1Control of microbial growth:Chemical and Physical methods

2Vocabulary

• Sterile: devoid of life. Something is either sterile or not.• Disinfect: kill most microbes, especially harmful ones, but

probably not necessarily spores which are resistant.• Disinfectant: chemical used on inanimate objects to kill

microorganisms.• Antiseptic: used to disinfect living tissue; must be gentler.• Bacteriostatic: keeps bacteria from growing.• Bactericidal: kills them. Sometimes subtle differences

between bacteriostatic and bactericidal.• Sanitize: no specific meaning; clean thoroughly so that

harmful bacteria are probably dead.

3Kinetics 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.

4How 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

5Microbes 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• Some fungi hard to kill, others easier

• Parallels levels of disinfectants:– High level, intermediate level, low level

6The 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!

7Disinfectants vary in effectiveness

Microbes vary in susceptibility• Difficult to develop standard tests for disinfectant

effectiveness suitable for all situations• Use-dilution test

– Metal cylinders coated with microbes are placed in different dilutions of disinfectant

– Number of surviving microbes determined.

• In-Use test– Swab surface of object to be disinfected, disinfect, then

swab again. – Determine number of bacteria before and after.

8Disinfectants -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

9Disinfectants -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

10Disinfectants -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; – Salts of benzoic, sorbic, and propionic

• Other stuff– Sulfites (control regrowth in wine), nitrites (botulism or

cancer?), various dyes (selective growth media)

11Chemical structures

Na+ OCl- sodium hypochlorite

Ethylene oxide

Benzalkonium chloride (quat)

SoapNa salt of a fatty acid

12Physical 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”

13More on temperature

• Sterilizing with moist heat: 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.

14Physical 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;

15Physical 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.• High efficiency particulate air = HEPA