Physiology and Growth of Bacteria

Bacterial growth
• It involves - an increase in the size of the cell and an
increase in the number of individual cells
• Bacteria increase in number (multiply) by binary fission. This
involves:
– Replication of chromosomes and elongation of cells
– Separation of chromosomes and septum formation
– Cell division begins with the invagination of the cytoplasmic membrane
between approximately equal amounts of protoplasm each containing at
least one nucleus.
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 …

• A visible group of bacteria growing on a solid medium,

presumably arising from a single bacterium known as a colony
– One cell becomes colony of millions of cells.
Binary fission

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Generation time
• The rate of bacterial reproduction is measured as the
generation time, the length of time required for a single
bacterial cell to yield two daughter cells.
• Bacterial pathogens have generation time ranging from 30
minutes to 30 hours.
• Escherichia coli, a common enteric organism (originating in
the intestine), has a generation time of approximately 20
minutes.
– Depend on chemical and physical conditions
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 …

Organism Generation Time

(optimal condition)
Bacillus cereus 28 min

Escherichia coli 12.5 min

Staphylococcus aureus 27-30 min

Mycobacterium tuberculosis 18 – 24 hrs

Treponema pallidum (agent of Syphilis) 30 hrs

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 …
Requirements for Growth of bacteria

• Physical
– Temperature
– pH
– Osmotic pressure
– Moisture

• Chemical
– Carbon
– Nitrogen, sulfur and phosphorus
– Trace elements
– Oxygen
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 …

Temperature

• Each bacteria has its own minimum, optimum and maximum

temperature requirements
• Minimum
– temperature below which bacterial growth will not take place
– metabolic rates are very slow
• Optimum
– Temperature at which organisms grow best
– Enzymatic reactions inside the cell proceed at faster rates, and
growth also become more rapid
• Maximum
– temperature above which bacterial growth will not take place
– Proteins, DNA, and RNA become irreversibly denatured, and the
growth rate falls rapidly to zero
– Continued increase in temperature will kill the microbe
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 …
• Four categories of microbes based on temperature ranges for growth

1. Psychrophiles: two groups

– True Psychrophiles (-10 to 20)
• Sensitive to temperatures over 20oC
• Optimum growth at 15oC or below
• Seldom cause disease or food spoilage
• Eg. Desulfofrigus oceanense (Arctic and Antarctic Oceans)

– Psychrotrophs (0-30)
• Optimum growth at 20 to 30oC
• Responsible for most low temperature food spoilage
• Eg. Yersinia species grow well at room temperature (25oC)

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 …

Mesophiles (10-50)
– Most bacteria (eg. E. coli)
– Include most pathogens and common spoilage organisms
– Optimum temperature for most mammalian pathogens ranges
from 35oC to 37oC
– Many have adapted to live in the bodies of animals

3. Thermophiles (40-70)
– Optimum growth between 50 to 60oC
– Many cannot grow below 45oC
– Some thermophiles form extremely heat resistant endospores
(eg.: Bacillus anthrasis )
4. Extreme Thermophiles (Hyperthermophiles) (70-110)
– Optimum growth at 80oC or higher (e.g. Archaebacteria)
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 …

• Categories of microbes based on the temperature ranges for growth

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H-ion Concentration (pH)

• Each microorganism has a pH range within which growth is possible

– Neutral or slightly alkaline pH (7.2 – 7.6)
• majority of pathogenic bacteria grow best

– Acidic pH (Acidophiles) : grow BEST at low pH (acid: pH 0 –

1.0)
• Eg. Lactobacilli

– Alkaline pH (Alkalophiles): grow BEST at high pH (alkaline:

pH 10.0)
• Eg. Vibrio cholerae

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 …

Osmotic pressure or Osmolarity

• A pressure that develops when two solutions of different

concentrations are separated by a semi-permeable membrane
• Microorganisms require water for growth and are made up of 80-
90% water
• Most bacteria require an isotonic environment or a hypotonic
environment for optimum growth (low osmotic pressure)

• Osmotolerant - organisms that can grow at relatively high salt

concentration (up to 10%)

• Halophiles – organisms that can grow at high salt concentration.

