O-F) medium

Glucose, maltose, and sucrose O-F media are
the most commonly used.

b. Sterile paraffin oil (liquid paraffin)

Method

Using a sterile straight wire, inoculate the test

organism to the bottom of two bottles (or more
if testing several carbohydrates) of sterile O-F
medium. Use a heavy inoculum.

Cover the inoculated medium in one of the

tubes (or one from each carbohydrate pair)
with a 10mm deep layer of sterile paraffin oil
or molten wax.

Incubate the tubes at 35-37°C for up to 14

days. Examine daily for carbohydrate
utilization as shown by acid production.

Interpretation
Yellow Green Oxidative organism

Yellow Yellow Fermentative organism

Green or blue Green No utilization of

carbohydrate

Controls

Oxidative control: Pseudomonas aeruginosa.

Fermentative control: Escherichia coli.

PHENYLALANINE DEAMINASE TEST

The test, which is also referred to as the

Phenylpyruvic acid (PPA) test, is used mainly
to assist in the identification of enterobacteria.
It is based on the ability of bacteria such as
Proteus species and some Providencia strains
to break down phenylalanine (by oxidative
deamination) with the production of
phenylpyruvic acid. Yersinia enterocolitica
(urease – producer is a phenylalanine negative.

Principle

The test organism is cultured on a slope of

phenylalanine medium.
After overnight incubation, the deamination of
phenylalanine to phenyl – pyruvic acid is
detected by adding iron III chloride (ferric

chloride) which produces a green colour on the

surface of the culture.

Requirement

a. Phenylalanine agar

b. Iron III chloride (ferric chloride), 100g/l

(10% w/v). The reagent must be freshly
prepared.

Method

. Inoculate a slope of phenylalanine agar with

the test organism, and incubate at 35-37°C
overnight.
. Add 4 or 5 drops of the freshly prepared iron
III chloride reagent to the culture, allowing the
reagent to run down the slope.

. Look for a green colour on the slope.

Results

Green colour ------------------------------------

Positive test

(Within 5 minutes) Phenylalanine deaminated

No green colour ---------------------------------

Negative test

No deamination of phenylalanine

Controls

Positive PPA control: Proteus species

Negative PPA control: Escherichia coli.

TWEEN 80 HYDROLYSIS TEST

This test is used mainly to differentiate slow–

growing Mycobacterium

Species.

Principle

The test organism is incubated in a Tween 80

buffered substrate

that contains the indicator neutral red. Tween

hydrolysis is detected
by a change in colour of the indicator from
amber to pink – red due

to the production of oleic acid.

Requirement

Tween 80 phosphate buffered substrate with

neutral red.

*The substrate requires storage at 4°C

Method

. Inoculate 4 ml of sterile Tween 80 phosphate

buffered

substrate with a loopful of growth of the test

organism.

. Incubate at 35-37°C for up to 18 days.

Examine at
5, 10, and 18 days for a change in colour of the
substrate

from amber to pink-red, as shown in colour.

Results

Pink-red substrate
-------------------------------------- Positive test

Tween 80 hydrolyzed

No change in colour
------------------------------------ Negative test

No hydrolysis of Tween 80
Controls

Positive Tween hydrolysis control:

Mycobacterium kansasii.

Negative Tween hydrolysis control: Use an

uninoculated tube of

substrate.

UREASE TEST

Testing for urease enzyme activity is important

in differentiating

entrobacteria. Proteus strains are strong urease

producers.

Yersinia enterocolitica also shows urease

activity (Weakly a 35-37°C)
Salmonellae and shigellae do not produce
urease.

Principle

The test organism is cultured in a medium

which contains urea and

the indicator phenol red. If the strain is urease-

producing, the

enzyme will leak down the urea (by

hydrolysis) to give ammonia

and carbon dioxide. With the release of

ammonia, the medium

becomes alkaline as shown by a change in

colour of the indicator to red-pink.

The method described is that which uses the

combined motility
indole urea (MIU) medium, urease production
can also be detected

by culturing the organisms in Christensen’s

urea broth.

Requirement

a. Motility indole urea (MIU) medium.

Method

. Using a sterile straight wire, inoculate a tube

of sterile

MIU medium with a smooth colony of the test

organism.

. Place an indole paper strip in the neck of the

MIU tube above the medium. Stopper the tube
and incubate at 35-37°Covernight
. Examine for urease production by looking for
a red pink colour in the medium as shown in
colour.

Results

Red-pink
medium----------------------------------------
Positive test

Urease produced

No red-pink colour
------------------------------------- Negative test

No urease produced

Controls

Positive urease control: Proteus vulgaris.

Negative urease control: Escherichia col.

Note: After overnight incubation, Yersinia
enterocolitica gives a weak urease reaction
and is non-motile at 35-37°C. At room
temperature (22-29°C), the species is motile
and shows a stronger urease reaction.

