Chapter 4 Culture Media and Culture Methods.pptx

Culture Media and
Culture Methods

Essentials of Medical Microbiology
At the end of the session, the students will be able to
• To know the classification of culture media
• Various culture methods
• Preservation of microorganisms
• And methods to isolate bacteria in pure culture
Learning objectives

CULTURE MEDIA
• Required to isolate the bacteria from the clinical
specimens.
• CONSTITUENTS OF CULTURE MEDIA
Constituent Explanation
1)Water • Distilled water or potable water with low mineral
content.
• Serves as the source of hydrogen and oxygen.
2)Electrolytes Sodium chloride or other electrolytes.

CONSTITUENTS OF CULTURE MEDIA
Peptone: • Complex mixture of partially digested proteins.
• Source- from lean meat or other protein material such as heart muscle,
casein or fibrin, or soya flour usually by digestion with proteolytic
enzymes such as pepsin.
• Constituents - proteoses, aminoacids, inorganic salts (phosphates,
potassium magnesium) and accessory growth factors like nicotinic acid
and riboflavin.

Agar • Used for solidifying agent.
• Agar (agar-agar) is prepared from the cell wall of variety of seaweeds.
• Components- Long-chain polysaccharide (D-galctopyranose units) and a
small amount of protein-like material, long chain fatty acids and traces of
inorganic salts (calcium and magnesium).
• Agar is preferred over gelatine
o Bacteriologically inert
o Melts at 98°C and usually solidifies at 42°C.
o Does not add any nutritive property to the culture medium.
o Whereas gelatin is liquefied by a number of bacteria and it melts at
24°C, and remains in liquid state at room temperature.
CONSTITUENTS OF CULTURE MEDIA (cont..)

Agar • Concentration of agar used:
o Solid agar preparation - 1-2%
o Semisolid agar- 0.5%
o Solid agar to inhibit Proteus swarming- 6%
• Preparation of agar media- The appropriate amount of agar powder is
added to water and the mixture is dissolved and sterilized by placing it in
an autoclave. When the temperature of the molten agar comes down to
450
C, it is poured to the petri dishes and then allowed to set for 20
minutes.

Meat extract Highly concentrated meat stock, usually made from beef, contains protein
degradation products, inorganic salts, carbohydrates and growth factors.
Yeast extract Prepared from washed cells of Baker’s yeast. It contains aminoacids,
inorganic salts (potassium and phosphates) and carbohydrates.
Malt extract- Consists of maltose (about 50%), starch, dextrin, glucose and 5% protein
products.

Blood and
serum
• Important components of enriched media and provide extra nutrition to
fastidious bacteria.
• Usually 5-10% of sheep blood is used. Horse, ox or human blood also
can be used.
• Blood should be collected aseptically and rendered non-coagulable by
defibrillation (by shaking the blood in a bottle containing sterile glass
beads) or adding oxalate or citrate.
• Serum is sterilized by filtration after collection.

TYPES OF CULTURE MEDIA
• Routine laboratory media-They are prepared from
nutrients such as aqueous extract of meat, peptone etc.
o Simple/ basal media
o Enriched media
o Enrichment broth
o Selective media
o Differential media
o Transport media
o Anaerobic media
• Defined or synthetic
media
o Simple synthetic
media
o Complex synthetic
media
1. Based on consistency, culture media are grouped into:
o Liquid media (or broth)
o Semisolid media
o Solid media
2. Based on growth requirements, culture media are classified as-

Simple/ basal media
• Contain minimum ingredients that support the
growth of non-fastidious bacteria.
o Peptone water-It contains peptone (1%) +
NaCl (0.5%) + water
o Nutrient broth- It is made up of peptone
water + meat extract (1%).
o Nutrient agar-It is made up of nutrient broth
+ 2% agar.
o Semisolid medium: It is prepared by reducing
the concentration of agar to 0.2–0.5 %.

o Testing the non-fastidiousness of bacteria
o They serve as the base for the preparation of many other media.
o Nutrient broth is used for studying the bacterial growth curve
o Nutrient agar is the preferred medium for-
o Performing the biochemical tests such as oxidase, catalase and
slide agglutination test etc.
o To study the colony character
o Pigment demonstration
Uses of basal media

Enriched Media
• Blood agar -
o Prepared by adding 5-10% of sheep blood to the
molten nutrient agar at 450
C.
o Tests the hemolytic property of the bacteria, which
may be either- i) partial or α (green) hemolysis and
ii) complete or β hemolysis
• Basal medium is added with additional nutrients such as blood,
serum or egg.
• In addition to non-fastidious organisms, they also support the
growth of fastidious nutritionally exacting bacteria.

