Chapter 7

he title of this chapter, Control of Microbial Growth, is essentially a contradiction in terms. We

live in a world where about one-half of the total biomass is microbial, and our bodies contain more
microbial cells than human cells. In reality, we coexist with microorganisms. We attempt to keep
the harmful ones at bay and foster the growth of those that are helpful. But regardless of our efforts,
they are always around, and some can pose a serious threat. Their growth is never really under
total control.
Microbes do play an indispensable role in the cycles of matter, and many of these contributions
are presented in Chapter 12. This positive role is a consequence of broad metabolic diversity among
microorganisms. Microorganisms have evolved as recyclers and, as a consequence, can grow on
and destroy our food, our clothing, our buildings, and even our bodies.
They have a virtually unlimited ability to metabolize organic compounds, thus returning car-
bon dioxide to the atmosphere. Some microorganisms are obligately parasitic, and these survive
by feeding on plants, animals, or other microbes. Others are opportunists that, under appropriate
conditions, cause disease.
To counteract a constant threat of destruction by microorganisms, humans have spent much
money and effort in devising preservation methods and have met with moderate success. Human
populations have historically suffered from the inability to understand or gain more than a limited
amount of control over microorganisms. In early times before microorganisms were known and
understood, whatever anyone did to thwart the destructive microorganism was the result of an
empirical observation that something worked. Thus, Nicholas Appert discovered in 1810 that one
could preserve food by heating it in a closed container (canning), American Indians dried meat,
Norsemen salted fish, and many civilizations made cheese.
During the last 130 years, humans have come to understand the microbe, and our strategies have
changed. We have developed procedures whereby, to a limited extent, microbes can be controlled.
This chapter discusses many of the procedures and processes that are employed to control the
microbe in areas where this is desirable. We sterilize with heat and chemicals, we filter air or liq-
uids, and antibacterial chemicals are now available to cure disease. We have developed immu-
nization methods and public health measures to survive in a world of the ubiquitous microbe.
In order to use chemotherapy successfully, we must search for substances which have an affin-
ity to the cells of the parasites and a power of killing them greater than the damage such sub-
stances cause to the organism itself, so that the destruction of the parasites will be possible
without seriously hurting the organism.
PAUL EHRLICH,
FATHER OF CHEMOTHERAPY, 18541915
HISTORICAL PERSPECTIVE
The early civilizations used many methods to protect
food from microbial destruction. These methods includ-
ed the salting of meat, fish, and vegetables. As discussed
in the previous chapter, salting removes available water
and prevents bacterial growth. Certain bacterial species
either alone or combined with salt proved effective in
preserving vegetables, such as with cabbage to produce
sauerkraut. Long ago, people found that fermented milk
products such as cheese, butter, and yogurt were stable
for some length of time even without refrigeration.
Acidification with vinegar (pickling) was also effective
in preserving selected food products.
The development of effective control measures for
microorganisms in general parallels the evolution
of microbiology as a science (see Chapter 2). Several
important early workers in medicine discovered micro-
bial control techniques that significantly decreased
the number of deaths during medical procedures. It is
generally acknowledged that Ignaz Semmelweis (1816
1865) pioneered the use of disinfectants. He insisted that
all individuals working in his hospital in Vienna wash
their hands in chlorinated lime before working with
patients. This brought about a marked decrease in dis-
ease and death in the obstetrics ward. Joseph Lister
(18271912) was able to conduct aseptic surgery by ster-
ilizing instruments with carbolic acid (phenol) and
spraying phenol around the room during surgery. As
discussed in Chapter 2, Robert Koch demonstrated that
mercuric chloride would kill all bacteria tested, includ-
ing the highly resistant endospore.
John Tyndall (see Chapter 2) developed an early and
effective sterilization procedure, termed tyndallization.
In this procedure, a broth would be steamed for a few
minutes on three or four successive occasions separated
by 12- to 18-hour intervals of incubation at a favorable
temperature. This would permit dormant spores to
become vegetative cells and be killed by boiling.
