1. Lab #10

2. 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

3. 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

4. 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.)

5. 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!

6. 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.

7. 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?

8. 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

9. 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?

10. 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

11. 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

12. 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