Control of microorganisms

Many bacteria cause disease and food spoilage

Need exists to kill or inhibit the growth of these bacteria

Frequently used terms

Sterilization - removal or destruction of all living cells, viable spores, viruses and viriods

Disinfection - removal or destruction of pathogens (spores and some other microorganisms remain)

Sanitization - reduction of microbial population to safe levels

Antisepsis - prevention of infection (accomplished by antiseptics)

Frequently used terms

Bactericide - substance that kills bacteria

Bacteriostatic - substance that prevents growth of bacteria

Pattern of microbial death

Microorganisms usually die logarithmically (i.e. the population will be reduced by the same fraction at regular intervals)

Pattern of microbial death

Microorganisms usually die logarithmically (i.e. the population will be reduced by the same fraction at regular intervals)

Conditions influencing effectiveness of antimicrobial agents

Population size

Population composition

Concentration or intensity of agent

Duration of exposure to agent

Temperature

Local environment (e.g. pH, presence of organic material)

Physical methods of control

Heat

Low temperature

Filtration

Radiation

Measuring heat-killing efficiency

Thermal death point (TDP) - lowest temperature at which all microorganisms in a suspension are killed in 10 minutes

Decimal reduction time (D value) - time it takes to kill 90% of the cells or spores in a sample at a specific temperature

Decimal reduction time (D value)

Measuring heat-killing efficiency

Z value - the increase in temperature required to reduce D to 1/10 its value

F value - time in minutes at a specific temperature required to kill a population of spores or cells

Measuring heat-killing efficiency

Moist heat

Effective against all types of microorganisms

Degrades nucleic acids, denatures proteins, and disrupts membranes

Moist heat

Autoclaves are used to kill endospores

Uses steam under pressure to achieve temperatures above boiling

Pasteurization

Controlled heating at temperatures below boiling

Does not sterilize

Kills pathogens and reduces levels of spoilage microorganisms

First used to preserve wine, now used for milk, beer, juice and other beverages

Pasteurization

Traditional method: 63 ºC for 30 minutes

Flash pasteurization: 72 ºC for 15 seconds

Ultrahigh temperature (UHT) sterilization

Sometimes used by the milk industry

Milk heated at 140 to 150 ºC for 1 to 3 seconds

Products can be stored at room temperature for 1 to 2 months

Dry heat sterilization

Less effective, requiring higher temperatures and longer exposure times

Does not corrode metal and glass instruments

Oxidizes cell constituents and denatures proteins

Low temperature

Refrigeration- storage at 4 ºC slows microbial growth (only used for short-term storage)

Freezing- storage at - 20 ºC stops microbial growth (does not kill microorganisms)

Freezing at -30 to -70 ºC used to preserve microbial samples

Filtration

Can be used to sterilized or reduce the microbial population of heat-sensitive liquids

Removes microorganisms rather than destroying them

Solutions often forced through filters by pressure or a vacuum

Depth filters

Thick layers of fibrous or granular material with small diameter channels

Made of various materials (e.g. diatomaceous earth or asbestos)

Membrane filters

Porous membrane about 0.1 mm thick

Entrap microbes on their surface

Pore size of 0.2 um diameter removes most cells but not viruses

Air filtration

Surgical masks

Cotton plugs on culture vessels

Air filtration

Laminar flow biological safety cabinets

Employ high efficiency particulate air (HEPA) filters

Remove 99.97 % of particles larger than 0.3 um

Air forced through filter forms a curtain of sterile air across the opening of the cabinet

Laminar flow biological safety cabinets

Radiation

Ultraviolet (UV) radiation

Ionizing radiation

Ultraviolet (UV) radiation

Wavelength of about 260 nM

Lethal but does not penetrate glass

Used to sterilize air or exposed surfaces

Can sterilize water in a thin layer

Burns the skin

Ionizing radiation

Penetrates deep into objects

Not always effective against viruses

Gamma radiation from Cobalt 60 often used

Used to treat meat, fruits, vegetables and spices

Chemical agents

Are most commonly used agents for disinfection and antisepsis

Phenolics

Phenol first used by Lister

Phenol and derivatives used as disinfectants in hospitals and labs

Effective in the presence of organic material

Can cause skin irritation

Phenolics

Act by denaturing proteins and disrupting cell membranes

Alcohols

Not effective against spores or lipid-containing viruses

Ethanol and isopropanol most commonly used (at 70-80 %)

Act by denaturing proteins and possibly dissolving membrane lipids

Halogens

Include fluorine, chlorine, bromine, iodine and astatine

Iodine used as a skin disinfectant

Chlorine used to disinfect water

Both act by oxidizing cell material and iodinating or chlorinating molecules

Aldehydes

Formaldehyde and gutaraldehyde are the most commonly used

Are highly reactive molecules

Inactivate proteins and DNA by cross-linking alkylating molecules

Quaternary ammonium compounds (QUATS)

Are a class of detergents

Consist of a positively charged quaternary nitrogen with a long hydrophobic chain

Act by disrupting membranes and denaturing proteins

Sterilizing gases

Ethylene oxide (EtO) is used to sterilize heat-sensitive material

Kills by combining with proteins

Rapidly penetrates packing material

Sterilizing gases

Betapropiolactone (BPL) is occasionally used

Less difficult to eliminate and kills more quickly

Does not penetrate well and may be carcinogenic

Heavy metals

Ions of mercury, silver, arsenic, zinc and copper were used for many years as germicides (less toxic and more effective compounds have generally replaced them)

Silver nitrate can be used in the eyes of newborns (antibiotics more common now)

Silver sulfadiazine used on burns

Copper sulfate used as an algicide

Heavy metals

Act by combining with proteins and inactivating or precipitating them

Evaluation of antimicrobial agent effectiveness

The Environmental protection agency (EPA) regulates disinfectants

The Food and Drug Administration (FDA) regulates agents used on humans and animals

Phenol coefficient

Potency of disinfectant compared to phenol

Coefficient greater than 1 indicates agent is more potent than phenol

Not always indicative of potency during normal use

Other evaluation methods

Use dilution test

Measures rate at which selected bacteria are destroyed by various chemical agents

In use test

Testing done using conditions that approximate normal use of disinfectant