22 –Bacteriostatic Compounds, and Antibiotic Resistance Dr. Souki Principles & Applications of Microbiology Cerritos College

The Inhibitory Action of Antiseptics and Disinfectants

Purpose To determine the effects of known disinfectants and antiseptics on bacterial growth.

FDA-Approved Antiseptics Tincture of Iodine 2% free iodine Povidone-iodine Betadine E-Z Scrub Silver sulfadiazine Creams, wound dressing

Common Disinfectants Bleach Ethanol or isopropanol 1:10 dilution household bleach 5250-6150 ppm hypochlorite ion Biocidal on most vegetative bacteria in seconds Inactivates most endospores in under 10 minutes Ethanol or isopropanol 70% Biocidal Does not inactivate endospores

Procedure Prepare nutrient agar plates Staphylococcus aureus Escherichia coli Mark quadrants on plate bottom Use sterile swab to inoculate Heat-sterilize forceps Dip disk in solution Solution 1:______ Solution 2: ______ Solution 3: ______ Solution 4: ______ Drop disk in center of quadrant Incubate RT, 48 hours Measure zone of inhibition

How is a zone of inhibition measured? Measure the diameter of the disk Measure the diameter of the zone of inhibition

Diameters of Zones of Inhibition (mm) Results Organism Diameters of Zones of Inhibition (mm) Solution 1 Solution 2 Solution 3 Solution 4 Staphylococcus aureus Escherichia coli

The Effects of UV Light on Bacterial Growth

Purpose To determine the effects of UV light on bacterial growth.

What is Ultraviolet Radiation UV penetrates cells and damages DNA

Procedure Work as a table. Obtain 1 NA plate. Prepare 1 plates of either Bacillus megaterium Escherichia coli Inoculate using sterile swab Expose half the plate, using index card, with lid open to UV light for prescribed amount time Incubate 37 C, 24 hours Count colonies

Results Table UV Exposure (sec) # Colonies 1 15 3 30 5 60 7 90 Table B. megaterium Table UV Exposure (sec) # Colonies 1 15 3 30 5 60 7 90 E. coli Table UV Exposure (sec) # Colonies 2 15 4 30 6 60 8 90

Detection of Antibiotic Resistance

Purpose To determine the if various bacterial cultures are antibiotic resistant, using the Kirby-Bauer disk-diffusion method.

Kirby-Bauer Disk Diffusion Method Antibiotic Bactericidal or bacteriostatic Penicillium (fungus) First source of penicillin Alexander Fleming Beta-lactam antibiotic Resistance due to beta-lactamases Inhibits cell wall synthesis in G+ & G-

Procedure Work in pairs. Obtain 2 NA plate. Prepare 2 plates Escherichia coli Bacillus megaterium Proteus vulgaris Staphylococcus aureus Pseudomonas aeruginosa Inoculate using sterile swab Apply antibiotic discs Incubate 37 C, 24 hours Measure zone of inhibition

Results Collect class data. P. vulgaris S. aureus E. coli B. megaterium P C Te S Data set 1 Data set 2 Ave Zone (mm) Susceptibility Key: P = Penicillin 10 ug C = Chloramphenicol 30 ug Te = Tetracycline 30 ug S = Streptomycin 10 ug Susceptibility: S = Susceptible I = Intermediate R = Resistant

Procedure Use the data you collected to determine if the bacteria are: Resistant Intermediate Susceptible