TERMS:
1. Sterilization—destruction
of all forms of microbial life including endospores, which are the most
resistant form. This is most often done with heat.
2. Commercial sterilization—term used for canned food—it must be heated
enough to kill endospores of Clostridium
botulinum. More resistant endospores may survive.
3. Disinfection—destruction of vegetative pathogens. Most
often this term refers to application of a chemical called a disinfectant to an
inert surface (floors, countertops, etc.), but ultraviolet radiation and boiling are other
methods used.
4. Antisepsis—chemical called an antiseptic is applied to living tissue.
Some microbes are killed, but not all.
5.
Degerming—mechanical removal of microbes from a limited area, such
as wiping an injection site with alcohol.
6.
Sanitization—high-temperature washing or washing and then dipping
into a chemical disinfectant. This is used to lower microbial counts on eating
or drinking utensils.
Treatments that kill microbes end in ---cide.
Germicide—kills
microbes but possibly not endospores
Fungicide—kills fungi
Virucide—kills viruses
Bacteriocide—kills bacteria
Treatments
ending in ----static stop growth (no
increase in numbers) but do not kill
Bacteriostatic
Microbistatic
Sepsis—indicates
bacterial contamination
Asepsis—absence
of significant contamination
important: death in microbes is loss of the ability to reproduce
When
bacterial populations are heated or treated with a chemical, they usually die
at a constant rate. If the population begins at 1 million and the agent kills
90 % of the microbes per minute of exposure:
Begin
1,000,000
After 1 minute 100,000
After 2 minutes 10,000
After 3 minutes 1,000
After 4 minutes 100
After 5 minutes 10
After 6 minutes 1
After 7 minutes Theoretically, all are dead, but a few
may survive for much longer
1. Number of microbes—the more there are to
begin with, the longer it takes to eliminate them all.
2. Microbial characteristics—susceptibility
to different agents varies among microbes. Also, endospores are much more
difficult to eliminate.
3. Environmental factors:
a. Organic matter (blood, feces, etc.)
often interferes with chemical antimicrobials, and also to a lesser extent with
heat treatment. Any medium containing fats or proteins tends to protect
bacteria.
b. pH—heat is
more effective in an acid pH.
4. Time of exposure
a. The lower the temperature used for
heat treatment, the longer the time required.
b. Effects of irradiation are also
related to time.
c. Chemical antimicrobials require
extended exposure to kill resistant bacteria or endospores.
1. Alteration
of membrane permeability—the plasma membrane regulates the entry of
materials into and the exit of wastes out of the cell. Damage to the plasma
membrane causes leakage of cell contents into the surroundings, killing the
cell or at least preventing cell division. Remember, death in microbes is loss
of the ability to reproduce.
2. Damage to proteins—enzymes and
other proteins are essential for cell function.
a. Hydrogen bonds hold proteins in the
characteristic 3-dimensional shape required for their functions. Heat and
certain chemicals break these bonds and the shape is lost. This is called
denaturation.
b. Covalent bonds, which are also part of
protein structure, may be broken by chemicals or heat even though they are
stronger than hydrogen bonds.
3.
Damage to nucleic acids—DNA
and RNA carry the cell’s genetic information and function in protein synthesis.
Damage to these by heat, radiation, or chemicals usually kills the cell.
Drying and salting were the earliest methods of food
preservation, so these were also the earliest means of controlling bacterial
growth. Physical means of control include:
Heat
Osmotic pressure
Filtration
Radiation
Low temperatures
Desiccation
When
choosing among modern physical methods, several factors must be considered:
1. Substance must not be inactivated by the
treatment—certain injectables such as vitamins and hormones are inactivated by
heat, for example
2. Material must not be destroyed—rubber
seals, latex tubing, etc. are damaged by heat
3. Economics—labor involved in cleaning
and sterilizing may make presterilized disposables more economic
Canned
foods are an example of control of microbial growth by heating. In the medical
world, lab media, glassware, and surgical instruments are examples of things
usually sterilized by heat. Heat resistance varies among microbes.
Thermal
death point is the lowest temperature at which all microbes in a liquid
culture will be killed in 10 minutes.
