Medical Microbiology Laboratory Techniques

Author: Rao Bangzhong
Compiler:
Publisher:
Publish Date: 2003-01-01
Features: Materials 1. Bacteria: Staphylococcus aureus, E. coli 18-24 hours broth culture. 2. Medium: Ordinary agar plate. 3. Chemical disinfectants: 0.1% mercury chloride, 5% carbolic acid, 2% iodine tincture, 2% mercuric chloride. 4. Others: Sterile filter paper discs (diameter 6 mm), forceps, etc. Methods 1. Using an inoculating loop, pick up several loops of Staphylococcus aureus and E. coli broth culture separately and streak them back and forth on two ordinary agar plates to make the broth spread densely over the surface of the agar plates. 2. Using sterile forceps, pick up several sterile circular filter paper discs and immerse them in mercury chloride, carbolic acid, iodine tincture, and mercuric chloride disinfectants, respectively. Then remove them and place them in the center and around the edges of the agar plates inoculated with bacteria. The distance between the discs should be roughly equal. 3. After culturing at 37°C for 24 hours, observe whether there are inhibitory zones around each disinfectant disc and compare the sizes of the inhibitory zones.
II. Killing Test of Carbolic Acid on Different Bacteria
Different chemical disinfectants have varying abilities to kill bacteria, and the same disinfectant may have inconsistent effects on different bacteria. Generally, bacteria with spores have stronger resistance to disinfectants than those without spores.
Materials 1. Bacteria: E. coli broth culture 18-24 hours, Bacillus subtilis broth culture 18-24 hours. 2. Medium: Broth medium. 3. 2% carbolic acid tubes, normal saline tubes, droppers, etc. Methods 1. Using a dropper, 0.2 mL of E. coli broth culture and add 0.1 mL to a 2% carbolic acid tube and another 0.1 mL to a normal saline tube. Mix well. Repeat the same procedure for Bacillus subtilis broth culture in another carbolic acid and normal saline tube. When adding the broth culture, add it directly into the disinfectant to avoid some of the broth clinging to the tube wall and not coming into contact with the disinfectant, which could lead to incorrect results. 2. After 5, 10, 20, and 40 minutes, take one loop of liquid from each carbolic acid tube inoculated with different bacteria and inoculate it into broth medium. At 40 minutes, take one loop of liquid from the normal saline tube and inoculate it into broth medium. 3. Place all 10 inoculated broth tubes in a 37°C incubator for 24 hours and observe the.
III. Killing Test of Different Concentrations of Alcohol
The bactericidal effect of alcohol is due to alcohol causing the dehydration and denaturation of bacterial body proteins. Alcohols with a concentration higher than 70-80% may rapidly dehydrate and quickly solidify the surface of bacteria, hindering further penetration of alcohol and thus reducing their bactericidal efficacy.
Materials 1. Bacteria: Filter strips (5 mm × 15 mm) coated with dried Staphylococcus aureus. 2. Medium: Broth medium. 3. 30%, 50%, 75%, and 95% alcohol solutions, forceps, etc. Methods 1. Using sterile forceps, pick up filter strips coated with dried Staphylococcus aureus and place them in tubes containing different concentrations of alcohol, with three strips per tube. Place one broth tube as a control. 2. After 5, 15, and 30 minutes, take one filter strip from each alcohol tube and place it in the broth tube. 3. Place all inoculated broth tubes in a 37°C incubator for 24 hours and observe the.
Reflection Questions 1. Why are spores more resistant to physical and chemical factors than vegetative cells? 2. How can you prove that the bacteria in the inhibitory zone are inhibited rather than killed? (Liu Binbo)
Experiment Twelve: Bacterial Drug Sensitivity Test
Various pathogenic bacteria have different sensitivities to chemical drugs and antibiotics. Even different strains of the same bacteria may exhibit varying sensitivities to drugs, and drug-resistant strains may even emerge, indicating drug resistance mutations. Therefore, determining the sensitivity of bacteria to drugs is of great significance for clinical treatment and timely control of infections. Common methods include the disk diffusion method and the tube dilution method.
Objective: To understand the operational methods and judgment criteria for bacterial drug sensitivity tests.
Projects I. Disk Diffusion Method
Materials 1. Bacteria: No. 1 and No. 2 Staphylococcus aureus broth culture 16-18 hours, Shigella broth culture 16-18 hours. 2. Medium: Ordinary agar plates. 3. Drug disks: Penicillin, streptomycin, chloramphenicol, gentamicin disks. 4. Others: Sterile cotton swabs, forceps, alcohol containers, etc. Methods 1. Using a sterile cotton swab, pick up Staphylococcus aureus No. 1 and No. 2 and Shigella broth culture separately and spread them on the agar surface. Each type of bacteria should be spread on one plate (after use, place the contaminated cotton swab in an alcohol container and do not discard carelessly). 2. Using forceps, flame the tips to sterilize them, wait for them to cool, pick up one type of drug disk, and place it on the plate inoculated with bacteria. Then flame the forceps again, pick up another type of drug disk, and place it in another area of the agar plate. Repeat this process for all drug disks, ensuring they are roughly equidistant. 3. Place the prepared drug sensitivity agar plates in a 37°C incubator for 18-24 hours and observe the inhibitory effects of each drug disk. Specifically, observe whether there are inhibitory zones around the drug disks. If there are, measure the diameter of the inhibitory zones on the bottom of the plates with a ruler to determine the drug sensitivity. The inhibitory zone diameters for various antibiotics and their sensitivity standards are shown in Table 12-1.