Chapter 3

Cultivation of Microorganisms
Dr. Honeylet J. Nicolas
TCA-IVM
 Introduction

❑ Cultivation of microorganisms
requires the creation of an
artificial environment where all
the requirements for growth
are met.

❑ This environment is otherwise

called “culture medium”.
Basic Requirements of all Organisms

Water – major component of cells of living

organisms (70-85%).
❑ It is responsible for the transport of nutrients,
secretions, and excretions, and all biochemical
reactions rely on water.
❑ The quality of water used in preparing media is
very important.
❑ Insoluble phosphates of calcium and magnesium
may precipitate in the presence of peptones and
beef extract in bacterial media.
Basic Requirements of all Organisms

Carbon – a major and essential element in all living

things. Organisms may 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.
Basic Requirements of all Organisms

Energy – the various life forms may be

categorized as being either chemotrophs or
phototrophs regarding the source of energy
used in ATP generation.
ATP

❑ 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.
Basic Requirements of all Organisms

Nitrogen – protein synthesis requires

considerable amounts of nitrogen.
❑ It is used primarily to form the amino group of the
amino acids of proteins.
❑ Heterotrophs get their nitrogen requirements from
intermediate protein compounds such as peptides,
peptones and proteases.
Basic Requirements of all Organisms

Minerals – all microorganisms require trace

amounts of several elements for normal growth like
- sulfur (S),
- sodium (Na),
- potassium (K),
- manganese (Mn),
- iron (Fe),
- magnesium (Mg),
- zinc (Zn),
- copper (Cu),
- phosphorus (P) and
- cobalt (Co).
Basic Requirements of all Organisms

Growth Factor – any essential component of

cell material that an organism is unable to synthesize
from its basic carbon and nitrogen sources is
classified as growth factor.
❑ These may include amino acids, fatty acids, nucleic
acids, or vitamins.
❑ Organisms termed fastidious tend to require a variety
of growth factors.
Basic Requirements of all Organisms

Oxygen – microbes that grow in the presence of

oxygen (free or atmospheric oxygen) are called aerobes
while those that do not are called anaerobes.
❑ Obligate aerobes require free oxygen to live.
❑ Anaerobes obtain oxygen from oxygen-containing
compounds (e.g. organic sulfates, nitrates,
carbonates, or water).
❑ Obligate anaerobes are unable to use and is harmed
by oxygen when present.
❑ Facultative anaerobes can use oxygen when present
but are able to grow by fermentation or anaerobic
respiration when oxygen is not available.
Basic Requirements of all Organisms
Hydrogen ion concentration – the
enzymes of microorganisms is greatly affected by
the pH of the medium.
❑ Most bacteria grow best at around pH 7 (6.5-7.5)
while molds grow best at ph 5-6 and have a
wider pH range.
pH

❑ Organisms themselves may change the pH of their

immediate environment. For example, the pH of a
medium tends to decrease when microbial
fermentations take place, producing acidic
products.
❑ Buffers, such as phosphates and calcium
carbonate, are often utilized to help stabilize the
pH during the growth of the cultures studied.
Incubation Condition

❑ Incubation conditions must be appropriate for the

organism under study.
❑ Considerations include the provision of a suitable
atmosphere, a suitable temperature, and anything
else which may be required such as a light source
for the cultivation of phototrophs.
❑ Psycrophilic (“cold-loving”) organisms require
temperatures less than 10°C.
❑ Mesophilic organisms require 20-40°C.
❑ Thermophilic (“heat-loving”) organisms need 50-55°
C.
I. Cultivation of Bacteria
 Culture Media
Chemically-defined medium – all the
ingredients of a culture medium are
known, both qualitatively and
quantitatively. These media are of great
value in studying the nutritional
requirements of microorganisms or in
studying a great variety of their
metabolic activities.

Complex medium – the exact chemical

composition is not known, and such a
medium is often prepared from very
complex materials, e.g., body fluids,
tissue extracts and infusions, and
peptones.
 Culture Media
3. Basic nutritive medium – support
the basic needs of ordinary bacteria
(e.g. nutrient agar, nutrient broth,
peptone).

4. Enriched medium – ordinary basal

medium enriched with the addition
of blood, serum, egg yolk and other
ingredients for the growth of
fastidious bacteria (e.g. blood agar).

