Chapter 38. Microbiology Experiment
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3. Antibiotic Susceptibility Test
4. Ames Test
6. Interrupted Mating Technique
a. Microbiological Testing and Genetic Monitoring
1. Streak Plate Method (streak plate)
⑴ Definition: Method used for isolating a single colony
⑵ Procedure
Figure. 1. Streak Plate Method Procedure
① 1st. Flame to sterilize inoculation loop.
② 2nd. Collect bacteria onto loop.
③ 3rd. Inoculate plate.
④ 4th. Inoculation procedure (flame loop between streaks)
⑤ 5th. Formation of discrete colonies
2. Gram Staining
⑴ Gram Staining: Staining technique developed by Gram
⑵ Gram Staining: Crystal violet → Safranin O
① 1st. Heat-fix the bacteria to the slide glass by passing it 2–3 times through an alcohol lamp flame.
② 2nd. Flood the smeared sample with crystal violet stain and leave for 20 seconds.
○ At this point, all cells appear purple.
○ Because crystal violet is a basic dye carrying a (+) charge, it binds strongly to the teichoic acids of Gram-positive bacteria.
③ 3rd. Briefly rinse the stain off in a tube filled with distilled water and remove any remaining moisture.
④ 4th. Flood the smear with iodine solution (I2–KI) and leave for 1 minute.
○ Iodine forms a crystal violet–iodine complex, preventing color change and decolorization (acts as a mordant agent).
⑤ 5th. Dip the slide in a tube containing 95% EtOH to wash off the iodine solution, moving it in and out.
○ Only Gram-positive bacteria remain purple.
○ In Gram-positive bacteria, alcohol shrinks the cell wall, trapping the I2–crystal violet complex within the cytoplasm.
⑥ 6th. Gently rinse the slide by dipping it in distilled water to stop decolorization.
⑦ 7th. Flood the smear with safranin and leave for 1 minute.
○ Gram-positive bacteria remain purple.
○ Gram-negative bacteria take up safranin and appear red.
⑧ 8th. Rinse carefully with water for a few seconds, then remove moisture.
⑶ Gram-positive bacteria
① Structure: protoplasm → cell membrane → thick peptidoglycan layer.
② Peptidoglycan: alternating N-acetylglucosamine (N-AG) and N-acetylmuramic acid (N-AM) linked by β 1→4 bonds.
③ Form endospores, enabling resistance to high temperature and pressure.
④ Teichoic acids protruding beyond the peptidoglycan are covalently linked and observed only in Gram-positives.
○ Teichoic acids contain glutamic acid and thus carry a negative charge.
⑤ Examples: Bacillus subtilis, Bacillus anthracis, Staphylococcus.
⑷ Gram-negative bacteria
① Structure: protoplasm → cell membrane → periplasmic space → thin peptidoglycan → periplasmic space → LPS, outer membrane.
② LPS (lipopolysaccharide): toxic and a target of the immune system.
○ The toxicity of botox is also attributed to LPS.
○ Penicillin cannot pass through LPS.
○ Causes blood coagulation and high fever.
③ Outer membrane: contains porins and is more permeable than the cell membrane.
④ Periplasmic space:
○ The compartment separating the peptidoglycan layer from the cell membrane and outer membrane.
○ A cellular space important for functions such as metabolism and transport.
⑤ Examples: Escherichia coli, Helicobacter pylori.
3. Antibiotic Susceptibility Test (AST)
⑴ Overview
① CFU (colony forming unit): Number of colonies formed by bacteria or fungal spores in a sample
○ Formula can be used if necessary: Sample’s CFU = CFU of diluted sample × dilution factor
② MIC (minimum inhibitory concentration): Minimum concentration of an antibiotic that inhibits microbial growth
③ MBC (minimum bactericidal concentration): Minimum concentration at which no microorganisms appear even after subculture on a nutrient medium
○ Essentially, the minimum concentration at which bacteria are killed
○ MBC ≤ MIC
④ Can provide information about the bacteria’s identity
⑵ Broth dilution test (broth microdilution method)
① 1st. Prepare varying antibiotic concentrations, then add microbes at the same CFU.
② 2nd. MIC determination: after overnight incubation, determine the lowest antibiotic concentration at which no visible turbidity appears.
③ 3rd. MBC determination: subculture each sample onto nutrient medium and determine the lowest antibiotic concentration that yields no colonies.
④ A limitation is that it relies on optical density.
⑶ Disc diffusion method
① Measure the diameter of the inhibition zone produced by the drug.
② Follow the EUCAST guidelines.
⑷ Epsilometer test (E-test)
① By generating a gradient of antibiotic concentrations on a single plate, the MIC can be measured directly and conveniently.
