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  2. 2. CULTURE MEDIA ▰ Liquid or solid substance - contains nutrients to support the growth, and survival of microorganisms. 2
  3. 3. Constituents of Culture Media 3 Constituent Explanation Water and Electrolytes Sodium chloride Peptone Mixture of partially digested proteins - obtained from various sources - heart muscle, casein or fibrin, or soya. Agar Used for solidifying the culture media Source: Prepared from - cell wall of seaweeds and available commercially in powder form Preparation: Agar powder is dissolved in water and subjected to sterilization by autoclave. When the temperature of the molten agar comes down to 45°C, it is poured into the Petri dishes and then allowed to set for 20 minutes. Concentration: Solid agar preparation - 1-2% Semisolid agar- 0.5% Solid agar to inhibit Proteus swarming- 6%
  4. 4. Constituents of Culture Media (Cont..) 4 Constituent Explanation Meat extract Highly concentrated meat stock, usually made from beef. Yeast extract (Prepared from Baker’s yeast) and malt extract (contains maltose) Blood and serum Important components of enriched media; provide extra nutrition to fastidious bacteria. 5–10% of sheep blood is used. Alternatively, horse, ox or human blood can also be used.
  5. 5. Types of Culture Media Based on consistency, culture media are grouped into: ▰ Liquid (or broth) media ▰ semisolid media ▰ solid media 5
  6. 6. Types of Culture Media Based on the growth detection, culture media are classified as: 6 Conventional culture media: Prepared from nutrients - aqueous extract of meat, peptone.  Simple/basal media  Enriched media  Enrichment broth  Selective media  Differential media  Transport media  Anaerobic media. Automated culture media: Mainly available for blood and sterile body fluid culture.
  8. 8. Con Consistency Decided by Agar concentration : - Solid media: 2% Agar - Semisolid media: 0.5% Agar - Liquid media: Agar not added - Biphasic media : Solid and a liquid phase in the same media container (Bottle)
  9. 9. Semisolid Media Mannitol motility media Used to demonstrate motile organisms – indicated by a haziness spreading away from the stab line
  10. 10. Brain heart infusion agar - Both liquid & solid phase -Reduces contamination and isolate even a low count of pathogen
  11. 11. Simple/Basal Media Contain minimum ingredients that support the growth of non-fastidious bacteria. ▰ Peptone water: Contains peptone (1%) + NaCl (0.5%) + water ▰ Nutrient broth: Peptone water + meat extract (1%). ▰ Nutrient agar: Nutrient broth + 2% agar ▰ Semisolid medium: Concentration of agar - reduced to 0.2–0.5 %. 11
  12. 12. Uses of Basal Media ▰ Testing the non-fastidiousness of bacteria. ▰ Serve as base for the preparation of many other media. ▰ Nutrient broth - used for studying the bacterial growth curve. ▰ Nutrient agar - preferred medium for:  Performing the biochemical tests, such as oxidase, catalase, etc.  Study the colony morphology  Pigment demonstration. 12
  13. 13. Enriched Media ▰ Basal medium added with additional nutrients - blood, serum or egg ▰ In addition to non-fastidious organisms, support the growth of fastidious nutritionally exacting bacteria. 13
  14. 14. Enriched Media (Cont..) Blood agar – ▰ Prepared by adding 5-10% of sheep blood to the molten nutrient agar at 450C. ▰ Tests the hemolytic property of the bacteria, which may be either- i) partial or α (green) hemolysis and ii) complete or β hemolysis 14
  15. 15. Enriched Media (Cont..) Chocolate agar: ▰ Heated blood agar, prepared by adding 5 -10% of sheep blood to the molten nutrient agar at 70°C. ▰ More nutritious than blood agar - supports certain highly fastidious bacteria - Haemophilus influenzae that does not grow on blood agar. 15
  16. 16. Enriched Media (Cont..) Loeffler’s serum slope: Contains serum - used for isolation of Corynebacterium diphtheriae Blood culture media: Used for isolating microorganisms from blood - available as conventional or automated blood culture media. 16
  17. 17. Enrichment Broth ▰ Liquid media added with inhibitory agents which selectively allow certain organism to grow and inhibit others. ▰ Important for isolation of pathogens from clinical specimens which also contain normal flora (e.g. stool and sputum specimen). 17
  18. 18. Enrichment Broth (Cont..) Examples : ▰ Tetrathionate broth—Used for Salmonella Typhi ▰ Gram-negative broth—Used for Shigella ▰ Selenite F broth—Used for Shigella ▰ Alkaline peptone water (APW)—Used for Vibrio cholerae. 18
  19. 19. Selective Media Solid media containing inhibitory substances - inhibit the normal flora present in the specimen and allow the pathogens to grow. 19 Media Used for isolation of Lowenstein Jensen (LJ) medium Mycobacterium tuberculosis Thiosulphate Citrate Bile salt Sucrose (TCBS) Vibrio species DCA (Deoxycholate Citrate Agar) Salmonella and Shigella from stool XLD (Xylose Lysine Deoxycholate) agar Salmonella and Shigella from stool Potassium tellurite agar (PTA) Corynebacterium diphtheriae
  20. 20. Selective Media (Cont..) 20 Lowenstein–Jensen medium TCBS agar
  21. 21. Lowenstein Jensen (JL) Medium: Used for the growth of Mycobacterium tuberculosis Contains malachite green as an inhibiting substance Mycobacterium tuberculosis colonies are described as rough, tough, buff
