Lab #10

Microbial Control Control by changing optimal growth environments Temperature, pH, osmotic pressure, oxygen Control by physical methods UV radiation Heat Filtration Control by chemical methods Dyes - GCV Disinfectants and antiseptics Antibiotics

Physical Methods of Control UV Radiation ~260nm wavelength absorbed by nucleic acid  DNA forms thymine dimers  lethal genetic mutations! ~280nm wavelength denatures proteins Weak penetration power Procedure Three agar plates are inoculated with E. coli Plate 1 – lid is replaced by a cut out  exposed to UV for 2 min Plate 2 - lid is replaced by a cut out  exposed to UV for 10 min Plate 3 – lid was kept on the plate  exposed to UV for 2 min

Physical Methods of Control What effect does time of exposure to UV have on bacterial survival? What effect does a paper vs. plastic barrier have on the efficacy of UV treatment? Applications of UV? Biological hoods Sterilizing equipment (hospitals, dental, etc.)

Physical Methods of Control Heat One of the most effective means of killing microbes Wet or dry heat Examples: Fire/incineration Boiling water: 100°C Autoclave: 121°C moist heat at 15psi pressure Pasteurization: 63°C for 30min or ~73°C for 15-30seconds – kills most pathogenic organisms (milk and wine) Most bacteria are killed at temperatures exceeding 50°C (proteins and vital molecules denature) Spores are very hard to kill!

Physical Methods of Control Heat – Procedure: Nutrient broths of E. coli and nutrient broths of B. subtilis are placed in water baths: 50°C, 65°C and 85°C A loopful of culture was aseptically transferred onto a nutrient agar plate from each broth every 5 min for 20min (5 samples) 3 plates total: 50°C, 65°C and 85°C Negative control? Positive control? Quantify relative abundance of growth in each section: (+), (++), (+++), or (-) Effect of temperature? Effect of exposure to a particular temperature? 0 min 5 min 10 min 15 min 20 min Uninoc.

Chemical Methods of Control Gram’s Crystal Violet (GCV) Germicidal for G+ bacteria Used in selective media for G- bacteria Two agar plates are inoculated – E. coli (-) and S. aureus (+)  bacterial lawn Sterile filter paper discs are dipped into different concentrations of GCV dye Paper discs are placed onto agar plates  incubated Measure and compare the zone of inhibition Effect of GCV concentration on growth? G+ vs. G- bacteria?

Chemical Methods of Control Antiseptic vs Disinfectant Antiseptic – antimicrobial agent safe to use on human body/ surfaces Disinfectant – antimicrobial agent effective for sterilizing inanimate objects (not safe for human use!) We can use paper disc to measure the effectiveness of an antimicrobial agent Same method as GCV test  paper discs soaked in antimicrobial agents placed on a seeded plate of E. coli or S. aureus Measure and compare zones of inhibition

Chemical Methods of Control Antiseptic vs Disinfectant Broad spectrum – target more microorganisms Narrow spectrum – target specific microorganism Effect of antimicrobials on G+ vs G- bacteria?

Chemical Methods of Control Antibiotics It is important to determine which antibiotic will work best in controlling the growth of a particular microorganisms Microorganisms can be: Resistant – R Intermediate – I Sensitive – S Test is performed using antibiotic discs coated with the antibiotic Zone of inhibition is measured and compared to a table of known/ standard values Antibiotics can be broad spectrum or narrow spectrum

Chemical Methods of Control Antibiotics Three nutrient agar plates seeded: E. coli, S. aureus, and M. luteus Six antibiotic discs are placed on each plate Penicillin Streptomycin Tetracycline Measure zone of inhibition for each antibiotic Use Antibiotic Table on pg. 156 to determine if bacteria is R, I, or S Neomycin Erythromycin Sulfadiazine

Chemical Methods of Control Skin Cleanliness and Antisepsis Skin is the first barrier of defense Skin becomes broken  portal of entry Counteract the potential for disease  hand washing, antiseptics, gloves, face masks, etc. Three nutrient agar plates were inoculated with samples: Hands not washed Hands washed once Hands washed two times Hands washed three times Swab from area treated with antiseptic for 2 min Scraping from area treated with antiseptic for 4 min Record the # of colonies for each sample