The invention of the autoclave in Pasteurs laborato-
ry revolutionized laboratory work, as it permitted one
to sterilize media and to work under aseptic conditions.
HEAT STERILIZATION AND
PASTEURIZATION
Sterilization is the destruction of all viable forms of life,
including endospores. Aproperly packaged sterile mate-
rial should be devoid of all life until opened and
exposed to the outside environment. Sterilization can be
attained by various procedures including fire, moist
heat, dry heat, radiation, filtering, and toxic chemicals.
Any procedure that disrupts an essential component of
the cellprotein, nucleic acid, or cytoplasmic mem-
branemay lead to a loss of cell function. The more
extensive the disruption, the greater the chance that the
damaged microorganism will die. When you watch an
egg fry, you are witnessing the effectiveness of heat in
denaturing (breaking down) egg protein. Some proteins
are more stable than others at high temperatures, but
this is only a matter of degree. Given sufficient heat, the
structural and intrinsic function of any protein will be
lost. Heat, in some form, is the agent most often
employed for routine sterilization.
Studies in a microbiology laboratory are dependent
on the ability to sterilize media, inoculating loops, glass-
ware, and all material involved in culturing microor-
ganisms. Most studies are performed with pure cultures
(only one strain present), and this can be accomplished
only when all manipulations are done under sterile
conditions. Sterility is also an important requirement for
surgical instruments, bandages, and in most medical
procedures.
Several factors can affect any sterilization procedure.
First, the presence of organic matter can protect Bacteria
or Archaea from the destructive effects of heat, chemi-
cals, or radiation. Second, a material to be sterilized usu-
ally harbors a mixed population of microorganisms.
Consequently, the regimen selected for sterilization must
be designed to kill all of the most resistant organisms
present and under the conditions that exist.
The pH of the suspending medium is also a significant
factor. In an acidic medium, heat is considerably more
effective in killing microorganisms than at neutrality. This
has been a major factor in home canning, where acidic
foods, such as fruits and tomatoes, rarely spoil. Foods that
have a more neutral pH, such as corn, peas, and beans,
spoil more often because on harvesting they harbor
endospore-forming organisms that may survive the can-
ning process. On rare occasions, the anaerobic spore-
forming organism Clostridium botulinum may survive in
improperly canned foods. This organism then may ger-
minate and grow, producing botulinum toxin, which caus-
es botulism. Ingestion of minute amounts of botulinum
toxin can lead to death. This was a danger in the days of
extensive home canning, when inefficient or improperly
used pressure cookers were sometimes employed.
Technique of Heat Sterilization
Microbial death by heating occurs at an exponential rate
(Figure 7.1); that, is a fraction of the cells present in a
heated solution will be killed per unit of time. The effect
of heat on the survival of a bacterium is illustrated in the
figure and affirms that a higher temperature (95C) kills
more rapidly than the lower temperature (75C). Asur-
vival curve can be obtained by suspending bacterial
cells in water and heating to a selected temperature. At
intervals, an aliquot can be removed and the number of
viable cells present determined by a plate count as
described in Chapter 6.
148 CHAPTER 7
Three general techniques are available for killing
microorganisms by heat: boiling, steam under pressure,
and dry heat. Boiling for 15 minutes will kill virtually
all vegetative bacterial and archaeal cells, viruses, and
fungi, but it does not generally kill bacterial endospores.
The length of time required to reduce a population ten-
fold (for example, from 10
6
to 10
5
) is the decimal reduc-
tion time, designated D. Endospores of various bacter-
ial species have differing resistances to boiling (100C),
as indicated by the D values (Table 7.1).
Acomparison of the decimal reduction times can dis-
tinguish between a heat-sensitive microor-
ganism and one that is relatively resistant
(Figure 7.2). The time required to sterilize a
suspension of endospores varies over a wide
range, with Bacillus anthracis requiring less
than 5 minutes. In contrast, a suspension of
Clostridium botulinum endospores can be heat-
ed for 5 hours or more at 100C, and some of
the endospores will survive. If placed in an
appropriate medium, they can germinate and
grow. It is clear that boiling water has its limi-
tations for sterilization.
