Microbial Control and Biosafety

Sterilization

• destruction of all forms of life including bacterial spores

• chemical or physical methods

Disinfection

• destruction of some forms of life except bacterial spores

• chemical or physical methods

Disinfectants

applied to inanimate objects

Antiseptic

applied to skin; cannot be used as disinfectants

Type of Organisms

• presents variability to withstand chemical and physical treatment

• biochemical composition and mechanisms to protect themselves

Bacterial Spores

• rich in proteins, lipids, and carbohydrates

• rich in dipicolinic acid and calcium

dipicolinic acid and calcium

bacterial spores are rich in?

lipids

Cell walls of mycobacteria are rich in?

Microbial Load

total number of organisms present

Microbial Load

• most likely composed of organisms with varying degrees of susceptibility to killing agents

• not all the organisms die at the same time

longer exposure times

higher number of organisms require?

Concentration of Disinfecting Agent

• amount of disinfectant needed to destroy microorganisms

• varies with the different agents

Organic material

___ affects killing activity by inactivating the disinfecting agent

blood, mucus, and pus

examples of organic materials are?

Bleach

easily inactivated by organic material

Contact/ Exposure Time

• amount of time a disinfectant or sterilant is in contact with the object

Heat

• Advantages: reliable effects, ease of use, shorter time and cost effective

• most common method of disinfection and sterilization

autoclave

heat under steam pressure

sterilization

application of autoclave

heat-stable objects

autoclave is the sterilization/disinfection method for?

Dry Heat

• longer exposure times & higher temperature

• Sterilization method for heat-stable substances that are not penetrated by moist heat

glass wares

Dry heat is the sterilization method for?

10 minutes

Boiling kills most microorganisms in approximately ___ minutes

Pasteurization

• eliminates food-borne pathogens & organisms responsible for food spoilage

• reduces spoilage of food without affecting its taste

63 or 72 C

Pasteurization is done at ___ and ___ C

121 C

Autoclave temperature

15 minutes at 15 psi

Duration time of autoclave

160 to 180 C

Temperature of oven (dry heat)

1.5-3 hours

duration time of oven

100 C

Temperature of Boiling

15 mins

duration time of boiling

72 C

temperature of pasteurization (flash method)

63 C

temperature of pasteurization (batch method)

15 seconds

Duration time of pasteurization (flash method)

30 mins

Duration time of pasteurization (batch method)

sterilizes

Does an autoclave application sterilize or disinfect?

sterilizes

Dry oven application

disinfection

Boiling steam application

disinfection

Pasteurization flash method application is a form of?

disinfection

Pasteurization batch method application is a form of?

liquid filtration

thin membrane filters composed of plastic polymers or cellulose esters containing pores

0.45 and 0.80 µm

pore size for bacteria, yeasts, and molds

0.22 µm

pore size for critical sterilizing

0.01 µm

pore sizes for small viruses

heat-sensitive solutions

Liquid filtration is the sterilization method for?

Air Filtration

• use of high-efficiency particulate air (HEPA) filters

• used in laboratory hoods and in air systems

0.3 µm

air filtration removes microorganisms larger than?

Radiation

• short wavelength and high energy

Ionizing

Gamma rays or electron beam

Disposable supplies

Radiation is the sterilizing method for?

non-ionizing

UV rays are?

non-ionizing

long wavelength and low energy

surfaces

non-ionizing is the sterilization method for?

Chemical Method

• for sterilization

• used mainly as disinfectants

Chemosterilizers

chemical agents that may be used to sterilize

Alcohols (50-70%)

Denatures proteins; makes proteins soluble

Tincture in Iodine (2% in 70% of alcohol)

Skin disinfectants (iodophor)

Chlorine and Chlorine Compounds

Used to disinfect drinking water and surface disinfectants

Tincture in Iodine (2% in 70% of alcohol)

Inactivates proteins

Chlorine and Chlorine Compounds

Reacts with water to form hyperchlorous acid (HClO); oxidizing agent

Silver Nitrate (AgNO2)

• Precipitates Proteins

• Eyedrops (1% Solution)

Mercuric Chloride (HgCl2)

• Reacts with -SH group; lyses cell membrane

• Disinfectant; toxic at high concentrations

Quarternary ammonium compounds

• Skin Antiseptics; disinfectants

• Disrupt cell membrane

Phenol, carbolic acid, lysol, hexachlorophene

• Disinfectant at high concentrations; used in soap at lower concentration

• Denatures proteins; disrupt cell membranes

Ethylene Oxide

• Sterilization of heat-sensitive objects

• Alkylating agent

Alcohol

• antiseptics and disinfectants

• principle: inactivate microorganisms by denaturing protein

• disadvantage: inactivated by organic materials

60-90% ethyl or isopropyl alcohol

Alcohol used for disinfection/antiseptic?

