CULTURE
METHODS
G.HARIPRASAD M.Sc.Med Micro M.Phil
LECTURER
THOOTHUKUDI GOVT. MEDICAL COLLEGE
THOOTHUKUDI
PURPOSE OF CULTURE
 To isolate bacteria in pure culture.
 Demonstrate their properties.
 Obtain sufficient growth for
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preparation of antigens and for
other tests.
Type isolates by methods such as
bacteriophage and bacteriocin
susceptibility.
Determine sensitivity to
antibiotics.
Estimate viable counts and
Maintain stock cultures.
METHODS
Streak culture.
Lawn culture.
Stroke culture.
Stab culture.
Pour plate culture.
Liquid culture.
STREAK CULTURE
Routinely used method to isolate
bacteria.
One loopful of culture is made as a
primary inoculum and is then distributed
thinly over the plate by streaking it with
the loop in a series of parallel lines in
different segments of the plate.
Loop flamed and cooled between the
different sets of streaks.
On incubation growth may be confluent
at the site of the original inoculation but
becomes progressively thinner and well
separated colonies are obtained over the
final series of streaks
LAWN CULTURE
Also called as carpet culture.
Provides a uniform, growth of the bacterium.
Useful for bacteriophage typing and antibiotic sensitivity testing.
Also used in the preparation of bacterial antigens and vaccines.
Prepared by flooding the surface of the plate with a liquid culture or
suspension of the bacterium, pipetting off the excess inoculum and
incubating the plate.
Alternatively the surface of the plate may be inoculated by applying a
swab soaked in the bacterial culture or suspension.
COTTON SWAB IS DIPPED IN
CULTURE NEAR FLAME
SWAB CHARGED WITH
CULTURE IS SWABBED ON
PLATE
ANTIBIOTIC SENSITIVITY TESTING
STROKE CULTURE
 Made in tubes containing
agar slopes (slant).
 Employed for providing a
pure growth of the bacterium
for slide agglutination and
other diagnostic tests.
STAB CULTURE
Prepared by puncturing with a long straight,
charged wire in a suitable medium such as
nutrient gelatin or glucose agar.
Medium is allowed to set with the tube in the
upright position, providing a flat surface at the
top of the medium.
Employed mainly for demonstration of gelatin
liquefaction and oxygen requirement of the
bacterium under study.
Also used in the maintenance of stock culture.
POUR PLATE CULTURE
Tubes containing 15 ml of the agar medium are melted
and left to cool in a water bath at 45ºC-50ºC.
Dilutions of the inoculum are added in 1 ml volume to
the molten agar, mixed well.
Contents poured in sterile petri dishes and allowed to set.
After incubation colonies will be seen well distributed
throughout the depth of the medium.
Enumerated using colony counters.
Gives an estimate of the viable bacterial count in a
suspension and is the recommended method for
quantitative urine cultures.
COLONY NUMBERS DECREASES WITH SERIAL DILUTION
COLONY COUNTER
LIQUID CULTURES
 Inoculated by touching with a charged
loop or by adding the inoculum with
pipettes or syringes.
 Method employed for blood culture &
for sterility tests.
 Preferable for inocula containing
antibiotics and other antibacterial
substances.
 Preferred when large yields are desired.
ANAEROBIC CULTIVATION
METHODS
 To isolate anaerobic bacteria – which grow only in the
absence of oxygen but grow only in the presence of
carbon-di-oxide (20%)
 Creating Anaerobic condition called anaerobiosis
established by various methods.
 In one way, Anaerobic condition can be created in a airtight closed container – anaerobic jar (McIntosh & Filde’s
jar)- made upof either stainless steel or
polycarbonate/polypropylene
 In another way, anaerobic condition can be created by
using anaerobic media in test tubes (contain reducing
substance) provide anaerobic condition.
Methods establishing anaerobic condition
in anaerobic jar
 Anaerobic gaspak method
 Evacuation – replacement method
Anaerobic gaspak method
 Gas pak is commercially used and never be used again
 Gas-pak is a disposable packet containing pellets of
sodium borohydride, cobalt chloride, citric acid and sodium
bicarbonate.
 These chemicals generate hydrogen and carbon dioxide
when water is added (about 10 ml) ( some gaspak do not
need water)
 Hydrogen combines with oxygen in the presence of a
catalyst.
 After the inoculated plates are placed inside an air-tight jar,
the packet of “Gas-pak” with water added, is kept inside
and the lid is tightly closed.
METAL JAR
PLASTIC JAR
McIntosh jar
GAS PAK
CATALYST
EVACUATIONREPLACEMENT METHOD
 This is performed by evacuating preexisting air from
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jar and filling of hydrogen and carbon dioxide.
The anaerobic jar is provided with inlet and outlet
and pressure gauze
Through inlet gases are passed inside the jar – but
through outlet gas is evacuated.
After placing the plates and palladium catalyst the jar
is tightly closed
The remaining air (preexisting oxygen) is evacuated
by vacuum pump
First Hydrogen gas (90%) is passed, followed by
passing carbon dioxide(10%) and incubated.
ANAEROBIC SETUP
ANAEROBIC MEDIA
Robertson’s Cooked Meat (RCM)
Broth:
 Cooked meat particles used as
a reducing agent which
absorbs oxygen.
 Anaerobes attack meat –
proteolytic
Eg. Cl. tetani
 Anaerobes attack
carbohydrates in meat –
saccharolyic
Eg. Clostridium perfringens
THIOGLYCOLLATE BROTH
 Thioglycollate used
as a reducing agent
which absorbs
oxygen.
 Bacterial growth
identified by
turbidity.
ANAEROBIC CULTURE METHODS
3.ABSORPTION OF CHEMICALS WITH CHEMICALS
Alkaline pyrogallol – absorbs oxygen.
Pyrogallic acid added to a solution of
sodim hydroxide in a large test tube
placed inside an air-tight jar – provides
anerobiosis.
QUALITY ASSURANCE
 Checking whether the process is working
properly or not.
Chemical indicators:
 Methylene blue indicator – turns white when
reduced i.e.during anaerobiosis.
Biological indicators:
 Pseudomonas aeruginosa ( aerobic
bacteria) will grow if anaerobiosis is not
maintained.
 CANDLE JAR METHOD:-
inoculated plates are placed inside a
large air-tight container and a
lighted candle kept in it before the
lid is sealed – provides a
concentration of carbondioxide
which stimulates the growth of most
bacteria.
References:
www.slideshare.net