Cultivation of Microorganisms

CULTIVATION OF MICROORGANISMS

OBJECTIVES:
 To know the proper technique of cultivating microorganisms
 To determine the factors that affect the growth of microbes
 To familiarize oneself with the cultural characteristics of some
microorganisms

INTRODUCTION

The survival of microorganisms in the laboratory, as well as in nature,

depends on their ability to grow under certain chemical and physical
conditions. An understanding of these conditions enables us to characterize
isolates and differentiate between different types of bacteria. Such
knowledge can also be applied to control the growth of microorganisms in
practical situations.

Methods of Culturing Microorganisms

5 Basic Techniques: - to manipulate, grow, examine and characterize
microorganisms
1. inoculation - to culture, one introduces a tiny sample (inoculum) into
a container of nutrient medium, which provides an environment in
which they multiply
2. incubation
3. isolation
4. inspection
5. identification

Culture
– a culture is the microorganisms that grow in a culture medium
– the observable growth that appears in or on the medium

Colony
– a pile or mass of a sufficiently large number of cells, growing on or
in solid medium, that they are visible to the naked eye

Types of Culture

1. Pure Culture – a container of medium that grows only a single known

species or type of microorganism
– most frequently used for laboratory study because it
allows the systematic examination and control of one
microorganism by itself
2. Mixed Culture – a container that holds two or more identified, easily
differentiated species of microorganisms

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 Cultivation of Microorganisms

3. Contaminated Culture – was once pure or mixed but has since had
contaminants introduced into it like weeds into a garden.

Techniques for isolating cells

The isolation methods most commonly used to get a pure culture include:
streak plates and pour plates.

• Streak Plate Method – a small droplet of culture or sample is spread

over the surface of the medium according to a pattern that gradually
thins out the sample and separates the cells spatially over several
sections of the plate
o most commonly used isolation technique in microbiological
laboratories
o involves techniques for physically separating microorganisms on
an agar surface
o Streaking is a method of applying cultures to solid medium:
 a sterile loop is cooled and brought into contact with a
culture
 the loop is then brought into contact with the surface of
solid medium whereupon it is streaked (i.e., dragged)
along the surface of the solid medium
 colonies grow along the points of the streak
o Colony isolation:
 A petri dish is streaked in manner such that individual
colonies may be isolated.

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 Cultivation of Microorganisms

• Loop dilution or Pour Plate – the sample is inoculated serially into a

series of cooled but still liquid agar tubes so as to dilute the number of
cells in each successive tube in the series.
o involves diluting organisms in fluid and adding a dilution to
melted agar, and pouring agar into plate

Culture medium [pl. media]

– Culture media are solutions containing all of the nutrients and
necessary physical growth parameters necessary for microbial
growth.
– nutrient material prepared for the growth of microorganisms

Criteria for culture medium:

1. contain nutrients for growth
2. proper moisture, oxygen and pH
3. sterile (initially)
4. incubated proper temperature

– Medium is always the singular form of the word, and media is

always and only the plural form. This can be classified on three
primary levels:
(1)physical form
(2)chemical characteristics
(3)functional type

PHYSICAL STATE
1. Liquid Media
− water-based solutions that do not solidify at temperatures above
freezing and that tend to flow freely when the container is tilted
2. Semi-solid Media
− exhibit a clot-like consistency; contains an amount of solidifying
agent (agar or gelatin) that thickens them but does not produce
firm substrate.
− used to determine the motility of bacteria and to localize a
reaction at a specific site
3. Solid Media
− provide a firm surface on which cells can form discrete colonies
− advantageous for isolating and subculturing bacteria and fungi
– has physical structure (broth lacks structure) and this allows
bacteria to grow in physically informative or useful ways (e.g., as
colonies or in streaks).
– usually used as:
 slants

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 Cultivation of Microorganisms

 stabs
 petri dishes

a. Liquefiable Solid Media

− sometimes called reversible solid media
− contain a solidifying agent that is thermoplastic – its physical
properties change in response to temperature
Agar
– complex polysaccharide isolated from the red algae (Gelidium)
– the major solidifying agent used in bacteriological media
– dissolves at approximately 100°C, and an agar-containing medium thus
heated will not solidify until the temperature is brought down to about
43°C. Once solidified, the medium will not melt until brought
back up to about 100°C
– among the advantages of this interesting temperature-related property
are the following:
 medium can be inoculated while in a liquid state at a low
enough temperature (approx. 43-50°C) such that the cells will
not die off
 the medium, once solidified, will stay solid over a wide range
of incubation conditions
– resistance to degradation by nearly all organisms and its relative
clarity, permitting easy viewing of growth on or in the medium
– very difficult, if not impossible, to purify it fully of trace impurities.
Thus, when agar is added to a chemically-defined liquid medium, the
medium must be considered complex. If an absolutely chemically-
defined solid medium is required, silicon-based solidifying agents can
be employed.
– In 1881, Fanny Eilshemius Hesse, a technician in the laboratory of
Robert Koch in Germany, introduced the concept of agar to
bacteriology, having used it for many years in the preparation of
homemade jellies.
– Short History:
 Agar, as a culture medium, was suggested by Frau Hesse, the
wife of a co-worker of Dr. Robert Koch. Walther Hesse apparently
discussed his work with his wife, who was not a scientist, but did
know her way around the kitchen. Lina Hesse - originally Fanny
Angelina Eilshemius from NY - suggested that they try agar-agar
when asked why her jellies and puddings stayed solid in the hot
summers in Dresden. Walther told Koch about it. Dr. Robert Koch
used it in 1881 to culture bacteria. It has been used ever since.