Moist HeatSteam under Pressure The most
effective sterilization system is steam under
pressure. This method is widely used in the
research laboratory and throughout the food
and pharmaceutical industries. The instrument avail-
able for accomplishing this is the autoclave. An auto-
clave is a closable metal vessel that can be filled with
steam at greater than atmospheric pressure. The auto-
clave is designed so that the air present inside when the
door is closed and sealed is driven out by steam. A
valve closes automatically when the air passing out-
ward reaches 100C, an indication that steam now fills
the entire inner space. Laboratory autoclaves operate at
15-psi (pounds per square inch) pressure, and at this
pressure, the temperature rises to 121.6C. The auto-
clave must be constructed to withstand the internal
pressure produced when water boils. At this tempera-
ture, sterilization of routine material can be achieved
within 10 to 15 minutes.
Large batches of material must be autoclaved for a
longer time because heat transfer to the interior can be
slow. The denser the material, the slower the heat is
CONTROL OF MICROBIAL GROWTH 149
Perry / Staley Lory
Microbiology 2/e, Sinauer Associates
Figure 07.01 Date 11/27/01
5
4
6
3
L
o
g
n

o
f

t
o
t
a
l

s
u
r
v
i
v
o
r
s
2
1
0
2 0 4 6
Exposure time (minutes)
95C
75C
8 10 12
A decimal reduction has occurred at this
point: 90 percent of the microbes initially
present have been killed. At 75C, the
decimal reduction time is 90 seconds.
Figure 7.1 Effect of temperature on survival
of bacterial cells
An exponential plot of number of surviving cells versus time
exposed to heat, at two different temperatures, for the vege-
tative cells of a mesophilic bacterium.
100
D
e
c
i
m
a
l

r
e
d
u
c
t
i
o
n

t
i
m
e

(
m
i
n
u
t
e
s
)
10
1.0
0.1
Temperature (C)
A B
125 120 115 110 105 100
Figure 7.2 Decimal reduction time and heat sensitivity
The decimal reduction times for two organism: one relative-
ly heat sensitive (A), the other more heat resistant (B). The
rate of killing increases at higher temperatures.
Heat resistance of bacterial endospores
obtained from various genera and species in
the genus Bacillus. Decimal reduction time at
100
o
C
DValue
a
Organism (minutes)
Bacillus cereus 0.8
Bacillus megaterium 2.1
Bacillus cereus var. mycoides 10.0
Bacillus licheniformis 24.1
Bacillus coagulans 270.0
Bacillus stearothermophilus 459.0
a
Time required to reduce the population tenfold.
Table 7.1
transferred and the longer the time required for sterili-
zation. Thus, it takes longer to sterilize a flask contain-
ing 1 liter of liquid than it does when the same flask is
empty. The autoclave is effective for the sterilization of
most laboratory growth media. Selected organic com-
pounds such as vitamins and antibiotics may not be sta-
ble at autoclave temperatures and should be sterilized
by filtration or other less-destructive means, as discussed
later.
Dry Heat Dry heat is effective in sterilizing glassware,
pipettes, and other heat-stable solid materials. Pipettes
can be placed in metal canisters, glassware wrapped in
paper, and flasks and other vessels capped with alu-
minum foil. This protects the objects from contamination
when they are handled subsequent to sterilization. An
oven that can be heated to 160C to 180C is used for dry
heat sterilization, and the materials are retained at this
temperature for 2 to 4 hours. Dry heat has been preferred
for sterilizing pipettes, syringes, and other heat-
stable objects, as autoclaving left them moist. However,
most laboratory autoclaves now have a vacuum cycle that
will remove moisture from such objects after sterilization.
Pasteurization
Pasteurization is a process that employs low heat and
is often applied to milk products and other heat-sensi-
tive foods. Pasteurization reduces the microbial popu-
lation but does not sterilize the product. The method
was devised by Louis Pasteur to control wine spoilage.
