Cultivation of baceria

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

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

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

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

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

8. 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

9. Nutrient agar Dr. Emad AbdElhameed Morad

10. Nutrient agar showing pseudomonas Dr. Emad AbdElhameed Morad

11. 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

12. 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

13. Bacterial motility Dr. Emad AbdElhameed Morad

14. 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, alpha and gamma hemolysis. Composition Uses Dr. Emad AbdElhameed Morad

15. Blood agar Dr. Emad AbdElhameed Morad

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

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

18. 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

19. Chocolate agar Dr. Emad AbdElhameed Morad

20. Composition • Loffler’ serum: • 3 parts horse serum + 1 part glucose broth. • Serum is the solidifying agent. • Cultivation of diphtheria bacilli. Uses Dr. Emad AbdElhameed Morad

21. Loffler’s serum Dr. Emad AbdElhameed Morad

22. Selective Solid media • Lowenstein-Jensen medium: • Beaten eggs + mineral salts + malachite green. • Malachite green inhibits bacteria other than tubercle bacilli. • Cultivation of tubercle bacilli. Composition Uses Dr. Emad AbdElhameed Morad

23. Lownstein Jensen medium Dr. Emad AbdElhameed Morad

24. 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

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

26. 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 colonies. Uses Dr. Emad AbdElhameed Morad

27. MacConkey’s agar Dr. Emad AbdElhameed Morad

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

29. 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

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

31. (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

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

33. 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

34. 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

35. 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

36. Cultivation of anaerobes Culture media for anaerobes Anaerobic jar Dr. Emad AbdElhameed Morad

37. Culture media for anaerobes • Examples of anaerobic bacteria: clostridia, bacteriodes, lactobacilli. • Meat contains hematin, glutathione and lipids which reduce the oxygen in the medium. Robertson cooked meat medium Thioglycolate broth Deep soft agar Dr. Emad AbdElhameed Morad

38. Robertson cooked meat medium Dr. Emad AbdElhameed Morad

39. Anaerobic jar • It is a plastic jar with tightly fitted lid. • Hydrogen is introduced from hydrogen generator envelope. • Hydrogen reacts with the O2 in the jar in the presence of a catalyst suspended from the lid forming water. • Anaerobic indicator such as methylene blue is placed in the jar. It is colorless under anaerobic conditions and changes into blue in the presence of O2. Dr. Emad AbdElhameed Morad

40. Anaerobic jar Dr. Emad AbdElhameed Morad

41. Thank You Dr. Emad AbdElhameed Morad