CULTURE MEDIA & CULTURE METHODS
Bacteria have to be grown (cultured) for them to be identified.
By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study.
The original media used by Louis Pasteur – urine or meat broth
Liquid medium – diffuse growth
Solid medium – discrete colonies.
Colony – macroscopically visible collection of millions of bacteria originating from a single bacterial cell.
Cooked cut potato by Robert Koch – earliest solid medium
Gelatin – not satisfactory
– liquefy at 24oC
Used for preparing solid medium
Obtained from seaweeds.
No nutritive value
Not affected by the growth of the bacteria.
Melts at 98oC & sets at 42oC
2% agar is employed in solid medium
Types of culture media
I.Based on their consistency
a) solid medium
b) liquid medium
c) semi solid medium
II.Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
Media for biochemical reactions
III.Based on Oxygen requirement
– Aerobic media
– Anaerobic media
Solid media – contains 2% agar
Colony morphology, pigmentation, hemolysis can be appreciated.
Eg: Nutrient agar, Blood agar
Liquid media – no agar.
For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture.
Eg: Nutrient broth
Semi solid medium – 0.5% agar.
Eg: Motility medium
Simple media / basal media
– Eg: NB, NA
– NB consists of peptone, meat extract, NaCl,
– NB + 2% agar = Nutrient agar
Media other than basal media.
They have added ingredients.
Provide special nutrients
Synthetic or defined media
Media prepared from pure chemical substances and its exact composition is known
Eg: peptone water – 1% peptone + 0.5% NaCl in water
Substances like blood, serum, egg are added to the basal medium.
Used to grow bacteria that are exacting in their nutritional needs.
Eg: Blood agar, Chocolate agar
Liquid media used to isolate pathogens from a mixed culture.
Media is incorporated with inhibitory substances to suppress the unwanted organism.
Selenite F Broth – for the isolation of Salmonella, Shigella
Alkaline Peptone Water – for Vibrio cholerae
The inhibitory substance is added to a solid media.
Mac Conkey’s medium for gram negative bacteria
TCBS – for V.cholerae
LJ medium – M.tuberculosis
Wilson and Blair medium – S.typhi
Potassium tellurite medium – Diphtheria bacilli
These media contain an indicator which changes its colour when a bacterium grows in them.
Mac Conkey’s medium
Christensen’s urease medium
A media which has substances incorporated in it enabling it to distinguish between bacteria.
Eg: Mac Conkey’s medium
Distinguish between lactose fermenters & non lactose fermenters.
Lactose fermenters – Pink colonies
Non lactose fermenters – colourless colonies
Media containing any fermentable substance.
Eg: glucose, arabinose, lactose, starch etc.
Media consists of 1% of the sugar in peptone water.
Contain a small tube (Durham’s tube) for the detection of gas by the bacteria.
Media used for transporting the samples.
Delicate organisms may not survive the time taken for transporting the specimen without a transport media.
Stuart’s medium – non nutrient soft agar gel containing a reducing agent
Buffered glycerol saline – enteric bacilli
These media are used to grow anaerobic organisms.
Eg: Robertson’s cooked meat medium, Thioglycolate medium.
BIOCHEMICAL TEST & REACTIONS
They provide additional information for the identification of the bacterium.
The tests include:
Triple sugar iron agar (TSI)
Detects the presence of an enzyme “oxidase” produced by certain bacteria which will reduce the dye – tetramethyl-p-phenylene diamine dihydrochloride.
Positive test is indicated by the development of a purple colour.
Oxidase positive – Pseudomonas, Vibrio, Neisseriae
Oxidase negative – Salmonella, Shigella
TRIPLE SUGAR IRON AGAR (TSI)
It is a composite media used to study different properties of a bacterium – sugar fermentation, gas production and H2S production.
In addition to peptone, yeast extract & agar, it contains 3 sugars – Glucose, Lactose, Sucrose.
The Iron salt – Ferric citrate indicates H2S production.
Phenol red is the indicator.
It is an orange red medium with a slant and a butt.
pH of the medium – 7.4
Yellow – Acid
Pink – Alkaline
Yellow slant / Yellow butt (A/A) – Lactose fermenters.
