Experiment 3 Culture of Microbe & Microbial

Antibiotic Susceptibility Test

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 ）

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.

• 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 .

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.

Colonies of bacterial L-form

(‘fried egg’ appearance)

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.

• [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.

Zones of growth inhibition

Microbial Antibiotic Susceptibility Test （ The agar diffusion test ）

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

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

[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.

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

[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

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。

• 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）

Experiment report