Important properties:
– few microbes can degrade it; remains solid even when bacteria and
fungi are growing on them

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 Cultivation of Microorganisms

– melts at boiling point of water; does not melt below 100oC; can be
used to culture many thermophiles
– once solidified can be incubated at temperatures approaching 100oC

b. Nonliquefiable Solid Media

− less versatile applications than agar media because they are not
thermoplastic

CHEMICAL CONTENT OF MEDIA

1. Defined/Synthetic Media
− media whose compositions are chemically defined
− contain highly pure organic and inorganic compounds that vary little
from one source from another
− have a molecular content specified by means of an exact formula
− all the ingredients of a culture medium are known, both qualitatively
and quantitatively
− great value in studying the nutritional requirements of
microorganisms or in studying a great variety of their metabolic
activities
2. Complex/Nonsynthetic Media
− contain at least one ingredient that is not chemically definable
− not a simple, pure compound and not representable by an exact
chemical formula
− exact chemical composition is not known, and such a medium is
often prepared from very complex materials
− contains most of the organic compounds-sugars, amino acids, and
nucleotides-necessary for growth
− contains nutrients released by the partial digestion of yeast, beef,
soy or proteins
− e.g. blood, serum, and meat extracts or infusions, body
fluids, tissue extracts and infusions, and peptones, nutrient
broth, Tryticase soy agar, MacConkey agar
3. Enrichment media - media used to enhance the growth of the
desired organism in a mixed population; similar to selective media but
designed to increase the numbers of desired microorganisms to a
detectable level without stimulating the rest of the bacterial population

Peptone
– is a commercially-available digest of a particular plant or animal
protein, made available to organisms as peptides and amino acids to
help satisfy requirements for nitrogen, sulfur, carbon and energy
– short chains of amino acids produced by enzymatic digestion of
proteins

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 Cultivation of Microorganisms

SELECTIVE AND DIFFERENTIAL MEDIA

1. Selective Medium
– contain one or more agents that inhibits the growth of a certain
microbe or microbes (A, B, C) but not others (D), and thereby
encourages or selects microbe D and allows it to grow
– very important in primary isolation of a specific type of
microorganisms from sample containing a highly mixed population
– growth media that contains substances that inhibit the growth of
unwanted organisms but permit the growth of the desired organism
- uses certain dyes, high salt concentration, pH or antibiotics
– Examples of selective media include:
 mannitol salts agar (selects against non-skin flora)
 MacConkey agar (selects against gram-positives)
 eosin-methylene blue agar (selects against gram-positives)
 phenylehyl alcohol agar (selects against gram-negatives)
 Eosin, methylene blue, crystal violet dyes (inhibit the growth
of gram-positive bacteria without affecting gram-negative)
– supports the growth of desired organisms while inhibiting the
growth of many or most of the unwanted ones – either by purposely
adding one or more selective agents which "poison" certain types
of organisms or by including or deleting certain nutrients such that
the desired organisms and few others are able to grow
2. Differential Medium
– can grow several types of microorganisms but it is designed to
highlight differences among these microorganisms
– allow the growth of more than one microorganism of interest but
with morphologically distinguishable colonies.
– contains a combination of nutrient and pH indicators to visually
differentiate bacteria that grow on or in it
– frequently a solid medium on which colonies of a particular species
will have a distinctive color or cause a change in the color of the
medium.
– Examples of differential media include:
 mannitol salts agar (mannitol fermentation = yellow)
 blood agar (various kinds of hemolysis)
 MacConkey agar (lactose fermentation = yellow)
 eosin-methylene blue agar (various kinds of differentiation)
– allows two or more different organisms to grow, but it contains dyes
and/or other components upon which different organisms act in
various ways to produce a variety of end products or effects, often
detected by variations in color. These differences are often very
apparent among colonies of a mixed culture growing in a petri dish.
Pure cultures, growing in separate tubes of the same differential
medium, may also be characterized and differentiated from one
another according to a particular biochemical characteristic.

