Culture media and methods

Culture media and methods

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  Dr. Amit kumar amitkumarshahi90@gmail.com
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  • Culture media: Clinical specimenseg pus ,urine Inoculat e BACTERI A Isolation CULTURE MEDIA
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  CONSTITUENTS OF CULTUREMEDIA SODIUM CHLORIDE AND OTHERS PEPTONE MEAT EXTRACT
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  • Peptone: • sourceof nitrogen • digested protein • SOURCE: lean meat or other protein material (heart muscles , casein) and digested by pepsin. • Constituents: proteoses , aminoacids , inorganic salts (phosphates , potassium and magnesium) and nicotinic acid and riboflavin. • Neopaptone , proteose peptone , mycological peptone
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  • 0.5% sodiumchloride : maintain the salt concentration of media equal to cytoplasm of microorganism
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  • Agar /agaragar : • solidifying agent (powder form) • Melt in water after boiling • Jellifies after cooling • Seaweeds • Components : long chain polysaccharides , protein , long chain fatty acids, inorganic salt (calcium and magnesium) • Concentration : • Solid : 1-2% • Semisolid : 0.5% • 6% to inhibit proteus swarming
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  • Meat extract: • Prepared : beef • Constituent : protein degradation product , inorganic salts , carbohydrates and growth factors • Yeast extract: • Prepared : washed cells of Baker’s yeast • Constituent : aminoacids , inorganic salts (potassium and phosphates) and carbohydrates • vitamins, carbohydrates, organic nitrogen compounds and salts.
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  • Blood andserum : • Enriched media • Fastidious bacteria • 5-10% sheep blood • Serum is sterilized after collection
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  I BASED ONCONSISTENCY
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  II) BASED ONINGRADIENTS Peptone water
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  Peptone:1% peptone +Nacl (0.5%) + D/W PEPTONE NB
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  • USE OFBASAL MEDIA : • preparation of enriched media • NB (study bacterial growth curve) • Study colony morphology • Pigment demonstration • Semisolid media: motility of bacteria , maintaining stock culture NA +BLOOD PSEUDOMONAS ON NA
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  • B) complexmedia : • other than basal media • added with complex ingredients such as yeast extract or casein hydrolyse • mixture of many chemical species in unknown properties.
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  C
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  D) SPECIAL MEDIA: Loeffler’s serum slop Blood culture media i)
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  5-10% SRBC +molten NA at 47o c = BA Type of media : differential media and enriched media Blood agar
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  • Chocolate agar:5-10% SRBC + molten NA at 70o c = CA • haemophilus influenza
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  – Loeffler serumslope: isolation of corynebacterium diphtheriae Beef serum: solidifying agent – + – Heart muscle: amino acids – + – Peptic digest of animal tissue: amino acids – + • Sodium chloride: ions. – + – Dextrose: carbohydrate – + – Egg: solidifying agent
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  • Blood culturebottle: microorganism from blood (SEPTECIMEA , BACTERIMEA) sodium polyanetholsulphonate (SPS) : • inactivate serum factors toxic to organism beef heart : amino acids + calf brain infusion : amino acids + disodium phosphate: buffer + sodium chloride : maintain salt = cytoplasm + glucose • • BRUCELLA BHI AGAR + BHI BROTH
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  • Ii) Enrichmentmedia • Liquid media + inhibitory substances • used isolation of organisms from mixed culture (stool , sputum) • Tetrathionate browth : salmonella typhae • Selenite F broth : shigella • Alkaline peptone water : vibrio cholerae
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  iii)
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  Magnesium Sulfate Glycerol L- Asparaginenitrogen and vitamins Monopotassium Phosphate carbon source , favorable to the growth of the human type tubercle Prevent contaminants coagulates enhance organism growth and act as buffer enhance organism growth and act as buffers
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  phenyl red Citrate :bromothymol blue Triple sugar iron : phenyl red iv)
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  iv)
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  • CLED (Cysteinelactose electrolyte-deficient agar): differentiate LF and NLF • Inhibit swarming of proteus • Specially use for urine specimen
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  v)
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  • Vi) Transport medium •Use for transporting of samples • Used for delicate organism • Eg : stuart’s medium : Gonococci • Buffer glycerol saline: enteric bacilli
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  VII)
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  METHODS OF CULTURE SPECIMENSCULTURE MEDIA
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  PLATINUM WIRE : 1)Bacteriological loop: 2) Bacteriological straight wire: stroke stab
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  Streak culture Use :to obtained isolated colonies
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  LAWN CULTURE/CARPET CULTURE USE: AST , BACTERIAL TYPING
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  STROKE CULTURE USE :URESE TEST TO OBTAINED PURE GROWTH
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  STAB CULTURE
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  POUR –PLATE CULTURE ANDSPREAD METHOD USE : TO OBTAINED VIABLE COUNT OF BACTERIA 1 ml 9ml nb + 1ml bs 9ml nb 9ml nb 9ml nb 1 ml 1 ml 1 ml
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  LIQUID CULTURE DISADVANTAGE : DONOTPROVIDE PURE CULTURE FROM MIXED GROWTH IDENTIFICATION NOT POSSIBLE EXAMPLE : BLOOD CULTURE TURBIDITY
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  • Metal orglass jar with metal lid with a screw (to close airtight) • Pressure gauge and outlet and inlet • Air evacuate through outlet and hydrogen induce through inlet • Residual oxygen remove with hydrogen gas • sachet containing aluminium pellets coated with palladium react with hydrogen and replace o2 in the form of water(h2o) Chemical indicator : methylene blue
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  Gaspak system sachet containingaluminium pellets coated with palladium HYDROGEN TO FORM H2O (OXYGEN REMOVED) OXYGEN REMOVAL sachet containing aluminium pellets coated with palladium