Example, some of the Archea (20% or higher)
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Moisture and drying

• Water – essential ingredient of bacterial protoplasm. Hence drying is

lethal to cells

• Maximum, optimum and minimum requirement for all

microorganisms

• Effect of drying varies

– T. pallidum – highly sensitive
– Staphylococcus spp– stand for months
– Spores – resistant to desiccation, may survive for several
decades

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Radiation and stress

• Radiation
– X rays and gamma rays exposure – lethal

• Mechanical and sonic Stress

– May be ruptured

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Chemical requirements
• Microorganisms may be divided into two major categories according to
their ability to use various forms of energy and carbon for
biosynthesis. These are autotrophs and heterotrophs

1. Autotrophs: two major categories

– Photoautotrophs: are capable of using light energy as a sole energy
source and either carbon dioxide or more reduced organic
molecules as a carbon source for growth

– Chemolithotrophs (autotrophic): are those that can not use light as

an energy source but that can use inorganic molecules as a sole
source of energy, and they may use either carbon dioxide or more
reduced organic molecules as a carbon source for synthesis and
growth
• There are no known strict autotrophic microorganisms that are
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animal pathogens 15
 …
2. Heterotrophic Bacteria
• In contrast to autotrophs, hetrotrophs cannot use light or inorganic
compounds for energy, and their carbon source for growth needs to
be obtained directly from their environment in the form of
biomolecules

• Especially, the heterotrophs use reduced organic molecules (such as

sugars, aminoacids, fatty acids, and nucleic acids) both as a source
of energy and as a source of carbon for synthesis and growth
– All pathogenic microorganisms, both opportunistic and strict
pathogens, are heterotrophs

– Most cultivated in artificial media

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 …

• Carbon
– One of the most important requirements for microbial growth
– structural backbone of living matter
– needed for all the organic compounds that make up a living cell
– ½ of the “dry weight” of a bacterial cell is carbon

• Nitrogen, Sulfur, and Phosphorus

– needed by microorganisms for the synthesis of cellular material
– e.g. protein, DNA, RNA, ATP

• Trace Elements
– iron, copper, and zinc
– essential for the function of certain enzymes
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Oxygen
• Depending on their oxygen requirements microorganisms can
be classified into 4 groups:
– Obligate aerobes - require oxygen for their growth e.g.
Micrococcus, spore forming bacilli
– Obligate anaerobes - do not require oxygen for their growth.
Oxygen is toxic substance, which either kills or inhibits their
growth. They live by fermentation or anaerobic respiration e. g.
Bacteriodes, Fusobacterium
– Facultative anaerobes (or facultative aerobes) - they grow in
the presence or absence of oxygen. Under anaerobic condition
they grow by fermentation or anaerobic respiration but in the
presence of oxygen they switch to aerobic respiration e.g.
Escherchia coli
– Micro-aerophilic - require very low oxygen for their growth e.g.
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1: Obligate aerobic bacteria gather at top of test tube to absorb maximal amount of oxygen.
2: Obligate anaerobic bacteria gather at bottom to avoid oxygen.
3: Facultative anaerobes gather mostly at the top, since aerobic respiration is most
beneficial; but as lack of oxygen does not hurt them, they can be found all along the test
tube.
4: Microaerophiles gather at upper part of test tube, not at top. Require O 2, but at low
concentration.
5: Aerotolerant bacteria are not affected by oxygen, and they are evenly spread along the
test tube.
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 Growth curve of bacteria

• Following inoculation of bacterial cells into fresh broth

medium, the growth curve of the culture exhibits:
– lag phase,
– exponential phase (logarithmic phase),

– stationary phase and

– decline phase (dead phase).