② The MBC cannot be measured.
⑸ SCMA (single-cell morphological analysis)
① Motivation: when determining the MIC from blood samples, the result strongly depends on the initial bacterial concentration.
② MIC can be determined easily using optical microscopy.
4. Ames Test
⑴ Overview
① Genetic toxicity assessment method developed by Dr. Bruce Ames’ team at the University of California, Berkeley
② Used to identify mutagenic and carcinogenic agents: Shows a strong correlation with carcinogenicity test results; quick and simple
③ Commonly used for screening before phase 1 of drug development
④ Verify if back mutation occurs using auxotrophic mutations
○ Reversion mutation: Requires the same mutation mechanism, typically a point mutation
⑤ Uses Salmonella typhimurium sensitive to mutagens
⑥ Colonies decrease when using strains with normal DNA repair functions
⑦ Commonly used test strains
○ Salmonella typhimurium TA98
○ Salmonella typhimurium TA100
○ Salmonella typhimurium TA1535
○ Salmonella typhimurium TA1537
○ Escherichia coli WP2 uvrA (pKM101)
⑵ Experimental procedure
① Prepare S. typhimurium mutants by strain.
② Prepare solid minimal-medium agar plates and spread the strains uniformly.
③ Place, at the center of each plate, a disk impregnated with one of the following:
○ Water: negative control
○ Liver enzymes: negative control
○ Chemical: experimental group
○ Water + liver enzymes: negative control
○ Chemical + liver enzymes: If the colony count is similar to that of the chemical-only group, the chemical is a direct mutagen, i.e., it does not require metabolic activation in the pathway to become mutagenic.
④ Incubate at 37 °C for 16 hours.
⑤ If colonies appear, they are revertants that have lost the auxotrophic requirement due to a reverse mutation.
Figure 2. Example of Ames Test Results
5. Cross-Feeding Experiment
⑴ Overview: Experiment to determine the sequence of metabolic actions
⑵ Premises
① Mutant A has a mutation in gene a, B has a mutation in gene b, C has a mutation in gene c
○ For convenience, let’s denote the substance produced by gene x as X
② In mutants unable to synthesize proline, the intermediates of proline biosynthesis accumulate inside the cell.
③ The accumulated proline-biosynthesis intermediates diffuse through the agar medium.
⑶ Results
① 22 °C: A—only some strains grow well; B—all strains grow well; C—no strains grow.
② 30 °C: A—no strains grow; B and C—all strains grow well.
③ 42 °C: A—no strains grow; B—only some strains grow well; C—all strains grow well.
Figure 3. Experiment under normal conditions
Figure 4. Experiment for temperature dependency assessment
⑷ Interpretation
① Analysis at 22 °C
○ Substance C acts later than substance A: ?? → A → C; the A strain can survive because it has enzyme c.
○ The B strain is prototrophic (non-auxotrophic).
② At 30 °C
○ The B strain is prototrophic.
○ The C strain is prototrophic.
○ Given point (②) above, the C strain does not accumulate the substance; therefore nothing is transferred to the A strain.
③ At 42 °C
○ The B strain is auxotrophic; it is a temperature-sensitive mutant.
○ Substance A acts later than substance B: ?? → B → A; the B strain can survive because it has enzyme a.
○ The C strain is prototrophic.
⑸ Tips for interpretation
① The later an enzyme acts in the pathway, the more severe the mutation.
② From the 22 °C experiment, we infer A → C.
③ From the 42 °C experiment, we infer B → C.
**6. Interrupted Mating Experiment
Figure 5. Interrupted Mating Experiment Process
Figure 6. Interrupted Mating Experiment Results
⑴ 1st. Hfr strain has strs leu+ thr+ azir tonr lac+ gal+
① Conditions: Antibiotic sensitivity, nutritional auxotrophy
⑵ 2nd. F- strain has strr leu- thr- azis tons lac- gal-
① Conditions: Antibiotic resistance, nutritional requirements
⑶ 3rd. Cultured in a medium with streptomycin and without threonine and leucine: Only strains with strr leu+ thr+ survive
⑷ 4th. After 8 minutes of mating, azir survives additionally: Adjacent to strr leu+ thr+, there’s azir
⑸ 5th. After 10 minutes of mating, tons survives additionally: Adjacent to strr leu+ thr+ azir, there’s tons
⑹ 6th. After 16 minutes of mating, lac+ survives additionally: Adjacent to strr leu+ thr+ azir tons, there’s lac+
⑺ 7th. After 25 minutes of mating, gal+ survives additionally: Adjacent to strr leu+ thr+ azir tons lac+, there’s gal+
⑻ In this way, it is applied in making genetic map.
Input: 2019.03.17 16:28