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CONTENTS
1.INTRODUCTION
2.HISTORY
3.CULTIVATION
4.EQUIPMENT
5.MEDIA CLASSIFICATION AND
FUNCTION
6.FREQUENTLY USED MEDIA
7.TYPES OF CULTURE MEDIA
8.CULTURE METHODS
9.CONCLUSION
10.REFERENCES
INTRODUCTION:-
Direct laboratory methods, such as microscopy provide
preliminary information about the bacteria involved in an
infection, but bacterial growth is usually required for definitive
identification and characterization,hence the culture method
is needed.
HISTORY
Hippocrates is credited with being the first person to believe
that diseases were caused naturally, not because of
superstition and gods.
Antonie Van Leeuwenhoek (1632–1723) was one of the first
person to observe microorganisms using a microscope of his
own design and named them as “little beasties”
Robert Hooke was the first person who made key observations
on living things with the use a microscope.
• Lazzaro Spallanzani (1729–1799) found that boiling broth
would sterilise it and kill any microorganisms in it.
• Louis Pasteur and the germ theory of disease, which states
that microorganisms are the causes of infectious disease.
• In 1876, Robert Koch (1843–1910) established that microbes
can cause disease.
CULTIVATION:
Cultivation is the process of growing
microorganisms in culture by taking bacteria from
the infection site (i.e., the in vivo environment) by
some means of specimen collection and growing
them in the artificial environment of the laboratory
(i.e., the in vitro environment).
PRINCIPLES OF BACTERIAL CULTIVATION:
● To grow and isolate all bacteria present in a clinical
specimen
● To determine which of the bacteria that grow are most
likely causing infection and which are likely contaminants
or colonizers
● To obtain sufficient growth of clinically relevant bacteria
to allow identification and characterization
EQUIPMENT :
1.Petri dish :
2.Bunsen Burner :
3.Inoculating loop :
Need for Culture media:
Used to grow bacteria
Can be used to enrich the numbers of bacteria
Select for certain bacteria and suppress others
Differentiate among different kinds of bacteria
WATER.CARBON SOURCE.NITROGEN SOURCE.INORGANIC SALTS
Phosphates. Sulfates.Sodium. Potassium.Magnesium. Iron.Manganese. Calcium.
BACTERIAL VITAMINS
Thiamine riboflavinnicotinic acid pyridoxinefolic acid vitamine B12
BASIC NUTRITIONAL REQUIREMENTS
GASEOUS REQUIREMENTS
OXYGEN
Obligate aerobesFacultative anaerobes Microaerophillic.Obligate anaerobes.
.
CARBONDIOXIDE:
Capnophillic
TEMPERATURE
• Psychrophiles: with optimum growth T
around 20 C
• Mesopihles: between 15 and 45 with
optimum around 37 C
• Thermophiles: between 30 and 75 with
optimum around 55 C
• Hyperthermophiles: T grater than 100C
pH
• Most bacteria grow between pH 6.5 and
7.5
• Molds and yeasts grow between pH
5 and 6
COLONIES - MAKE A OBSERVATION
• Shape
• Size
• Elevation
• Edge
• Surface
• Opacity
• Consistency
Types of culture media
1.Solid
2.liquid
Liquid medium:
• bacteria in small numbers grow
only in liquid media e.g. blood
culture.
• used for biochemical test
• Earliest liquid medium: urine or
meat broth - Louis Pasteur.
• Isolation and identification of
bacteria in pure culture is not
possible.
Solid medium:
• Distinct colony morphology
• Characteristics → easy to
identify
• Colony – macroscopically
visible collection of millions
of bacteria originating from
a single bacterial cell
Earliest solid medium:
Cooked cut potato by Robert Koch
Gelatin - not satisfactory
- liquefy at 24oC
Agar
Frau Hesse
Universally used for preparing solid medium
Obtained from seaweed: Gelidium
No nutritive value
Long chain polysaccharides.
- Also contains varying amounts of inorganic salts and
small quantities of a protein – like substance
Not affected by the growth of the bacteria.
Melts at 98°C & sets at 42°C
2% agar is employed in solid medium
FREQUENTLY USED MEDIA:
Brain-Heart Infusion.
Sheep Blood Agar.
Chocolate Agar.
Columbia CNA with Blood.
MacConkey Agar.
Thioglycollate Broth.
Brain-Heart Infusion Nutritionally rich medium
Allows the growth of aerobic organisms
Key ingredients:
Infusion from several animal tissue sources
Added peptone (protein)
Phosphate buffer
Dextrose
uses :1. a major component of the media developed for culturing a patient’s blood for bacteria
2. to determine bacterial susceptibility to antimicrobial agents
An enrichment media for
certain fastidious bacteria like N.gonorrhoeae, haemophilous spp
These bacteria utilize
haemoglobin,
hemin(x factor), coenzyme
Nicotinamide adenine
dinucleotide,
(v factor) released from lysis of
RBC
Red blood cell lysis gives the
medium a chocolate-brown
colour
from which this agar gets its
name
Chocolate Agar
MacConkey Agar Frequently used primary selective
and differential medium
Ingredients:
crystal violet: supresses +ve & fungi
neutral red: pH indicator provide this medium with a differential capacity
MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red to pink.