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 Cultivation of Microorganisms

CULTURAL CHARACTERISTICS OF THE COLONIES IN PLATES:

Plate Color Form Margin Elevation

A
B
C (1)
(2)
(3)

A. TSI Reaction: Butt –

Slant –
Gas Production –

Possible Identity of the Sample:

COLONY MORPHOLOGY
Differentiating colonies:
– Colony morphology gives important clues as to the identity of their
constituent microorganisms.
– Important classes of characteristics include:
 size
 type of margin
 colony elevation
 colony texture
 colony pigmentation
Colony size
– Colony size is dependent not just on the type of organism but also
on the growth medium and the number of colonies present on a
plate (that is, colonies tend to be smaller when greater than
ascertain amount are present) and on culture medium
characteristics.

Usually stabilizes after few days:

– Colony size usually stabilizes after a day or two of incubation.
– Exceptions include:
 slow growing microorganisms
 during growth under conditions that promote slow growth
 With slow growth colonies may continue to experience growth
past this time, especially if an effort is made to prevent solid
medium from drying out.

Type of margin
– Colonies can vary in the shape of their margins.

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 Cultivation of Microorganisms

Illustration, variation in colony form

Colony elevation
– Colonies can vary in their elevations both between microorganisms
and growth conditions, and within individual colonies themselves.

Illustration, variations in colony elevation

Colony texture
Surface appearance:
– Colonies can vary in their texture.
– Possible textures include:
 shiny to dull
 smooth to wrinkled
 rough
 granular
 mucoid
 A shiny, smooth, and/or mucoid appearance tends to be
associated with the presence of capsular material.

Colony pigmentation
– Colonies can come in a rainbow of colors.

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Cultivation of Microorganisms

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 Cultivation of Microorganisms

Examples of various colony morphologies. The appearance of colonies on a

plate is species specific and can be very helpful in identifying isolates.

NUTRITIONAL AND ENVIRONMENTAL FACTORS INFLUENCING

MICROBIAL GROWTH
Growth requirements - two categories:
o Environmental - temperature, pH, osmotic pressure, oxygen
o Chemical - water, sources of carbon and nitrogen, minerals, and
organic growth factors

Chemical Requirements - Nutritional Classification of

Microorganisms

Organisms can be divided into groups based on their energy and

carbon sources. Regarding the source of energy which becomes trapped in

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 Cultivation of Microorganisms

an organism's ATP, the various life forms may be categorized as either

chemotrophs or phototrophs.
• Chemotrophs obtain their energy purely from the oxidation of
chemical compounds.
• Phototrophs use light as the ultimate source of energy. Phototrophs
include plants, algae, cyanobacteria, and the purple and green
anoxygenic bacteria.

As carbon is a major and essential element in all living things,

organisms may also be classified according to the nature of their source of
carbon. Organisms which assimilate organic compounds for their carbon
needs are termed heterotrophs. Those which utilize carbon dioxide are
called autotrophs.

Another method of classifying organisms nutritionally is by the source

of reducing power utilized. All organisms need reducing power in the form
of electrons for biosynthesis. Organisms that oxidize organic compounds
are called organotrophs and those that oxidize inorganic compounds are
called lithotrophs.

Considering the various requirements for carbon and energy described

above, nearly all living things can be placed in one of the following
categories:

• CHEMOHETEROTROPHS. As these organisms are generally

organotrophic, they may also be called chemoorganotrophs. These
organisms may use a variety of organic compounds as both carbon
and energy sources. A common sugar so used is glucose. ATP is
generated by either substrate-level or oxidative phosphorylation.
These also require organic molecules such as glucose, amino acids and
vitamins, which supply carbon and energy or are vital growth factors.
• CHEMOAUTOTROPHS. As these organisms are generally lithotrophic,
they may also be called chemolithotrophs. ATP is usually generated
by oxidative phosphorylation.
• PHOTOHETEROTROPHS. Green nonsulfur and purple nonsulfur
bacteria
• PHOTOAUTOTROPHS. Plants, algae and cyanobacteria use water to
reduce carbon dioxide, producing oxygen as a byproduct

Energy source
 Phototroph - light
 Chemotroph - chemical

Carbon source
 Heterotroph -- "feeder on others" organic

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 Cultivation of Microorganisms

 Autotroph - "self-feeder" carbon dioxide

* Most medically important microorganisms are chemoheterotrophs.

Environmental Factors affecting Microbial Growth

The physical-chemical factors of natural environment determine the

rates of microbial growth and the nature and size of the indigenous
population.

