Control of microbial growth:
Chemical and Physical methods
1
Vocabulary
• 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.
2
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.
3
How do antimicrobial agents work?
4
• 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
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
Some fungi hard to kill, others easier
• Parallels levels of disinfectants:
– High level, intermediate level, low level
5
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!
6
Disinfectants 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.
7
8
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
phenol
– 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
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
9
Disinfectants -3
10
• 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)
Chemical structures
Na+ OCl- sodium hypochlorite
Ethylene oxide
Soap
Na salt of a fatty acid
Benzalkonium chloride (quat)
11
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”
12
More on temperature
13
• 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.
Physical methods-2
14
• 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;
Physical methods-3
15
• 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