5. Enrichment broth – liquid media

that are selective for a particular
bacterium (e.g. selenite broth).
 Culture Media
6. Selective medium – supports growth of a group of bacteria
of interest while inhibiting others. Contain inhibitory
substances for unwanted bacterial species (e.g. MacConkey
agar is selective for Gram – bacteria).
 Culture Media
7. Indicator (differential) medium – medium that contain
fermentable sugars and pH indicators; they are designed to
provide presumptive identification of some bacteria due to
some biochemical reactions in the media
(e.g. MacConkey agar where lactose-fermenting bacteria
grow as pink colonies while non-lactose fermenters are
colorless).
 Culture Media

8. Maintenance medium – maintains the viability

and physiological characteristics of a culture
over time.

9. Reduced (anaerobic) medium – contains

reducing agents such as cysteine, thioglycollate,
Na2S, and sodium ascorbate which chemically
reduce the concentration of dissolved oxygen,
hence decreases the oxidation-reduction potential
of the medium (e.g. cooked meat, thioglycollate
medium).
 Commonly-Used Constituents in
Microbiological Media

s a solidifying agent in media. It is an

aride gum obtained from certain
r dissolves and melts around 100°C
und 43°C. Generally agar itself is not
by microorganisms.

rinated blood, plasma, serum or

are frequently added to culture
ation and cultivation of many
fluids contribute many growth
bstances which detoxify certain
 Commonly-Used Constituents in
Microbiological Media
BUFFERS.
These compounds are incorporated to maintain the
optimum pH range of the organism; e.g. sodium and
potassium phosphates and calcium carbonate

EXTRACTS.
Eukaryotic tissues (yeast, beef muscle, liver, brain,
heart, etc.) are extracted by boiling and then concentrated
to a paste or dried to a powder. These extracts are
frequently used as a source of amino acids, vitamins and
coenzymes.
 Commonly-Used Constituents in
Microbiological Media
PEPTONES.
These complex mixtures of organic and inorganic
compounds are obtained by digestion of
protein-containing tissues of animals and plants such as
meat scraps, beef muscle, gelatin, milk protein (casein)
and soybean meal. These materials are then dried
down to a powder and made commercially available to
microbiology laboratories.

Peptone in a concentration of 0.1% is often used as

a diluent.
 Commonly-Used Constituents in
Microbiological Media
REDUCING AGENTS. They stimulate growth by reducing the
oxidation-reduction potential in the environment; e.g. cystine
and thioglycollate.

SELECTIVE AGENTS. Antimicrobial agents such as crystal violet,

bile salts, brilliant green, potassium tellurite, sodium azide and
antibiotics can be employed in selective media to suppress or
inhibit the growth of certain groups of microorganisms while
allowing growth of desired organisms. These agents are usually
bacteriostatic.

pH INDICATORS. An acid-base indicator is often added to

differential media to detect changes in hydrogen ion
concentration during the growth of an organism
 pH Indicators Diagnostic media and tests
pH indicator pH Range Color Change Media and Biochemical
Alk Acid Alk Acid test
Andrade’s 7.2 - 5.5 Colorless - pink Peptone water sugars
indicator
Bromocresol 6.8 - 5.2 Purple - yellow Purple agar base, lysine
purple decarboxylase broth
Bromothymol 7.6 – 7.0 – 6.0 Blue – green – yellow O-F medium, Simmons
blue citrate, Smith-Baskerville
Litmus 8.3 - 4.5 Blue - red Litmus milk medium
Methyl red 6.4 - 4.4 - red MR test
Neutral red 8.0 - 6.8 Pale yellow - red MacConkey agar, Yersinia
selective medium
Phenol red 8.4 - 6.8 Red - yellow XLD, BGA, TSI, PWS,
Christensen urea agar
pH indicator pH Range Alk Color Change Media and Biochemical
Acid Alk Acid test
Andrade’s 7.2 - 5.5 Colorless - pink Peptone water sugars
indicator
Examples of Media used in Diagnostic Bacteriology

1) Nutrient agar – basic

medium for growth of
non-fastidious bacteria;
suitable for
demonstrating colonial
morphology and pigment
production, also used for
viable counting methods.
Examples of Media used in Diagnostic Bacteriology

2. Bloodagar – enriched medium for the growth of most bacteria

including fastidious ones. Red blood cells are used as substrate
to show hemolysis reactions particularly in Streptococcal spp.
which can be differentiated through the presence and/or type
hemolysis exhibited.
Examples of Media used in Diagnostic Bacteriology

3.MacConkey agar – for growth of most bacteria in the family

Enterobacteriaceae and some other G- bacteria. It has bile salts
that inhibit the growth of G+ bacteria. Its pH indicator is neutral
red and the substrate is lactose. Lactose-fermenting bacteria
grow as pink colonies while non-lactose fermenters are colorless
Examples of Media used in Diagnostic Bacteriology