1. Temperature
– one of the most important factors affecting the growth rate of
microbes
– a profound effect on microorganisms
 For many bacteria both extremely high and extremely low
temperatures can be quite harmful, the former due to
protein denaturation, the latter due to intracellular ice
crystal formation upon freezing.
 There neverthless exist microorganisms whose optimum
growth temperatures might be considered extreme.
– as the temperature rises, there is a minimum temperature, below
which growth does not occur
– as we rise above the minimum, rate of growth increases in
accordance with the laws governing the effect of temperature on
the chemical reactions that make up growth
– reactions are mostly enzyme catalyzed
– a point is reached the optimum temperature when there is also a
very rapid increase the rate of inactivation of heat sensitive cell
components, like enzymes, ribosomes, DNA, membranes etc.
– above an optimum temperature, this heat denaturation will occur
so rapidly that there is a corresponding rapid drop in the rate of
growth to give a maximum temperature for growth for that
particular microorganism
– there is an increase in the growth rate due to increasing the
rates of enzyme reactions; eventually a temperature becomes
too high and microorganisms are damaged by enzyme
denaturation, membrane disruption, and other phenomena
– organisms can be divided into groups on the basis of their
preferred temperature range
 psychrophile --- optimum <15oC; can even grow at
temperatures below O oC; die at temperatures much above
20 oC, live in surface of glaciers, snowfields, ice and cold
water
--- do not cause disease to humans because
they cannot survive body temperature

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 Cultivation of Microorganisms

 mesophile --- optimum 20-45oC; human pathogens are

mesophiles
o e.g. Thermoduric organisms – mesophiles that
capable of short exposures to relatively high
temperatures; organisms that survive following
inadequate heating of foods and may thereby
contribute to the spoilage of foods that have
been heated (e.g., Pasteurization) to kill
microorganisms
 thermophile --- optimum 55-65oC; above 45 oC; found in
compost heaps or hot springs
--- do not cause disease because they freeze at
body temperature
 hyperthermophile --- optimum > 80oC; e.g. Pyrodictium;
usually members of the Archae and are found growing near
hydrothermal vents at great depths in the ocean
– Organisms exhibit distinct cardinal temperatures (minimal,
maximal, and optimal growth temperatures)
 minimum temperature - temperature below which no
growth occurs
 optimum temperature - maximum growth; highest
growth rate
 maximum temperature - temperature above which no
growth occurs

2. pH
– the negative logarithm of the hydrogen ion concentration
– organisms are sensitive to changes in acidity because hydrogen
ions and hydroxyl ions interfere with hydrogen bonding within
the molecules of proteins and nucleic acids
– Most microbes grow best at pH near neutrality, bacteria usually
slightly on the alkaline side and algae and fungi on the acid side.
– some can grow at extreme values of low or high environmental
pH. For example, a few bacteria that oxidize inorganic sulphur
compounds to H2SO4 can grow at pH 0 (i.e. 1M H2SO4)
– other bacteria, as those causing human urinary, tract infections
(that hydrolyze urea to produce excess of NH3 causing a rise in
pH) grow at high pH of 11.0
– Each species has a pH growth range and pH growth optimum
 acidophiles - grow best between pH 0 and 5.5;
organisms that grow best in acidic habitats
e.g. chemoautotrophic bacteria – live in mines
 neutrophiles - grow best between pH 5.5 and 8.0; pH
range of most tissues and organs in the human body
 alkalophiles - grow best between pH 8.5 and 11.5

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 Cultivation of Microorganisms

e.g. Vibrio cholerae – grows best outside of the body

in water at ph 9.0
– Microorganisms can usually adjust to changes in environmental
pH by maintaining an internal pH that is near neutrality; some
bacteria also synthesize
protective proteins (acid shock
proteins) in response to pH
– When bacteria are cultured in
the lab, they produce acids as
part of their metabolism. The
acids can interfere with bacterial
growth. Buffers (chemicals) are
added to growth media to
maintain proper pH.
– measure of the amount of acidic
ions vs. basic ions
Scale of 1-14
1. most grow at pH of 7
(neutral)
2. acidophilic (acid-loving)
e.g. Helicobacter pylori – acid-tolerant
bacterium; neutralizes stomach acid by
secreting bicarbonate and urease, an
enzyme that converts urea to ammonia,
which is alkaline

3. Oxygen concentration
Aerobe
– An aerobe is a microorganism that can utilize molecular oxygen
as its final electron acceptor, i.e., as in cellular (aerobic)
respiration.
– produce enzymes catalase and superoxide dismutase that
protect them from toxic forms of oxygen

 Obligate aerobe
– No fermentation:
i. An obligate aerobe is a microorganism that cannot live in
the absence of molecular oxygen.
ii. This basically means that they cannot obtain energy via
fermentative processes.
iii. More precisely, obligate aerobes are organisms that
1. have an electron transport system
2. are able to grow in the presence of atmospheric
oxygen concentrations
3. can use O2 as a final electron acceptor
4. cannot ferment

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 Cultivation of Microorganisms

– Examples:
i. Bacillus subtilis
ii. Bdellovibrio spp.
iii. Bordetella pertussis
iv. Legionella spp.
v. Mycobacterium leprae
vi. Mycobacterium tuberculosis
vii. Neisseria gonorrhoeae
viii. Neisseria meningitidis
ix. Pseudomonas spp.

