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
2. Gram Staining
⑴ Gram Staining : Staining technique developed by Gram
⑵ Gram Staining : Crystal violet → Safranine O
① 1st. Heat bacterial cells on a slide glass using an alcohol lamp for 2-3 times for fixation
② 2nd. Sprinkle Crystal Violet stain reagent on the sample with Crystal Violet, let it sit for 20 seconds
○ All cells are violet at this point
○ Crystal Violet, being a positively charged basic dye, binds strongly to the teichoic acid of Gram-positive bacteria’s peptidoglycan
③ 3rd. Wash off the stain with distilled water in a tube for a short time and remove excess water
④ 4th. Sprinkle Iodine solution (I2-KI) on the sample with Iodine, let it sit for 1 minute
○ Iodine forms a complex with crystal violet preventing decolorization (mordant function)
⑤ 5th. Dip the slide in a tube with 95% EtOH and remove it, washing off the Iodine solution
○ Only Gram-positive bacteria retain the violet color
○ In the case of Gram-positive bacteria, alcohol contracts the cell wall, trapping I2-crystal violet within the cytoplasm
⑥ 6th. Dip the slide gently in a tube with distilled water to stop the decolorization
⑦ 7th. Sprinkle Safranine on the sample, let it sit for 1 minute
○ Gram-positive bacteria maintain the violet color
○ Gram-negative bacteria appear red due to Safranine
⑧ 8th. Rinse gently with water for a few seconds, then remove excess water
⑶ Gram-positive bacteria
① Structure : Cytoplasm - Cell membrane - Thick peptidoglycan layer
② Peptidoglycan : Alternating N-acetylglucosamine (N-AG) and N-acetylmuramic acid (N-AM) with β 1→4 linkage
③ Form endospores for high-temperature and high-pressure resistance
④ Teichoic acid protrudes from peptidoglycan and is connected by covalent bonding, observed only in Gram-positive
○ Teichoic acid contains glycotamine and carries a negative charge
⑤ Examples : Bacillus subtilis, Bacillus anthracis, Staphylococcus
⑷ Gram-negative bacteria
① Structure : Cytoplasm - Cell membrane - Periplasmic space - Thin peptidoglycan - Periplasmic space - LPS, Outer membrane
② LPS (lipopolysaccharide) : Toxic and an immune system target
○ Toxicity of botulinum toxin is derived from LPS
○ Penicillin cannot pass through LPS
○ Blood clotting, fever
③ Outer membrane : More permeable than cell membrane due to porins
④ Periplasmic space
○ Separates peptidoglycan layer, cell membrane, and outer membrane
○ Important cellular space for metabolism and material 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. Different concentrations of antibiotics added to microorganisms with the same CFU
② 2nd. MIC determination : Determine the lowest antibiotic concentration that doesn’t cause visual turbidity after overnight incubation
③ 3rd. MBC determination : Determine the lowest antibiotic concentration at which no colonies appear after subculture on a nutrient medium
④ Limited by using optical density
⑶ Disc Diffusion Method
① Measure the diameter of the inhibition zone caused by the drug
⑷ Epsilometer Test
① Measure MIC directly and conveniently by establishing a gradient of antibiotic concentrations within a single plate
② Cannot measure MBC
⑸ SCMA (Single-Cell Morphological Analysis)
① Problem : Highly dependent on initial bacterial concentration when measuring MIC in blood samples
② MIC can be easily determined using an optical microscope
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 reversion 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 Process
① Prepare mutant strains of S. typhimurium for each strain
③ Culture on minimal medium agar and spread them
④ Place discs with the following on the agar’s center
○ Water: Negative control
○ Enzyme: Negative control
○ Chemical substance: Experimental group
○ Water + Enzyme: Negative control
○ Chemical substance + Enzyme: If colony count is similar to the experiment group treated solely with the chemical substance, then the chemical substance is a mutagen on its own
④ Cultivate at 37°C for 16 hours
⑤ If colonies appear, it indicates no nutritional requirements and reversion mutations occurred
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 Intermediate substances of proline biosynthesis that mutants unable to synthesize proline accumulate within the cell
③ Accumulated proline biosynthesis intermediate substances diffuse through a single medium
⑶ Results
Figure. 3. Experiment under normal conditions
Figure. 4. Experiment for temperature dependency assessment
⑷ Interpretation
① Interpretation of the 22°C experiment
○ Substance C acts later than substance A : ?? → Substance A → Substance C, Strain A survives because enzyme c is present
○ Strain B has nutritional requirements
② Interpretation of the 30°C experiment
○ Strain B has nutritional requirements
○ Strain C has nutritional requirements
○ According to premise ②, strain C doesn’t accumulate substances, hence there’s no substance transferred to strain A
③ Interpretation of the 42°C experiment
○ Strain B has nutritional requirements : Strain B is temperature-sensitive
○ Substance A acts later than substance B : ?? → Substance B → Substance A, Strain B survives because enzyme a is present
○ Strain C has nutritional requirements
⑸ Interpretation Know-How
① The more delayed the enzyme’s action, the more serious the mutation
② Experiment at 22°C shows Substance A → Substance C
③ Experiment at 42°C shows Substance B → Substance C
6. Interrupted Mating Experiment **: Applied in genetic mapping
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+
Input: 2019.03.17 16:28