Exam 3 - Chapter 13

Clean

is a relative term

reduced by washing,vacuuming and dishwashing but, is this clean enough? - well, it all depends on the intended use of an object or surface

Microbial load can be

Sterilization often requires time, is labor intensive and, may degrade the quality of the item being treated, or even have toxic effects on users

Why clean and not sterilize everything?

Biosafety levels

…ranked by the severity of disease and ease of transmission

Biological Safety Level (BSL) - 4

Examples: Ebola and Marburg viruses

Microbes are dangerous and exotic (foreign), posing a risk of aerosol-transmitted infections, which are frequently fatal without treatment or vaccines. Few labs are at this level

BSL-3

Examples: Mycobacterium tuberculosis

Microbes are indigenous or exotic and cause serious or potentially lethal diseases through respiratory transmission

BSL-2

Example: Staphylococcus aureus

Microbes are typically indigenous and are associated with diseases of varying severity. They pose moderate risk to workers and the environment

BSL-1

Examples: Nonpathogenic strains of Escherichia coli

Microbes are not known to cause disease in healthy hosts and pose minimal risk to workers and the environment

BSL-1 → BSL-2 → BSL-3 → BSL-4

CDC Classification low-risk microbes to high-risk microbes

sterilization

process by which ALL living cells, spores, and viruses are destroyed on an object

disinfection

the killing or removal of DISEASE-PRODUCING organisms from inanimate surfaces; it does not necessarily result in sterilization

antisepsis

similar to disinfection but applies to removing pathogens from the surface of LIVING tissues, such as skin

sanitation

consists of reducing the microbial population to safe levels and usually involves cleaning an object as well as disinfection

cidal agents and static agents

antimicrobials fall into 2 broad classes:

kill microbes

• Bactericidal, algicidal, fungicidal, virucidal, depending on what type of microbe is killed

cidal agents

inhibit or control growth

• bacteriostatic, algistatic, fungistatic, virustatic, depending on what type of microbe is inhibited

static agents

1. Must be fast-acting in the presence of organic materials

2. Must be effective against all microorganisms without destorying tissue or acting as a toxin if ingested

3. Easily penetrate the material to be disinfected without discoloration or damage

4. Easy to prepare and stable in the environment where it is to be used

5. Inexpensive and easy to use

6. Not have an unpleasanr odor

There is no perfect disinfectant that meets all criteria

selection of disinfectant

microbial death curve to describe the progress and effectiveness of a particular protocol

The degree of microbial control can be evaluated using a

a fixed percentage of the microbes within the population will die.

When exposed to a particular microbial control protocol,

the percentage killed is more useful information than the absolute number of microbes killed

Because the rate of killing remains constant even when the population size varies,

semilog plots just like microbial growth curves because the reduction in microorganisms is typically logarithmic

Death curves are often plotted as

decimal reduction time (DRT) or D-value

The amount of time it takes for a specific protocol to produce a one order-of-magnitude decrease in the number of organisms, or the death of 90% of the population, is called the

• the initial population size (the larger the population, the longer it takes to decrease it to a specific number)

• population composition (are spores involved?)

• agent concentration or dose for radiation

• duration of exposure

• presence of organic material (blood, feces) that can inhibit disinfectant action; organic load

several factors influence the speed at which lethal damage accumulates

physical agents are often used to kill microbes or control their growth

Physical methods to control microorganisms

• Temperature extremes

• Pressure (usually combined with temperature)

• Filtration

• Irradiation

• Sonication

Commonly used physical control measures include:

• moist heat is very effective at killing microbes

• dry heat is less effective. but sometimes required

• to kill spores and thermophiles, a combination of heat and pressure is usually required (steam autoclave)

• standard autoclave conditions are 121 degrees Celsius at 15 psi (pounds per square inch) for 20 minutes

High Temperature and Pressure

Preferred sterilizing agent as long as it doesn’t damage the materials

Heat killing

Thermal death point (TDP)

lowest temperature that kills all the bacteria in a 24 hour old culture/ 10 min

Thermal death time (TDT)

Time required to kill all the bacteria in a culture at a specified temperature

Decimal reduction time 9D value)

length of time required to kill 90% of the organisms in a population at a specific temperature

is not to sterilize, but to kill pathogens without affecting the texture, color, or taste of the product

Different time and temperature combinations can be used:

• low temperature, long time (LTLT) - process involves bringing the temperature to 63 degrees Celsius (146 degrees Fahrenheit) for 30 minutes

• high temperature, short time (HTST) - process (also called flash pasteurization), brings the temperature to 72 degrees Celsius (162 degrees Fahrenheit) for only 15 seconds

• ultra high temperature (UHT) - brings the temperature to 138 degrees Celsius (273 degrees Fahrenheit) for 2 seconds, can actually produce sterilized products with unrefrigerated shelf lives up to 6 months