 Obligate [strict] anaerobe

a. O2 intolerant:
i. Many anaerobes not only can't utilize molecular oxygen but
are harmed by it as well.
ii. One usage of the term obligate anaerobe is to describe
only those microorganisms which are unable to grow (and,
for that matter, even survive) in the presence of molecular
oxygen.
iii. The other, less strict usage of the term obligate anaerobe
is simply to distinguish the term "anaerobe" from the term
"facultative anaerobe."
iv. Of bacterium, which are incapable of survival in the
presence of O2 are included Clostridium botulinum and
Clostridium tetani.

– Facultative anaerobes - organisms that have the

ability to use oxygen when it is present but are able to
continue to grow by using fermentation or anaerobic
respiration when oxygen is not available though their
metabolic efficiency is often reduced in the absence of
oxygen
e.g. Escherichia coli

– Aerotolerant anaerobes – do not use aerobic

metabolism but they tolerate oxygen by having some of
the enzymes that detoxify oxygen’s poisonous forms
e.g. lactobacilli that transform cucumbers into
pickles; milk into cheese; Streptococcus pyrogenes

– Microaerophile
a. Low O2 requirement and tolerance:
i. Microaerophiles are microorganisms which are unable to
grow when oxygen concentrations reach those found in air
(20%) but nevertheless whose growth requires the
presence of some oxygen (2% to 10%).

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 Cultivation of Microorganisms

ii. Microaerophiles are damaged by the 21% concentration of

oxygen in the atmosphere because they have limited
tolerance due to sensitivity to toxic forms of oxygen, low
levels of detoxifying enzymes or enzymes that don't work
adequately.
iii. Microaerophiles appear to grow best in the presence of a
small amount of free oxygen. They grow below the surface
of the medium in a culture tube at the level where oxygen
availability matches their needs.
b. Examples:
i. Borrelia burgdorferi
ii. Helicobacter pylori

4. Osmotic pressure
– is the diffusion of water across a membrane from an area of
higher water concentration (lower solute concentration) to
lower water concentration (higher solute concentration)
– is related to the concentration of dissolved molecules and ions in
a solution
– a cell can find itself in one of three environments: isotonic,
hypertonic, or hypotonic. (The prefixes iso-, hyper-, and hypo-
refer to the solute concentration)
 In an isotonic environment, both the water and solute
concentration are the same inside and outside the cell
and water goes into and out of the cell at an equal rate.
 If the environment is hypertonic, the water
concentration is greater inside the cell while the solute
concentration is higher outside (the interior of the cell is
hypotonic to the surrounding hypertonic environment).
Water goes out of the cell.
 In an environment that is hypotonic, the water
concentration is greater outside the cell and the solute
concentration is higher inside (the interior of the cell is
hypertonic to the hypotonic surroundings). Water goes
into the cell.
– different relationships with O2 are due to several factors including
inactivation of proteins and the effect of toxic O2 derivatives
(superoxide radical, hydrogen peroxide, and hydroxyl radical),
which oxidize and destroy cellular constituents; many
microorganisms possess enzymes that protect against toxic O2
derivatives (superoxide dismutase and catalase)
– most bacteria require an isotonic environment or a
hypotonic environment for optimum growth. Organisms that
can grow at relatively high salt concentration (up to 10%) are
said to be osmotolerant. Those that require relatively high salt

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 Cultivation of Microorganisms

concentrations for growth, like some of the Archea that require

sodium chloride concentrations of 20 % or higher halophiles.
i. Obligate halophile – adapted to growth under high osmotic
pressure
ii. Facultative halophile – do not require high salt
concentrations but they can tolerate them
– Plasmolysis - osmotic loss of water resulting in shrinkage of
the cell; growth of the cell is inhibited as cytoplasmic membrane
pulls away from the cell wall.
- Environments containing large concentrations of
dissolved substances draw water out of cells, causing
shrinkage of the cytoplasm volume
- interferes with growth and this is why highly
osmotic environments prevent bacterial growth (e.g.,
brine, the high sugar concentrations in jellies and
jams, salting of meats)
- addition of salts (or other solutes) to solution results in an
increase in osmotic pressure, used to preserve food (salted
fish, honey, and sweetened condensed milk are preserved
largely by this mechanism)
- high salt or sugar concentrations draw water out of
microbial cells and prevent growth

DIFFERENT CULTURE MEDIA

1. Sabourauds’ Dextrose Agar
− was formulated by Sabouraud in 1892
− a solid medium used for isolation, cultivation and maintenance of
pathogenic and non pathogenic yeasts and molds
 recommended for the cultivation and growth of fungi,
particularly those associated with skin infections
− peptone medium supplemented with dextrose to support the growth
of fungi, and is inhibitory to contaminating bacteria in clinical
specimens. The acidic pH, however, also may inhibit some fungal
species. Emmons modified the original formulation by adjusting the
pH close to neutral to increase the recovery of fungi and by
reducing the dextrose content from 40 to 20 g/L
− pH is adjusted to approximately 5.6 in order to enhance the growth
of fungi, especially dermatophytes, and to slightly inhibit bacterial
growth in clinical specimens
− peptones are sources of nitrogenous growth factors while dextrose
provides an energy source for the growth of microorganisms. This
formulation serves as an excellent basal medium to which
antibiotics and other inhibitors may be added for the selective
cultivation of various groups of microorganisms.

