1. Control of Microbial Growth Tim Ho University of Alberta, Canada * The materials are mostly based on Dr. Brian Lanoil’s Microb 265 tim3@ualberta.ca 1 Part I

2. Objectives 1.Know 3 methods of microbial control 2.Know the strategies on how drugs control the growth of microorganisms. 3.Understand how do bacteria become resistant to antibiotics. tim3@ualberta.ca 2 Physical agents Mechanical removal methods Chemical agents

3. tim3@ualberta.ca 3 Terminology of Microbial Control Sterilization: -The complete destruction or removal of all viable organisms, except bacteria endospores, from an environment. - Autoclave or high energy ionizing radiation (x ray, gamma) Disinfection: - Killing, inhibition, or removal of viable microorganisms from inanimate objects. - Phenolics, boiling water, UV irradiation Sanitization: - Reduction of microbial population to a safe level. - Soaps Antisepsis: - Prevention of infection of host tissue. - Iodine, ethanol

4. 6 Factors that Affect Antimicrobial Activities 1.Population size: larger populations require longer time to kill 2.Population composition: different microorganisms have different range of sensitivity to antimicrobial agents G- bacteria vs. G+ bacteria 3.Concentration of antimicrobial agent: higher concentrations of agents have greater efficient in killing microorganisms tim3@ualberta.ca 4 4.Exposure (time): longer exposure lead more organisms get kill 5.Temperature: Higher temperature usually increases effectiveness in killing 6.Environment: Environmental factors play an important role in affecting effectiveness of antimicrobial agents pH, humidity, concentration of organic matter

5. tim3@ualberta.ca 5 D value - Time at which kill 90% of the original population - Applies to all methods Z value - Increase in temperature needed to reduce the original D value by one log (90%) 20 Decimal reduction time (D) Z value of (i) = 89-75, or 14 degree Z value of (ii) = 95-85, or 10 degree

6. tim3@ualberta.ca 6 3 Methods of Microbial Control 1.PHYSICAL AGENTS Heat Dry Incineration Dry oven Moist Autoclaving Boilling water Pasteurization Tyndalization The temperature of 100 °C is effective against vegetative cells and eukaryotic spores - 15 sec. at 71°C - Used for heat-sensitive materials eg. Wine, and milk - Not effective against gram positive pathogens due to endospores - 15 min. at 2 atm at 121°C - Effective against vegetative and endospore cell - Serilize glassware - Twice boiling - First boiling: kill vegetative cells, but not spores - Remaining spores germinate to form new vegetative cells after incubation - Second boiling: kill remaining vegetative cells -> nearly sterilized Moist heat is more effective than dry heat

7. tim3@ualberta.ca 7 3 Methods of Microbial Control 1.PHYSICAL AGENTS Radiation X ray, cathode gamma UV - Sterilization - Produce oxygen radicals that react with DNA - Disinfection (surface sterilization only) - Thiamine dimer formation - Works best near 260 nm to destroy pyrimidine bases Corrin, N. Damage to DNA from Toxins. Washngton: Nicholas Corrin. 2012 Jul. [Cited 2013 Dec 22]. Available from: http://radiation-remedies.com/inflammation-and-double-break-dna-rupture/why2 /

8. tim3@ualberta.ca 8 2. MECHANICAL REMOVAL AGENTS Filtration Liquids Air High-efficiency particulate air (HEPA) filters 3 Methods of Microbial Control - Remove (but not kill) microorganisms from serum containing media - Membrane filters can remove most bacteria but not viruses - Remove microorganisms in laminar flow biological safety cabinets and clean rooms Figure. HEPA filter eliminates over 99% of microorgansims and allergens. Image: CleanCraft Products, Inc.

9. 3 Methods of Microbial Control 3. Chemical agents GasesLiquids Animate Chemotherapy 1 Antisepsis Inanimate Disinfection Sterilization eg. Ethylene oxide, Vapor-phase hydrogen peroxide - Antibiotics - Antifungals - Antivirals 1 More information about chemotherapy can be found in Control of Microbial Growth – Part II. To kill or inhibit microorganisms within host tissue tim3@ualberta.ca 9

10. 3. Chemical agents Liquids Animate Chemotherapy Antisepsis Inanimate Disinfection Sterilization tim3@ualberta.ca 10 Phenolics - denature parteins - target sites: cell wall and membrane - used as disinfectants Alcohols - denature proteins; do not kill endospores - target sites: membrane - used as disinfecants and antiseptics Heavy metals (mercury, silver) - inactivate proteins - target sites: ribosomes - used as antiseptics Halogens (Cl 2 and I 2 ) - oxidize cell constituents - target sites: ribosomes - used as disinfectants and antiseptics