CULTURE MEDIA

Major A.K.Mishra

DEFINITION
Culture media are artificial media in which essential nutrients are added to give satisfactory growth of bacteria. We need culture media to isolate pathogenic microorganisms from clinical samplesurine,faeces,sputum,blood,CSF,pus etc

DISCOVERY

Original media introduced by LOUIS PASTEUR. All were liquid media. Solid media introduced by ROBERT KOCH. Agar-agar was used as the solidifying agent.

LOUIS PASTEUR.

ROBERT KOCH.

AGAR-AGAR
Obtained from sea-weeds. Long chain polysaccharide. It has no nutrient value. Not affected by growth of bacteria. Melts at 98degrees Sets at 42degrees 2% agar solid media 0.2-0.5% agar-semi solid medium 6% agar-prevents swarming

UNIVERSAL INGREDIENTS

Agar-agar Peptone-partially digested proteins Meat extract- lab-lemco Yeast extract Blood-sheep,rabbit,human Serum Egg-albumin

Basic requirements of culture media

Nutrients - Energy source - Carbon source - Nitrogen source Mineral salts Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca. A suitable pH 7.2 7.4 Accessory growth factors - Tryptophan for Salmonella typhi - X & V factors for H. influenzae

Petri dish with Media

Plate: provide large surface for isolation and observation of colonies Using a sterile loop or a sterile swab streak your sample on the petri plate Important let your sterilized loop cool before you pick up your sample

Classification of Culture media

Based on the consistency: Liquid -- Peptone water, Nutrient broth Semisolid -- Nutrient agar stabs Solid -- Blood agar, Serum agar Based on Oxygen requirement: -- Aerobic medium -- Anaerobic media

Liquid Medium
Difficulat to identify all types of organisms Suitable for isolation of bacteria from Blood culturing and water analysis

TYPES OF MEDIA
1. 2. 3. 4. 5. 6. 7. 8. 9. Simple or basal medium Enriched medium Enrichment medium Selective medium Indicator medium Differential medium Sugar medium Transport medium Anaerobic medium

SIMPLE OR BASAL MEDIUM

Peptone water-peptone+NaCl+distilled water-pH 7.2-7.6 Nutrient broth-peptone water+meat extract Nutrient agar-nutrient broth+2% agar Most common medium used in routine diagnostic laboratories for doing biochemical tests for identification of bacteria.

ENRICHED MEDIUM
Addition of blood or serum or egg albumin to basal medium.eg:blood agar,chocolate agar,egg media. Blood agar-nutrient agar+5-10%blood In blood agar,bacteria produce 2 types of hemolysis-alpha and beta. Alpha hemolysis-partial-streptococcus viridans,pneumococci. Beta-complete-staphylococci,strep.pyogenes

blood agar

CHOCOLATE AGAR
Heated blood agar Prepared by heating blood agar at 75-80 degrees in a water bath until it becomes chocolate brown in colour. Required for the growth of Haemophilus influenzae. Egg medium is prepared by addition of egg albumin to basal medium

Loefflers serum slope

Lowenstein-Jensens medium
Mineral salt soln - 600ml Malachite green soln - 20ml (2gm% in D.water) Beaten egg - 1000ml (20-22 eggs) Mix the above Distribute in Mc Cartney bottles Sterilize by Inspissation Use: To cultivate Mycobacteria

Lowenstein Jensen Medium

ENRICHMENT MEDIUM
Eg:Selenite F broth, Tetrathionate broth, Alkaline peptone water.-LIQUID MEDIUM In mixed cultures or materials containing more than one bacterium to be isolated is overgrown by unwanted bacteria. Certain substances are added into the medium which will stimulate the growth of pathogenic bacteria and inhibit the growth of unwanted bacteria. Pathogenic bacteria isolated in pure culture.

SELECTIVE MEDIUM
Solid medium in which inhibitory substances are added to inhibit the growth of unwanted bacteria. Deoxycholate citrate agar(DCA) and xylose lysin deoxycholate for salmonella and shigella. DCA--It is a heat sensitive medium It should not be autoclaved or remelted Thiosulfate citrate bile salt sucrose agar(TCBS)-for vibrio cholerae. TCBS agar need not be sterilized

INDICATOR MEDIUM
Certain indicator substances are added into the medium which changes colour when a bacterium grows on the medium WILSON AND BLAIR MEDIUM for Salmonella typhi-sulphite is reduced to sulphide-black colonies. Mc Leods medium(potassium tellurite agar)Corynebacterium diphtheriae -tellurite is reduced to tellurium-black colonies

DIFFERENTIAL MEDIUM
Certain substances are incorporated in the medium to bring out different characteristics of bacteria to distinguish between them. Mac Conkeys medium which contains peptone,lactose,agar,nuetral red and sodium taurocholate. Lactose fermenting bacteria produce pink colonies-E.coli and klebsiella. Non-lactose fermenting bacteria produce colourless colonies-Salmonella,Shigella.

TRANSPORT MEDIUM
Medium used for transporting clinical specimens containing delicate organisms which do not survive the time taken for transporting the material from the site of collection to the laboratory. Bacteria remain viable without multiplication. Stuarts medium-gonococci Pikes medium-streptococci Cary-Blair medium-vibrio cholerae Buffered glycerol saline-shigella

SUGAR MEDIUM
Peptone water containing 1% sugarglucose,lactose,sucrose,maltose, and mannitol. Fermentation of sugars produce acid and gas. Acid production detected by colour change. Gas production detected by Durhams tube.

ANAEROBIC MEDIUM
Medium used for growing anaerobic bacteriaClostridium species. Robertsons cooked meat medium contains glucose broth+cooked meat. Proteolytic organisms-the colour of meatblack.Eg:Clostridium tetani. Saccharolytic organisms-meat-pink.Eg:Clostridium perfringens Thioglycollate broth is also an anaerobic culture medium

Robertson's cooked meat medium

STERILISATION OF CULTURE MEDIA

AUTOCLAVING at 121 degree for 15 min. Media which cannot be autoclaved-Selenite F broth and tetrathionate broth are sterilised by steaming at100 degrees. Lowenstein-Jensens medium for mycobacterium tuberculosis and Loefflers serum slope for Corynebacterium diphtheriae are sterilised by inspissation using inspissator at 80-85 degrees.

inspissator

AUTOCLAVE

GROWTH OF BACTERIA
In liquid medium,the bacterial growth is detected by turbidity. In solid medium,bacteria produce colonies. A colony means visible cluster of bacteria arising from a single parent cell. Pure culture is obtained by using solid medium.

Storage of culture media

Prepared media in individual screw capped bottles can be stored for weeks at room temp Poured plates deteriorate quickly and often contaminated, hence cold storage is necessary For smaller labs domestic refrigerators & for larger labs insulated cold room(4-5oc)

Deep freeze refrigerators for preservation of sera, antibiotics & amino acids (-10 to - 400c)

Sabouraud's Dextrose agar commonly used Fungal Isolation Medium

Sabouraud's Dextrose Agar

Dextrose - 4 gm% Neopeptone - 1 gm% Agar - 1.5 gm% Distilled water - 100 ml Dissolve the ingredients by heating in a water bath, cool and adjust pH to 5.4 Autoclave and dispense 20 ml amount in test tubes Use: For the cultivation of Fungi