3. Laked Kanamycin-vancomycin blood agar (LKV): It is
selective for isolation of Prevotella and Bacteriodes spp.
Anaerobic phenylethyl alcohol agar (PEA):
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4. Egg-yolk agar (EYA): Determination of lecithinase and
lipase production by clostridia
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5. • Sodium thioglycolate consumes oxygen and
allows the growth of obligate anaerobes
• Glucose, tryptone, yeast extract, L-cystine
provide the growth factors necessary for
bacterial multiplication
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6. • To isolate bacteria in pure culture.
• Demonstrate their properties.
• Obtain sufficient growth for preparation of antigens and for
other tests.
• Type isolates by methods such as bacteriophage and
bacteriocin susceptibility.
• Determine sensitivity to antibiotics
• Estimate viable counts
• Maintain stock cultures
9. STREAK CULTURE
• Most widely used method for isolation of cultures.
• 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.
10.
11. • 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
11
12.
13. LAWN CULTURE
• Carpet culture
• Provides a uniform, growth of the bacterium.
• 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.
15. • Alternatively the surface of the plate may be
inoculated by applying a swab soaked in the bacterial
culture or suspension.
16.
17. 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.
18.
19. • Employed mainly for demonstration of gelatin
liquefaction and oxygen requirement of the bacterium
under study.
• Also used in the maintenance of stock culture.
20. LIQUID CULTURE
• Test tubes
• Screw capped bottles
• Flask
• Adding innoculum with pipettes or loop
23. • Advantages :
• Small quantity of bacteria
• Blood
• Disadvantages:
• Not provide pure culture from mixed innoculum
• Identification of bacteria may not be possible
24. POUR PLATE METHODS:
• The main principle of this method is dilution of the
inoculums in successive tubes containing liquefied
agar medium so as to permit a thorough distribution
of bacterial cells within the medium.
25.
26. LOOP DILUTION TECHNIQUE
• In this method, the mixed culture of bacteria is
diluted directly in tubes of liquid (cooled) agar
medium and mixed well.
• The medium is maintained in a liquid state at a
temperature of 45°C-50°C to allow thorough
distribution of the inoculums.
27. • The content of each tube are poured into separate
Petri plates and then allowed to solidify.
• Incubated to develop bacterial colonies in both within
the agar medium (subsurface colonies) and on the
medium (surface colonies).
• A series of agar plates showing decreasing number
of colonies resulting from the loop dilution technique.
28.
29. SERIAL DILUTION TECHNIQUE:-
• In this method the original inoculums may be diluted
by using sterile water or a saline solution, so that the
concentration of the microbes gradually becomes
less.
• A micro-organism that predominates in a mixed
culture can be isolated in pure form by a series of
dilutions.
• The aim of this dilution is to inoculate a series of
tubes with a microbial suspension so dilute that
there are some tubes showing growth of only one
individual microbe
31. DISADVANTAGES
• Organisms are trapped beneath the surface of the medium
when it solidifies. Hence, surface as well as subsurface
colonies are developed and it is very difficult to isolate and
count the subsurface colonies.
• Time consuming and requires skill.
• The microorganisms are subjected to heat
• Liquid medium is maintained at given temperature may be
difficult.
32. SPREAD PLATE METHOD
• Involves using a sterilized spreader with a smooth surface.
• Add the bacteria suspension over a plate that needs to be dry at
room temperature so that the agar can absorb the bacteria more
readily.
• The plates are incubated and the isolated colonies are observed
after 24 hours and count
33.
34.
35. Advantage
• Simple method
• Only surface colonies are formed.
• This method is also used for counting the
microorganisms present in the inoculums.
• Microorganism are not exposed to higher
temperature.
36. 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
– Reducing agents
– Anaerobic glove box
– Prereduced anaerobically sterilized(PRAS)
37. Production of vacuum:
Incubate the cultures in a vacuum desiccator.
Displacement of oxygen with other gases
Displacement of oxygen with hydrogen, nitrogen,
helium or CO2
38.
39. Chemical methods
• Petri plates can be incubated in an anaerobic jar
• Sodium bicarbonate and sodium borohydride are mixed with a small amount of
water to produce CO2 and H+. A palladium catalyst in the jar combines with the O2
in the jar and the H+ to remove O2.
QUALITY ASSURANCE
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.
39
44. 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 carbon dioxide which stimulates
the growth of most bacteria.
• 3-5% CO2
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