  22. 22. TCBS Agar Thiosulphate Citrate Bile salt Sucrose (TCBS) agar: Used for the isolation of Vibrio cholerae Contains bile salt as an inhibiting substance Large yellow coloured colonies
  23. 23. Potassium tellurite agar: Used for the isolation of Corynebacterium diphtheria Contains potassium tellurite as an inhibiting substance
  24. 24. Selective media for Salmonella typhi : Wilson Blair bismuth sulfite medium – jet black colonies with a metallic sheen Deoxycholate citrate agar – NLF colonies with a black centre Xylose lysine deoxycholate agar – Red colonies with a black centre Salmonella Shigella agar – Colourless colonies with a black centre
  25. 25. Selective Media (Cont..) 25 DCA (Deoxycholate Citrate Agar) XLD (Xylose Lysine Deoxycholate) agar
  26. 26. ▰ WILSON BLAIR ▰ XLD DCA 26
  27. 27. Transport Media ▰ Used for the transport of the clinical specimens suspected to contain delicate organism or when delay is expected while transporting the specimens from the site of collection to the laboratory ▰ Bacteria do not multiply in the transport media - they only remain viable. 27
  28. 28. Transport Media (Cont..) 28 Organism Transport media Neisseria Amies medium, Stuart’s medium Vibrio cholerae VR (Venkatraman-Ramakrishnan) medium Autoclaved sea water Cary Blair medium Shigella, Salmonella Buffered glycerol saline Cary Blair medium
  29. 29. Differential Media ▰ Differentiate between two groups of bacteria - by using an indicator. 29 Differential media Features MacConkey agar  Differential and low selective medium - used for the isolation of enteric GNB.  Differentiates organisms into LF (pink colonies, e.g. Escherichia coli) and NLF or (colorless colonies, e.g. Shigella).  Composition- Peptone, lactose, agar, neutral red (indicator) and taurocholate
  30. 30. Differential Media (Cont..) 30 Differential media Features CLED agar (cysteine lactose electrolyte- deficient agar) Capable of differentiating between LF and NLF. Used as an alternative to combination of blood agar and MacConkey agar, for the processing of urine specimens
  31. 31. Differential Media (Cont..) 31 MacConkey agar CLED agar
  32. 32. Anaerobic Culture Media ▰ Contain reducing substances which take up oxygen and create lower redox potential - permit the growth of obligate anaerobes, such as Clostridium. 32 Media Features Robertson’s cooked meat (RCM) broth  Contains chopped meat particles (beef heart), which provide glutathione (a sulfhydryl group containing reducing substance) and unsaturated fatty acids.  Widely used  Used for maintenance of stock cultures.
  33. 33. Anaerobic Culture Media (Cont..) Other anaerobic media include: ▰ Thioglycollate broth ▰ Anaerobic blood agar ▰ BHIS agar (Brain-heart infusion agar) with supplements (vitamin K and hemin) ▰ Neomycin blood agar ▰ Egg yolk agar ▰ Phenyl ethyl agar ▰ Bacteroides bile esculin agar (BBE agar). 33
  34. 34. Blood Culture Media ▰ Recovery of bacteria from blood is difficult - they are usually present in lesser quantity and many of the blood pathogens are fastidious. ▰ Therefore, enriched media - used for isolating microorganisms from blood. ▰ Available either as conventional or automated media 34
  35. 35. Conventional Blood Culture Media Two types: ▰ Monophasic medium: Contains brain–heart infusion (BHI) broth. ▰ Biphasic medium: Liquid phase containing BHI broth and a solid agar slope made up of BHI agar 35
  36. 36. Disadvantages of conventional media Subcultures are made manually - performed less-frequently (once a day) as it is cumbersome. 36 Broth Disadvantages Monophasic BHI broth  Subcultures are made, periodically for 1 week.  Higher risk of contamination - due to opening of the cap of the bottle every time when subcultures are made. Biphasic BHI broth  Subcultures - made just by tilting the bottles so that the broth runs over the agar slope.  Lower risk of contamination as it obviates the opening of the cap of the bottle
  37. 37. Automated Blood Culture Techniques (Cont..) ▰ Specimens: Used for culture of blood, bone marrow and sterile body fluids. ▰ More sensitive: Higher yield of positive cultures from clinical specimens ▰ Rapid: Less time than conventional methods ▰ Less labor intensive: fully-automated 37
  38. 38. Automated Systems BacT/ALERT 3D: ▰ Principle - colorimetric detection of growth. ▰ When bacteria multiply - produce CO2 - increases the pH - changes the color of a blue-green sensor present at the bottom of the bottle to yellow 38
  39. 39. Automated Blood Culture Techniques ▰ Continuous automated monitoring: Blood culture bottles - periodically monitored for the microbial growth - every 10 minutes by the instrument. ▰ Composition: Tryptic soy broth and/or brain heart infusion broth + polymeric resin beads - adsorb and neutralize the antimicrobials present in blood specimen. 39
  40. 40. Automated Systems (Cont..) BacT/ALERT VIRTUO (bioMerieux): Advanced form of BacT/ALERT – has several advantages, such as: ▰ Automatic loading and unloading of bottles, ▰ Faster detection of growth, ▰ Determine volume of blood present in bottle 40
  41. 41. Automated Systems (Cont..) BACTEC (BD Diagnostics) ▰ Principle - fluorometric detection of growth. ▰ Uses an oxygen-sensitive fluorescent dye present in the medium. 41
  42. 42. Disadvantages of automated systems ▰ High cost of the instrument and culture bottles, ▰ Inability to observe the colony morphology as liquid medium is used. 42
  43. 43. THANK YOU