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Experiment 3 Culture of Microbe & Microbial
Antibiotic Susceptibility Test
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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 )
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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.
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• 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 .
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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.
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Colonies of bacterial L-form
(‘fried egg’ appearance)
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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.
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• [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.
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Zones of growth inhibition
Microbial Antibiotic Susceptibility Test ( The agar diffusion test )
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Report-- Microbial Antibiotic Susceptibility Test ( The agar diffusion test ) principle procedure result analysis
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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
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[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.
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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
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[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
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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。
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• 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)
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Experiment report