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Cultivation of baceria

Cultivation of baceria. By. Dr. Emad AbdElhameed Morad. Lecturer of Medical Microbiology and Immunology. INOCULATION PROCEDURES. P lating out technique: Aims at spreading out the bacteria on the surface of the medium. Each bacterium will divide to give a separate colony.

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Cultivation of baceria

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  1. Cultivation of baceria By Dr. Emad AbdElhameed Morad Lecturer of Medical Microbiology and Immunology

  2. INOCULATION PROCEDURES • Plating out technique: • Aims at spreading out the bacteria on the surface of the medium. • Each bacterium will divide to give a separate colony. • The loop is sterilized and loaded with the bacterial mixture present in the culture or the specimen. Then, it is smeared over area A to give the main inoculum. • The loop is then sterilized and after cooling, drawn into three parallel lines B across a fresh part of the medium. • The procedure is repeated as shown in the figure. • The plate is incubated at 37 degree for 24 hours. Dr. Emad AbdElhameed Morad

  3. Plating out technique Dr. Emad AbdElhameed Morad

  4. Subculture: • Subculture is a term which means transferring bacteria from one culture to another culture. • Is done for purification of cultures containing mixture of bacteria. • Done by picking one colony from a culture plate by the sterile loop and inoculating it on another fresh culture plate. • By this, pure cultures are obtained which could be used in identification and antibiotic sensitivity testing. Dr. Emad AbdElhameed Morad

  5. CULTURE MEDIA • Liquid media. • Solid media. • Simple media • Enriched media • Selective media • Differential (indicator media) • Sugar media. • Enrichment media. • Litmus milk. • Cultivation of anaerobic bacteria (culture media & anaerobic jar) Dr. Emad AbdElhameed Morad

  6. Liquid media • Peptone water: • 1% peptone + 0.5%NaCl. • Base for indole test. • Base for sugar media. Composition Uses Dr. Emad AbdElhameed Morad

  7. Composition • Nutrient broth (meat infusion broth): • Meat extract + 1% peptone + 0.5% NaCl. • pH is adjusted to 7.2 and sterilized in the autoclave. • Cultivation of many microorganisms. • Base for nutrient agar medium. Uses Dr. Emad AbdElhameed Morad

  8. Nutrient broth with bacterial growth Dr. Emad AbdElhameed Morad

  9. Simple Solid media • Nutrient agar: • Nutrient broth + 2 – 3% agar. • Agar is sea weeds that is dissolved by heating at 100 degree and become s solidified at 45 degree. • Base for blood agar. • Detect exopigment production by pseudomonas. Composition Uses Dr. Emad AbdElhameed Morad

  10. Nutrient agar Dr. Emad AbdElhameed Morad

  11. Nutrient agar showing pseudomonas Dr. Emad AbdElhameed Morad

  12. Composition • Nutrient slope agar: • Nutrient broth + 2– 3% agar. • Poured in tubes. It has a sloping surface. • Preservation of bacteria. Uses Dr. Emad AbdElhameed Morad

  13. Composition • Deep soft agar: • Nutrient broth + 0.5 – 1% agar. • Poured in tubes. It has horizontal upper surface. • Detection of bacterial motility. • Cultivation of anaerobes. Uses Dr. Emad AbdElhameed Morad

  14. Bacterial motility Dr. Emad AbdElhameed Morad

  15. Composition • Nutrient gelatin: • Nutrient broth +15% gelatin. • Poured in tubes. • Sterilized in the steamer for 45 min for 3 successive days. • Study the proteolytic activity of bacteria. • Vibrio cholerae liquefies gelatin. Not autoclavable Uses Dr. Emad AbdElhameed Morad

  16. Nutrient gelatin Dr. Emad AbdElhameed Morad

  17. Enriched Solid media • Blood agar: • Nutrient agar + 10% sheep or horse blood. • The sterile blood is added to the nutrient agar at 55 degree to avoid hemolysis of red blood cells. • Base for chocolate agar. • Differentiates between bacteria according to the type of hemolysis produced: beta hemolysis (Staph. aureus), alpha (Pneumococci) and gamma (Enterococci). Composition Uses Dr. Emad AbdElhameed Morad

  18. Blood agar Dr. Emad AbdElhameed Morad

  19. Blood agar showing beta hemolysis Dr. Emad AbdElhameed Morad

  20. Blood agar showing alpha hemolysis Dr. Emad AbdElhameed Morad

  21. Composition • Chocolate agar: • The same as blood agar. • But, the temperature of the medium is raised to 100 degree. During heating, red blood cells become ruptured with release of hematin. • Cultivation of highly fastidious bacteria as neisseria and hemophilus. Uses Dr. Emad AbdElhameed Morad

  22. Chocolate agar Dr. Emad AbdElhameed Morad

  23. Composition • Loffler’ serum: • 3 parts horse serum + 1 part glucose broth. • Sterilized in hot air inspissator at 75 - 80 degree for 2 hours on 2 successive days. This leads to coagulation of the serum and solidification of the medium. • Cultivation of diphtheria bacilli. Not autoclavable Uses Dr. Emad AbdElhameed Morad

  24. Loffler’s serum Dr. Emad AbdElhameed Morad

  25. Selective Solid media • Lowenstein-Jensen medium: • Beaten eggs + mineral salts + malachite green. • Malachite green inhibits bacteria other than tubercle bacilli. • Sterilized in hot air inspissator at 80 degree for 1 hour. • Cultivation of tubercle bacilli. Composition Not autoclavable Uses Dr. Emad AbdElhameed Morad

