1. Experiment 3 Culture of Microbe & Microbial Antibiotic Susceptibility Test

2. Ⅰ. Culture of Microbe ( Ⅱ ) * Culture of non-routine bacteria ： culture of anaerobic bacteria culture of bacterial L-forms * Microbial Antibiotic Susceptibility Test （ The agar diffusion test ）

3. Culture of anaerobic bacteria --- Anaerobic medium Obligate anaerobic bacteria (spore-forming anaerobic bacteria ;non- spore-forming anaerobic bacteria) can’t grow in free oxygen condition. Beef medium ( biological method ): Beef as a reducing agent absorb the free oxygen, forming an anaerobic enviroment. Anaerobic jar ( physical method ) The O2 in the jar is removed with a high vaculum pump, then the jar is refilled with N2 or CO2 and sealed.

4. Metagallic acid methed ( chemical method ) The chemicals absorb free O2, producing anaerobic environment. 1. Streak anaerobes on a blood agar plate, invert the dish; 2. Put a little powder of pyrogallic acid on a thin layer of cotton which has been stick to a square of glass; 3. Add several drops of 10% NaOH, immediately remove the cover of the plate and place the dish on the square of glass, seal it tightly with wax, then incubate.

5. Culture of bacterial L-forms Bacterial L-form can’t grow in general medium. Bacterial L-form can propagate in nutrient media of an appropriate osmotic pressure. Colonies of bacterial L-form on agar are small and have a characteristic ‘fried egg’ appearance.

6. Colonies of bacterial L-form (‘fried egg’ appearance)

7. Microbial Antibiotic Susceptibility Test （ The agar diffusion test ） Principle: Zones of growth inhibition surrounding each type of disc were correlated with the minimum inhibitory concentrations of each antimicrobial agent. Significant: antibiotic susceptibility testing is often essential in order to determine which antibiotics to use against a specific strain of bacterium.

8. [Procedure] 1. Using your wax marker, divide each plate into fourth to guide your streaking. 2. Streak the S. aureus and E. coli. on the plate ， overlapping the streaks to assure complete coverage of the entire agar surface with inoculum. 3.Touch each disc lightly with sterile forceps to make sure it adheres to the agar surface. 4. Incubate the 2 plates upside-down at 37°C for 24 hors. 5.Using a metric ruler, measure the diameter of the zone of inhibition around each disc on each plate in mm by placing the ruler on the bottom of the plate. 6.Determine whether each organism is susceptible, intermediate, moderately susceptible or resistant to each antibiotics using the standardized table and record your results.

9. Zones of growth inhibition Microbial Antibiotic Susceptibility Test （ The agar diffusion test ）

12. Report-- Microbial Antibiotic Susceptibility Test （ The agar diffusion test ） principle procedure result analysis

13. Bacterial Culture from Finger Skin [Principle] Comparing the number of bacterial colonies before disinfecting finger with those after, you will obtain such conclusion: bacteria exist normally on the skin and the disinfectants have the effect of killing and inhibiting bacteria. [Material] 1.agar plate 2.75% alcohol,2%iodine tincture 3.sterile cotton swab

14. [Procedure] 1.Mark the plate into four sectors with a glass pencil. 2.Student press slightly on two sectors with forefinger, then disinfect the finger with iodine tincture and alcohol, press on the other sectors. 3.Incubate agar plate at 37 ℃, for 18-24 hrs, observe the result.

15. Bacterial Culture from Throat There are lots of normal flora in human body, and they can cause diseases in special conditions. [Material] 1.blood agar plate 2.sterile cotton swab

16. [Procedure] 1. Take the secretion in the throat by sterile cotton swab and isolate bacteria by continuous streak method. 2.The blood agar plate are incubated for 18-24 hours at 37 ℃ 。 3.Observe the Hemolytic crisis of colony on medium,and observe the Morphology of bacteria by gram stain

17. 实验材料： 1. 大肠杆菌和金黄色葡萄球菌斜面各 1 管 / 小组。 2. 琼脂平板 3 个 / 小组，血平板 2 个 / 小组。 3. 抗生素小瓶 1 套 / 小组。 4. 无菌棉拭子 2 个 / 小组。

18. 实验报告内容： 1. 细菌对抗菌药物的敏感试验（本次实验） 2. 皮肤、咽部细菌的培养及咽部细菌的鉴 定（下次实验鉴定后）

19. Experiment report