VOGES – PROSKAUER (V-P) TEST

This test is occasionally used to assist in the

differentiation of enterobacteria. K.
pneumoniae, Vibrio cholerae biovar el tor, and
some strains of Enterobacter, ferment glucose
with the production of acetylmethylcarbinol
(acetoin) which can be detected by an
oxidation reaction.
Principle

The test organism is cultured in a glucose

phosphate peptone water for 48 hours. Sodium
hydroxide and a small amount of creatinine are
then added. Under alkaline conditions and
exposure to the air, the

acetione produced from the fermentation of the

glucose is oxidized to diacetyl which forms a
pink compound with the creatinine.

Requirements

a. Glucose phosphate peptone water.

b. Sodium hydroxide, 400g/l.

c. Creatinine powder.

Method
. Inoculate 2ml of sterile glucose phosphate
peptone water with the test organism. Incubate
at 35-37 °c for 48hours.

. Add a very small amount (knife point) of

creatinine and mix.

. Add about 3ml of the sodium hydroxide

reagent and shake well,

Caution: The sodium hydroxide reagent is

corrosive, therefore handle with care and do
not mouth – pipette.

. Remove the bottle cap, and leave for 1 hour

at room

temperature. Look for the slow development

of a pink – red.

Results
Pink – red colour
-------------------------------------- Positive test

Acetoine produced

No pink – red colour

---------------------------------- Negative test

No acetoin produced

Controls

V-P Positive control: Enterobacter aerogenes

or

Klebsiella pneumoniae

V-P Negative control: Escherichia coli

8. BACTERIAL GENETICS

Genetics is the study of inheritance. Bacterial

inherited characteristics are encoded in DNA.

Bacteria have two types of DNA that contain

their genes. These are :

. Chromosome

. Extra chromosome: Plasmid

The bacterial chromosome is circular, double

stranded DNA attached to bacterial cell
membrane.

DNA replication in bacteria is semi-

conservative i.e. each strand of DNA is
conserved intact during replication and
becomes one of the two strands of the new
daughter molecules.
Plasmids are self-replicating extra
chromosomal DNA molecules.

They multiply independent of the host cell.

Multiple copies of the same plasmid may be

present in each bacterial cell.

Different plasmids are also often present in the

same bacterial cell.

Plasmid types

There are many types of plasmid types. The

following are examples.
a. R factors: Plasmids which contain genes
that code for antibiotic resistance.

b. Col factors: Plasmids which contain genes

that code for extracellular toxin (colicines)
production that inhibit strains of the same and
different species of bacteria.

c. F(fertility) factors: Plasmids that can

recombine itself with the bacterial
chromosome. It promotes transfer of the
chromosome at a high frequency of
recombination into the chromosome of a
second (recipient) bacterial cell during mating.
Genetic variation in Bacteria

Mechanisms: Mutation and Gene transfer

1. Mutation: It is due to a chemical alteration

in DNA. It could be spontaneous or induced
by chemical and physical means. Mutants are
variants in which one or more bases in their
DNA are altered; which are heritable and
irreversible

Types of mutation

1. Substitution: Change of a single base.

2. Deletion: Loss of a base.

3. Insertion: Addition of a base.

2. Gene transfer

There are three types of gene transfer that alter

the DNA gene content of bacteria.

These are:

. Transformation

. Transduction

. Conjugation

1. Transformation occurs when fragments of

exogenous bacterial DNA are taken up and
absorbed into recipient bacterial cells.

Transformation of genes from one bacterium

to another results in

. Change in pathogenicity of the bacterium.

. Change in antibiotic sensitivity pattern of

bacterium.
Competence: The recipient bacterium must be
competent to absorb the exogenous fragments
of bacterial DNA.

Frequency: The frequency of transformation

is low.

Transformation; gene transfer by the uptake &

subsequent recombination of a fragment of
exogenous bacterial DNA
2. Transduction occurs when fragments of
chromosomal DNA is transferred or
transduced into a second bacterium by phage.

During phage replication, the bacterial DNA

may be accidentally enclosed instead of the
normal phage DNA, and when this particle

which enclosed the bacterial DNA infects a

second bacterial cell, the DNA from the first
bacterium is released and incorporated into the
chromosome of the second bacterium.
3. Conjugation occurs when plasmid DNA is
transferred from donor

to recipient bacterium by direct contact via a

sex pilus.
4. Transposition

Mechanism which enhances genetic flexibility

among plasmids and bacterial chromosomes.

Transposons (Jumping genes) are segments of

DNA that can transpose or move extremely
readily, from plasmid to plasmid or from
plasmid to chromosome (and viceversa).In

this way, plasmid genes become part of the

chromosomal component of genes.

When transposons transfer to a new site, it is

usually a copy of the transposon that moves,
the original transposon remaining in site.