4. Brilliant green agar – for Salmonella isolation from fecal samples,

a few other G- bacteria also grow. Substrates are lactose and
sucrose, pH indicator is phenol red, and the inhibitor is brilliant green
dye. BG dye suppresses G+ bacteria. Lactose- or sucrose-fermenting
colonies are yellowish or greenish. Salmonella (except S. typhi) form
reddish to white colonies.
Examples of Media used in Diagnostic Bacteriology

5. Xylose lysine deoxycholate agar –

isolates enteric pathogens such as
Shigella (appear as red colonies).
- Differentiation of bacteria is based
on fermentation of the substrates
xylose, lactose or sucrose.
Producers of hydrogen sulfide form
black colonies. Lysine may be
decarboxylated to neutralize acidic
reaction of Salmonella (appear as
red colonies with black centers).
- Other coliforms have yellow to orange
colonies, and Pseudomonas
aeruginosa has pink, flat, rough
colonies.
Examples of Media used in Diagnostic Bacteriology

6) Triple sugar iron agar – differentiates

isolates of G- enteric bacteria on the
ability to ferment lactose, sucrose and
glucose. Producers of hydrogen sulfide
cause black reaction. Results may be:
alkaline slant, acid butt (K/A), alkaline
slant and butt (K/K), acid slant and butt
(A/A), etc.

7) Selenite broth – an enrichment medium

for the isolation of Salmonella that may
be present in small numbers and
competing with intestinal flora. Sodium
selenite inhibits all G+ and many G-
bacteria.
Examples of Media used in Diagnostic Bacteriology

Eosin methylene blue agar – selective and differential medium

used to isolate fecal coliforms. Eosin Y and methylene blue are
pH indicator dyes which combine to form a dark purple
precipitate at low pH; they also serve to inhibit the growth of
most G+ bacteria.
Examples of Media used in Diagnostic Bacteriology

Chocolate agar – enriched medium for Haemophilus species. Has

lysed red cells as substrates and gives the X factor (hemin) and V
factor (Nicotinamide adenine dinucleotide) needed for growth
by Haemophilus.
Examples of Media used in Diagnostic Bacteriology

10. Edwards medium – selective for Streptococci; indicates aesculin

hydrolysis and hemolysis. Substrates are aesculin and RBS,
inhibitors are crystal violet and thallous sulphate.
Incubation Temperature

37°C – most of the microorganisms

pathogenic for animals including
Mycoplasmas and fungi that cause
systemic mycoses
42°C – Campylobacter jejuni,
Brachyspira hyodysenteriae
28-30°C – Leptospira interrogans
serovars
4°C – for cold enrichment of Listeria
monocytogenes (from brain samples)
and Yersinia enterocolitica and Y.
pseudotuberculosis (from fecal
samples).
Incubation Time

❖ 24-48 hrs – most of the rapidly

growing bacteria
❖ 48-72 hrs – the rapidly growing
bacteria when plated on selective
media
❖ 4-6 days – Brucella spp.,
Campylobacter spp., Nocardia
asteroides, Mycoplasma spp., and the
relatively fast-growing fungi
❖ 2-3 wks – Mycobacterium bovis
❖ 4-16 wks – Mycobacterium
paratuberculosis
 Incubation Atmosphere
✔ Normal atmosphere (aerobic) – for most of the pathogenic
bacteria and all fungi
✔ 5-10% CO2 – for the following bacteria:
• Actinobacillus pleuropneumoniae
• Actinomyces viscosus
• Brucella app.
• Haemophilus spp.
• Taylorella equigenitalis
✔ Anaerobic – for the following bacteria:
• Actinomyces bovis
• Bacteroides spp.
• Campylobacter mucosalis
• Clostridium spp.
• Eubacterium spp.
• Fusobacterium spp.
• Peptostreptococcus spp.
• Brachyspira hyodysenteriae
 Anaerobic Growth Media and Methods

The cultivation of anaerobic bacteria poses a

special problem. Because anaerobes might be killed
by exposure to oxygen, special media called reducing
media must be used.