• More nutritious than blood agar, and even
supports certain highly fastidious bacteria
such as Haemophilus influenzae that does not
grow on blood agar.
Enriched Media (cont..)
Chocolate agar
• Heated blood agar, prepared by adding 5-10% of sheep blood
to the molten nutrient agar at 700
C

• Blood culture media- Culturing microorganisms from
blood specimen. They are either monophasic or
biphasic media.
o Monophasic medium -brain heart infusion (BHI)
broth.
o Biphasic medium has a liquid phase containing BHI
broth and a solid agar slope made up of BHI agar.
Enriched Media (cont..)
• Loeffler's serum slope- is used for isolation of
Corynebacterium diphtheriae.

Enrichment broth
• Liquid media added with some inhibitory agents which selectively
allow certain organism to grow and inhibit others.
• Important for isolation of the pathogens from clinical specimens
which also contain normal flora (e.g. stool and sputum specimen).
o Tetrathionate broth (used for Salmonella Typhi)
o Gram negative broth- for isolation of Shigella
o Selenite F broth–for isolation of Shigella
o Alkaline peptone water (APW)- for Vibrio cholerae

Selective media
Media Used for isolation of
Lowenstein Jensen (LJ) medium Mycobacterium tuberculosis
Thiosulphate Citrate Bile salt Sucrose (TCBS) Vibrio species
DCA (Deoxycholate Citrate Agar) Salmonella and Shigella from stool
XLD (Xylose Lysine Deoxycholate) agar Salmonella and Shigella from stool
Potassium tellurite agar (PTA) Corynebacterium diphtheriae
Wilson Blair bismuth sulphite medium Salmonella Typhi.
Solid media containing inhibitory substances that inhibit the normal flora present in the
specimen and allow the pathogens to grow.

• A. Lowenstein–Jensen medium; B. TCBS agar
Selective media (cont..)

Transport media
• Used for the transport of the clinical specimens suspected to contain
delicate organism or when the delay is expected while transporting
the specimens.
• Bacteria do not multiply, they only remain viable.
Organism Transport media
Streptococcus Pike’s medium
Neisseria Amies medium, Stuart’s medium
Vibrio cholerae VR (Venkatraman-Ramakrishnan) medium
Autoclaved sea water
Cary Blair medium
Shigella,
Salmonella
Buffered glycerol saline
Cary Blair medium

Differential media
• Differentiate between two groups of bacteria by using an
indicator.
Differential media Features
1)MacConkey agar • Differential and low selective medium -used for the
isolation of enteric GNB.
• Differentiates organisms into LF (pink colonies, e.g.
Escherichia coli) and NLF or (colorless colonies, e.g.
Shigella).
• Composition- Peptone, lactose, agar, neutral red (indicator)
and taurocholate

Differential media (cont..)
Differential media Features
2)CLED agar -
Cysteine lactose
electrolyte-deficient
agar
• Similar to MacConkey agar.
• Used as an alternative to combination of blood agar and
MacConkey agar, for the processing of urine specimens
• Advantages over MacConkey agar-It is less inhibitory than
MacConkey agar, supports the growth of Gram positive
bacteria (except β hemolytic Streptococcus) and Candida.
• Advantage over blood agar -It can prevent the swarming of
Proteus.

Culture media
A. DCA; B. XLD agar; C. MacConkey agar; D. CLED agar

Anaerobic Culture Media
• Contain reducing substances which take-up oxygen and create lower
redox potential and thus permit the growth of obligate anaerobes such
as Clostridium.
• Robertson’s cooked meat (RCM) broth
o Contains chopped meat particles (beef heart), which provide
glutathione (a sulfhydryl group containing reducing substance) and
unsaturated fatty acids.
o Most widely used anaerobic culture medium.
o Also used for maintenance of stock cultures.

Anaerobic Culture Media (cont..)
• Other anaerobic media include
o Thioglycollate broth
o Anaerobic blood agar
o BHIS agar-Brain heart infusion agar with supplements (vitamin K
and hemin)
o Neomycin blood agar
o Egg yolk agar
o Phenyl ethyl agar
o Bacteroides bile esculin agar (BBE agar)

Defined or synthetic media
• Chemically defined media are used for various
experimental purposes, prepared exclusively from pure
chemical substances and their composition i.e exact
quantity of each chemical used is known.

