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Chapter 38. Microbiology Experiment

Recommended articles : 【Biology】 Biology Table of Contents, 【Biology】 Chapter 37. Biology Experiment


1. Streak Plate Method

2. Gram Staining

3. Antibiotic Susceptibility Test

4. Ames Test

5. Cross-Feeding Experiment

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

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

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