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1. Lag phase
– Period of adjustment to new conditions
– Little or no cell division occurs, population size doesn’t increase
– Phase of intense metabolic activity, in which individual
organisms grow in size
– May last from one hour to several days

2. Log phase (exponential growth)

– Cells begin to divide
– Period of most rapid growth
– Number of cells produced > Number of cells dying
– Cells are at highest metabolic activity
– Most sensitive to drugs and radiation during this period

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 …

3. Stationary phase
– Number of cells produced = Number of cells dying
– Cell division begins to slow down
– Factors that slow down microbial growth
• Accumulation of toxic waste materials
• Acidic pH of media
• Limited nutrients
• Insufficient oxygen supply
4. Death or Decline phase
– Population size begins to decrease
– Number of cells dying > Number of cells produced
– Cells lose their ability to divide
– Few cells may remain alive

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• Morphological and physiological alterations during growth
– Lag phase – maximum cell size towards the end of lag phase

– Log phase – smaller cells, stain uniformly

– Stationary phase – irregular staining, sporulation and production

of exotoxins

– Phase of Decline –involution forms (with ageing)

• irregular/atypical bacteria. Bacteria that do not color with
gram stain, but remains colorless

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 Microbial genetics and variation

• Microbial genetics is concerned with the transmission of

hereditary characters in microorganisms.
• Because of their relative simplicity,

– microbes are ideally suited for combined biochemical

and genetic studies and
– have been successful in providing information on
the genetic code and the regulation of gene activity.

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• The many applications of microbial genetics in medicine
and the pharmaceutical industry emerge from the fact that
microbes are both the causes of disease and the producers
of antibiotics .

• Genetic studies have been used to understand variation in

pathogenic microbes and also to increase the yield of
antibiotics from other microbes.
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• Hereditary processes in microorganisms are analogous to
those in multicellular organisms.

• In both prokaryotic and eukaryotic microbes, the genetic

material is DNA ; the only known exceptions to this rule are
the RNA viruses. 

• Mutations - heritable changes in the DNA, occur

spontaneously

• the rate of mutation can be increased by mutagenic agents.

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 Bacterial genetics and mechanisms of
genetic variation
• Bacteria are haploid with one circular chromosome consisting
of double-stranded DNA.
• The chromosome, which is free in the cytoplasm in a coiled
configuration, is much longer than the parent cell and
contains a large number of genes coding proteins which are
essential for the metabolic process.
• Plasmids, bacteriophages and transposable elements may
contribute additional genetic information, some of which may
influence phenotypic expression.
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 Bacterial DNA replication

• As bacteria replicate by binary fission, daughter cells are

usually identical genetically.
• During replication, the sequence of purine and
pyrimidine nucleotides in the DNA is copied into two
double stranded daughter molecules.

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 Mechanisms contributing to genetic
variation
• Genetic variation may occur following mutation, in
which a change occurs in the nucleotide sequence of
a gene, or
• By recombination, in which new groups of genes are
introduced into the genome.
– new genetic material may be introduced by conjugation,
transduction or transformation collectively known as
horizontal gene transfer.
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 …

• Transformation - a cell takes up isolated DNA

molecules from the medium surrounding it
• Conjugation - involves the direct transfer of DNA
from one cell to another
• Transduction - the transfer is mediated by bacterial
viruses (bacteriophages)

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 Laboratory diagnosis of bacterial disease
• Laboratory investigation of bacterial disease is necessary
for
– identifying the etiological agent and,

– determining the antimicrobial susceptibility of

pathogens.
• A full clinical history including the age, sex, species and
number of animals affected and treatment administered
should accompany the specimens, together with a
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tentative clinical diagnosis.
Selection, collection and transportation of
specimens
• Results of laboratory examination is influenced by

– the selection, collection and submission of samples to

the laboratory.
• Ideally, specimens should be obtained from live animals
before administration of antimicrobial therapy.
• Samples from dead animals should be collected, if
possible, before autolytic or putrefactive changes occur.
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 …
• Specimens, from a site most likely to yield a pathogen,
should be collected using procedures which minimize
contamination.
• In warm weather, refrigeration of samples may be required.
• Samples must be submitted in separate leak-proof
containers.
• Each container should be labeled with the identity of the
animal, the type of specimen and the date of collection.
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 Identification of pathogenic bacteria

• The presence of pathogenic bacteria can be

confirmed by
– examination of stained smears
– cultural and biochemical characteristics and
– detection by immunological and molecular
methods.

