Culture Media

¢ Culture media

Media which are designed to provide all the essential nutrients in solution for bacterial growth

—  nutrients or nutritional requirements- chemicals and elements of the environment that are utilized for bacterial growth

¢ Culture bacteria- why?

—  For their identification

—  Cannot be recognised by their morphology alone

—  Have to be isolated on culture media and obtained as pure cultures for study

¢ Types

¢ Classified in many ways

¢ According to physical state

—  Solid media , liquid media and semisolid media

¢ According to nutritional factors

—  Simple media and complex media, synthetic media , semidefined media and special media

¢ According to use for different organisms

—  Aerobic and anaerobic media

¢ History

¢ Original media

—  Louis Pasteur:  urine and meat broth

¢ Solid media 

—  by Robert Koch: cooked potato

—  Gelatin to solidify liquid media ( by Koch) but no useful because liquefied at 24^OC and by many proteolytic bacteria

¢ Frau hesse (wife of one of the investigator in Koch’s laboratory)- used agar to solidify culture media

¢ Constituents of basal culture media

¢ Water

—  sources of hydrogen and oxygen, low mineral content preferable

—  Copper distilled water should not be used

¢ Electrolyte

—  sodium chloride or other elctrolytes

¢ Peptone

—  complex mixture of partially digested protein

—  Contains water soluble products eg pepsin, trypsin or papain

—  should  allow the growth of moderately exacting bacteria, absence of fermentable carbohydrate, low content of copper

¢ Meat extract, yeast extract: contain protein degradation products, carboydrates, inorganic salt and certain growth factors

¢ Agar

—  used for preparing solid media.

—   obtained from seaweeds

—   chief constituent is long chain polysaccharide(complex polysaccharide)

—  Also contain inorganic salts and ,small amount of protein like material and some traces of long chain fatty acid.

—  Virtually no nutritive value and is not affected by the growth of bacteria

—  Melts at 95 ^OC and solidifies at 42 ^OC

—  Hydrolysed by high alkaline and acidic pH

¢ Act as solidifying agent of culture media

¢ Different types of agar eg. New Zealand agar and Japanese agar( jellifying property different)

¢ Manufactured either as dried fibres or as powder

      Nutrient agar

                             

  Nutrient broth

¢ Simple media( basal media)

¢ Simplest media used commonly in diagnostic laboratories

¢ Nutrient broth (liquid) , nutrient agar(solid), peptone water

¢ Peptone water -– 1% peptone, 0.5 %  sodium chloride and  distilled water and pH  (7.4-7.5)

¢ Nutrient broth  – 1% peptone, 0.5 %  sodium chloride and  distilled water and pH (7.4-7.5) + 1% meat extract

                                     Liquid media

¢ Nutrient agar- nutrient broth + 2%agar

¢ 0.2-0.5 % of agar –semi solid agar- used for motile organisms to spread.

¢ 6% agar prevent spreading or swarming by organisms like Proteus.

¢ Complex media

¢ Have added ingredients  for special purposes or for bringing out certain characteristics or providing special nutrients required for the growth of the bacterium under study

1) general purpose medium- eg. Blood agar

     capable of detecting most aerobic and facultatively anaerobic organisms

       Commonly used for the general isolation of microorganisms directly from primary specimens inoculated in the agar

¢ 2) Enriched media – substances like blood, serum or eggs are added

—   media that allow growth of fastidious organisms ( presence of haemin, cystein)

—  eg,. Blood agar chocholate agar, loeffler’s serum

media

Chocolate Agar

¢ 3) enrichment media – liquid medium

—  Favours the growth of particular species of bacteria

—  Substances having stimulating effect

—  e.g.  Selenite broth, tetrathinate broth, alkaline peptone water and Robertson’s cooked meat medium

¢ Blood agar

¢ Chocolate agar

¢ 4) Selective media – contain additives that enhance the growth of the desired organisms and inhibit other organisms

—  Solid media

—  Use to isolate particular bacteria from mixed infection

—  e.g bile salt agar, Thiosulphate citrate bile salt sucrose (TCBS) : only vibrio grow

—  deoxycholate citrate agar, MacConkey’s agar, lowenstein jensen medium etc

¢ TCBS

¢ 5) Indicator media

—  Certain indicator is incorporated in media

—  eg neutral red , bromothymol blue, potassium tellurite

¢ 6) Differential medium- contains substances which help to distinguish differing properties of bacteria

—  eg MacConkey agar( also an indicator media and selective media), blood agar

¢ MacConkey agar

¢ Composition

—  Peptone, agar , lactose, sodium taurocholate and neutral red

—  Transport media

—  Those bacteria who may not survive for long time

—  Or may be overgrown by another nonpathogenic bacteria

—  eg: Stuart’s transport media

—  Bile peptone transport medium for stool (vibrios )

—  Glycerolsaline transport medium for stool( dysentery bacilli)

¢ Transport media

¢ Brain heart infusion (BHI) broth

¢ Contd….

Biphasic media:

 Castaneda medium – for Brucella

-        Two phases; solid and liquid

¢ Biochemical media

¢ Routinely used:

  - Triple Sugar Iron (TSI) agar

  - Citrate medium

  - Urease medium

  - Sulphide Indole Motility (SIM) medium* semisolid  medium

     

                            

¢ Sugar media-( fermentation substances)

¢ For biochemical tests

¢ Contains

¢ 1%sugar concerned in peptone water

¢ Anrades indicator

¢ Durham’s fermentation tube is also kept

¢ Detection –acid production by colour change and gas production by accumulation gas in the tube

¢ Glucose , sucrose lactose and mannitol are generally used for sugar fermentation tests

Mycobacterial culture media

¢ Eg Lowenstein-Jensen media, middlebrook or  based media

¢ Mycobacteria need special media

Anaerobic media

¢ eg –Robertson’s cooked meat media, thiglycolate broth etc

¢ Media used to grow anaerobic organism

¢ Nutritionally enriched

¢ Prereduced of molecular oxygen for better growth of anaerobes