微生物实验课件：disinfection and sterilization

1、Chen Qingxin Dept of Immunology and Microbiology,Disinfection and sterilization,Contents,1. Killing bacteria by moist heat,3. Antibiotic susceptibility test,2. Ultraviolet radiation on bacteria,Each student should be able to 1. Determine the effectiveness of some disinfectants on bacteria; 2. Apprec

2、iate the scope of antimicrobial activity of selected antibiotics; 3. Perform the method for determination of antibiotic sensitivity.,Learning Objectives,1. Killing bacteria by moist heat,Moist heat can denature protein in bacteria and then destroy the Bacteria.,Principle:,Materials:,E. Coli broth cu

3、ltures and B. Subtilis broth cultures. Agar plate. Marking pen, Alcohol lamp, Inoculating loop.,Heat the broth tubes of E. Coli and B. Subtilis at 100 for 20min;,Procedure,2. Transfer a loopful of E. coli and B. subtilis to the plate, respectively.Spread the culture to the full surface of plate;,3.

4、Invert the plate and incubate it at 37 for 24h;,4. Observe whether bacteria occurs on the plate.,Results,There were no bacteria colonies on the plate.,2. Ultraviolet radiation on bacteria,Ultraviolet radiation kill bacteria by causing DNA damage (forming thymine dimer). UV can not pass through paper

5、 or glass.,Principle:,Materials:,Agar plate. E. Coli broth cultures and B. Subtilis broth cultures. Sterilized cotton stick.,Procedure,Add a loopful of S. aureus or B. subtilis on the plate, spread the culture with a cotton stick.,2. Place the plate half open under UV for 30 min.,3. Invert the plate

6、 and incubate it at 37 for 24h.,4. Observe the results.,Result,There were no bacteria colonies in region being radiated by UV.,2. There were no bacteria colonies in region not being radiated by UV.,3. Antibiotic susceptibility test,Introduction:,Identification of a bacterial isolate from a patient p

7、rovides guidance in the choice of an appropriate antibiotic for treatment Many bacterial species are not uniformly susceptible to a particular anti-bacterial compound This is particularly evident among the Enterobacteriaceae, Staphylococcus spp., and Pseudomonas spp. The wide variation in susceptibi

8、lity and high frequencies of drug resistance among strains in many bacterial species necessitates the determination of levels of resistance or susceptibility as a basis for the selection of the proper antibiotic for chemotherapy,Antimicrobial Susceptibility testing can be down by three ways: Minimum

9、 Inhibitory Concentration (MIC) Disk Diffusion Method Minimum Bactericidal Concentration (MBC),Antibiotic-impregnated paper disks are placed on the agar plate which has been seeded with the bacteria. If the bacterium is susceptible to the antibiotic, its growth will be inhibited and a zone of inhibi

10、tion would be formed around the antibiotic disk. The diameter of the zone of inhibition is proportional to the susceptibility of bacteria to tested antibiotic.,Principle (disk diffusion) :,Materials:,Agar plate, Inoculating loop, Sterilized cotton stick, et al; Broth culture of S. aureus and E. coli

11、; Antibiotic-containing filter paper disks: (1) Penicillin: (destroy the cell wall of G+ bacteria) (2) Tetracyclin: (interfere with the protein synthesis of G+ and G- bacteria) (3) Norfloxacin: (inhibit DNA replication of G+ and G- bacteria) (4) Cefalothin sodium: (interfere with the protein synthes

12、is of G+ and G- bacteria) (5) Amikacin: (interfere with the protein synthesis of G- bacteria),Procedure:,1. Transfer a loopful of culture to the plate and then spread the culture to the full surface of the plate. Label the plate as E. coli or S. aureus.,2. Sterilize you forceps, pick up the paper disk from the tube, put it on the plate and then gently tap it to the surface of plate.,3. Invert the plate and incubate it at 37 for 24h. 4. Measure the diameter of the inhibition zone.,Result:,1. 5 mm: Resistance.,2. 5-10mm: Moderate susceptible.,3. 10-15mm: Intermediate susc