Thermal
death time is the minimum length of time in which all bacteria in a
liquid culture will be killed at a given temperature.
Decimal
reduction time (D value) is the time in minutes in which 90 % of bacteria will
be killed at a given temperature.
1. MOIST HEAT—all moist heat methods
kill microbes by coagulating or denaturing their proteins, including enzymes.
This occurs faster in the presence of water, so moist heat requires lower
temperatures and less time of exposure than dry heat.
a. BOILING (100o
C)—kills all vegetative bacterial pathogens, almost all viruses, and fungi and their
spores in 10 minutes or less (often much less). Endospores and a few viruses
are much more resistant to boiling.
Hepatitis A virus—30
minutes
Endospores—up to 20 hours
Although
boiling will kill most pathogens, it is not a dependable means of
sterilization.
b. AUTOCLAVE—this device uses steam under pressure for
effective sterilization. Items to be sterilized are placed in a chamber, which
is then sealed. All air is exhausted and steam under pressure is injected. This
achieves higher temperatures than boiling. It is the preferred means of
sterilization for all materials that can withstand it.
The
most common setting uses steam under 15 lbs pressure and reaches a temperature
of 121o C. This kills all organisms and their endospores in about 15
- 20 minutes.
Here
we use an autoclave to sterilize culture media. In hospitals, doctors’ offices,
dentists’ offices, etc. this methods is used to sterilize instruments.
Materials
being autoclaved are often wrapped in paper, so that after sterilization the
outside of the package can be handled without contaminating the sterile item
inside.
The
size of the container, the volume of a liquid, and the type of wrapping can
influence the time and temperature required for sterilization.
c. PASTEURIZATION—Pasteur used
mild heat to kill microbes in beer and wine ingredients before fermentation.
The same general idea is now used for milk. Pasteurization kills pathogenic
bacteria and viruses, although some harmless bacteria do survive. (Heat
sufficient to kill all microbes changes the character of the milk.) Bacteria
that are left eventually cause the milk to spoil, but
the life is greatly prolonged.
Originally,
milk was heated to 63o C for 30 minutes. Today, new methods use 72o
C for 15 seconds (high-temperature short-time pasteurization). Milk can be sterilized so that it can be
sealed in a carton and stored without refrigeration. This requires a
temperature of 140o C for about 3 seconds (ultra-high temperature
treatment). This process gives the milk an “off” taste.
2. DRY HEAT—this kills by burning to
ashes or by oxidation
a. FLAMING—we use this on loops in the lab
b. INCINERATION—burning
contaminated paper disposables in a controlled chamber
c. HOT-AIR STERILIZATION—items are
placed in an oven. Typical procedure would be 170o C for 2 hours. It
takes much longer for dry heat to kill than moist heat. This method sterilizes
by oxidation. It is mainly used for items that the autoclave is not suitable
for. This may be due to size or quantity--lots of lab glassware might be
sterilized in a hot-air oven. Other things that cannot be sterilized in the
autoclave:
·
Oily
substances
·
Powders
·
Sharps
(it is used sometimes for these but it dulls them)
This
is the passage of a liquid or gas through a screenlike material with pores
small enough to retain microbes. This method is used to sterilize items that
would be destroyed by heat. Some examples are:
Certain culture media
Enzymes
Vaccines
Injectable antibiotics
HEPA
(high-efficiency particulate air filters) trap microbes larger than 0.3 mm in diameter.
They are sometimes used in operating rooms and rooms of transplant patients to
lower numbers of bacteria.
To
filter liquids, filters of unglazed porcelain were originally used. Bacteria
were removed but viruses went through. For this reason, they were called
filterable viruses. In recent years, membrane filters have been developed.
These are made of cellulose or plastic and the size of the pores can be
selected, down to a size that will even retain most viruses.
These
are much more often bacteriostatic that bacteriocidal. Although some species
are fairly easily damaged, low temperatures are never a dependable means of
killing microbes.
