culture media

20/10/2015

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Culture Media Preparation
Practical Medical Microbiology
Culture Media Preparation

Culture Media Preparation

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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.
Broth Media :Liquid medium – diffuse growth

Solid medium (Agar Media)

discrete colonies.

• 20/10/2015

TYPES OF CULTURE MEDIA
BASED ON THE AGAR CONCENTRATION (Consistency):1. Solid media – 2% agar.2. Liquid media – broth media absence of agar.3. Semi solid media – 0.2-0.5% agar

BASIC NUTRITIONAL REQUIREMENTS

WATER.
CARBON SOURCE.
NITROGEN SOURCE.
INORGANIC SALTS.Phosphates. Sulfates.Sodium. Potassium.Magnesium. Iron.Manganese. Calcium.
BACTERIAL VITAMINS.Thiamine riboflavinnicotinic acid pyridoxinefolic acid vitamine B12.

GASEOUS REQUIREMENTS

OXYGEN Obligate aerobesFacultative anaerobes.Microaerophillic.Obligate anaerobes.
CARBONDIOXIDE:Capnophillic.

TEMPERATURE

Psychrophillic – bacteria that grows at lower temeperature – below 20ºC.
Mesophillic – bacteria that grows at 25ºC-40ºC. optimum temp range - 37ºC.
Thermophillic – bacteria that grows at high temperatures - 55ºC-80ºC.


CULTURE MEDIABASIC INGREDIENTS
PROTEIN SOURCE – peptone (a complex mixture of partially digested proteins).- special brands – Neopeptone & proteose peptone- digest broths can also be used.- meat extract (Lab Lemco).
MINERAL SALT – sodium chloride.

WATER- source of hydrogen and oxygen.

Culture Media Preparation
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Agar
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

Culture Media Preparation

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• Based on their consistency
• a) solid medium
• b) liquid medium
• c) semi solid medium
• Based on the constituents/ ingredients
• a) simple medium
• b) complex medium
• c) synthetic or defined medium
• d) Special media
Types of culture media


Culture Media Preparation
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Special media
Enriched media
• Enriched with Blood,serum,extrapeptones,egg& vitamins
• Used for: cultivation of all fastidious organisms,
• (haemophilus influenza&Streptococcus spp.)
• Examples: Blood agar &chocolate agar
Enrichment media
Contains substances that stimulates the desired bacteria or has inhibitory substances that inhibits the growth of unwanted bacteria.
Or otherwise promotes the growth of a particular organism and prevent the growth of normal competitors.
Differential media”:
are specialized media that allow identification of organisms based on their growth
and appearance on the media
Selective media
are specialized media that allows the growth of one type or certain types of bacteria ,fungi , while inhibiting the growth of other organism







Enriched media
Such as blood agar

Culture Media Preparation

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which is differential for mannitol

fermentation
Like Staphylococcus aureus change
the color of agar from pink to yellow
Differential media
Mannitol salt agar

MacConkey agar

which is differential for lactose fermentation Ex: E.coli
From non lactose fermenters
Ex: salmonella,shigella
MacConkey agar
Differential media


Selective media

Thiosulphate citrate bile salt sucrose (TCBS) which is selective for Vibrio cholerae

MacConkey agar which is selective for Enterobacteriaceae

TCBS agar

Culture Media
Chemically defined media: Exact chemical composition is known
Complex media: Extracts and digests of yeasts, meat, or plants
Nutrient broth
Nutrient agar

SYNTHETIC & SEMISYNTHETIC MEDIUM

SYNTHETIC OR DEFINED MEDIUM:Prepared from pure chemical substances and the exact composition of the medium is known.Used for various special studies such as metabolic requirements.EXAMPLE: Dubo’s medium
SEMISYNTHETIC OR SEMIDEFINED MEDIUM:Composition is approximately known.


COMPLEX MEDIUM
Exact chemical composition of ingredients of culture medium is not known.
Widely used for the cultivation of bacteria for diagnostic purposes.

11/28/2010

Culture Media Preparation
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Solid media – contains 2% agar
Colony morphology, pigmentation, hemolysis can be appreciated.
Eg: Nutrient agar, Blood agar


Liquid media(Broth 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

Culture Media Preparation

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Blood agar

Chocolate agar

Urease medium

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Culture Media Preparation
STEP 1:
Correct amount of Dehydrated medium is weighed on balance.

Culture Media Preparation

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Dehydrated medium is dissolved in
a measured amount of
distilled water.
STEP 2:

Culture Media Preparation

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Tubes and conical of media are sterilized in an autoclave for 15 to 20 minutes at 15 psi steam pressure or (1 bar).

STEP 3:

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Culture Media Preparation
STEP 4:

After sterilization of media .Pour the media agar into the petri dish bottom