Pasteurization was originally accomplished by heating
the material to 63C to 66C and holding it at this tem-
perature for 30 minutes. This
method will kill 79% to 99% of
the microorganisms present
in milk. A short-term flash
method is now generally
employed, whereby milk is
passed through coils where it
is heated quickly to a temper-
ature of 71.6C for 15 seconds.
Pasteurization of milk was
adopted as a common practice
in the dairy industry because
raw milk often contained
Mycobacterium tuberculosis, the
causative agent of tubercul-
osis (TB), and/or Brucella abort-
us, which causes brucellosis.
These organisms have now
been mostly eliminated from
dairy herds in the United
States through mandatory
testing. However, pasteuriza-
tion is still employed as a pre-
caution and to extend the shelf life of certain food prod-
ucts, including beer and milk. Due to the widespread
consumption of dairy products, the potential spread of
disease by this consumable is a constant threat.
STERILIZATION BY RADIATION
Selected wavelengths of the electromagnetic spectrum
(Figure 7.3) can cause the death of living organisms, thus
making radiation a practical method of sterilization. The
two types of radiation that are effective microbe killers
are ultraviolet (UV) light and ionizing radiation. Light
in the UV range can disrupt cellular DNA and some
other cell constituents, whereas ionizing radiation can
cause harm directly and indirectly to all constituents of
a cell. Gamma () rays, X-rays, alpha () and beta () par-
ticles, and neutrons are all forms of ionizing radiation.
Ionizing Radiation
Gamma rays, as with other forms of ionizing radiation,
kill cells by causing the formation of highly reactive free
radicals (see Chapter 6). These free radicals, such as the
hydroxyl radical (OH), can react with and inactivate
any of the various macromolecules present in a cell. It is
probable that all macromolecules in a cell are equally
susceptible to damage from ionizing radiation. Because
there are multiple copies of most cellular macromole-
cules, damage to a limited number of the copies of a
protein will not cause cell death. Generally, however, the
cellular DNA will contain a single copy of a specific
gene, and disruption of a gene can cause cell death.
Ionizing radiation is destructive to genetic material
150 CHAPTER 7
400 nm 700 nm 600 nm
Radio waves Microwaves Infrared
UV
light
X rays Gamma rays
500 nm
Increasing wavelength
Increasing energy
Violet Blue
Blue-
green Green
Yellow-
green
Visible light
Yellow
100 m 1 m 1 cm 0.01 cm 1000 nm 10 nm 1 nm 0.001 nm
Orange Red
Figure 7.3 The electromagnetic spectrum
Humans can detect visible wavelengths, which range from about 400 nm (violet)
to 750 nm (red).
because it causes single- and double-strand breaks in the
DNAdouble helix.
The sensitivity of different organisms to ionizing
radiation varies considerably. Endospores of Clostridium
or Bacillus species are highly resistant, as are cells of
Deinococcus radiodurans. D. radiodurans is exceedingly
resistant to UV or gamma radiation and also to most
mutagens. Materials that have been exposed to ionizing
radiation often bear this organism as the sole survivor.
The ability to repair DNAdamage efficiently and rap-
idly is a major survival factor in radiation-resistant
microorganisms.
Gamma rays are an effective sterilizing agent because
they can penetrate deeply into objects such as contain-
ers. Consequently, sterilization can be accomplished
after a product is packaged. Major sources of usable
radiation for sterilization include cobalt-60 (gamma
rays), X rays, and the neutron piles that are available at
nuclear plant sites. The radiation source most often used
commercially is cobalt-60. Generally, items that are heat
sensitive and nonfilterable are sterilized by radiation.
Among the items sterilized in this way are plastics,
antibiotics, serums, various medicinals, and in some
cases, food.
Ultraviolet Light
Wavelengths of electromagnetic radiation in the ultra-
violet range (UV) between 220 and 320 nm are particu-
larly important biologically because cellular constituents
can absorb these wavelengths. More intense, shorter
wavelengths of UV (less than 220 nm) are emitted by the
Sun but are absorbed by the ozone layer in the Earths
upper atmosphere. This shorter wavelength UV radia-
tion is lethal to microorganisms and harmful to many
other organisms as well. This has caused the present
concern over the damage to the ozone layer over the
Arctic and Antarctic areas.
