Culture Methods-

Bacteria
Dr. Pragasam Viswanathan
Professor, SBST
Culture
Methods
Indications for culture -

 Isolate bacteria in pure cultures.

 Demonstrate their properties.

 Obtain sufficient growth for preparation of

antigens & for other tests.

 Typing bacterial isolates.

 Antibiotic sensitivity.

 Estimate viable counts.

 Types of culture methods
 Streak culture or surface plating
 Lawn or carpet culture
 Stroke culture
 Stab culture
 Pour plate method

 Anaerobic methods of culturing

Aseptic technique
 Streak Culture

 Routinely employed for isolation

 Platinum / Nichrome loops

Dr Ekta, Microbiology
 Lawn or Carpet Culture

 Uniform surface growth

 Bacteriophage typing

 Antibiotic sensitivity testing

 Preparation of bacterial antigens &

vaccines
Stroke Culture
 Tubes containing agar slopes
 F o r slide agglutination & other
diagnostic tests.
Stab Culture
 B y puncturing a
suitable medium
with a long,
straight charged
wire.

 For gelatin
liquefaction, stock
cultures &
motility
 Pour Plate Method
 1 ml of appropriately
diluted inoculum is added
to 15 ml of molten
agar and poured on
petridish.

 Colonies appear through

out the depth of
medium.

 Used to
estimate viable
count,
recommended
method for quantitative
urine cultures.
Broth Culture
 Inoculated by a
charged loop,
pipette or
syringes.

 For blood cultures

& sterility
testing.
Anaerobic Culture Methods

Anaerobic condition can

be achieved by
1. displacement of O2 with
other gases.
2. absorption of O2 by
chemical or biological means
3. reduction of O2
1. Displacement Method
 Displacement of O2
with gases like H2 , N2 ,
He or CO2 .

 Rarely produces
complete anaerobiosis.
e.g. Candle jar
2. Chemical or Biological Method
 Alkaline
pyrogallol ( pyrogallic acid
in NaOH) absorbs O2.
 Yellow phosphorous.
 Rosenthalmethod - Mixture of
chromium & sulphuric acid.
 McIntosh - Fildes’ Jar.
 Gaspak
 McIntosh - Fildes’ Jar

 Most reliable &

widely used anaerobic
method

 Complete
anaerobiosis

 Catalyst –
palladinised asbestos
 Gaspak
 Method of choice for preparing
anaerobic jars.

 Commercially available as
disposable envelope, containing
chemicals which generate H2 ,
CO2 with the addition of
water.

 Reduced methylene blue is used

as indicator. Remains colorless
anaerobically but turns blue on
exposure to O2
 3. Reduction of Oxygen
 Usingreducing agents like 1% glucose,
0.1% thioglycollate, 0.1% ascorbic
acid.

Robertson’s Cooked Meat Medium

 most widely used fluid medium
 Fat free minced cooked meat in
broth, with a layer of sterile vaseline
over it.
Robertson’s Cooked Meat Medium
05.10.08 Dr Ekta, Microbiology
 Automated Methods
 Bactec - blood culture method
◦ The sample to be tested is
inoculated into one or more
vials which are inserted into
the BACTEC fluorescent
series instrument for
incubation and periodic
reading.

◦ Each vial contains a chemical

sensor which can detect
increases in CO2 produced by
the growth of
microorganisms.
Automated Methods

◦ The sensor is monitored

by the instrument
every
ten minutes for an
increase in its
fluorescence, which is
proportional to the amount
of CO2 present.

◦ A positive reading
indicates the presumptive
presence of viable
microorganisms in the vial.
Virus culturing and
assaying methods
 Viruses are obligate intracellular
parasites
 They multiply only inside the living host cells
 Animals, plants, humans, bacteria, fungus,
protozoa and algae are the natural hosts of
viruses
Viruses are host specific and grow only in
selective hosts. Virologists use only a suitable
host system for cultivation of a virus

Viruses cannot grow in artificial media

.They cannot grown in non-living culture
or on agar plates alone , they must require
living cells to support their replication

There is no universal cell that will

support all viruses.
 Main purpose of virus cultivation

 To isolate and identify viruses in clinical

samples.
 To prepare viruses for
vaccine production.
 To do research on viral structure, replication,
geneticsand effects on host cells.
METHODS FOR CULTIVATION OF VIRUSES

Inoculation of virus
into animals

Inoculation of virus into

embryonated eggs

Tissue culture
 1. Inoculation of Virus in animals

 Viruses which are not cultivated in

embryonated egg and tissue culture are
cultivated in laboratory animals. e.g: mice,
guinea pig, hamester, rabbits and primates are
used
 The selected animals should be healthy and
free from any communicable diseases
 Suckling mice (less than 48 hours old) are most
commonly used
Advantages and disadvantages of animal
inoculation

Advantages :
 Production of antibodies can be identified
 Diagnosis, pathogenesis and
clinical symptoms are determined
 Primary isolation of certain viruses
 Mice provide a reliable model
for studying viral replication
 Used for the studyof immune responses,
epidemology and oncogenesis
Disadvantages :
 Expensive and difficulties in maintaince of
animals.
 Difficulty in choosing of animals for particular
virus.
 Some human viruses cannot be grown in
animals or can be grown but do not cause
diseases.
 Mice do not provide models for vaccine
development.
2. Inoculation of virus into embryonated
egg

The process of cultivation of viruses in

embryonated eggs depend upon the type of
egg being used.
Egg provide a suitable means for :
i. The primary isolation and identification
of viruses.
ii. The production of vaccines.
iii. The maintainance of stock culture.
Inoculation of virus into
embryonated eggs
 Virus growth and multiplication in the
egg embryo is indicated by the death of the
embryo , by embryo cell damage , or by the
formation of typical pocks or lesions on the
egg membrane.
 Viruses can be cultivated in various
pats of egg like :
1) Chorioallantoic membrane (CAM)
2) Allantoic cavity
3) Amniotic sac
4) Yolk sac Pock
 Advantages of inoculation into
embryonated egg
 Widely used method for the isolation of virus and
growth.
 Cost effective and maintenance is much easier.
 The embryonated eggs are readily available.
 They are free from contaminating bacteria
and many latent viruses.
 Ideal substrate for the viral growth and replication.
 less labor is needed.
 Widely used method to grow virus for
some vaccine production.
 Defense mechanisms are not involved
in embryonated eggs.
Disadvantage of inoculation into
embryonated egg

• The site of inoculation for varies

with different virus . That is , each
virus have different sites for growth
and replication.
 3) Tissue culture
Cultivation of bits of tissues and organs
in vitro had been used by physiologists
and surgeons for the study of
morphogenesis and wound healing
 Before the advent of cell culture, animal

viruses could be propagated only on whole

animals or embryonated chicken eggs

  Cell cultures have replaced
Embryonated eggs as
preferred type of growth
medium for many viruses.
 Cell culture consists of cells
grown in culture media in the
laboratory.
Advantages of cell culture
 Relative ease, broad spectrum, cheaper and
sensitivity
Disadvantage of cell culture
 The process requires trained technicians with
experience in working on a full time basis.
 State health laboratories and hospital
laboratories do not isolate and identify
viruses in clinical work.
 Tissue or serum for analysis is sent to central
laboratories to identify virus
 Detection of virus growth
The following methods are available to detect
the virus growth in the cell or tissue cultures

a) Cytopathic effect
b) Haemadsorption
c) Interference
d) Transformation
e) Immunofluorescence
f) Metabolic inhibition