1. NORMAL REFRIGERATOR TEMPERATURES —very few pathogens can grow. Psychrotrophs
grow slowly, and with time can cause food spoilage. This is a means of
preservation of foods, drugs, microbiological cultures, etc. Remember,
refrigerator temperatures do not kill many pathogens, so food allowed to sit at
room temperature for prolonged times before refrigeration can be dangerous.
2. FREEZING IN REGULAR HOME FREEZERS—this may kill some bacteria, but at least
some will usually survive even a year of freezing. Some bacteria are even able
to grow very slowly at several degrees below freezing.
3.
QUICK-FREEZING IN LIQUID NITROGEN AND FREEZE-DRYING (LYOPHILIZATION)—these
are means of preserving cultures, not of killing bacteria.
DESICCATION
Desiccation
or drying can kill microbes. Loss of moisture causes cells to either become
dormant or die because there is insufficient water for cellular reactions to
proceed. Species vary in their susceptibility. Dry surroundings kill some vegetative
cells in one hour or less. Others can survive for months or even years.
Bacteria that produce endospores are extremely resistant to drying. Many
viruses are also resistant to drying.
Placing
a high concentration of salt or sugar in bacterial surroundings causes water to
leave the cell. This is very similar to plain desiccation. It causes the plasma
membrane to shrink away from the cell wall---plasmolysis. The cell stops
growing and eventually dies.
This is a way of preserving foods by preventing bacterial
growth. Ham, bacon, salted fish, etc. use high concentrations of salt. Jellies
and fruit preserves use sugar.
Yeasts
and molds are better able to resist effects of both regular desiccation and
osmotic pressure, so they commonly cause spoilage of jelly, baked goods, etc.
Bacteria grow poorly if at all in these foods.
1. IONIZING RADIATION—this includes X
rays, gamma rays, and high-energy electron beams. These all have very short wavelengths and
high levels of energy. They cause ionization of water within cells, which results
in formation of hydroxyl radicals. These destroy cell components, especially
DNA. This process is used to sterilize wrapped plastic disposables such as
syringes, catheters, gloves, suture materials, vials of injectables, disposable
Petri dishes, pipettes, etc. It is also
used to sterilize spices. Recently, approval has been granted for use of low
level radiation of fruits and meats. The post office is now using this method
to sterilize some mail.
This
process does involve the use of dangerous radiation and can only be used in a
properly shielded room, so it is mostly used in factories where widescale use
of the setup makes it economical.
2. NONIONIZING RADIATION—this has a longer wavelength and less
energy. Ultraviolet (UV) light is the common example. It causes the formation
of thymine dimers, which interferes with DNA replication and formation of mRNA.
UV
lamps are used in hospitals and in food service. This method does not
sterilize, but it does reduce bacterial growth. Penetrating power is very low,
so any type of covering protects microbes.
Sunlight
has some weak antimicrobial effects, but the wavelengths of sunlight are too
long to work well.
SEE SUMMARY OF PHYSICAL METHODS TABLE 7.5 P.
197
CHEMICALS
Bacterial
species vary in their susceptibility to chemical as well as physical agents.
Some factors to consider:
1. Type of microbe
a. In general, gram-negative bacteria
tend to be less susceptible to chemical agents than gram-positive bacteria
b. Members of the genus Pseudomonas are often highly resistant,
and have even been found growing in disinfectant solutions
c. Members of the genus Mycobacterium are also resistant to many
chemical antimicrobials
d. Bacterial endospores and cysts of
protozoa are very highly resistant
e. Some viruses are resistant
2.
Environment
a. Presence of organic matter interferes
with most chemical agents
b. Temperature—many chemical agents work
better in warm temperatures
Most
chemical agents are disinfectants or antiseptics, although there are a few
chemical sterilants.
Chemical
agents have labels which indicate the proper dilution and use of the agent.
Factors such as pH, temperature, and the presence of organic matter must be
considered. Few disinfectants achieve maximum effect in less than 10 minutes,
and some require more.
1.
Use-dilution tests—this is the current official test for a chemical agent.