Pink slant / Yellow butt (K/A) – Non lactose fermenters.
Pink slant / no colour change (K/K) – Non fermenters
Black colour – H2S production.
Gas bubbles or crack in the medium – gas production.
LF – E.coli, Klebsiella
NLF – Salmonella, Shigella
H2S – Proteus
Done in Simmon’s Citrate medium.
To detect the ability of certain bacteria to utilize citrate as the sole source of carbon.
Contains Sodium citrate and bromothymol blue as the indicator.
If citrate is utilized, alkali is produced which turns the medium to blue.
Citrate positive – blue colour
Citrate negative – green colour
Positive – Klebsiella
Negative – E.coli
Done in Christensen’s urease medium.
This test is used to detect organisms that produce urease.
Urease produced by the organisms split urea into ammonia and CO2.
Urease positive – pink colour
Urease negative – yellow colour
Positive – Proteus, Klebsiella
Negative – E.coli, Salmonella
Culture methods employed depend on the purpose for which they are intended.
The indications for culture are:
To isolate bacteria in pure cultures.
To demonstrate their properties.
To obtain sufficient growth for the preparation of antigens and for other tests.
For bacteriophage & bacteriocin susceptibility.
To determine sensitivity to antibiotics.
To estimate viable counts.
Maintain stock cultures.
Culture methods include:
Pour plate method
Anaerobic culture methods
Used for the isolation of bacteria in pure culture from clinical specimens.
Platinum wire or Nichrome wire is used.
One loopful of the specimen is transferred onto the surface of a well dried plate.
Spread over a small area at the periphery.
The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate.
On incubation, separated colonies are obtained over the last series of streaks.
Provides a uniform surface growth of the bacterium.
For bacteriophage typing.
Antibiotic sensitivity testing.
In the preparation of bacterial antigens and vaccines.
Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium.
Stroke culture is made in tubes containing agar slope / slant.
Provide a pure growth of bacterium for slide agglutination and other diagnostic tests.
Prepared by puncturing a suitable medium – gelatin or glucose agar with a long, straight, charged wire.
Demonstration of gelatin liquefaction.
Oxygen requirements of the bacterium under study.
Maintenance of stoke cultures.
POUR PLATE CULTURE
Agar medium is melted (15 ml) and cooled to 45oC.
1 ml of the inoculum is added to the molten agar.
Mix well and pour to a sterile petri dish.
Allow it to set.
Incubate at 37oC, colonies will be distributed throughout the depth of the medium.
Gives an estimate of the viable bacterial count in a suspension.
For the quantitative urine cultures.
Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes.
Continuous culture methods
It does not provide a pure culture from mixed inocula.
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.
Production of vacuum
Displacement of oxygen with other gases
Reduction of medium
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.
Eg: Candle jar
Alkaline pyrogallol absorbs oxygen.
McIntosh – Fildes’ anaerobic jar
Consists of a metal jar or glass jar with a metal lid which can be clamped air tight.
The lid has 2 tubes – gas inlet and gas outlet
The lid has two terminals – connected to electrical supply.
Under the lid – small grooved porcelain spool, wrapped with a layer of palladinised asbestos.
Inoculated plates are placed inside the jar and the lid clamped air tight.
The outlet tube is connected to a vacuum pump and the air inside is evacuated.
The outlet tap is then closed and the inlet tube is connected to a hydrogen supply.
After the jar is filled with hydrogen, the electric terminals are connected to a current supply, so that the palladinised asbestos is heated.
Act as a catalyst for the combination of hydrogen with residual oxygen.
Commercially available disposable envelope.
Contains chemicals which generate H2 and CO2 on addition of water.
Cold catalyst – in the envelope
Indicator is used – reduced methylene blue.
Colourless – anaerobically
Blue colour – on exposure to oxygen
Absorption of oxygen by incubation with aerobic bacteria, germinating seeds or chopped vegetables.
Reduction of oxygen
By using reducing agents – 1% glucose, 0.1% Thioglycolate