Culture methods

The indications for culture are :

1) isolate bacteria in pure culture
2) demonstrate their properties
3) preparation of antigens & other tests
4) type isolates by bacteriophage & bacteriocin
susceptibility
5) sensitivity to antibiotics
6) estimate viable counts
7) maintain stock cultures
 Methods

Streak, lawn, stroke, stab, pour plate &

liquid cultures

Special method for anaerobic bacteria

Sweep plate method for bacteria in the

dust on clothing
Streak culture (surface plating)
 Routinely employed
 A platinum loop (high
cost) or nichrome
resistance wire
charged with the
specimen
 A loopful transferred
to the periphery of a
well dried plate
Streak culture contd…
 Distributed thinly by
streaking parallel lines
 Then to different
segments of the plate
 The loop should be
flamed & cooled between
the different sets of
streaks
 Incubation
 Growth confluent at the
original inoculation
 Well separated colonies
over the final streak
 The lawn or carpet culture
 Provides a uniform surface growth
 Useful for phage typing & antibiotic sensitivity
testing (disc method)
 To prepare bacterial antigens & vaccines
 Prepared by flooding the surface with a liquid
culture
 Pipetted off the excess inoculum
 Incubate
 Inoculation by a swab soaked in bacterial
suspension-alternate method
Lawn or carpet culture
 Stroke culture
Made in tubes containing agar slope

Employed for providing pure growth for

slide agglutination & other diagnostic
tests
Stab culture
 Medium punctured with
a long ,straight charged
wire

 Employed for
demonstration of gelatin
liquefaction & oxygen
requirement

 To maintain stock
cultures
 Pour plate culture
 Tubes containing 15 ml of the agar
 Melted & left to cool in water bath at 45 – 50 c
 Appropriate dilutions of the inoculum are
added in 1 ml volume to the molten agar
 Mixed well & poured into a sterile petridish
 Allowed to set & incubate
 Colonies enumerated using colony counters
 Gives an estimation of viable bacterial count in
a suspension
 Recommended method for quantitative urine
cultures
Pour plate method
 Sweep plate method
 Edges of the petridishes containing the medium
are rubbed over the fabric

 Colonies develop on incubation

 Counted & estimates made

 Liquid cultures
 Made by touching with a charged loop in a
broth
 Large inocula can be employed
 Method adopted for blood culture & for
sterility tests – concentration of bacteria are
small
 Used for inocula containing antibiotics & other
antibacterial substances – rendered ineffective
by dilution in the medium
 Preferred when large yields are desired
 Disadvantage, does not provide a pure culture
from mixed inocula.
 Anaerobic culture methods

Principles :
1.exclusion of oxygen or production of a vacuum
2.displacement of oxygen with other gases
3.absorption of oxygen by chemical or biological
means
4.reduction of oxygen
 Cultivation in vacuum
 Inoculating cultures in a vacuum desiccator
 Unsatisfactory as some oxygen always remains
behind
 Fluid cultures may boil over
 The media may get detached from the plates
 Not in use now
Displacement of oxygen with gases
 H2, N2, helium or CO2
 Popular but ineffective method is candle jar
 Inoculated plates placed in a airtight container
 Lighted candle kept & the lid sealed
 Lighted candle expected to use oxygen
 But some always left behind
 Candle jar provides CO2 which stimulates the
growth of most bacteria
Candle jar
 Chemical methods
 Alkaline pyragallol absorbs oxygen
 Pyrogallic acid added to NAOH in a test tube
 Placed inside an airtight jar
 Disadvantage – small amount of CO formed -
inhibitory to some bacteria
 Mixture of chromium & sulphuric acid
(Rosenthal method)
 Mixture of chromium & yellow phosphorous
 Mc Intosh & Fildes jar
 stout glass or metal jar with a metal lid
 Clamped airtight with screw
 Lid has 2 tubes with taps
 One act as the gas inlet,other as the outlet
 2 terminals to connect electrical supply
 Underside of the terminals of the lid a small
grooved porcelain spool
 A palledinised asbestos wraps around it
 Inoculated plates placed inside the jar
 Outlet tube connected to vacuum pump & air
inside evacuated
Mc Intosh & Fildes jar
 Mc Intosh contd……
 The outlet tap closed
 The inlet tube connected to a H2 supply & fill
with it
 Terminals are connected to a current supply
 Heated asbestos acts as a catalyst,combination
of H2 with the residual O2
 Risk of explosion but rare
 eliminated by alumina pellets coated with
palladium,contained in a gauze sachet act as
catalyst at room temperature
Gas pack
 Method of choice
 Disposable envelop has
chemicals,generates H2 &
CO2 on the addition of water
 Inoculated plates are kept in
the jar with the envelop
 Water added
 The lid screwed tight
 Simple & effective
 Indicator - reduced methylene
blue,colorless anaerobically
 Biological methods

Incubation along with aerobic bacteria,

germinating seeds or chopped vegetables

Anaerobiosis is slow & ineffective with

biological methods
 Reduction of oxygen
 Various reducing agents – 1% glucose, 1%
thyoglycolate, 0.1% ascorbic acid & 0.05% cysteine
 Pieces of red hot iron in broth – easy method
 Broth containing many animal tissue supports the
growth of anaerobes
 Robertson’s cooked meat medium is most widely used,
consists of fat free minced cooked meat in broth
 Even strict anaerobes grow
 Indicates saccharolytic activity – red
 Proteolytic activity - black
 Glove box

 Air-tight, glass-
fronted cabinet filled
with inert gas
 An entry lock for the
introduction &
removal of materials
 Gloves for the
insertion of hands for
working
Methods of isolating pure cultures
1. Surface plating
2. Enrichment, selective & indicator media for the
isolation of of pathogens from specimens like feces
3. Pretreatment of specimens with bactericidal substances
which destroy unwanted bacteria
E.g :isolation of T.B bacilli from sputum by treatment
with alkali
4. Obligate aerobes & anaerobes,cultivation under aerobic
& anaerobic conditions
5. Separation of bacteria with different temperature
optima
E.g :only thermophiles grow at 60c
 Pure culture contd….
6. By heating at 80 c vegetative forms can be
eliminated
E.g : isolation of tetanus bacilli from dust
7. Separation of motile & nonmotile bacteria by
Craigie’s tube.Motile traverse & appear at the
top of medium
8. Inoculation into animals, pathogens produce
fatal septicemia.Cultured pure from the heart
blood
9. By using selective filters
10.Use of micromanipulators by which a single
bacterium can be separated