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 Cultivation of Microorganisms

2. Blood/Chocolate Agar
– a nutrient medium which is used in culturing fastidious organisms
such as Haemophilus species and
Neisseria
– comprised of sheep blood that
provides the X and V factors
necessary for Haemophilus growth
– in addition to 5% blood, it contains a
rich amino acid source but minimal
carbohydrates (fermentation of
these would produce acids that
would readily lyse the red blood
cells).
– some pathogens secrete products
that damage the membranes of host cells. Production of these
hemolysins can be detected on blood agar.
– heat-lysed red-blood cells in agar
– HEMOLYSIS: Appearance of culture in blood agar.
 alpha-HEMOLYSIS: Partial hemolysis, appearing green.
 beta-HEMOLYSIS: Full hemolysis. A "halo" appears around
the colony.
 gamma-HEMOLYSIS: No hemolysis.

Hemolysis on Blood Agar

– used for the preliminary or confirmatory identification of many types
of clinically important bacteria. While it is factored into the
differential diagnosis of a specific infectious agent, hemolysis type
is not specific enough to be a final diagnosis criterion.

 Alpha-hemolysis is a greenish
discoloration of the blood agar
surrounding a bacterial colony;
it is a characteristic of
Streptococcus pneumoniae.
This is an incomplete lysis or
"greening" of red blood cells

 Beta-hemolysis indicates a
zone of clearing in the blood
agar in the area surrounding a
bacterial colony. It is a
characteristic of Streptococcus pyogenes as well as some
strains of Staphylococcus aureus. This is a complete lysis
(hemolysis) of red blood cells surrounding the colonies

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 Cultivation of Microorganisms

 Gamma-hemolysis is
actually a lack of
hemolysis in the area
surrounding a bacterial
colony growing on blood
agar. In fact, culture of
bacteria on blood agar
for the purpose of
hemolysis classification
is performed at 37oC in
the presence of 5% CO2. This results in an overall brownish
discoloration of the blood agar, from its original blood-red
hue. An uninoculated blood agar plate (BAP) is shown on
the left, above. Gamma-hemolysis would therefore
describe bacterial growth that results in neither a greenish
tinge to the discoloration (alpha-hemolysis) nor a clear
zone that the observer "could read a newspaper through"
(beta-hemolysis). This has no lysis of red blood cells.
Staphylococcus epidermidis is gamma hemolytic.
– Purpose:
 a non-selective medium for the primary isolation of
fastidious bacteria such as Neisseria meningitidis and
Haemophilus spp.
 recommended as a primary plating medium for spinal
fluids, eye cultures, gonococcal cultures, and any other
specimen which may contain fastidious organisms
3. Coliform Test
a. Triple Sugar Iron Agar (TSI)
– used to differentiate enterics based on the ability to reduce sulfur
and ferment carbohydrates
– a differential media used for detection of glucose, lactose, and
sucrose fermentation and the production of gas and H2S. It contains
the pH indicator phenol red. The media will turn yellow in an acid
pH. This test is normally used for fermentative gram-negative rods.
– Purpose:
– used for the differentiation of Gram-negative enteric bacilli
based on carbohydrate fermentation and the production of
hydrogen sulfide
– to determine the ability of an organism to attack a specific
carbohydrate incorporated into a basal growth medium,
with or without the production of gas, along with the
determination of possible hydrogen sulphide (H2S)
production
– Principle:

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 Cultivation of Microorganisms

– contains three sugars (dextrose, lactose and sucrose),

phenol red for detecting carbohydrate fermentation and
ferrous sulfate for detection of hydrogen sulfide production
(indicated by blackening in the butt of the tube)
– a differential medium that can distinguish between a number of
Gram-negative enteric bacteria based on their physiological ability
(or lack thereof) to:
a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S

INTERPRETATION OF RESULTS:

A/A = yellow throughout

K/A = red slant, yellow butt
K/K = red or red/orange throughout

carbon dioxide = bubbles or breaks in medium

black precipitate = presence of hydrogen sulfide

SLANT Code
Interpretation
COLOR: letter:
does not ferment either lactose or
RED R
sucrose
YELLOW Y ferments lactose and/or sucrose

SCORING THE BUTT COLOR AND CONDITION:

Code
BUTT
Lette Interpretation
COLOR/CONDITION
r
no fermentation, the
RED R bacterium is an obligate
aerobe
some fermentation has
occurred, acid has been
YELLOW Y
produced, it is a facultative
anaerobe.