Germicidal lamps and lights can be constructed to
emit the short, damaging UV wavelengths. UV lamps
with this potential may be placed in sterile rooms and
safety cabinets to sterilize the air, but cannot be operat-
ed when people are present. These wavelengths burn
skin and may damage the eyes. UV radiation does not
penetrate glass, water, or nongaseous substances to a
significant extent. This limits the effective use of UV
radiation to sterilization of surface areas and air.
Cellular components that strongly absorb UV light
are the purines and pyrimidines. Both are present in
DNAand RNAand absorb UV maximally at 260 nm. A
UV lamp will produce levels of radiation that can effec-
tively penetrate bacterial, archaeal, or other viable cells
and cause alterations in cellular nucleic acids. The pro-
duction of thymine dimers is probably the most impor-
tant result of UV damage to DNA. Adimer is formed by
the linkage of two adjacent thymine molecules on a sin-
gle strand of DNA. Such linkages prevent the replica-
tion of DNA, and this can be lethal to a cell. Thymine-
cytosine or cytosine-cytosine dimerization occurs less
frequently and can disrupt DNAreplication.
Microbes have DNArepair systems that can excise
the dimers and replace them with unaltered thymine
molecules. UV light is lethal if a sufficient number of
dimers are created such that all of them cannot be
repaired. In some cases, the enzyme repair system will
make an error, which leads to mutations or death.
STERILIZATION BY FILTRATION
The ability of filters to retain bacterial and archaeal cells
and allow viruses and other small particles to pass
through has been recognized for over 100 years. In 1892,
Dmitri Ivanowsky, a Russian scientist, demonstrated
that tobacco mosaic virus would pass through a filter
that would retain the smallest bacteria. This was con-
firmed and expanded by Martinus Beijerinck a few
years later (see Chapter 2).
Filters have proven useful for the selective steriliza-
tion of liquids and gases. Heat-sensitive materials, such
as enzymes, vitamins, and sugars, can be filtered to
remove contaminating bacteria. Beer and wine are now
filtered in bulk to remove bacteria that would oxidize
the ethanol to acetic acid and render these drinks unac-
ceptable (see Chapter 31). Virologists routinely filter
blood serum and other heat-sensitive culture media to
prevent unwanted bacterial growth. Media for plant or
animal cell cultures are generally complex, and compo-
nents break down at high temperatures. They, too, are
sterilized by filtration.
Basically, a filter is constructed with a pore size that
permits liquids to pass, but the pores are small enough
to retain Bacteria and Archaea. These organisms, in gen-
eral, are longer than 1 m and are greater than 0.5 m in
diameter. Because some bacterial cells are smaller than
this, effective filters must be able to retain cells that are
quite small.
In practice, a filter can also be employed to separate
or concentrate microbes based on their size. Bacteria can
be separated from yeasts by employing a membrane
with a pore diameter of about 3 m. Most bacteria can
pass through a filter such as this, but yeasts would be
retained. Small bacteria such Bdellovibrio, an organism
that parasitizes other bacteria, can be concentrated by
passing water or dilutions of soil through membrane fil-
ters with a pore diameter of 0.45 m. The Bdellovibrio are
only 0.25 m in width and would pass through, where-
as most other microorganisms would be retained. The
organisms in the filtrate can then be concentrated by
centrifugation.
Most early filters were composed of stacks of
asbestos, sintered glass (glass heated to fuse without
CONTROL OF MICROBIAL GROWTH 151
melting), or diatomaceous earth. These materials formed
circuitous passageways that retained microbial cells.
Several different types of filters are used today. Aglass
fiber depth filter is a random stacking of glass fibers
that forms a barrier impenetrable by bacteria and other
particles (Figure 7.4A).