Formaldehyde

• principle: Reaction with the thiol (–SH) groups of enzymes

• disadvantage: carcinogenic agent

• not recommended (in any form) be used as a disinfectant or sterilant on a routine basis

37% aqueous solution or formaldehyde gas

Formaldehyde used?

Biosafety Cabinets

Formaldehyde is the sterilization method for?

Glutaraldehyde

• principle: inactivation of DNA and RNA through alkylation of sulfhydryl and amino groups

• advantage: rapid killing action; remains active in the presence of organic materials (active at alkaline)

• disadvantage: extremely susceptible to pH changes

2% solution for 10 mins

glutaraldehyde disinfectant uses ___% of solution for ___ mins

2% solution for 3-10 hrs

glutaraldehyde sterilant solution?

• heat labile medical equipment

• materials that cannot be sterilized in gas

glutaraldehyde is used as sterilant for?

Iodophors

• principle: degrades microbial cell walls and cytoplasm, denatures enzymes, and coagulates chromosomal material

• antiseptic

• advantage: less irritating, nonstaining, and more stable

polyvidone or povidone

Iodine can be used in two forms as antiseptics:

>30 seconds

Contact time for iodophors

iodine (5-10%) + neutral polymer carrier

iodophor solution?

1. liqiud sodium hypochlorite “household bleach”

2. solid calcium hypochlorite

forms of hypochlorite

Hypochlorite

• principle: oxidative effects of hypochlorous acid

• disadvantages: long exposure time required for sporicidal action; inactivated by organic materials

0.5% to 1% sodium hypochlorite

hypochlorite disinfectant

atleast 30 minutes

contact time for hypochlorite

1 : 10 dilution of a 5.25% concentration of sodium hypochlorite

recommendation of CDC for blood spills

water

Hypochlorite is the disinfection method for?

Detergents: Quaternary Ammonium Compound

• principles: reducing the surface tension of molecules in a liquid; disruption of the cellular membrane, resulting in leakage of cell contents

• disadvantages: effectiveness is reduced by hard water & soap; inactivated by organic materials

noncritical surfaces (benches and floors)

Quarternary Ammonium Compound is the disinfection method for?

Phenolics

principle: disruption of cell walls leading to precipitation of proteins

• commonly found as ingredients of germicidal soaps

lower concentration of phenolics

disruption of enzyme system

surfaces in hospitals and household environments

Phenolics disinfects?

Chlorhexidine Gluconate (0.5% to 4%)

• topical antiseptic

• principle: disrupts the microbial cell membrane and precipitates cellular contents

• advantages: strong affinity to the skin and mucous membranes; not significantly affected by organic materials

• disadvantage: affected by pH

• hands of surgical personnel

• body of patients undergoing surgery

Chlorhexidine Gluconate is used to disinfect?

Hexachlorophene

• effective against gram-positive bacteria (3% for 15-30 seconds)

• principle: interrupts bacterial electron transport

Low Concentration Hexachlorophene

inhibits membrane-bound enzymes

High Concentration Hexachlorophene

ruptures bacterial membranes

Chloroxylenol (0.5% to 4%)

• principle: microbial cell wall disruption and enzyme inactivation

• advantage: unaffected by organic materials

• disadvantage: neutralized by nonionic surfactants andpolyethylene glycol

** low antimicrobial efficacy compared with iodines, iodophors, and CHG in reducing skin flora

• health care personnel handwash

• surgical hand scrub

Chloroxylenol is the disinfection method for?

Triclosan

• advantage: not significantly affected by organic materials

• disadvantage:

• affected by pH, surfactants and emollients

• formulation significantly affects activity

• absorbed through intact skin

• consumer and professional health care products

gram (+) & (-) bacteria & viruses

Triclosan good activity

M. tuberculosis

Triclosan has fair activity on?

fungi

Triclosan poor activity