Biology 216 - Lect 38 - Disinfection/Sterilization)
Principles, uses, limitations. BRING Amphyl bottle
I. Definitions and basics
A. Selective toxicity - not applicable here. Agents go on surface, 
	not inside, Ex. Amphyl not orally.
B. Sterilization - destruction or removal of all life. 
	Ex. flame loop, autoclave, harmful products behind (LPS)
	"Pyrogen free"
C. Disinfection - Destruction or removal of pathogens. 
	Ex. Pasteurization.
D. Antiseptic - disinfection of skin surface
	 (topical application of chemical - Bactine, alcohol...)
E. Rate of killing exponential instead of arithmetic
	(Log viable organisms drop with time)
	Follows single hit kinetics. 
	Rate independent of original concentration.
	Graph Conc vs time. Show two different initial 
		concentrations. Use 90 % rate.
	Salk polio vaccine improperly killed.
	What affects rate: Disinfection, organism, 
		concentration of agent, temp, medium (organic)
II. Physical methods
A. Heat - most reliable (not always practical). Rate is time and
	 temp dependent (few degrees critical)
	Ex. TB killed: 58 C/30min; 59 C/20 min; 65 C/2 min
	Vegetative cells not major concern. Spores dictate the rules.
	1. Moist heat - most effective: MOA - coagulate 
		and denature protein, melt DNA and lipids
		a. Autoclave - 121 C/20 min - longer if larger volumes.
		Pressure cooker to prevent evaporation (15 lbs)
		Uses - Stable liquids (canning, surgical eqpt, media)
		QC - B. stearothermophilus 
		b. Tyndallization - successive heating/cooling to 
		destroy vegetative cells, encourage germination
		c. Pasteurization - not sterilization; 
		62 C/30 min, 72 C/15 sec. 
		Preserve quality of product/killpathogen
		TB, Q-fever, Salmonella, Brucella, Campy, Strept. 
		Sporeformers survive -> spoilage.
		Applications: Milk, wine, beer
	2. Dry heat - Less effective. 180 C/2 hr. Denature protein.... 
		Uses: glassware, pipettes, metals.
B. Radiation - energy waves determine effect
	1. uv light - 240-280 optimal (max abs for DNA) - 
		differentiate short vs long wavelength
		a. MOA - Molecular excitation -> covalent bond pyrimidine bases 
		(Thymine and Cytosine) and aromatic AA
		b. Repair - Photoreactivation (photolyase - light repair) 
		- hydrolysis of dimer.
		Dark repair - several enzymes cut out and replace damage.
		c. Uses - surface only. decrease airborne infection in hosp rooms.
	2. Ionizing radiation - x-ray/gamma rays - 
		much higher energy; penetration.
		a. MOA - cause ionization, highly reactive radicals from water, 
		break sugar/P DNA backbone, protein.
		b. Uses - Surgical eqpt (gloves, sutures....), 
		foods (Package, then irradiate; require label)
C. Filtration - for labile liquids (antibiotics, 
		protein products, beer, pharmaceuticals)
	Common pore size: 0.22 - 0.45 um - exclude bacteria 
		by size and charge. Virus pass.
	Uses: Sterilize labile liquids; 
	Concentrate bacteria (ex. coliform/Legionella)
III. Chemical disinfectants
A. Test for efficiency - Phenol coefficient 
	(comparison with phenol standard)
	Standardize test condition: temp, 
		organism (S. aureus, S. typhi, Ps aer)
	Determine dilution which kills at 10 min/ but not at 5 min.
	Ex. Phenol 1:10 kills in 10 min/ but not 5 min
	Test disinf 1:30 kills in 10 min/ but not 5 min
	i.e. phenol coefficient = 30/10 = 3; Show Amphyl bottle.
B. Cell membrane acting (some have multiple sites) - > 
		structural disorganization -> leakage
	1. Surface acting agents (surfactants/detergents) -
		 fat soluble polar compound (philic & phobic)
		MOA - intercalate between lipids in membrane
		a. Quaternary ammonium compounds (quats) - NH3-(CH2)x
			Low human toxicity; Less effective vs virus and spores
			Activity decreased by organic material
			Ex. Zephiran, Phemerol
		b. Anionic detergents - SDS (PLAX), soaps
	2. Phenols & cresols - activity enhanced by side chains
		 (increase activity , decrease toxicity)
		Semmilweis noted: physicians and students did autopsies, 
		then attended patients -> puerperal sepsis (GpA)
		Midwives had less problems with sepsis (since no autopsies)
		Joseph Lister - Used phenol to sterilize surgical room.
		a. Lysol (meta cresol) -  
		b. Bisphenols (diphenol linked by S or CH2 - 
		Ex. Hexachlorophene/Phisohex)
		Phisohex used in 1950s in high concentration to control 
			Staph in hospital premature babies (dip in it).
			Neurotoxic -> learning disabled
			Now prescription
	3. Alcohols - not real effective. 70 % ethanol>95 %
C. Protein acting - Alter tertiary configuration, 
	alter functional group (active site)
	1. Denaturation - acids/bases. Benzoic acid preservative.
	2. Alter functional groups (SH at active site)
		a. Heavy metals - Bind sulfhydryls
			Ex. Mercury (toxic) - combine with organic -> 
			useful less toxic form (mercurichrome)
			Copper sulfate - fungicide
		b. Halogens (Chlorine, Iodine) - oxidize SH
			i. Iodine 
			Tincture of iodine (2 % iodine in 50 % ethanol);
			 Iodophores (iodine in detergent)
			Hydroperiodide - highly soluble, stable,
			Giardia cysts affected.
			ii. Chlorine - affected by organic load
			Sewage and water treatment
		c. H2O2 - oxidant
	3. Aldehydes - irreversibly replace H of NH3, OH, or SH
		Formaldehyde - 0.1 % used to treat toxins ->
		 toxoid or virus inactivation. 
		Glutaraldehyde - dialdehyde - used to 
			conjugate/cross-link proteins (ELISA reagents)
	4. Ethylene oxide - Used as gas for sterilization of 
		heat labile solid materials (plastic...)
		90 % CO2 included so non-explosive. 
		Stuff placed in chamber 4 - 6 hours - 
		Allow time for diffusion out
	--> epoxy ring: H2C---------CH2
					O