Standard liquid cultures of 3 types of bacteria are used:
Salmonella
cholerasuis
Staphylococcus aureus
Pseudomonas aeruginosa
Metal
carrier rings are dipped into the cultures, removed, and dried briefly at 37o
C. Rings are then placed in the manufacturer’s recommended concentration of the
agent and left for 10 minutes at 20o C. The rings are then placed in
culture media and incubated to see if there are any survivors.
If
there is particular interest in a certain chemical for control of endospores,
mycobacteria, etc. these organisms may also be used in tests.
2.
Disk-diffusion method—a disk of filter paper is dipped into the chemical and
then placed on an agar plate that has already been heavily inoculated with
bacteria. The plate is incubated and observed. If the agent if effective, a
clear zone with no bacterial growth will surround the disk. This is called a
zone of inhibition, and can be measured to compare effectiveness.
1. PHENOLS and PHENOLICS
a. PHENOL (carbolic acid) was used by Lister to
reduce the incidence of surgical infections. It is irritating to skin and
mucous membranes and has a bad odor, so it is rarely used today. Its main use
now is in throat lozenges and sprays, but the concentration is so low that
there is little antimicrobial effect, although there is some local anesthetic
action. Some throat sprays may have a concentration above 1% and these may show
antibacterial action.
b. PHENOLICS—chemicals derived from phenol—the molecule
has been chemically altered to make it less irritating and more effective.
These agents act in several ways, damaging plasma membranes, inactivating
enzymes, and denaturing proteins. Phenolics are often used as disinfectants
because they remain active in the presence of organic matter. Original Lysol
products contain O-phenylphenol, but many of the newer ones do not.
c. BISPHENOLS--two phenolic groups are connected by a
bridge. Hexachlorophene (pHisoHex) is an example. It is very effective against gram-positive
cocci, which often cause skin infections, so it makes a good skin antiseptic,
but excessive use can result in toxicity.
Triclosan is a bisphenol found in antibacterial soaps.
2. BIGUANIDES—chlorhexidine is an example. These are
similar to phenolics, but are less toxic. Biguanides act by disrupting the
plasma membrane and are excellent as surgical scrubs and for patient preps,
although they must be kept away from the eyes. They are also used in dental
treatments.
They
are effective against most vegetative bacteria and fungi, but not against
endospores and many viruses.
3. HALOGENS
a. IODINE—one of the oldest and most effective
antiseptics. Works against all bacteria and many endospores,
fungi, and viruses. It acts by combining with amino acids, especially
tyrosine, and inhibits the functions of microbial proteins. It also alters
plasma membranes.
1) Tincture—iodine combined with
alcohol
2) Iodophor—iodine combined with an
organic carrier molecule. These are still quite effective, but they are less
toxic and do not stain as badly. Betadine is an example.
In
addition to use as a skin disinfectant and wound treatment, iodine can be used
to purify drinking water.
b. CHLORINE—when chlorine is added to water,
hypochlorous acid forms. This is a strong oxidizing agent that interferes with
cellular enzymes.
1) A liquid form of compressed chlorine
gas is used for treating water systems, swimming pools, and sewage.
2) Calcium hypochlorite is used
to disinfect dairy equipment and restaurant equipment. It can also be called
chloride of lime and was the disinfectant Semmelweis used.
3) Sodium hypochlorite (Chlorox)
is used as a household disinfectant and bleach. Adding 2 - 4 drops of Chlorox
to a quart of water can make it safe as drinking water (let sit 30 minutes
before using). A solution of Chlorox is the recommended disinfectant for
killing the AIDS virus in households.
4) Chloramines—combine chlorine
and ammonia, used to sanitize glassware, eating utensils, and food-handling
equipment as well as in water systems.
4. ALCOHOLS—these kill bacteria and
fungi but not endospores and most viruses. Alcohol acts mainly by denaturing
proteins, but it can also disrupt membranes and dissolve lipids. Alcohols
evaporate rapidly, leaving no residue. Alcohols are frequently used as skin degerming
agents. Wiping with alcohol mostly wipes away microbes, skin oils, and dirt,
although some microbes may be killed.
