Chapter 7 Control of Microbial Growth Sterilization Disinfection

Chapter 7 Control of Microbial Growth Sterilization & Disinfection

Sterility n Sterilization - destruction of ALL life. n Commercial Sterilization - enough to kill C. botulinum spores. n Disinfection - kills all pathogens. n Antisepsis - destruction of pathogens in living tissue. n Degerming- removal of microbes from a limited area. n Sanitization - treatment to lower bacterial load.

Sterility • Bacteriostatic - inhibits bacterial growth (but doesn’t kill). • Bacteriocidal - kills bacteria. • Germicide - kills microbes (broader group). • Viricide - inactivates viruses. • Fungicide - kills fungus. • Sporocide - kills bacterial and/or fungal spores.

Disinfectants • Phenol Coefficient - the effectiveness of a disinfectant compared to phenol. { Disinfectant introduced by Joseph Lister} • A value > 1. 0 means it’s more effective. • A value of < 1. 0 means it’s less effective. • Testing Disinfectants • in solution - dilution method • in filter paper - disc diffusion method.

Disinfectants Dilution Zone of Inhibition Disc Diffusion

Disinfectants • • • Effects on potency • • Number of Microbes (Heavier load takes longer) Time (Slower agents take longer. ) Temp & p. H - (warmer works faster) Concentration - (higher works better- usually) Environment - (Organic matter may reduce effectiveness). Disinfectant Stability - (stable works longer) Microbial Characteristics (Capsules, OM, waxy cell walls, spore coats. . )

Disinfectants 7 Classes • Phenols • Halogens • Alcohols and Solvents • Heavy Metals • Quaternary Ammonium Salts • Aldehydes • Peroxygen

Disinfectants • Phenol • • • damage membranes stable, persistent active in the presence of organics o-phenyl phenol – Lysol Bisphenols • hexachlorophene

Disinfectants • Halogens - oxidizers • bleach – HOCl • iodine • tincture, Betadine • chlorine agents not very stable. • React with aromatic amino acids (denature proteins). Cl 2 +H 2 O < === > H+ +Cl- + HOCl < = > H+ + OCl. Iodophors - combination of iodine with an organic molecule usually a detergent. Doesn’t stain, less irritating and slow release for long lasting effect.

Disinfectants • Alcohols and Solvents • lipid solvents • alcohol (70%); acetone • detergents • slow required long exposure • organics diminish activity • membrane disruption

Disinfectants • Heavy metals • silver • Mercury – mercurichrome • copper • Oligodynamic action • interacts with sulfhydryl groups on proteins • Salvarsan- arsenic – used to treat syphilis and early 1900 s.

Disinfectants • Quaternary ammonium salts • positively charged – cleansing ability. • Damages membranes. • Potent against gram-positive organisms, fungi, amoeba, and envelope viruses. • not effective against spores or mycobacteria and

Disinfectants • Aldehydes - alkylating agents. • glutaraldehyde better than formaldehyde. • Cross-links proteins (teenagers). • 2% solution is bactericidal, viricidal, tuberculocidal in 10 minutes • sporicidal in three hours.

Disinfectants • Peroxygens • O zone O. • hydrogen peroxide H O. • Oxidizes membrane lipids, proteins and DNA. • Not very stable. • Benzyl peroxide – aromatic ring – OOH 3 2 2

Disinfectants • Peroxygens • Ethylene oxide – propylene oxide. How collates proteins • kills all bacteria and spores but takes 4 to 18 hours. • Used to sterilize plastics (plates, tubing, etc. ). • Suspected carcinogens.

Disinfectants y d u t S • Spores & Mycobacteria most resistant • Enveloped viruses the least. (HIV) • Gram + easier to destroy than most Gram –

Disinfectants • Killing curve • the steeper the line the faster the killing. • Tinctures contain alcohol. • tincture of iodine – best • soap and water – least

Sterilization • Heat • T D P – lowest T˚C to kill all in 10 minutes. • T D T - time to kill at a specific temperature. • Wet heat – 121°C, 15 psi, 15 minutes. • Dry heat – 170°C for two hours.

Sterilization • Autoclave • free-flowing steam – 121°C. • 15 psi • 15 – 20 minutes • large volumes require longer time • 10 L carboy – one hour. • Cannot use for heat sensitive chemicals like vitamins, antibodies, serum components, antibodies, etc.

Pasteurization • Not sterile. • Originally to kill Brucella abortus at 65°C for 30 minutes. • today use 72°C for 15 seconds – HTST.

Sterilization • Hot air ovens • 170 180–°C – two hours. • Flaming (wire loop) • Flaming with alcohol • glass or large metal objects But NOT Customers !

Sterilization • Filtration • filters with 0. 45 µ or 0. 22 µ pores. • Filter vitamins, protein solutions, serum, any heat sensitive chemicals!

Sterilization • Bacteria trapped on membrane. • Can place on agar plate and they will grow.

Sterilization • HEPA - High efficiency particulate air filters. • Sterilization of air. Biological Safety Cabinet (hood)

Sterilization • Radiation • ionizing – gamma rays, x-rays. • Non-ionizing – UV light, microwaves.

Thymine Dimers • Thymine dimers are formed from 2 adjacent thymine bases activated by UV light. • Causes bulge in DNA backbone & mutations.

Preservation • Refrigeration and freezing. • Low temperatures slows or stops growth; usually does not kill. 4˚C -20˚C

Preservation • Desiccation– drying or freeze drying.

Preservation • Canning - greatly reduces bacterial load by heat. • Stored in anaerobic conditions – prevents oxidation.

Preservation • Osmotic pressure – salt, high sugar. Salt pork Jellies & Jams

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