Simple synthetic media
• Provide the basic essentials for the growth of many
non-fastidious heterotrophs but they will not support
growth of fastidious bacteria.

Complex synthetic media -
• In addition to the above, certain aminoacids, purines,
pyrimidines, and other growth factors are
incorporated.
• Hence, they can also support the growth of more
exacting bacteria.

CULTURE METHODS

Purpose of bacteriological culture
• Isolating bacteria in pure culture from the clinical specimens
• To perform biochemical tests for the identification of bacteria
• To perform antimicrobial susceptibility testing of the isolated
bacteria.
• To maintain stock cultures
• To obtain sufficient growth for the preparation of antigens
• For typing of bacterial isolates.
• To estimate the viable bacterial count.

METHODS OF CULTURE
• Loops and straight wires-
• Inoculation of specimen onto
the culture media is carried out

METHODS OF CULTURE (cont..)
• The inoculating straight wire or loop is
first heated in the Bunsen flame by
making it red hot and then made cool
waiting for 10 sec.

Streak culture
• Method of streaking- A loopful of
specimen is smeared onto the
surface of a dried solid culture plate
near the peripheral area with the
help of a sterile bacteriological loop
to form the primary or mother
inoculum.

Streak culture (cont..)
• The culture plate is incubated at 37°C for 12-
18 hrs (overnight).
• Confluent growth occurs at the primary
inoculum and well separated colonies are
obtained on the final streak.
• Obtaining isolated colonies is the prerequisite
to perform various biochemical tests to
confirm the identification of bacteria.

Lawn or carpet culture
• Lawn culture provides uniformly thick surface growth of
the bacterium on the solid medium.
• Done by inoculating a sterile swab soaked in liquid
bacterial culture on to the culture plate and then
incubated at 37°C overnight.

Lawn culture
• Uses
o AST
o Bacteriophage typing.
o Producing large amount of bacterial
growth required for preparation of
bacterial antigens and vaccines.

Stab culture
• Stab culture is performed by stabbing the semisolid agar
butt by a straight wire.
• Stab culture is used for i) maintaining stock cultures, ii)
for demonstration of oxygen requirement of the
bacteria by oxidative-fermentative (OF) test, iii) for
motility testing using semi solid agar.

Liquid culture inoclulation
• Inoculated by touching with a loop or by adding the
inoculum with pipettes or syringes.
• Bacterial growth is detected by observing the turbidity in the
medium; i) some bacteria produce uniform turbidity, ii) some
produce granular turbidity with sediment at the bottom of
the tube, ii) some aerobic bacteria form surface pellicles.
• Uses-Liquid cultures are useful for i)blood culture, ii) for
sterility testing.

Pour-plate culture
• Quantitative culture method, used to estimate viable bacterial count.
• One of the best methods to determine the number of bacteria present
per mL of liquid broth/specimen.
• Serial 10 fold dilutions of the original bacterial suspension are made.
This is achieved by-
o 9 ml of nutrient broth is poured into a set of test tubes.
o 1 ml of the bacterial suspension added to the first test tube, mixed
and then 1ml is serially transferred to the subsequent tube and so on.

Pour-plate culture

Spread-plate method
• After serial dilution of the sample, known volume of
individual dilutions are spread evenly on the surface of
a suitable agar plate to obtain a lawn culture.
• After incubation, colonies are counted and multiplied
by dilution factor to estimate the colony count.

Incubation of Culture Media
• Most of the pathogenic organisms grow best at 37°C i.e. body temperature of human beings.
• For aerobic bacteria, inoculated culture plates are incubated at 37 ºC for overnight in an
incubator.
Bacteriological incubator
• Device used to grow and maintain
microbiological cultures or cell.
• The incubator maintains optimal temperature.
Some incubators are specially designed to
maintain other conditions such as humidity and
the carbon dioxide (CO2).

• Provides an atmosphere of approximately 3-5%
CO2.
• Useful for capnophilic bacteria such as Brucella
abortus, Streptococcus, pneumococcus and
gonococcus.
Incubation of Culture Media (cont..)
Candle jar- Inoculated media are placed in a jar with a
lighted candle and the jar is sealed. The burning candle
reduces oxygen to a point where the flame goes off.