Alcohols
are not satisfactory for cleaning open wounds, because they coagulate a surface
layer of protein and leave bacteria unharmed beneath it.
a. ETHANOL (ethyl alcohol)—70 %
concentration is ideal, although concentrations of 60 - 95 % are
effective. 100% is not effective because some water
must be present for denaturation to occur.
b. ISOPROPANOL (isopropyl alcohol or rubbing alcohol)—more
commonly used because it is cheaper and more effective. Usual concentration is
90%.
Both
of these alcohols may be mixed with other agents to enhance activity. When
another agent is mixed with alcohol, the solution is called a tincture.
5. HEAVY METALS—very low
concentrations of heavy metals can be effective against microbes. This is called oligodynamic action, and works
by denaturing proteins, including enzymes.
a. SILVER NITRATE (1% solution)—this was once used to swab
sore throats (in the days before antibiotics). It was also used in the eyes of
newborns, to prevent an eye infection caused by the gonorrhea bacteria.
Antibiotic ointments are now used for this purpose.
b. PURE SILVER
INCORPORATED INTO DRESSINGS (ACTICOAT)--In recent years, dressings containing silver have been used in
treating infections caused by antibiotic-resistant bacteria. The silver seems
to shut down energy production in bacteria and little resistance has been
found, and tissue damage does not occur as with other means of delivering
silver.
c. OTHER FORMS OF SILVER—Cream containing silver combined with a
sulfa drug, catheters impregnated with silver, a silver-containing product for
surfaces.
c. MERCURY---compounds such as mercuric chloride were
probably the earliest disinfectant. They are bacteriostatic. The drawbacks are
toxicity, corrosiveness and inactivation by organic matter. Mercurochrome and merthiolate were once widely used, but contained such tiny
amounts of mercury that they had little effect. Mercury compounds may be used
in paint to prevent mildew.
d. COPPER---copper sulfate is used
to kill algae (algicide) in bodies of water or aquariums. Copper compounds may
also be used in paint to prevent mildew.
e. ZINC---zinc chloride is found in some mouthwashes and zinc is often
used as an antifungal in paint. Zinc lozenges & such are sold as treatment
for colds, but their effect is questionable.
6. SURFACE-ACTIVE AGENTS (SURFACTANTS)---these
agents decrease surface tension and include soaps and detergents.
a. SOAPS---main value is in
causing microbes to be mechanically removed. Washing with soap breaks up the
oily film that covers skin and allows microbes and dirt to be washed away.
Deodorant soaps have antimicrobial ingredients added.
b. ACID-ANIONIC SURFACE-ACTIVE SANITIZERS---used
in cleaning food and dairy equipment. The negatively charged portion of the
molecule (anion) reacts with the plasma membrane of microbes.
7.
QUATERNARY AMMONIUM
COMPOUNDS---these are surface-active cationic detergents.
These are very pleasant to use---non-irritating, non-corrosive, no unpleasant
odor, etc. They are strongly bacteriocidal against gram-positive bacteria but somewhat less
effective against gram-negative. They
also kill fungi, amoebas, and some viruses. Unfortunately, they are not
effective against endospores, tuberculosis bacteria, or some strains of Pseudomonas.
They
are believed kill microbes by disruption of the plasma membrane, enzyme inhibition,
and protein denaturation. Organic matter interferes with their activity.
Examples
are Zephiran (benzalkonium chloride) and Cepacol (cetylpyridinium chloride).
8. CHEMICAL FOOD PRESERVATIVES---these
are frequently added to retard spoilage, and are believed to be safe for
consumption.
a. ORGANIC ACIDS---they interfere with the metabolism of
molds or damage their plasma membranes.
1) Sorbic acid, potassium sorbate,and sodium benzoate are added to prevent growth of mold
in acid foods such as cheese and soft drinks.
2) Calcium propionate---used in bread
b. SODIUM NITRITE OR NITRATE---added to meat products such as ham,
bacon, and hot dogs. It preserves the red color of the meat and prevents
germination and growth of botulism endospores.
As
nitrites react with amino acids, compounds called nitrosamines are formed.
These are carcinogens. Because of this, the amount added to meat has been
reduced.