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 Cultivation of Microorganisms

Seen as cracks in the agar,

bubbles, or the entire slant
may be pushed out of the
GAS FORMED YG tube. (Caution:these gassy
fermenters may have
bacteria close to the
opening.)
BLACK "+" H2S has been produced

Slant/Butt Color(Slant/Bu
Utilization Examples
Reaction tt)
Glucose,
Pseudomon
Alkaline/Alkalin lactose,
Red/Red as
e (K/K) sucrose not
aeruginosa
fermented
Alkaline/Acid Glucose only
Red/Yellow Shigella
(K/A) fermented
Glucose
fermented, Escherichia
Acid/Acid (A/A) Yellow/Yellow lactose+ orcoli,
sucrose Klebsiella
fermented
Alkaline/Acid Blackening of
Glucose onlyProteus,
(K/A) with H2Smedium may be
fermented Salmonella
produced throughout tube

b. IMViC Test
A. IMViC represents the first letter of each individual test within the
series:
1. Indole test (tryptone broth)
2. Methyl Red (MR-VP broth)
3. Voges-Proskauer (MR-VP broth)
4. Citrate (Citrate agar slants)

B. This series of tests has been used for many years to differentiate
gram-negative enteric bacteria (Enterobacteriaceae).
a. Pathogenic (Salmonella, Shigella)
b. Occasionally pathogenic (Proteus, Klebsiella)
c. Normal Flora (Escherichia, Enterobacter)

Enterobacteriaeae (enterics)
– are Gram-negative bacteria that grow in the intestinal tract of
humans and other animals

21
 Cultivation of Microorganisms

– IMViC tests are frequently employed for identification of this

group of microbes which includes such organisms as Klebsiella,
Enterobacter, and Escherichia coli
– presence of E. coli is used by public health officials as an
indicator of fecal contamination of food and water supplies
– Enterobacter and Klebsiella resemble E.coli in being lactose
fermenters, their presence does not necessarily indicate fecal
contamination because they are widespread in soil and grass

C. The significance of these tests is that when testing drinking water for
the presence of the sewage indicator E. coli, one must be able to rule
out Enterobacter aerogenes. E. aerogenes is not always associated
with sewage, and its presence in water would not necessarily indicate
sewage contamination.

==============================================
========================
Indole Methyl Red
VP Citrate
E. coli + + - -
E. aerogenes - - + +
==============================================
========================

C. Results are recorded in the same order as the name sequence. For
example: ++-- or --++.

D. Individual tests of the IMViC:

1. Indole Test
a. Tryptophane, an essential amino acid, can be oxidized by
some organisms.

b. Test: SIM (Sulfide, Indole, Motility) Agar, which contains

tryptophan, is used. Kovac’s reagent (P-Dimethly-
aminobenzaldehyde) is added to growth. Organisms that
produce indole yield a red layer at the top of tube (positive
reaction).
c. test organism is inoculated into tryptone broth, a rich source
of the amino acid tryptophan

22
 Cultivation of Microorganisms

d. indole positive bacteria such as Escherichia coli produce

tryptophanase, an enzyme that cleaves tryptophan, producing
indole and other products
e. when Kovac's reagent (p-dimethylaminobenzaldehyde) is
added to a broth with indole in it, a dark pink color develops
f. indole test must be read by 48 hours of incubation because
the indole can be further degraded if prolonged incubation
occurs. The acidic pH produced by Escherichia coli limits its
growth.
g. Description:
– tryptophan hydrolysis -Some bacteria split tryptophan
into indole and pyruvic acid using the hydrolase called
tryptophanase
– can be detected with Kovac's reagent (Indole reagent)
– very important in differentiating E. coli (indole positive)
from some closely related enteric bacteria
– also differentiates Proteus mirabilis (indole negative)
from all other Proteus species (indole positive)
h. Interpretation: After incubation: The broth must be turbid. A
clear broth indicates that the organism did not grow and
cannot be tested. Add a few drops of Indole reagent to the
broth culture (tryptone broth). A positive result has a red layer
at the top. A negative result has a yellow or brown layer.

2. Methyl Red Test

a. qualitative test of acid produced from oxidation of glucose
b. used primarily to differentiate E. coli from E. aerogenes
– E. coli – produces large amounts of acid.
– E. aerogenes -- produces neutral or non-acidic end
products.

c. Test: Organism is grown in MR-VP broth. Methyl red indicator is

added to culture to identify amount of acid present. When
organism is E. coli, medium pH=4 and is red (positive). When
acid is present, but at a much lower concentration, pH = 6,
the medium turns yellow.
d. contains glucose and peptone. All enterics oxidize glucose for
energy; however the end products vary depending on
bacterial enzymes.

