Selective media,types,Composition andpplications

Culture media

1. Selective media

2. Selective media: used to select (isolate) specific groups of bacteria; chemical substances in the media inhibit the growth of one type of bacteria while permitting growth of another (MSA, EMB, MacConkey )

3. Differential media: distinguishes among morphologically and biochemically related groups of organisms; chemical compounds (following inoculation and incubation) produce a characteristic change in the appearance of bacterial growth and/or the medium surrounding the colonies (MSA, EMB, MacConkey )

4. MACCONKEY AGAR A Selective and differential medium used for the cultivation of enterobacteria Content: peptone,Sodium taurocholate,water , agar ,Neutral red soln,2 % in 50% ethanol, Lactose 10% soln Adjust ph to 7.5

5. Bile salts and crystal violet inhibit growth of G+ organisms (selective) Neutral red is a pH indicator that is colorless, but yellow above pH 8 and red at pH less than 6.8 (differential)

6. Acid accumulating from lactose fermentation turns the colorless neutral red to a red color—therefore coliforms produce a red “halo” on the medium ( E.coli , E.aerogenes ) Lactose nonfermenters will grow, but don’t produce acid. Therefore, the neutral red remains colorless ( P. vulgaris ) No growth indicates a Gram + organism ( S.aureus ) Macconkey agar with lactose(left) and non-lactose(right) fermenters

7. MacConkey’s Agar

8. Eosin Methylene Blue Agar (EMB) Eosin methylene blue agar is a selective and differential medium Eosin methylene blue agar is a selective and Typically used for the family Enterobacteriaceae enteric (gut) bacteria Selective: EMB contains the dyes methylene blue and eosin which inhibit Gram + bacteria, thus favoring growth of Gram – )

9. Differential: EMB contains lactose, thus allowing for the distinction between lactose fermenters and nonferments Large amounts of acid from lactose fermentation cause the dyes to precipitate on the colony surface, producing a black center or a “green metallic sheen” (E. coli)

10. Smaller amounts of acid production result in pink coloration of the growth (E. aerogenes ) Nonfermenting enterics do not produce acid so their colonies remain colorless or take on the color of the media (P. vulgaris ) No growth indicates a Gram + organism ( S.aureus )

11. EMB

12. Large amounts of acid from lactose fermentation cause the dyes to precipitate on the colony surface, producing a black center or a “green metallic sheen” ( E. coli)

13. Mannitol Salt Agar (MSA) Mannitol salt agar is both selective and differential Selective: It favors organisms capable of tolerating high salt concentrations (7.5 % NaCl ) Differential: It distinguishes bacteria based on their ability to ferment mannitol Differentiates Staphylococcus species, by mannitol fermentation . S. aureus ferments, S. epidermidis and s.lugdunensis does not Phenol red is the pH indicator. Basic pHred at 7.4 to 8.Acidic ph yellow below 6.8

14. Positive Results: The development of “yellow halos” around the bacterial growth means mannitol has been fermented and acid end products have been produced ( S. aureus ) Negative Results: No color change in the medium is a negative result ( S. epidermidis,s.lugdunensis ) No growth on the medium indicates a Gram- organism ( E. coli)

15. Xylose -lysine deoxycholate agar Xylose -Lysine Deoxycholate Agar (XLD Agar) is a selective medium recommended for the isolation and enumeration of Salmonella Typhi and other Salmonella species. Yeast extract , L-Lysine, Lactose, Sucrose, Xylose , Sodium chloride,Sodium deoxycholate Sodium thiosulphate , Ferric ammonium citrate, Phenol redAgar Final pH ( at 25°C) 7.4±0.2

16. The medium contains yeast extract, which provides nitrogen and vitamins required for growth. It utilizes sodium deoxycholate as the selective agent and therefore it is inhibitory to gram-positive microorganisms an H2S indicator system, consisting of sodium thiosulphate and ferric ammonium citrate, is included for the visualization of hydrogen sulphide produced

17. . S. Paratyphi A, S.Choleraesuis , S. Pullorum and S. Gallinarum may form red colonies without H2S, thus resembling Shigella species Lysine is included to differentiate the Salmonella group from the non-pathogens. S

18. Hektoen enteric agar Selective media for enteric pathogens Bile salts,lactose,sucrose,bromothymol blue,acid fuschin,ferric ammonium citrate Lactose fermenters appear green colonies H2s production indicates black color

