2. NAS Lecturer: Mr. M. Zivuku LECTURE 4 1

3. Objectives By the end of this unit, you should be able to ; Explain the cultivation of bacteria in terms of in vivo and in vitro. Describe why is it important to cultivate microorganisms. Describe and explain the different types of culture media (Solid, semisolid, liquid, biphasic). Define the terms(i) selective media,(ii) differential media. list the composition of each of these culture media (i) and (ii) above and explain why they are selective/differential. 2

4. Cultivation of Bacteria The process of growing microorganisms in culture by: –Taking bacteria from an infection site by specimen collection - in vivo –Growing bacteria in the artificial environment of the laboratory - in vitro 3

5. Why cultivate bacteria? Obtain definitive identification and characterization Grow and isolate all bacteria present in an infection Determine which bacteria is most likely causing infection Determine which bacteria is likely a contaminant or colonizer 4

6. Types of Culture Media -Solid, semisolid, liquid, biphasic -Simple media, special media (enriched, selective, enrichment, indicator/ differential, transport) -Aerobic and anaerobic media -Cell culture for obligate intracellular bacteria (e.g., Chlamydia spp) 5

7. Biphasic Culture Medium Broth (liquid medium) Agar slope (solid medium) 6

8. Selective and Differential Media Selective- These are culture media in which chemical substances are added to prevent growth one group of bacteria without affecting the growth of the other bacteria e.g. crystal violet will inhibit the growth all G+ve organism Example: Mac Conkey agar, Crystal violet agar 7

9. Selective Media Example: Bismuth sulfite for Salmonella typhi (inhibits gram-positive and most gram- negative intestinal bacteria) It use glucose as a primary source of carbon Bismuth will stop G+ve growth Selectivity, utilize ferrous sulphate and convert to Hydrogen sulphide 8

10. Composition of Bismuth sulfite Bismuth Sulfate Bi2(SO3)3-1.6% Pancreatic digest of casein-1.0% Pancreatic digest of animal tissue-1.0% Beef extract-1.0% Glucose-1.0% Dibasic sodium phosphate-0.8% Ferrous sulphate.7 water-0.06% pH adjusted 7.7 at 25C 9

11. Differential These are media to which dyes or other substances are added to differentiate microorganism. Such changes may include color, pH, haemolysis, coagulation, hydrolysis or fermentation – Example: Blood agar plates for Streptococcus pyogenes 10

12. Selective and differential Media Differential Blood agar plates for Streptococcus pyogenes Type of hemolysis reaction, identification of Identification S. pyogenes 11

13. Selective and differential Selective & differential Mannitol salt agar Selective medium for pathogenic for Staphylococcus aureus isolation in clinical samples and biological and pharmaceutical products. 12

14. Composition TYPICAL FORMULA (g/l) Beef Extract ……………………. 1.0 Peptospecial …………………..10.0 Sodium Chloride ……………..75.0 Mannitol …………………………10.0 Phenol Red …………………… 0.025 Agar ………………………………15.0 Final pH = 7.4 ± 0.2 at 25°C. 13

15. Mannitol salt agar The high concentration of sodium chloride (7,5%) generally inhibits the growth of other bacteria. Fermentation of the mannitol causes an acidification of the medium and a colour change from red to yellow. 14

16. Mannitol salt agar The coagulase positive staphylococci cultivate with yellow colonies, surrounded by a yellow zone. The coagulase negative staphylococci grow less luxuriantly forming small red-purple colonies. 15

17. Principle of MSA Summary This tests for the bacteria’s ability to tolerate 7% salt concentration and ferment mannitol. The media is selective because it selects for salt tolerant bacteria. The media is also differential because it differentiates the salt tolerant organisms on their ability to ferment mannitol 16

18. Solid, Semi-solid and Fluid culture media Solid Culture Media Media are solidified by incorporating a gelling agent such as agar or gelatin Agar- a pollysacharide extract obtained from seaweed and is commonly used to solidify culture media because of high gelling strength, its setting temp 32-39 Deg C and melting temp 90-95Deg C 17

19. Solid Media Solid media are used mainly in Petri dishes as plate cultures. Also in bottles or tubes as stab (deeps) or slope cultures NB The purpose of culturing on a solid medium is principally to isolate discrete colonies of each organism present in the specimen. 18

20. Semi Solid Culture Media This form of culture medium is prepared by adding a small amount of agar (0.4-0.5%) to a fluid medium Semi solid are used mainly as transport media and for motility and biochemical tests 19

21. Fluid culture Media Fluid media are most commonly used as enrichment where organisms are likely to be few e.g blood culture Some organisms produce a surface growth on the in which they are growing e.g Vibrio cholerae when grown in alkaline peptone water 20