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 presumptive identification of bacterial
pathogens
• Colonial morphology and color

• Presence or absence of hemolysis on blood agar

• Appearance when stained by the Gram method

• Motility

• Ability to grow on MacConkey agar

• Reaction in the oxidation-fermentation test

• Reactions in catalase and oxidase tests

• And various biochemical tests
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 Bacterial colony and characteristics

Bacteria grow on solid media as colonies

– A colony is defined as a visible mass of
microorganisms all originating from a single
mother cell.

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 Colony morphology

 Morphology of bacterial colony can be described by

 Size Odor
 Shape Pigmentation
 Color Density
 Texture Hemolysis
 Elevation Surface appearance
 Edge/ margin
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 Types of media for cultivating Bacteria

• Culture media generally provide source of carbon,

energy, and nitrogen in the form of available
carbohydrates and amino acids

• Special media provide specific requirements like

inorganic salts and growth factors

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 Classification of bacterial culture media on
the basis of consistency

• Solid medium- 1.5-2% solidifying agent (agar)

• Semi solid- <=0.5% solidifying agent
• Liquid – no solidifying agent

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 Based on purpose/function culture media can
be grouped in to

• Basal media

• Enriched media

• Selective media

• Enrichment media

• Indicator(differential media)

• Transport media
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 …

Basic media
• Simple media used to support the growth of
microorganisms that does not have additional
nutritional requirement
• Nutrient broth, nutrient agar, peptone water

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Enriched media
• Adding extra nutrients, such as blood, serum, egg yolk,
to the basal medium makes an enriched medium.
• Enriched media are used to grow nutritionally exacting
(fastidious) bacteria.
• Blood agar, chocolate agar, Loeffler’s serum slope

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Selective Media
• Selective medium is designed to suppress some
microorganisms’ growth while allowing others’ growth.
• Selective medium is an agar-based (solid) medium so that
individual colonies may be isolated.
• Manintol salt agar, Macconkey agar, Lowenstein Jensen
(LJ) Medium 

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Enrichment Media

• The enrichment medium increases the relative

concentration of specific microorganisms in the culture
before plating on a solid selective medium.

• Unlike selective media, enrichment culture is typically

used as a broth medium.

• Tetrathionate broth and alkaline peptone water (APW)

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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
color during their growth.
• Various approaches include incorporation of dyes/
metabolic substrates so that those bacteria that utilize
them appear as differently colored colonies.
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 …

• Such media are called differential media or indicator

media.
• Differential media allow the growth of more than
one microorganism of interest but with
morphologically distinguishable colonies
• Blood agar, macConkey agar, Mannitol salt agar

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 Blood agar
Macconkey agar

MSA

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 …
Transport Media
• Clinical specimens must be transported to the
laboratory immediately after collection to prevent
overgrowth of contaminating organisms or
commensals and maintain the viability of the potential
pathogens.
• This can be achieved by using transport media
• Cary Blair transport medium
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Culture methods

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 …

Culture methods
– The different culturing techniques used in
bacteriology
• Culture methods employed depend on the purpose
for which they are intended.

