9. Stroke Culture
• Tubes containing agar slopes
• For slide agglutination & other
diagnostic tests.
10. Stab Culture
• By puncturing a
suitable medium
with a long,
straight charged
wire.
• For gelatin
liquefaction, stock
cultures & motility
11. 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.
14. Anaerobic Culture Methods
Anaerobic condition can be achieved by:
• Cultivation in vacuum
• Displacement of oxygen with other gases
• Chemical or biological methods
• By displacement and combustion of oxygen
• By reducing agents
• Anaerobic chamber
16. Chemical or Biological Methods
• Alkaline pyrogallol ( pyrogallic acid in
NaOH) absorbs O2
• Yellow phosphorous
• Rosenthal method - Mixture of
chromium & sulphuric acid
• Gaspak
17. BIOLOGICAL METHODS
Absorption of oxygen from small closed systems has been attempted
by incubation along with
Aerobic bacteria EXAMPLE:- Pseudomonas aeruginosa
Anaerobiosis produced by this method is slow and ineffective.
21. Gaspak
• Method of choice for preparing
anaerobic jars.
• Commercially available as
disposable envelope, containing
chemicals which generate H2 ,
CO2 with the addition of water.
22. Reduction of Oxygen
Using reducing agents:
• 1% glucose
• 0.1% thioglycollate
• 0.1% ascorbic acid
• 0.05% cysteine
23. ANAEROBIC CHAMBER
• It is an anaerobic incubation
system
• It provides oxygen free
atmosphere for inoculating culture
media and for incubation
• It is fitted with airtight rubber
gloves to insert hands for
working with specimens
• The anaerobic chamber contains
catalyst, desiccant, hydrogen gas,
carbon dioxide gas, nitrogen gas
and an indicator
Cultivation in vacuum was attempted by incubating cultures in a vacuum desiccators but it proved to be unsatisfactory. This method is not in use now .
Displacement of oxygen
Displacement of oxygen by inert gases like hydrogen, nitrogen,
carbon dioxide or helium is sometimes employed. Oxygen can never be removed completely by this method. A popular but ineffective method is use of candle
Chemical Methods
Pyrogallol
First introduced by Buchner (1888)
Principle:-Alkaline pyragollol absorbs oxygen
Procedure:- a large tube containing solution of NaOH and pyragollol acid placed inside air tight jar produce an anaerobic conditions
Disadvantages:- small amount of CO is formed during the reaction, may be inhibitory to some bacteria
Chromium and sulphuric acid (Rosenthal Method)
Mixture of chromium and sulphuric acid is used for producing anaerobiosis
Principle:- two chemicals react in presence of oxygen
[O2]
Chromium + Sulphuric acid Chromous + anaerobic
sulphate condition
Alumina pellets coated with palladium- catalyst at room temperature
Reduced methylene blue is used as indicator. Remains colorless anaerobically but turns blue on exposure to O2
Gas pack
Commerically available disposable packet containing pellets of sodium borohydride, cobalt chloride, citric acid and sodium bicarbonate
Principle :- These chemicals generate hydrogen and carbon dioxide when water is added then hydrogen combines with oxygen in the presence of a catalyst
Now this technique is widely used for preparing anaerobic jars
Procedure:- after the inoculated plates are placed inside an air tight jar, the packet of “gas pack “ with water added is kept inside and the lid is tightly closed
Anaerobic broth is an easily prepared anaerobic medium into which pieces of red hot metallic iron are introduced. It is then layered over with sterile vaseline.
Anaerobic broth containing fresh animal tissue, such as rabbit kidney, spleen, testes or heart called Smith-Noguchi medium, supports the growth of many anaerobes.
The most widely employed anaerobic liquid culture are:-
-Thioglycollate broth
-Robertson’s cooked meat medium
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.
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.
