Chapter
11 - Controlling Microbes (Sterilization & Disinfection)
sterilization - treatment to destroy all
microbial life (even destroys bacterial endospores and fungal spores); there are no
degrees of sterility!
disinfection (sanitation) - treatment to reduce
the number of pathogens to a level at which they pose no danger of disease; disinfectants are used to kill microbes on
inanimate objects (most disinfectants are too harsh for use on delicate tissue); most
disinfectants do not kill spores.
antisepsis - kill microbes or inhibit their
growth on skin or other living tissue; antiseptics
are applied to living tissue.
sanitizer – typically used on
food-handling equipment and eating utensils to reduce bacterial numbers so as to meet
public health standards (may mean just washing with soap in some cases).
“-static” - treatments that
inhibit rather than kill; ex. refrigeration. (bacteriostatic,
fungistatic, etc.)
“-cidal” - treatments that kill. (bactericidal, fungicidal, viricidal, etc.) (germicidal is a more general)
chemotherapeutic agents - chemicals, incl.
antibiotics, used to treat disease (discussed in Chap. 12)
A. Heat
1. Advantages
- simple, inexpensive, effective penetrates to kill microbes throughout the object; best
method if material being treated is not damaged by heat.
2. Mode
of Action - denatures proteins.
3. Treatments
a. Dry
Heat Sterilization
- ex. flaming loops, tubes in lab & hot air ovens (171oC, 1hr., 160oC
for 2 hr., 121oC for 16 hrs.); used to sterilize materials that can withstand
high temps. & any materials damaged by moisture.
b. Moist
Heat Sterilization
- ex. boiling or in autoclaves; effective at a lower temperature than dry heat & it
penetrates more quickly; disadvantages of boiling - does not kill thermophiles,
endospores; autoclave is more effective than boiling- it uses pressure to raise the
temperature above that of boiling (121oC, 15psi, for 20 min.); used to
sterilize liquids and material easily charred; used in food canning & the lab to
sterilize glassware & media.
c. Pasteurization
- limits growth, but does not sterilize; used to slow spoilage of milk & dairy
products, wine, beer; advantage: causes minimal damage to the product; developed by Louis
Pasteur; standard treatment: heat to 63oC for 30 min. or 72oC for 15
sec.
B. Cold
1. Effect - microbiostatic; does not
sterilize; slows down enzymes.
a. Refrigeration
- preserves food because it stops the growth of most species of microbes (slows chemical
reactions); most disease-causing microbes are mesophiles, not psychrophiles; an exception
is Listeria spp., which causes listeriosis (food
poisoning).
b. Freezing
- kills most bacteria, but survivors can remain alive for long periods in the frozen
state; bacteria cultures can be preserved by rapid freezing, sometimes with the addition
of a compound called DMSO, milk, or glycerol to protect proteins.
C. Radiation
1. Electromagnetic
Spectrum
- Radiation is classified by wavelength with ionizing and UV light radiation at the
short-wavelength end, visible light in the middle, & radio waves at the
long-wavelength end. The shorter the
wavelength, the greater its energy, & the more lethal it is. Mode of Action: denatures DNA.
2. Two
types of radiation that kill bacteria directly are UV (ultraviolet) Light & Ionizing Radiation. The effect of both is sterilization.
a. UV
Light
- bacteria actually have special enzymes that can correct some damage done by UV light!;
in the lab mercury vapor lamps (germicidal lamps) are used; disadvantages: kills only on surfaces & these wavelengths can
also be harmful to humans.
b. Ionizing
Radiation
- 2 forms; both cause a chain of ionizations by stripping electrons from atoms, resulting
in cell death; disadvantages: technically complex; is being used to sterilize some
produce, much to the public's dismay.
1.) X rays
2.) Gamma rays
D. Membrane Filtration
1. Effect
- physically removes cellular organisms (not viruses - they are too small).
2. Uses
- in lab, used with media, antibiotics, & other heat sensitive materials; filtration
is replacing pasteurization in some causes, because filtration causes even less damage;
you may have heard of the new "cold filtered" beers.
E. Drying
1. Defined - the removal of water.
2. Two processes:
a. evaporation
involving heat -
effect - kills many microbes; rarely used in lab because the high heat causes chemical
changes (denaturation); is used in food industry.
b. lyophilization
[freeze drying] - removes water directly by converting water from a solid state (ice) to a
gaseous state; materials are frozen & placed in a chamber to which a partial vacuum is
applied; avoids the chemical changes caused by heat drying; effect - stops microbial
growth by stopping most chemical reactions (just like regular freezing) frequently used in
the microbiology lab to preserve perishable materials such as proteins, blood products,
& reference cultures of microbes; used in food industry to make instant coffee, etc.;
disadvantage - expensive.