Lactose fermenters – Pinkcolonies
Non lactose fermenters –colourless colonies
eg: Shigella spp.
Supportive medium for the
isolation and differentiation of
gram +ve cocci from clinical and
non-clinical specimens which
contain mixed flora
Three peptone sources
5% defibrinated sheep blood
Colistin
Nalidixic acids
can also be used to help
differentiate bacterial colonies
based on the hemolytic reactions
they produce
To suppress gram –
ve
Columbia CNA with Blood
Sheep Blood Agar. supports all but the most fastidious clinically significant bacteria
It has specific colony morphology which is easier to identify bacteria
Ingredients: tryptons
soyabean protein digest
NaCl
Agar
5% sheep blood
extracellular enzymes that lyse
red blood cells in the agar (hemolysis).
Alpha hemolysis
partial lysis --greenish discoloration around the colony.
Beta hemolysis
complete clearing of the red blood cells around the bacterial colony
gamma or nonhemolysis
no halo is produced around the colony
Thioglycollate Broth Most frequently used enrichment
medium
Ingredients:
1.nutrient factors, including casein,
yeast and beef extracts, and vitamins.
2. oxidation-reduction indicator
(resazurin), dextrose, phylloquinone
and hemin .
3. 0.075% agar to prevent water
currents to take O2 in to deeper layers.
Agar & thioglycollate (reducing
agent) allow anaerobic bacteria to
grow deeper in the tube.
Gram-positive cocci grow as“puff
balls.”
Strictly aerobic organisms such as
P.aeruginosa, grow toward the top of
the broth
Strictly anaerobic organisms grow in
the bottom of the broth
Types of culture media
I. Based on their consistency
a) Solid medium
b) Liquid medium
c) Semi solid medium
II. Based on the constituents/
ingredients
a) Simple medium
b) Complex medium
c) Synthetic or defined medium
d) Special media
Special media
Enriched media
Enrichment media
Selective media
Indicator media
Differential media
Sugar media
Transport media
Media for biochemical reactions
III.Based on Oxygen
requirement
- Aerobic media
- Anaerobic media
Simple media / basal media:
Most common in routine diagnostic laboratories
Eg: Nutrient Broth, Nutrient Agar
NB consists of peptone, meat extract, NaCl, water
NB + 0.5% Glucose = Glucose Broth
NB + 2% agar = Nutrient agar
Agar conc. Reduced (0.2 - 0.5%) = Semi-solid medium
Complex media
Media other than basal media.
They have added complex ingredients such as yeast extract or
casein hydrolysate, which consist of a mixture of many
chemical species in unknown proportions
Provide special nutrients
Synthetic or defined media
Media prepared from pure chemical substances
exact composition is known
Used for special studies, eg. metabolic requirements
Eg: peptone water- (1% peptone + 0.5% NaCl in water)
Enriched media
Substances like blood, serum, egg are added to the basal
medium.
Used to grow bacteria that are exacting in their nutritional
needs.
Eg: Blood agar, Chocolate agar
Chocolate
agar
Blood agar
Enrichment media
Liquid media used to isolate pathogens from a mixed culture.
Media is incorporated with inhibitory substances to suppress
the unwanted organism → increase in numbers of desired bacteria
Eg:
Selenite F Broth –for the isolation of salmonella,Shigella
Tetrathionate Broth ( inhibit coliforms)
Alkaline Peptone Water – for Vibrio cholerae
Selective media
The inhibitory substance is added to a solid media
Increase in number of colonies of desired bacterium
Eg:
Desoxycholate citrate medium for dysentery bacilli
Mac Conkey’s medium for gram negative bacteria
Thiosulfate citrate bile salts sucrose (TCBS) agar– for V.
cholerae
LJ medium – M. tuberculosis
Thiosulfate citrate
bile salts sucrose (TCBS) agarMac Conkey’s medium
Deoxycholate citrate agar LJ media
Indicator media
contain an indicator which changes its colour when a bacterium
grows in them
Eg:
Wilson-Blair medium – S. typhi forms black colonies
McLeod’s medium (Potassium tellurite)– Diphtheria bacilli
Wilson-Blair Medium McLeod’s medium
• Differential media
some factor that allows colonies of one bacterial species or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate.
• Eg:
MacConkey agar:
A. Deep purple
B. Light pink or colourless
Sugar media
Media containing any fermentable substance
Eg: glucose, arabinose, lactose, starch etc.
Media consists:
1% of the sugar in peptone water + Indicator
Contain a small tube (Durham’s tube) for the detection of gas by
the bacteria
Transport media
Media used for transporting the samples.
Delicate organisms may not survive the time taken for
transporting the specimen without a transport media.
Eg:
Stuart’s medium – non nutrient soft agar gel containing a
reducing agent & charcoal
used for Gonnococci
Buffered glycerol saline – enteric bacilli
Anaerobic media
These media are used to grow anaerobic organisms.
Eg: Robertson’s cooked meat medium, Thioglycolate
medium.