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 …
 Streak culture
 Lawn culture

 Stroke culture

 Stab culture

 Pour plate method

 Anaerobic culture methods

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 Streak Culture

Purpose
 used for the isolation of bacteria into pure culture of
the organisms from mixed population clinical
specimens

Principle
 As the original sample is diluted by streaking it over
successive quadrants, the number of organisms
decreases

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 Pour Plate

Purpose
 usually the method of choice for counting the
number of colony forming bacteria present in liquid
specimen
 provide a uniform surface growth of the bacterium

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 Spread Plate

Purpose
 A technique to plate a liquid sample containing
bacteria so that the bacteria are easy to count
and isolate
 A successful spread plate will have a
countable number of isolated bacterial colonies
evenly distributed on the plate
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 Lawn Culture

 Also called as carpet culture

 A term used to describe the appearance of bacterial
colonies when all the individual colonies on a petridish
agar plate merge to form a field or mat of bacteria

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 …
Purpose

 Provides a uniform growth of the bacterium

 Useful for bacteriophage typing and AST

 Also used in the preparation of bacterial antigens and

vaccines

 Bacterial lawns use in screens for antibiotic resistance

and bacteriophage tittering

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 Stroke Culture

 Stroke culture is made in tubes containing agar

slope / slant
Purpose
 Provide a pure growth of bacterium for slide
agglutination and other diagnostic tests
 To store a clone of bacteria to be used later

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 Stab Culture

 A culture produced by inserting an inoculating needle

with inoculum down the center of a solid medium
contained in a test tube

Purpose
 Demonstration of gelatin liquefaction
 Oxygen requirements of the bacterium under study
 Maintenance of stoke cultures
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 Anaerobic Culture

 Is a method used to grow anaerobes from a clinical

specimen
 Anaerobic bacteria differ in their requirement and
sensitivity to oxygen
 C. tetani is a strict anaerobe grows at an oxygen
tension < 2 mm Hg

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 …

Purpose
 To identify bacteria that grow only in the absence of
oxygen

 To identify anaerobic pathogens and institute

effective antibiotic treatment

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• Enumeration of bacteria?

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Biochemical tests

83
 …

  After a pure culture is obtained, relatively simple

and complex tests will be used to identify

bacterium to a generic/species level.

 Some of the tests _____________________________________

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 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.
 This enzyme converts hydrogen peroxide into water and
oxygen.

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 Catalase test

Results
• Active bubbling =
Positive catalase
test.
• No bubbles =
Negative catalase
test.

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 Oxidase Test

The oxidase test is used to assist in the identification of

Pseudomonas, Neisseria, Vibrio, Brucella, and Pasteurella
species, all of which produce the enzyme cytochrome oxidase.
 Principle

When the organism is oxidase-producing, the phenylenediamine

(oxidase reagent) will be oxidized to a deep purple color.

 Requirements:

1.Oxidase reagent strips.

2.Stick or glass rod.

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 Oxidase Test

• Deep purple color=

positive oxidase test
• no color= negative

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 Coagulase test
Principle:
• Differentiate Staphylococcus aureus (positive) from
coagulase negative Staphylococci. S. aureus
produces two forms of coagulase: bound and free.
• Bound coagulase or clumping factor, is bound to
the bacterial cell wall and reacts directly with
fibrinogen.
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 …
• When a bacterial suspension is mixed with plasma, this
enzyme causes alteration in fibrinogen of the plasma to
precipitate on the staphylococcal cells, causing the cells to
clump.

• Free coagulase is produced extra‐cellularly by the bacteria

that causes the formation of a clot when S. aureus colonies
are incubated with plasma.

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 ...
Method:

Slide test: (for bound coagulase)

Tube test: (for free coagulase)

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 ....
Slide test:
• Positive: Macroscopic clumping in
10 seconds or less in coagulated
plasma drop and no clumping in
saline or water drop.
• Negative: No clumping in either
drop.
• Note: All negative slide tests must
be confirmed using the tube test.
Tube test:
• Positive: Clot of any size (a)
• Negative: No clot (b)
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 ...

Indole Test
Principle:
• Indole test is performed to determine the ability of the
organism to split tryptophan molecule into Indole. Indole
is one of the metabolic degradation product of the
amino acid tryptophan.
• Bacteria that possess the enzyme tryptophanase are
capable of hydrolyzing and deaminating tryptophan with
the production of Indole, Pyruvic acid and ammonia.
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 …
Result
• Development of bright red color at the interface of
the reagent and the broth within seconds after
adding the reagent is indicative of the presence of
Indole and is a positive test

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 ...

