Control of microbial growth: Sterilization and disinfectants

1. Control of microbial growth:Sterilization and disinfectants

2. Vocabulary • Numerous terms are defined in the text: be familiar with them. Sterile, disinfection, antiseptic, bacteriostatic, and bactericidal, plus others. • Sterile: devoid of life. Something is either sterile or not. • Disinfect: kill most microbes, especially harmful ones, but probably not spores which are resistant. • Disinfectant: use on inanimate objects like benchtops, etc. • Antiseptic: used to disinfect living tissue; must be gentler. • Bacteriostatic: keeps bacteria from growing. • Bactericidal: kills them. Sometimes subtle differences between bacteriostatic and bactericidal.

3. Kinetics of bacterial death • Bacteria not only grow exponentially, but die that way too. • Factors that affect the rate of death include: temperature, pH, concentration of disinfectant, type of microbe, and presence of organic material. The longer the treatment is applied, the more that are killed. The more microbes there are, the longer it will take.

4. The Ideal Disinfectant • Fast and effective, even in the presence of organic material (like blood, vomit, feces..) • Effective, but non-toxic to humans. • Penetrate materials without damaging them. • Easy to prepare and stable over time. • Inexpensive and easy to apply. • Not stink!

5. How do antimicrobial agents work? • Attack proteins • Oxidize, hydrolyze, or bind to proteins. • Change 3-D structure, usually irreversibly; ruin protein. • Dissolve membranes or damages cell walls • Leaky membranes means vitamins, metabolites, escape • Proton gradient across membrane gone, little ATP made. • Wall destroyed, loss of osmotic protection • Damage to nucleic acids • DNA denatured or broken, cell can’t replicate • RNA molecules in ribosomes affected

6. Microbes vary in susceptibility • Bacterial endospores: hardest of all to kill • Resist high heat, dessiccation, chemicals, radiation • Cysts of protozoa • Resting cells of eukaryotes, similar to endospores • Mycobacteria: waxy cell wall provides resistance • Gram negative bacteria: more resistant than G+ • Viruses: some hard to kill, other easier Disinfectants vary in effectiveness Not all disinfectants work equally well against all microbes. These differences can be measured in different ways.

7. Disinfectants vary in effectiveness • Use-dilution test • Small glass cylinders coated with microbes are placed in different dilutions of disinfectant • Cylinders washed, then put into culture medium • Growth from survivors on cylinders noted. • Disk-diffusion method • Disks containing disinfectants placed on a lawn of bacteria; size of zone of inhibition is measured. • In-Use test: swab a surface in use before and after disinfection, note survivors.

8. Physical methods • Temperature • Cold: slows or prevents growth, may kill slowly • Ultra-cold used to preserve bacteria long term • Heat: denatures enzymes, kills cells • Moist heat • Traditional pasteurization does not sterilize; • Standard protocols, time and temperature e.g. 62.9°/ 30 min • flash pasteurization 71.6° / 15” • Newer UHT sterilizes: 74-140-74° in 5”

9. More on temperature • Sterilizing: boiling, autoclave • Boiling will not necessarily kill endospores • Autoclave is steam heat under pressure, so above boiling • 121°C, 19-21 psi. Very effective. • Compare moist heat and dry heat • Dry heat: 170 deg C, near 350 F, for 2 hours • Water conducts heat much more effectively, sterilizes at lower temperature for shorter time. • Dry heat not useful for liquids! • Incineration (e.g. flaming loop) has its place too.

10. Physical methods-2 • Drying: cells need water. Remove it, and no growth. • Freeze drying: lyophilization; also used to preserve cultures for long term storage. • Osmotic pressure/high salt • Sucks water out of cytoplasm; salted meat, jellies, etc. • Radiation • UV used to sterilize air, surfaces in hospitals, etc. • Ionizing radiation: x-rays (electron beam) and gamma rays; important treatment of plastics, various foods. • Irradiation of meat important tool in food safety. • Microwaves only boil; ultrasonics used to break cells.

11. Physical methods-3 • Filtration • Membrane filtration: thin plastic disks with holes of 0.22 or 0.45 micrometers, separate liquid from bacteria • Used to collect bacteria or sterilize liquids • Solutions of vitamins or proteins can be destroyed by heating • Air filtration: HEPA filters used in hospitals and also homes to help remove dust-borne bacteria, allergens.

12. Disinfectants -1 • Halogens: Cl, I, Br, Fl • Chlorine: Cl2 gas, hypochlorites (bleach), and chloramines (NH2Cl) • I as tincture or as iodophors such as betadine. • Phenols and phenolics • O-phenyl phenol (Lysol), hexachlorophene, triclosan, chlorhexidine • Alcohols: isopropanol, ethanol; best at 70-95% • Good at removing lipids w/ attached bacteria from skin • Weakly attacks proteins, cell membranes phenol

13. Disinfectants -2 • Hydrogen peroxide and ozone • H2O2, stored at 30%, used at 3% • new plasma gas sterilizers; read article • Soaps and detergents: cationic (Quats), anionic • Soaps are alkaline salts of fatty acids, weak • Quaternary ammonium compounds weak but useful. • Mostly wash away microbes or damage membranes. • Heavy metals: Hg, Ag, Se, Cu • Used less; toxic and corrosive. Hg and Ag historically

14. Disinfectants -3 • Alkylating agents • Formaldehyde, glutaraldehyde (effective at high pH) • Ethylene oxide: common, toxic, explosive • Acids and alkalis: often microbiostatic • Acetic and lactic acids in foods; • Benzoate, sorbic acid and propionate • Other stuff • Sulfites (control regrowth in wine), nitrites (botulism or cancer?), various dyes (selective growth media)

15. Chemical structures Na+ OCl- sodium hypochlorite Ethylene oxide SoapNa salt of a fatty acid Benzalkonium chloride (quat)