Experiment 2

1. Experiment 2 routine cultivation of bacteria The cultivation of bacteria (1)

2. Contents 1. Media for routine cultivation of bacteria 2. Techniques for bacterial cultivation and inoculation 3. Growth characteristics of bacteria on/in different media 4. Metabolic activities of bacteria 5. The physical effect on bacteria and commonly used sterilizers

3. 1. Media for routine cultivation of bacteria Media--- The basic ingredients of media include carbon source、nitrogen source、 inorganic salt 、growth factor and distilled water etc.

4. Classification of culture media According to function, in general, media can be classified five types: 1. basal medium ( agar plate medium) 2. nutrient medium (blood agar plate medium) 3. selective medium 4. differential medium 5. anaerobic medium.

5. According to physical properties, media can be classified three types: 1. solid medium ( 1.5～2.5% of agar) agar plate /slant medium 2. semi-solid medium (0.3～0.5% of agar) 3. liquid medium 2 1 3 1

6. 2. Techniques for bacterial inoculation Bacterial inoculation is a basic technique of microbiology. The operation must be under sterile condition. 1) isolation cultivation 2) pure cultivation

7. 1) Isolation cultivation streak a bacterial culture on a nutrient agar plate to separate the individual bacterial cells, each isolated cell will develop into a pure colony after incubation. continuous streak method quadrant streak dilution method method

11. 2) Pure cultivation transfer a single colony from agar plate or a pure slant culture to liquid medium, slant or semisolid medium to get a pure culture. subculture into liquid medium subculture into agar slant/plate subculture into agar deep tube (semi-solid)

12. 3. Phenomena of bacterial growth on agar plate (on blood agar plate) in liquid medium in semi-solid medium

13. 1) on agar plate observe the isolated colony on the surface of nutrient agar plate; note size, shape, elevation, margin, surface, transparency and pigment (color of colony).

14. on agar plate -- colony Note: Size 大小 Shape 形状 Elevation 突起 Margin 边缘 Surface 表面 Transparency 透明度 Pigment 色素

15. on blood agar plate observe hemolysis(complete and incomplete) phenomena around colony.

16. 3) in liquid medium three growth phenomena will be seen: surface growth pellicle 表面生长 uniformly turbid 均匀混浊生长 sediment in bottom 沉淀生长

17. In liquid medium surface growth pellicle uniformly turbid sediment in bottom

18. 4) in semi-solid medium nonmotile organism motile organism

19. 4. Metabolic Products and Biochemical Testing

20. Carbohydrate Fermentation Test 溴甲酚紫【pH 5(黄)－pH7(紫)】

21. Methyl Red (MR) Test

22. Voges-Proskauer (VP) Test

23. Indole Test

24. Citrate utilization test

25. I M Vi C test E.coli + + - - E.Aerogenes - - + +

26. HydrogenSulfide (H2S) Production sulfur amino acid sulfurinorganic compound Pb2+ PbS H2S （black ） FeS Fe2+

27. Urease Test

28. 5. The physical effect on bacteria and commonly used sterilizers 1) Physical factor: dry、moist、ultraviolet、ultrasonic wave2) Commonly used sterilizers: autoclave dry-air oven bacteriological filters

29. Experiment manipulation 1.Continuous streak method or quadrant streak method strain:S.aureus or E.coli one piece of plate/person 2. Observe phenomena of bacterial growth on agar plate in liquid medium in semi-solid medium

30. Experiment report 1. Continuous streak method or quadrant streak method: principle procedure result: describe the characteristics of colony analysis 2. Draw phenomena of bacterial growth