F. Osmotic Strength
1. Method
- high concentrations of salt or sugar.
2. Mode
of action - microbes cannot grow if they are deprived of water; also, crenation or
shrinkage can occur (you're placing the microbes in a hypertonic environment).
3. Disadvantage
- once added, solutes (such as salt or sugar) cannot be easily removed; not used in lab.
A. Testing
Germicides – 3 ways:
1. Phenol
coefficients:
Germicides can be tested by comparing their effectiveness to phenol, a traditional germicide. It was phenol that Lister first used - he called
it carbolic acid. The procedure involves
preparing several dilutions of a chemical agent, inoculating them with the bacteria Salmonella typhi (a digestive tract pathogen) or Staphylococcus
aureus (a wound pathogen), incubating the tubes, and then checking for cloudiness in
the tubes, indicating growth. The ratio of
the effective dilution of the chemical agent to the dilution of phenol that has the same
effect is the phenol coefficient. A disinfectant with a phenol coefficient of
1.0 has the same effectiveness as phenol. Less
than 1.0 means it’s less effective. Greater
than 1.0 means it’s more effective.
2. Paper
disc method
– paper discs are saturated with the chemical agent and placed on the surface of an
agar plate inoculated with a test organism. Clear
“zones of inhibition” appear around the discs if the chemical agent is
effective.
3. Use-dilution
test
– The test microbe is added to dilutions of the chemical agent. The highest dilution that remains clear after
incubation indicates a germicide’s effectiveness.
1. Affect
Proteins –
The alteration of protein structure is called denaturation. Denaturation can be permanent (bacteriocidal)
or temporary (normal structure can be restored – bacteriostatic). Mechanisms of denaturation include:
a.
Hydrolysis
– breakdown of a molecule by addition of water
b. Oxidation
– addition of oxygen or removal of hydrogen
c.
Attachment
of atoms or chemical groups –
ex. heavy metals (mercury), alkylating agents (ex. –CH
)
2. Affect
Membranes
a.
membrane
proteins – denaturation
(see above)
b. membrane
lipids –
can be dissolved.
3. Affect
Cell Wall Formation
4. Affect
Nucleic Acid Structure
5. Affect
Metabolism
C. Types
of Germicides
1. Surfactants
a. Structure
- compounds with hydrophilic & hydrophobic parts.
b. Mode
of action - Penetrate oily substances in water & break them apart into small droplets
that become coated with surfactant molecules. The
hydrophobic end of the surfactant stick into the droplets & the hydrophilic end is
attracted to the water. The result is an
emulsion, a fine suspension of oily droplets in water, which can now be rinsed away.
c. Effect
of soaps & detergents - wash away microbes, but do not kill them.
d. Wetting
agents are
surfactants that are often used with other chemical agents to help the agent penetrate
fatty substances. Surfactants are not
germicidal by themselves!
e. Quaternary
ammonium salts – four organic groups attached to a nitrogen atom. Effect: kill
all classes of cellular microbes & enveloped viruses by disrupting membranes. Uses: nontoxic & widely used in the home,
industry, labs, & hospitals. Their
effectiveness is decreased in the presence of soap. Actually
support Pseudomonas growth! Now being mixed with other agents to overcome some
of these problems.
2. Phenol & Phenolics
a. Structure
- compounds with hydroxyl groups (-0H) attached to a benzene ring.
Mode
of Action - denature cell proteins, disrupt cell membranes.
b. Effect
- kill most organisms; action is not impaired by organic materials (remain active even in
the presence of blood, feces, etc.)
c. Examples:
1.) Lysol
2.) Cresol
– found in creosote; plant derivative used to prevent the rotting of wooden posts,
fences, railroad ties.
3.) Hexachlorophene
- chlorinated phenolic; effective as an antiseptic; once widely used as an ingredient in
soaps & lotions; in 1970's was found to increase risk of brain damage in babies; has
now been replaced with chlorhexidine in
hospitals – good agent for surgical scrubs.
3. Alcohols
a. Structure
- compounds with a hydroxyl group (-OH).
b. Mode
of Action – when mixed with water disrupt lipids in cell membranes & denature
proteins.
c. Ethanol
& Isopropanol
- widely used as skin antiseptics; a 50 to 70% solution in water is the most effective
concentration (one of the few exceptions to the rule: increase effectiveness by increasing
concentration); does not sterilize skin because it evaporates quickly and does not
penetrate deeply enough into skin pores.
d. Main disadvantage - do not
kill endospores.