The goal of pasteurization

• excellent to preserve food and other materials - retards microbial growth

• but does not effectively kill microorganisms

• microorganisms can be stored in this way

• Long-term storage of bacteria usually requires placing solutions in glycerol at very low temperatures (-70 degrees Celsius or -94 degrees Fahrenheit). This deep freezing suspends growth altogether and keeps cells from dying

Cold - refrigeration (4-8 degrees Celsius (39-43 degrees Fahrenheit)), freezing

• cultures are quickly frozen at very low temperatures and placed under a vacuum, which causes the water to sublimate, removing all water from the cells

• many microorganisms are sensitive to drying - but, many are not

• freeze drying is used for storing many microorganisms

Cold - Freeze-drying (Lyophilization)

• many drugs/chemicals are sensitive to heat or other chemical sterilization methods

• these solutions can be sterilized by passing them through sterile filters with tiny pore sizes that effectively “sift” the microbes out of the fluid

• filtration through micropore filters of 0.2 µm can remove microbial cells, but not viruses, from solutions

• To remove viruses, pore sizes of 20 nm are necessary (1 nm = 0.001 µm)

• Air can also be sterilized through filtration. From simple surgical masks to sophisticated air purifiers, air is forced through filters to remove microbes

Filtration

sonication…rapid changes in pressue within the intracellular liquid

…is high-frequency ultrasound waves to disrupt cell structures, and the disruption is achieved due to the…

Irradiation - Ultraviolet (UV) light, Gamma rays, electron beams, X-rays

• Ultraviolet (UV) light: UV light is useful only for surface sterilization due to its poor penetrating ability

method in which objects are bombarded with high-energy electromagnetic radiation

Foods

…do not become radioactive when irradiated, and any reactive molecules produced when high-energy particles are absorbed by food dissipate almost immediately

• wheat flour to control mold

• white potatoes to inhibit sprouting

• pork to kill trichina (Trichinella) parasites

• fruit and vegetables to control insects; increases shelf life

• herbs and spices to sterilize

• poultry to reduce numbers of bacterial pathogens

• meat to reduce numbers of bacterial pathogens

• shellfish to reduce numbers of Vibrio species and other pathogens

• lettuce and spinach to reduce numbers of bacterial pathogens

Examples of foods approved for irradiation in the united states

• the presence of organic matter - chemicals will bind to inert organic material, lowering its effectiveness against microbes

• the kinds of organisms present - ideally should be effective against a broad range of pathogens

• corrosiveness - should not corrode the surface (nonliving or living)

• stability, odor, and surface tension - should be stable upon storage, neutral or pleasant odor, low surface tension

efficacy of a given chemical agent depends upon:

reduce or eliminate microbial content from commerical products

commerical disinfectants are all used to

ethanol, iodine, chlorine

highly reactive compounds that damage proteins, lipids, and DNA

surfactants (such as detergents)

help in the mechanical removal of microbes from surfaces

aldehydes

combine with and inactivate proteins and nucleic acids

phenolics

denature proteins and disrupt membranes

heavy metals

bind to proteins and inhibit enzymatic activity

peroxygens

strong oxidizers, produce free radicals that damage cellular macromolecules

• Disposable plastic ware such as petri dishes, syringes, sutures, and catheters is not amenable to heat sterilization or chemical disinfection

• These types of products are best sterilized by gamma irradiation or antimicrobial gases, such as ethylene oxide (EtO)

• Key characteristics of Gas sterilization:

  • destroys proteins by alkylation

  • microbicidal/sporicidal

  • rapidly penetrates packing materials, including plastic wraps

  • highly explosive

Gas Sterilization

can develop resistance to chemical disinfectants used to prevent infections

Bacteria

develop resistance to chemical agents that have multiple targets and can easily diffuse into a cell (example: iodine)

It is difficult for bacteria to

only have a single target at low concentrations - a situation that can foster resistance (example: triclosan

Disinfectants that have multiple targets at high concentrations may

involves heating a particular food (such as milk) to a moderately high temperature long enough to kill Coxiella burnetii, the causative agent of Q fever and the most heat-resistant spore-forming pathogen known

Today, pasteurization…

to temper growth and to preserve strains

Low temperatures have 2 basic purposes in microbiology:

more slowly in cold temperatures, but they also die more slowly

Bacteria grow

Laminar flow biological safety cabinets

are elaborate and effective ventilated workbenches in which air is forced through high-efficiency particulate air filters (HEPA filters) to remove more than 99.9% airborne particulate material 0.3 µm or larger

HEPA

removes microbes >0.3 µm

membrane filtration

removes microbes >0.22 µm

cavitation, the formation of bubbles inside the cell, which can disrupt cell structures and eventually cause the cell to lyse or collapse

Sonication leads to

in the laboratory for efficiently lysing cells to release their contents for further research

Sonication is useful

sonication is used for cleaning surgical instruments, lenses, and a variety of other objects such as coins, tools, and musical instruments

Outside the laboratory,