CULTURE METHODS
Culture methods employed depend on the purpose for
which they are intended.
Purposes:
Isolation
Properties of bacteria
To create antigens for
laboratory use
To test for Antibiotic sensitivity
Estimate viable counts
Maintain stock cultures
Culture methods include:
Streak culture
Lawn culture
Stroke culture
Stab culture
Pour plate method
Liquid culture
Anaerobic culture methods
STREAK CULTURE
Used for the isolation of bacteria in pure
culture from clinical specimens.
Platinum wire is used.
One loopful of the specimen is transferred
onto the surface of a well dried plate.
Spread over a small area at the periphery.
The inoculum is then distributed thinly over
the plate by streaking it with a loop in a
series of parallel lines in different
segments of the plate.
On incubation, separated colonies are
obtained over the last series of streaks.
LAWN CULTURE
Provides a uniform surface growth
of the bacterium.
Uses
For bacteriophage typing.
Antibiotic sensitivity testing.
In the preparation of bacterial antigens and vaccines.
Lawn cultures are prepared by flooding the surface of the plate
with a liquid suspension of the bacterium.
Antibiotic sensitivity testing
STROKE CULTURE
Stroke culture is made in tubes containing
agar slope.
Uses
Provide a pure growth of bacterium for slide
agglutination and other diagnostic tests.
STAB CULTURE
Prepared by puncturing a suitable medium – gelatin or glucose
agar with a long, straight, charged wire.
Uses
Demonstration of gelatin liquefaction.
Oxygen requirements of the bacterium under study.
Maintenance of stock cultures.
POUR PLATE CULTURE
Agar medium is melted (15 ml) and cooled to 45oC.
1 ml of the inoculum is added to the molten agar.
Mix well and pour to a sterile petri dish.
Allow it to set.
Incubate at 37oC, colonies will be distributed throughout the
depth of the medium.
Uses
Gives an estimate of the viable bacterial count in a
suspension.
For the quantitative urine cultures.
LIQUID CULTURES
Liquid cultures are inoculated by touching with a charged
loop or by adding the inoculum with pipettes or syringes.
Uses
Blood culture
Sterility tests
Continuous culture methods
Disadvantage
It does not provide a pure culture
from mixed inocula
ANAEROBIC CULTURE METHODS
Anaerobic bacteria differ in their requirement and sensitivity to
oxygen.
Cl. tetani is a strict anaerobe - grows at an oxygen tension < 2 mm
Hg.
Methods:
Production of vacuum
Displacement of oxygen with other gases
Chemical method
Biological method
Reduction of medium
Displacement of oxygen with other gases
Displacement of oxygen with hydrogen, nitrogen, helium or
CO2.
Eg: Candle jar
Inoculated plates are placed inside a large airtight container
And a lighted candle kept in it before the lid is sealed
The burning candle is expected to use up all the oxygen inside
Before it is extinguished ,but some oxygen is always left behind.
The candle jar provides a concentration of carbondioxide which
stimulates the growth of most bacteria.
Chemical method
Alkaline pyrogallol absorbs oxygen.pyrogallic acid added to a solution
of sodium hydroxide in a large test tubes placed inside an airtight jar povides
anaerobic environment but small amount of carbonmonoxide, which is formed
during reaction,may be inhibitory to some bacteria
Instead of alkaline pyrogallol,anaerobic environment has been produced
within jars with a mixture of Chromium and Sulphuric acid
McIntosh – Fildes’ anaerobic jar
Consists of a metal jar or glass jar with a metal lid
which can be clamped air tight.
The lid has 2 tubes – gas inlet and gas outlet
The lid has two terminals – connected to electrical supply.
Under the lid – small grooved porcelain spool, wrapped
with a layer of palladinised asbestos.
Working:
Inoculated plates are placed inside the jar and the lid
clamped air tight.
The outlet tube is connected to a vacuum pump and the air
inside is evacuated.
The outlet tap is then closed and the inlet tube is connected to a
hydrogen supply.
After the jar is filled with hydrogen, the electric terminals are
connected to a current supply, so that the palladinised asbestos
is heated.
Act as a catalyst for the combination of hydrogen with residual
oxygen.
Gaspak
Commercially available disposable envelope.
Contains chemicals which generate H2 and CO2 on addition of
water.
Cold catalyst – permits combination
of Hydrogen & Oxygen
Indicator is used – reduced methylene
blue.
Colourless – anaerobically
Blue colour – on exposure to oxygen
Biological method
Absorption of oxygen by incubation with aerobic bacteria,
germinating seeds or chopped vegetables.
Reduction of oxygen
By using reducing agents – 1% glucose, 0.1% Thioglycolate
CONCLUSION
Even though there are several methods for bacterial
identification, culture method of cultivating microorganisms is
considered the gold standard procedure because we can
isolate pure culture of bacteria in viable condition, which is a
major drawback with PCR.
1.BEILY & SCOTT diagnostic microbiology 12th edition.
2.R.ANANTHANARAYAN AND C.K.J PANIKER Text book of microbiology 7th
edition
3.mim’s MEDIAL MICROBIOLOGY 4th edition.
REFERENCES