• Indole Positive:
• E.coli
• Proteus vulgaris
• Indole Negative:
• Salmonella spp.
• Klebsiella spp.
• Enterobacter aerogens

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 Methyl Red/Voges‐Proskauer (MR/VP)
 Both tests are used to differentiate species of the family
Enterobacteriaceae.
 Media and Reagents Used:
• MR/VP Broth
• Methyl Red indicator for MR test
• Voges Proskauer reagents‐
– A: 5% Alpha‐Naphthol & ethanol,

– B: Potassium Hydroxide; (3:1 ratio) &

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 ....

Principle of MR test:
• To test the ability of the organism to produce
and maintain stable acid end products from
glucose fermentation and to overcome the
buffering capacity of the system.
• This is a qualitative test for acid production.
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 ....
Result interpretation:
• Positive result is red (indicating pH below 6)
• Negative result is yellow (indicating no acid
production).

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 ....

• Left: negative/Right:
positive

• MR Positive: E. coli
• MR Negative:
– Enterobacter aerogenes
– Enterobacter cloacae
• Klebsiella spp.

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 Voges Proskauer Test (acetoin production)

Principle:
• To determine the ability of the organisms to produce
neutral end product acetyl methyl carbinol (acetoin)
from glucose fermentation

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 Intrepretation:

• VP: left + and right –

• Positive
• Klebseilla pneumoniae
• Enterobacter
• Negative
• E.coli

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 Citrate Utilization test:

 This test is one of several technique used to assist in the

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

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 …

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 color of the
indicator from light green to blue, due to alkaline
reaction following citrate utilization.
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 ....
Result interpretation:
• Growth on the slant and change in color to blue
of the medium indicates positive result.

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 ....

• Positive:
Klebsiellapneumoniae
• Negative: E. coli

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 Oxidation‐Fermentation (OF) test(Hugh &
Leifson)
Principle:
• Oxidation‐Fermentation test is used to determine the
oxidative or fermentative metabolism of a carbohydrate
or its non utilization.
• Fermentation is a anaerobic process and bacterial
fermenters of carbohydrates are usually facultative
anaerobes. Oxidation is a aerobic process and bacterial
oxidisers are usually strict aerobes.
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 ....

Result interpretation:
• Oxidising organisms, eg Pseudomonas species, produce an
acid reaction in the open tube only.
• Fermenting organisms, eg Enterobacteriaceae, produce an
acid reaction throughout the medium in both tubes.
• Organisms that cannot break down the carbohydrate
aerobically or anaerobically, eg., Alcaligenes faecalis,
produce an alkaline reaction in the open tube and no change
in the covered tube.
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 Triple Sugar Iron Agar (TSI) test
Principle:
• TSI agar is used to determine whether a gram negative
rod utilizes glucose and lactose or sucrose
fermentatively and forms hydrogen sulphide(H2S).
• TSI contains 10 parts lactose: 10 parts sucrose: 1 part
glucose and peptone.
• Phenol red and ferrous sulphate serves as indicators of
acidification and H2S formation, respectively.
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 …
• The formation of CO2 and H2 is indicated by the
presence of bubbles or cracks in the agar or by
separation of the agar from the sides or bottom of the
tube.
• The production of H2S requires an acidic environment
and is indicated by blackening of the butt of the
medium in the tube.
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 Result interpretation:
• 1.Alkaline slant/no change in the butt (K/NC)
= Glucose, lactose and sucrose non‐utilizer
(alkaline slant/alkaline butt) [figure: 1(d)]
• 2.Alkaline slant/acid butt (K/A) = Glucose
fermentation only. [figure: 1(b)]
• 3.Acid slant/acid butt (A/A), with gas
production = Glucose, sucrose, and/or
lactose fermenter. [figure: 1(a)]
• 4.Alkaline slant/acid butt (K/A), H2S
production = Glucose fermentation only.
[figure: 1(c)]
Quality control:
• A/A, with gas: E. coli
• K/A, H2S: Salmonella typhi
• K/NC: Pseudomonas aeruginosa
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 UREASE TEST
 Purpose: The test use to détermine the ability of an organism to split
Urea, forming to molécules of ammonia by the action of the enzyme
urease.
 Principle
• Urease is an enzyme possessed by many species of microorganisms
that can hydrolyze urea with the formation of ammonia (alkaline)
 Procedure
• Add 2% urea on the urea agar base slant and apply heavy inoculums,
then incubate up to 24 hours at 370C.
• Add about 5 drops of Phenol red and leave for 1-2 hours
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Result