23
 Cultivation of Microorganisms

e. used to determine what end products result when the test

organism degrades glucose. E. coli is one of the bacteria that
produces acids, causing the pH to drop below 4.4. When the
pH indicator methyl red is added to this acidic broth it will be
cherry red (a positive MR test).
f. Klebsiella and Enterobacter produce more neutral products
from glucose (e.g. ethyl alcohol, acetyl methyl carbinol). In
this neutral pH the growth of the bacteria is not inhibited. The
bacteria thus begin to attack the peptone in the broth, causing
the pH to rise above 6.2. At this pH, methyl red indicator is a
yellow color (a negative MR test).
g. Description:
– Mixed acid fermentation - Many gram-negative
intestinal bacteria can be differentiated based on the
products produced when they ferment the glucose in
MR-VP medium. Escherichia, Salmonella, and Proteus
ferment glucose to produce lactic, acetic, succinic, and
formic acids and CO2, H2, and ethanol. The large
amounts of acids produced lowers the pH of the
medium - Methyl red (a pH indicator) will turn red
when added to the medium if the organism was a
mixed acid fermenter. Many of these organisms also
produce gas.
h. Interpretation: After incubation: The broth must be turbid. A
clear broth indicates that your organism did not grow and
cannot be tested. Remove 1 ml of broth and place into a
sterile tube before performing the methyl red test if you are
going to use the same broth for the VP test. Add 3-4 drops of
methyl red to the original broth. A positive result has a distinct
red layer at the top of the broth. A negative result has a yellow
layer.

3. Voges-Proskauer Test
a. Determines ability of an organism to produce non-acid or
neutral end products from organic acids present following
glucose metabolism.
b. Test: Organism is grown in MR-VP broth. Barritt’s reagent is
added to culture. Pink/red color indicates presence of
acetylmethylcarbinol (positive reaction), a natural product
formed from pyruvic acid in the course of glucose
fermentation.
c. contains glucose and peptone. All enterics oxidize glucose for
energy; however the end products vary depending on
bacterial enzymes.
d. reagents used for the VP test are Barritt's A (alpha-napthol)
and Barritt's B (potassium hydroxide). When these reagents

24
 Cultivation of Microorganisms

are added to a broth in which acetyl methyl carbinol is

present, they turn a pink-burgundy color (a positive VP test).
E. coli does not produce acetyl methyl carbinol, but
Enterobacter and Klebsiella do.
e. Principle:
– demonstrate an organisms ability to convert pyruvate to
acetoin. This property is a valuable tool in distinguishing
Klebsiella, Enterobacter, Ewingella and Serratia from
other members of Enterobacteraceae, as well as
distinguishing between Proteus vulgaris (V-P negative)
and P. mirabilis (V-P positive).

4. Citrate Test
a. lactose is not present; organisms have two primary enzymes --
citrate permease and citrase
b. Test: Simmon’s Agar Slants are used. Bromthymol blue
indicator is incorporated in the medium. If culture grows
(citrate positive, pH 7.6) media turns blue. If no growth,
medium is green (pH 6.9) and culture is citrate negative.
c. utilizes Simmon's citrate media to determine if a bacterium
can grow utilizing citrate as its sole carbon and energy source.
Simmon's media contains bromthymol blue, a pH indicator
with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic
pH's (around 6), and gradually changes to blue at more
alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar
has a pH of 6.9, so it is an intermediate green color. Growth of
bacteria in the media leads to development of a Prussian blue
color (positive citrate). Enterobacter and Klebsiella are citrate
positive while E.coli is negative.
– Thus E.coli gives ++-- results on the IMViC tests, while
Enterobacter and Klebsiella give the reverse: --++
d. Description:
– Simmon's citrate agar tests for the ability of an
organism to use citrate as its sole source of carbon. This
media contains a pH indicator called bromthymol blue.
The agar media changes from green to blue at an
alkaline pH.
e. Interpretation: After incubation: A positive reaction is
indicated by a slant with a Prussian blue color. A negative
slant will have no growth of bacteria and will remain green.

IMViC Tests Results

25
 Cultivation of Microorganisms

Indole Test
Bacteria were grown in tryptone
broth.
The uninoculated tube is on the
left.
The positive indole test is in the
center.
The negative indole test is on
the right.

Methyl Red (MR) Test

Bacteria were grown in MRVP
broth.
The uninoculated tube is on the
left.
The positive MR test is in the
center.
The negative MR test is on the
right.

Voges-Praskauer (VP) Test

Bacteria were grown in MRVP
broth.
The uninoculated tube is on the
left.
The positive VP test in the
center.
The negative VP test is on the
right.

26
 Cultivation of Microorganisms

Citrate test
Bacteria were grown on Citrate
Agar slants.
The uninoculated tube is on the
left.
The positive Citrate test in the
center.
The negative Citrate test is on
the right.

Give the nutritive value of the different ingredients of nutrient agar. ??? No
Answer Yet…???

27