  26. Lownstein Jensen medium Dr. Emad AbdElhameed Morad

  27. Composition • Blood tellurite medium: • Blood agar + 0.04 % tellurite. • Cultivation of diphtheria bacilli. • Differentiation between types of Corynebacterium diphtheria due to varying black to grey color they produce as a result of reduction of tellurite to tellurium. Uses Dr. Emad AbdElhameed Morad

  28. Composition • Modified Thayer-Martin medium: • Chocolate agar + antibiotics (vancomycin, colistin, nystatin). • Cultivation of Neisseria gonorrhea. Uses Dr. Emad AbdElhameed Morad

  29. Composition • TCBS medium: • Thiosulphate, citrate, bile as selective agents. • Sucrose as test sugar. • Thymol blue and bromothymol blue as pH indicators. • Cultivation of Vibrio cholerae. • Vibrio cholerae grow s as yellow colonies on TCBS. Uses Dr. Emad AbdElhameed Morad

  30. TCBS medium TCBS with Vibrio cholerae Dr. Emad AbdElhameed Morad

  31. Composition • Desoxycholate citrate agar (DCA): • Meat extract, peptone nutrients. • Desoxycholate inhibits intestinal flora. • Lactose as test sugar. • Neutral red as pH indicator. • Isolation of salmonella and shigella. • Salmonella and shigella give pale yellow colonies. Uses Dr. Emad AbdElhameed Morad

  32. DCA medium DCA medium with salmonella Dr. Emad AbdElhameed Morad

  33. Differential Solid media • MacConkey’s medium: • Peptone nutrient. • Sodium taurocholate inhibits non intestinal bacteria. • Lactose + neutral red indicator. • Differentiates enterobacteriacae into: • lactose fermenters which give rose pink colonies. • Non lactose fermenters which give pale yellow colonies. Uses Dr. Emad AbdElhameed Morad

  34. MacConkey’s agar Dr. Emad AbdElhameed Morad

  35. MacConkey’s agar with both lactose and non lactose fermenters Dr. Emad AbdElhameed Morad

  36. Composition • CLED medium: • Cystine lactose electrolyte deficient agar. • Lactose is test sugar. • Bromothymol blue is the pH indicator. • Differentiates between: • lactose fermenters which give yellow colonies. • Non lactose fermenters which give green colonies. Uses Dr. Emad AbdElhameed Morad

  37. CLED agar with lactose fermenters CLED agar with non lactose fermenters Dr. Emad AbdElhameed Morad

  38. Composition • XLD medium: • XLD = Xylose, lysine deoxycholate agar. • Lactose, sucrose, xylose are test sugars. • Phenol red is the pH indicator. • Ferric ammonium citrate to detect H2 S production. • Differentiates between: • lactose fermenters which give yellow colonies (as E. coli) • Non lactose fermenters which give red colonies (as shigella) • Non lactose fermenters with H2S production which give red colonies with black centers (as Salmonella typhi). Uses Dr. Emad AbdElhameed Morad

  39. XLD medium Dr. Emad AbdElhameed Morad

  40. XLD medium with lactose fermenters Dr. Emad AbdElhameed Morad

  41. XLD medium with non lactose fermenters Dr. Emad AbdElhameed Morad

  42. XLD medium with H2S producing organism Dr. Emad AbdElhameed Morad

  43. Composition • Triple sugar iron (TSI) agar: • Glucose 0.1%, Lactose 1%, sucrose 1% are the test sugars. • Phenol red is the pH indicator. • Ferrous sulphate to detect H2 S production. • 0.5% agar (soft agar cracks on gas production). • Differentiates between: • Bacteria according to their effect on sugars and production of H2S. Uses Dr. Emad AbdElhameed Morad

  44. Triple sugar iron A B C D E F G H Dr. Emad AbdElhameed Morad

  45. (From left to right) • Uninoculated control. • Red slant and red butt, no black color = no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced. • Red slant and black butt = no lactose or sucrose fermentation, H2S has been produced. • Red slant with yellow butt = no lactose or sucrose fermentation, glucose is fermented, no H2S has been produced. • Yellow slant, yellow butt and black coloration = Lactose, sucrose and glucose fermented, and H2S has been produced. F, G. Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration = Lactose, sucrose and glucose fermented, H2S has not been produced but gas has been produced. H. Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been produced nor has gas been. Dr. Emad AbdElhameed Morad

  46. Quiz A: B: C: D: Dr. Emad AbdElhameed Morad

  47. Sugar media • Six tubes. • Each tube contains peptone water + specific sugar in a concentration 1%. • The sugars are glucose, lactose, maltose, mannite, sucrose, salicin. • Each tube contains also Andrade’s indicator which changes to pink if acid is produced. • An inverted small tube (Durham’s tube) is immersed in the medium and gas production is revealed by collection of bubbles at its apex. Composition Dr. Emad AbdElhameed Morad

  48. Uses • Differentiate between bacteria according to their fermentative effect on different sugars and whether the fermentation is accompanied by production of gas or not. Fermentation of glucose, maltose, mannite with acid only Fermentation of all sugars with acid + gas Dr. Emad AbdElhameed Morad

  49. Enrichment media • These are fluid media that contain substances that enhance the growth of some bacteria and not others. • Examples: • Selenite broth: an enrichment medium used for isolation of salmonella and shigella. • Tetrathionate broth: an enrichment medium used for isoaltion of salmonella. Dr. Emad AbdElhameed Morad

  50. Litmus milk • Skimmed milk + litmus indicator. • Test the action of bacteria on milk. • acidified, alkalinized, clotted. Dr. Emad AbdElhameed Morad

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