Transcript RCC Lab 10 post

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?
0 min
• Positive control?
• Quantify relative abundance of
Uninoc.
5 min
growth in each section: (+), (++),
(+++), or (-)
20 min
10 min
• Effect of temperature?
• Effect of exposure to a particular
15 min
temperature?
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 (+)
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 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
• Neomycin
• Streptomycin
• Erythromycin
• Tetracycline
• Sulfadiazine
• Measure zone of inhibition for each antibiotic
• Use Antibiotic Table on pg. 156 to determine if bacteria is R, I, or S
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