These media contain ingredients such as sodium

thioglycolate that chemically combine with dissolved
oxygen and deplete oxygen of culture medium. The
medium is heated shortly before use so that any
absorbed oxygen is driven off, and then stored in
ordinary tightly capped test tubes.
1. Petri plates in special anaerobic jars

• The oxygen is removed by this process: a

packet of chemicals (sodium bicarbonate
and sodium borohydride) in the jar is
moistened with a few mL of water and the
jar is then sealed.
• Hydrogen and carbon dioxide are produced
by the reaction of the chemicals with the
water.
• A palladium catalyst in the jar combines with
oxygen in the air with the hydrogen
produced in the chemical reaction, and
water is formed.
• As a result, the oxygen disappears in a short
time. Moreover, the CO2 produced. An
anaerobic indicator is also used, such as
methylene blue (blue when oxidized,
colorless when oxygen is absent).
• A palladium catalyst
in the jar combines
with oxygen in the
air with the
hydrogen produced
in the chemical
reaction, and water
is formed.
• As a result, the
oxygen disappears
in a short time.
Moreover, the CO2
produced. An
anerobic indicator is
also used, such as
methylene blue
(blue when oxidized,
colorless when
oxygen is absent).
 2. Candle jar technique

• Plates and tubes inoculated with anaerobic bacteria

are placed in a jar with a lighted candle.
• The cap is placed on the jar.
• The candle will extinguish when the atmosphere
contains approximately 10% CO2.
 3. Anaerobic Chamber
• The transparent enclosure is filled with an inert,
oxygen-free gas.
• Organisms and materials enter and leave through
and air lock and manipulations are made by means
of glove ports.
Obligate anaerobes can be culture in special reducing
media such as sodium thioglycollate or in anaerobe
chambers and handled in anaerobe hoods
II. Cultivation of Fungi
 Cultivation of Fungi

Media
Whatever a particular fungus needs, it must always be supplied
with:
some form of organic carbon for energy,
a source of nitrogen for protein and vitamin synthesis,
and several minerals.

Liquid Media
Liquid media are employed in physiological studies, and in
laboratory work when the entire colony must be recovered for
weighing or chemical extraction. For identification purposes, liquid
media are seldom chosen because few moulds sporulate well on
them.
 Cultivation of Fungi

Solid media
Most culture media fit into one of three categories: (1)
synthetic, (2) semi-synthetic, and (3) natural:

❑ Synthetic media are composed of ingredients of known

chemical composition and concentration.
❑ Semi-synthetic media resemble synthetic media in containing
a known set of ingredients, but differ in that at least some of the
ingredients are of unknown or variable composition.
❑ Natural media are so called because they are partly or
completely composed of natural materials, such as ground-up
(or whole) plants or animals.
 Examples of Mycological Media

Czapek's Solution Agar - widely used synthetic

media in mycological laboratories.
• Many moulds produce very characteristic colonies
on it and may also exude pigmented substances.
• Aerial growth is often suppressed and sporulation
may be enhanced. Some moulds, however, grow
poorly on this medium and may even fail to
sporulate altogether, often because of their
inability to synthesize vitamins.
• As noted above, the addition of agar to this
medium makes it, in reality, a semi-synthetic one.
 Examples of Mycological Media

Penicillium Reference Medium - synthetic

medium prepared as three stock solutions that can
be stored and later combined to form the final
mixture. The complete mixture contains in a final
volume of l litre: 15 g agar, 100 ml each of major salts
and micronutrient stocks, and 10 ml of buffer stock.
• This medium was formulated by Dr. I. Ahmad for
the cultivation of Penicillium species.
• It can be modified in several ways to assess the
physiological activities of moulds.
 Examples of Mycological Media

Modified Leonian's Agar - semi-synthetic medium

devised by the American mycologist L.H. Leonian, and was
designed to promote sporulation in certain moulds.
• Later, at the University of Toronto, R.F. Cain found it to
be more suitable for ascomycetes if it contained a little
yeast extract; hence the term "modified" in its name.
• It is a good general purpose medium that will yield
good growth with most fungi.
• Certain fungi, such as many mycorrhizal forms, will not
grow on Leonian's Agar because they are unable to use
maltose as an energy source. For these fungi we use
Modified Melin-Norkran's medium (Marx 1969), which
has a glucose energy component.
 Examples of Mycological Media

Potato Dextrose Agar – semi-synthetic medium. Heat

potatoes at 60° C for 1 hour and filter through cheesecloth.
Make up volume to 1000 ml and add other ingredients. Cook
1 hour and then sterilize. It is an old formula used by plant
pathologists and many mycologists for general laboratory
use. It is a good all-purpose medium.
Sabouraud's Agar – semisynthetic; this is the standard
medium used in medical mycology. It is probably no better
for molds than Leonian's or PDA but is simply the traditional
choice and thus the medium that must be used if colonies
are to be compared with those described by medical
workers. It can be used as a general laboratory medium in
place of Leonian's or PDA.
Microsporum gypseum and Sporothrix schenkii on SDA
 Examples of Mycological Media
Martin's Rose Bengal Agar – semisynthetic. Ten
milliliters of a 3.3 g/l stock solution of rose bengal is added
to the medium after the other ingredients (except the
streptomycin) have been dissolved. After sterilization, the
streptomycin is added to the cooled medium. This medium
is useful in plating techniques when the aim is to slow down
the growth of colonies in a mixed culture. It discourages the
growth of bacteria and certain other organisms so that they
will not swamp isolates from natural materials.
Dextrose-peptone-yeast extract Agar (DPYA) –
semisynthetic. DPYA is an excellent medium for isolating
fungi from soil and other natural substrates. The oxgall and
sodium propionate restrict the growth of some rapidly
spreading fungi, while the chlortetracycline and
streptomycin discourage bacteria.
 Examples of Mycological Media