• Microaerophilic bacteria such as Campylobacter and
Helicobacter require- 5% oxygen for optimum growth.
Incubation of Culture Media (cont..)

ANAEROBIC CULTURE METHODS
• Obligate anaerobic bacteria can grow only in the absence of oxygen.
• Anaerobic culture methods includes:
o Production of vacuum
o By displacement and combustion of oxygen
o Absorption of oxygen by chemical methods-
o Anaerobic glove box
o By reducing agents
o PRAS (Pre-Reduced, Anaerobically Sterilized)

• Production of vacuum- Achieved by incubating cultures in a
vacuum desiccator. It is not an effective method, not used.
• By displacement and combustion of oxygen- Involves
evacuation of the air from jar and replacement with inert gas
like hydrogen followed by removal of the residual oxygen by
use of a catalyst. It is carried out by-
o McIntosh and Filde's anaerobic jar
o Anoxomat instrument
ANAEROBIC CULTURE METHODS

McIntosh and Filde’s anaerobic jar
• Most effective and popular method.
• Consists of a metal or glass jar with a metal lid with a screw (to
close airtight), pressure gauge and two openings (inlet and outlet).

Anoxomat
• Automated equipment which evacuates the
air from jar and replaces by hydrogen gas
from a cylinder.
• Same catalyst is used here to remove the
traces of oxygen.
• Easier to operate than McIntosh jar method
and claims to be highly effective for
creating anaerobiosis.

Absorption of oxygen by chemical methods
• Principle- Oxygen is removed by
chemical reactions in contrast to
evacuation and replacement technique
used in McIntosh Filde’s jar.
• Gas-pak- Most commonly used method
for anaerobiosis, simple to perform and is
perfect for a laboratory having less
sample load.

Gas-pak
• Sachet containing sodium bicarbonate and sodium
borohydride which react chemically in presence of water,
to produce hydrogen and CO2 gas.
• Traces of oxygen is removed by using same catalyst
(aluminium pellets coated with palladium) placed below
the jar.

Indicator of anaerobiosis
• Chemical indicator- Reduced methylene blue remains colourless
in anaerobic conditions, but turns blue on exposure to oxygen.
• Biological indicator-Plate inoculated with Pseudomonas is
incubated along with other inoculated plates for anaerobic
culture. Absence of growth of Pseudomonas (which is an obligate
aerobe) indicates that perfect anaerobiosis has been achieved.

Anaerobic glove box
• Also called anaerobic chamber.
• Self-contained anaerobic system that allows microbiologists to
process the specimen and perform most bacteriological
techniques for isolation and identification of anaerobic bacteria
without exposure to oxygen.

By reducing agents
• Oxygen in culture media can be reduced by various
reducing agents such as glucose, thioglycollate, cooked
meat pieces, cysteine and ascorbic acid.
• Robertson cooked meat broth is the most widely
employed anaerobic culture medium which uses
chopped meat particles (beef heart) as reducing agent.

PRAS (Pre-Reduced, Anaerobically Sterilized)
• PRAS media are prepared entirely under oxygen-free
conditions from initial sterilization to packaging in
sealed foil packages.

PRESERVATION OF MICROORGANISMS
• Preservation of organisms is necessary for -
epidemiological investigation, future research and
educational purposes.
• Both short term (weeks to months) and long term (up to
years) preservation methods are available.

• Cheaper, easy to perform but phenotypic and genotypic properties of
bacteria may get altered as organism is more liable to undergo mutations.
o Sub-culturing- Regular subculturing on to various medium.By this
method cultures can be preserved for not more than a few weeks.
Cooked-meat medium is used for the preservation of anaerobes.
• Other methods include-
o Preservation by immersing the culture in mineral oil, glycerol, or sterile
distilled water
o Freezing at -20 °C
o Drying- This may be useful for moulds and spore bearing bacteria.
Short term preservation methods

• Ultra temperature freezing
• Lyophilization (freeze-drying)
Short term preservation methods

METHODS OF ISOLATING BACTERIA IN PURE CULTURES
• Surface plating
• Selective media and enrichment broth
• Pre-treatment of specimens
• Anaerobiosis

Chapter 4 Culture Media and Culture Methods.pptx

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Chapter 4 Culture Media and Culture Methods.pptx