4. Halogens
a. Mode
of Action - inactivates enzymes by oxidation.
b. Examples
1.) Iodine
–
antiseptic
a.) Tincture
– iodine
in a dilute alcohol solution; one of first skin antiseptics.
b.) Iodophor
– mixture
of iodine and surfactants; ex. Betadine and Isodine (used for surgical scrubs and to prepare
skin for surgery)
2.) Chlorine
–
disinfectant; ingredient in household bleach; added to drinking water and swimming pools;
inactivated by the presence of organic materials.
5. Hydrogen peroxide
a.
Mode
of Action – oxidizing agent (denatures proteins)
b.
Uses
of H2O2: antiseptic for
cleaning wounds, disinfect medical instruments & soft contact lenses. When H2O2 comes into contact
with tissue, it bubbles producing oxygen gas. This
is because all aerobes (incl. eukaryotes) produce the enzymes catalase & peroxidase
which decompose H2O2 into oxygen & H2O. H2O2 generally kills
microbes before it is destroyed by catalase or peroxidase.
(You can differentiate between Staphylococcus & Streptococcus
using H2O2; Staph
is relatively resistant to H2O2 because of the large amounts of
catalase & peroxidase it produces.) May
be used to clean deep puncture wounds, because the oxygen produced kills obligate
anaerobes present in the wound (ex. Clostridium).
6. Heavy Metals
a. Mode
of Action - heavy metals (mercury, copper, silver) react with the sulfhydryl groups of
proteins (denaturation)
b. Effect
- kills many microbes.
c. Examples:
1.) Mercuric
chloride
- once widely used as an antiseptic; highly toxic; now merthiolate & mercurochrome are used (less toxic); merthiolate is prepared as a tincture; use - basic
first aid kit supplies for disinfecting skin & mucous membranes.
2.) Silver
Nitrate
- once applied to eyes of newborns to prevent gonorrhea; the trend for a while was toward
using antibiotics instead, but the development of antibiotic-resistant strains has
necessitated the use of silver nitrate again.
3.) Selenium
sulfide
– kills fungi, including spores; commonly used to treat fungal skin infections;
included in dandruff shampoos (dandruff is often caused by a fungus).
7. Alkylating Agents
1. Mode
of action - they alkylate (attach short chains
of carbon atoms) to proteins and nucleic acids. Must
not be used where they may effect human cells (these agents are carcinogenic).
2. Formalin
- 37% solution. of formaldehyde used to preserve tissues & to embalm; kills all
microbes, including spores; lower concentrations are used to inactivate microbes for
killed vaccines.
3. Glutaraldehyde
- used to sterilize surgical instruments if equipment for heat sterilization is not
readily available.
4. Ethylene
oxide
- gas; advantages: disappears from the object after treatment; disadvantage: extremely
toxic to humans so must be used in a sealed chamber; kills all bacteria, including
endospores; used to sterilize materials destroyed by heat (plastic, rubber gloves, animal
feed, mattresses, telephones).
8. Dyes
– Ex.
Crystal violet blocks cell wall synthesis. It
effectively inhibits growth of G(+) bacteria in cultures and in skin infections. It can be used to treat yeast infections.
A.
Temperature
- Environmental
factor most often used to preserve food. Canning
is the oldest method. Two factors, time &
temp., determine safe heat treatments for canning. Refrigeration
is low enough to stop the growth of most microbes. Psychrophilic
(ex. Listeria) microbes are the exception. See pg. 1 of this handout for more info. on temp.
B.
pH
- Acidity
(low pH) prevents the growth of most microbes, especially in an anaerobic environment. Ex. adding vinegar (acetic acid) to foods. Low pH also increases the effectiveness of heat
treatments (ex. acidic foods like tomatoes can be canned merely by boiling).
C.
Drying
- drying
& salting do not sterilize but preserve food by making it unable to support microbial
growth for lack of water, an essential nutrient. See
pg. 1 & 2 of this handout for more info. on drying & freeze drying.
D. Chemicals
- Various chemical preservatives are added to commercially prepared foods. Ex.:
1. calcium propionate - antifungal agent added to
bread.
2. sorbic acid - antifungal agent added to soft
drinks, salad dressings, cheeses.
4. sodium
benzoate
- antifungal agent added to soft drinks, salad dressings, cheeses.
5. sodium
nitrate (nitrite) - antibacterial agent that prevents
germination of Clostridium botulinum spores when added to bacon, ham, hot dogs.