Positive - Red (pink) color

throughout the medium.
 protease species
Negative - Yellow color
throughout the medium.
 E. coli

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 CARBOHYDRATE (SUGAR) DIGESTION TESTS 

Purpose

 To identify the isolated bacteria to specious level

Principle

 Some bacteria utilize carbohydrate and change the

PH and color of the Liquid media.

113
 Result

a. Un inoculated
b. no fermentation
c. Fermentation
d. Fermentation + gas

Positive - yellow (utilization of the carbohydrate in the

medium)
Negative - purple (no utilization of the carbohydrate in the
medium)
114
 Motility Test
 Property- This test is done to help differentiate species

of bacteria that are motile from non‐motile.

– Media and Reagents Used: Motility media contains

tryptose, sodium chloride, agar, and a color indicator.

– How to Perform Test: Stab motility media with

inoculating needle.

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 ....
• Results: If bacteria is
motile, there will be
growth going out away
from the stab line, and
test is positive. If bacteria
is not motile, there will
only be growth along the
stab line. A colored
indicator can be used to
make the results easier to
see.

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 Maintenance and preservation of pure cultures of bacteria

• Once a microorganism has been isolated and grown in

pure culture, it becomes necessary to maintain the
viability and purity of the microorganism by keeping the
pure cultures free from contamination

• Normally in laboratories, the pure cultures are transferred

periodically onto or into a fresh medium (sub culturing) to
allow continuous growth and viability of microorganisms.
 …

– Since repeated sub culturing is time consuming, it

becomes difficult to maintain a large number of pure

cultures successfully for a long time.

– In addition, there is a risk of genetic changes as well as

contamination

• Therefore, it is now being replaced by some modern

methods that do not need frequent sub culturing.

These include refrigeration, paraffin method,

cryopreservation, and lyophilization (freeze drying).

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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
bacteria and 3-4 months for fungi)
– because the metabolic activities of the
microorganisms greatly slow down but not stopped
– Thus, their growth continues slowly, nutrients are
utilized and waste products released in medium.
Finally this results in the death of the microbes after
sometime
Paraffin Method

• In this method, sterile liquid paraffin is 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 can
 …
• The advantage is that we can remove some of the
growth under the oil with a transfer needle, inoculate
a fresh medium, and still preserve the original culture

• The simplicity of the method makes it attractive, but

changes in the characteristics of a strain can still occur.
 

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Cryopreservation

• 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 or
Dimethyl Sulfoxide (DMSO) that
prevent the cell damage due to
formation of ice crystals and promote
cell survival
 …
• This liquid nitrogen method has been successful with
many species that cannot be preserved by lyophilization

• Most species can remain viable under these conditions

for 10 to 30 years without undergoing change in their
characteristics

• However this method is expensive.

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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

• Many species of bacteria preserved by this

method have remained viable and
unchanged in their characteristics for more
than 30 years. 
Lyophilization (Freeze-Drying)

• Advantage of Lyophilization
– Only minimal storage space is required; hundreds
of lyophilized cultures can be stored in a small area

– Small vials can be sent conveniently through the

mail to other microbiology laboratories when
packaged in a special sealed mailing containers

– Lyophilized cultures can be revived by opening the

vials, adding liquid medium, and transferring the
rehydrated culture to a suitable growth medium.