V-8 Agar – natural medium. The V-8 juice used in this

medium probably contains many nutrients that fungi can
use, but we have little idea what they may be. It is a medium
that is used routinely in plant pathology and seems to be a
good complement to Leonian's or PDA. Molds that fail to
sporulate on those media often sporulate heavily on V-8, or
vice versa.

Weitzman and Silva-Hutner's Agar – natural medium

designed to enhance sporulation in certain medically
important fungi, but is useful for a great many other molds
as well.
III. Cultivation of Viruses
 Cultivation of Viruses
✔ The fact that viruses cannot grow outside
of a living host cell complicates their
detection, enumeration and identification.
✔ It is necessary to provide viruses with
living cells instead of fairly simple
chemical medium.
✔ Living plants and animals are expensive to
maintain, and disease-causing viruses
that grow only in higher primates and
humans cause additional complications.
✔ On the other hand, viruses that use
bacterial cells as a host (bacteriophages)
are rather easily grown on bacterial
cultures.
Growth of Bacteriophages in the Laboratory

❖ Bacteriophages can be grown either in suspensions

of bacteria in liquid medium or in bacterial cultures
on solid medium.
❖ The use of solid medium makes possible the
plaque method for detecting and counting viruses.
❖ The plaque method mixes bacteriophages with host
bacteria and nutrient agar.
❖ After several viral multiplication cycles, the bacteria
in the area surrounding the original virus are
destroyed, and the area of lysis is called a plaque.
❖ Each plaque can originate with a single viral particle
or more than one; the number of viral particles
required to initiate a plaque is termed
plaque-forming unit.
Growth of Bacteriophages in the Laboratory

Figure 3a. Plaque assay of bacteriophage.

Growth of Animal Viruses in the Laboratory

A. In living animals

Cultivation of some animal viruses

requires whole animals, such as mice,
rabbits and guinea pigs. Generally,
animal inoculation is used as a
diagnostic procedure for identifying a
virus from a clinical specimen. After the
animal is inoculated with the specimen,
the animal is observed for signs of
disease or is killed so that infective
tissues can be examined and evaluated.
Growth of Animal Viruses in the Laboratory
B. In embryonated eggs
A hole is drilled in the shell of the embryonated egg, and a
viral suspension or suspected virus-containing tissue is injected
into the fluid of the egg. There are several membranes in the
egg on which the virus can be made to grow, and the virus is
injected into the proper location in the egg. Viral growth is
signaled by the death of the embryo, by embryo cell damage,
or by the formation in the membranes of the egg of typical
pocks or lesions that result from viral growth. This method,
discovered in 1931, was once the most widely used method of
viral isolation and growth and is still used to grow viruses for
some vaccines.
Growth of Animal Viruses in the Laboratory

C. In Cell Culture

Cell cultures are homogenous collections of animal

cells grown in culture media in the laboratory. Cell
culture lines are readily started by treatment of a slice
of animal tissue with enzymes that separate the
individual cells.
These cells are suspended in a solution that
provides the osmotic pressure, nutrients, and growth
factors needed for the cell to grow.
The cells tend to adhere to the glass or plastic
container and reproduce to form a monolayer. Viruses
infecting such a monolayer sometimes cause the cells
to deteriorate as they multiply. This is termed as
cytopathic effect.
Growth of Animal Viruses in the Laboratory

o Primary cell lines, derived from tissue slices, tend to die out
after only a few generations.
o When viruses are routinely grown in a laboratory, continuous
cell lines are used. These are “transformed” cells that can be
maintained through an indefinite number of generations, and
they are sometimes called “immortal” cells.
Growth of Animal Viruses in the Laboratory

o A major problem with cell culture is that the cell

lines must be kept free of microbial contamination.
o Identification of viral isolates is not an easy task.
o Viruses can be seen only with the use of electron
microscope.
o Viruses maybe identified by serological tests and
nucleic acid base sequencing.