9. ANTIBIOTICS---two which are not
used in treating disease are used as food preservatives.
a. NISIN---added to cheese to
prevent growth of endospores
b. NATAMYCIN---added to foods to
prevent growth of fungi
10 ALDEHYDES---these can act very effectively against
microbes. They inactivate proteins.
a. FORMALDEHYDE
1) FORMALDEHYDE GAS---can be
used as a disinfectant
2) FORMALIN, a 37% aqueous
solution of formaldehyde gas, has been used to preserve biological specimens
and inactivate bacteria and viruses in vaccines.
b. GLUTARALDEHYDE---this is more
effective and somewhat less irritating than formaldehyde. It is used to
disinfect medical equipment that cannot withstand autoclaving. This is the most
commonly used chemical sterilant.
A
2 % solution, such as CIDEX, kills bacteria including Mycobacterium tuberculosis and viruses in 10 minutes. Endospores
require 3 - 10 hours. If the 10 hours are allowed, this is considered to be
chemical sterilization. However, instruments must be rinsed before use, so
unless they are handled aseptically and rinsed with sterile water, this is more
likely to be disinfection.
c. ORTHO-PHTHALALDEHYDE (OPA)—this is a relatively new alternative to
glutaraldehyde. It is somewhat less effective against endospores, but works
well against vegetative pathogens. It is less irritating to skin.
11. GASEOUS CHEMICAL STERILANTS---these
are used in a closed container, which is sometimes called a gas autoclave.
a. ETHYLENE OXIDE---this is the one most frequently used. It
acts by denaturing proteins. It kills all microbes and endospores, but has
several disadvantages:
1) Long exposure time (4 - 18 hours)
2) Items must then be aired 12 - 24
hours before use
3) Highly toxic and explosive
In
spite of these drawbacks, the product is still used because:
1) Can be used on items that cannot
withstand autoclaving
2) Some large hospitals have ethylene
oxide chambers where even large items such as mattresses can be sterilized
3) Was used to sterilize spacecraft
returning to earth
4) Used to sterilize spacecraft that
landed on the moon and on Mars
b. PROPYLENE OXIDE
c. BETA-PROPRIOLACTONE
12. PEROXYGENS (oxidizing agents)---these oxidize cellular components of microbes.
a.
OZONE (O3)---highly reactive form of oxygen, often used
along with chlorine to disinfect water
b. HYDROGEN PEROXIDE---although
frequently used, it is a poor choice as an antiseptic for open wounds, because human
cells contain the enzyme catalase, which breaks down the peroxide before it has
much chance to act. (This is where the bubbles come from).
It
is an effective disinfectant for inanimate objects, where it can even kill
endospores. It is used to disinfect food packaging (before the food is put in)
and contact lenses.
Although
peroxide does not directly kill microbes well in wounds, it is used in deep
wounds because it releases oxygen as it breaks down, which makes conditions
unfavorable for anaerobic bacteria.
c. BENZOYL PEROXIDE---main ingredient in many acne treatments
and may be used in treating wound infections caused by anaerobes.
d.
PERACETIC ACID---excellent chemical sporicidal agent and is considered a
chemical sterilant which acts within 30 minutes (much faster than the others).
Vegetative bacteria and fungi are killed in 5 minutes. However, it is extremely
irritating to skin and mucous membranes, including the respiratory tacg when
inhaled. It is widely used in food
processing and can also be used for medical equipment. An advantage is that it
can act in the presence of organic matter.
Susceptibility
to chemical antimicrobials varies considerably among microbes:
1. Most gram-positive bacteria are relatively
susceptible to chemical agents, with the mycobacteria the major exception.
2. Gram-negative bacteria tend to be more
resistant, with Pseudomonas the worst
3. Endospores are most resistant life form.
4. Viruses
a. Some viruses have a lipid-containing
envelope surrounding them (enveloped viruses). These are much more susceptible
to chemical agents.
b. Nonenveloped viruses are much more
resistant.
5. Prions are the most resistant of all
infectious agents (they are not considered to be living)
BE SURE
TO STUDY:
TABLE 7.8 P. 207 - 208-----SUMMARY OF AGENTS,
USE AND GENERAL COMMENTS