19. H e agar

20. H e agar

21. Thayer-martin medium Used to find neisseria.gonorrhoeae Contains vancomycin,colistin,nystatin Plates incubated in 3-10% co2 Modified thayer –martin medium include trimethoprim

22. Thiosulfate citrate bile sucrose ( tcbs ) agar Selective and differential media for vibrio High ph value of 8.6 Contains peptone,yeast,sucrose,bromothymol blue,sodium citrate,ferric citrate Sucrose fermenting vibrio form yellow colonies Non- fermenters v.parahemolyticus are green

23. Hoylers tellurite blood agar For c.diptheria Contains agar base,lysed blood and tellurite soln ( potassium tellurite ) Potassium tellurite inhibits c.albicans , s.aureus &most oral commensals Colonies appear as dark slate –grey color and has matt surface

24. PLET MEDIUM Selective media for B.anthracis Contains ethylene diamine tetra acetate,thallous acetate,polymyxin,lysosyme Ph should be 7.35 PLET medium inhibits other bacillus series,enterobacteria & pseudomonas

25. Buffered charcoal yeast extract agar Selective media for Legionellas Contains N2-acetamino 2-amino ethane sulphonic acid,koh,activated charcoal,yeast extract,bacto agar,ketoglutarate,l-cysteine,ferric pyrophosphate,polymyxin,cycloheximide & vancomycin & anisomycin Charcoal- detoxify,remove co2,modify surface tension L- cysteine -growth of L.pneumophilia

26. Antibiotics inhibit G+,G-, yeast and fungi Colonies appear gray-white to blue-green glistening ,convex, circular

27. Sp-4 medium Media for mycoplasma Contains cmrl 1006 tissue culture media glutamine,yeast extract,yeasteolate,fetal bovine serum,penicillin,amphotericin – b,polymyxin – b,phenol red,mycoplasma agar Thallium acetate- M.pneumoniae SP4 glucose broth with urea- U.urealyticum SP4 glucose broth with arginine-M.hominis

28. pH Indicators and buffers

29. Why is pH important in biology? pH affects solubility of many substances. pH affects structure and function of most proteins - including enzymes. Many cells and organisms (esp. plants and aquatic animals) can only survive in a specific pH environment. Important point - pH is dependent upon temperature

30. pH Paper pH 0 1 2 3 4 5 6 pH 7 8 9 10 11 12 13

31. Common pH Indicators Zumdahl, Zumdahl, DeCoste, World of Chemistry 2002, page 520

32. Ways to measure pH Indicator dyes and test strips Less precise Each indicator is only good for a small pH range (1-2 pH units) But may be good for field usage, or measuring small volumes, or dealing with noxious samples.

33. Buffers Definition: a solution that resists change in pH Typically a mixture of the acid and base form of a chemical Can be adjusted to a particular pH value Why use them? Enzyme reactions and cell functions have optimum pH’s for performance Important anytime the structure and/or activity of a biological material must be maintained

34. How buffers work Equilibrium between acid and base. Example: Acetate buffer CH 3 COOH  CH 3 COO - + H + If more H + is added to this solution, it simply shifts the equilibrium to the left, absorbing H + , so the [H + ] remains unchanged. If H + is removed (e.g. by adding OH-) then the equilibrium shifts to the right, releasing H + to keep the pH constant

35. Factors in choosing a buffer Be sure it covers the pH range you need Generally: pK a of acid ± 1 pH unit Consult tables for ranges or pK a values Be sure it is not toxic to the cells or organisms you are working with. Be sure it would not confound the experiment (e.g. avoid phosphate buffers in experiments on plant mineral nutrition).

36. Calculating buffer recipes Henderson-Hasselbach equation pH = pKa - log 10 [acid]/[base] Rearrange the equation to get 10 (pKa-pH) = [acid]/[base] Look up pKa for acid in a table. Substitute this and the desired pH into equation above, and calculate the approximate ratio of acid to base. Because of the log, you want to pick a buffer with a pKa close to the pH you want.

37. Common buffers Acetate buffer Citrate buffer Citrate-phosphate buffer phosphate buffer Barbitone buffer tris HCL buffer Boric acid-borax buffer Bicarbonate-co2 buffer

38. THANK YOU