The filters that are now in common use are mem-
brane filters made of cellulose acetate, polycarbonates,
or cellulose nitrate (Figure 7.4B). These filters are quite
thin (200 m) and become relatively transparent when
wet, allowing direct microscopic examination of
microorganisms that are trapped on the filter. Membrane
filters can be produced with pore sizes ranging from 0.1
to 0.5 m, with 80% to 85% of the filter being open space.
This allows a rather high rate of fluid passage. With this
much open space available, a filter can be placed on the
upper surface of an agar medium, and nutrients will dif-
fuse to the cells through the filter by capillary action.
Following incubation and growth, one can count the
number of colonies that develop directly on the filter and
observe their colonial morphology (see Figure 6.5).
The Isopore membrane filter has widespread appli-
cation in research and industry. It contains clearly de-
fined cylindrical holes that pass vertically through the
membrane (Figure 7.4C). These filters are manufactured
by treating thin sheets of polycarbonate with nuclear
radiation. The radiation results in a random distribution
of minute holes in the polycarbonate that can be
enlarged by chemical etching. The length of time that
the membrane is etched determines the size of the pores.
Amajor advantage of these filters is that bacteria and
other objects remain on the surface of the membrane
and are thus readily observable with a scanning electron
microscope (Figure 7.4C). With other types of membrane
filter, the bacteria absorb to the sides of the pores and
are not so readily observable. One problem with the
Isopore filters, however, is the limited total space on the
filter that is porous. This may result in low flow rates or
clogging.
Presterilized disposable filter units available com-
mercially are now routinely employed in the laborato-
ry. In industry, much larger and more sophisticated fil-
tration systems are used for large volumes of liquid.
These larger volumes are sterilized by utilizing the fil-
ters placed in large stainless-steel cartridges. As an alter-
native to centrifugation, particularly for large volumes,
bacteria can also be harvested from culture fluids by
using membrane filters.
TOXIC CHEMICAL STERILIZATION
OR CONTROL
We previously mentioned that some of the nineteenth-
century scientists made use of chemical disinfectants.
Included were Koch (mercuric chloride), Semmelweis
(chlorinated lime), and Lister (phenol). Today we have
a vast number of chemical agents available for use as
disinfectants or to control the growth of undesirable
microorganisms. These range from toxic gases to the
medically important antibiotics. Following is a discus-
sion of the chemicals now employed to kill or control
microorganisms.
152 CHAPTER 7
Figure 7.4 Scanning electron micrographs of filters
Scanning electron micrographs of (A) a glass fiber depth filter; (B) a membrane filter;
(C) Thermoleophilum album trapped on an isopore filter. Courtesy of: AB, Millipore
Corporation; C, Jerome Perry.
(A) (B) (C)
Types of Antimicrobial Agents
An array of chemical agents are now available
that can be used in the control of undesirable
microorganisms. These chemicals are called
antimicrobial agents, or antimicrobials (Table
7.2). Some of the agents are naturally occur-
ring, some are synthetic, and others are a
combination of the two.
An effective method for sterilizing surgical
instruments and some heat-sensitive materi-
als is to expose them to toxic compounds in
the form of gases. Sheets and bedding in a
hospital are sterilized in this manner. Toxic
gases are also used to sterilize plastic Petri
dishes and other plastic laboratory containers.
Ethylene oxide and propylene oxide are gen-
erally the compounds of choice. Both function
as alkylating agents in disrupting cellular
DNA. Because these gases are exceedingly
toxic to humans, their use in the sterilization
process must be carefully controlled.
Amuch-desired property of an antimicro-
bial agent is selective toxicity. That is, the
agent will eliminate a pest without doing sig-
nificant harm to other species in the environ-
ment. Spraying plants with a chemical that
kills invading bacteria or fungi but also harms
the plant is of little value. Humans cannot be
cured of disease by treating them with medi-
cines that are overly harmful to the host (see
the quote at beginning of this chapter).
In addition, humankind has become more and more
concerned with the effects of chemical agents on the
total environment. Experience has taught us the harm
that can be done by careless application of antimicrobial
substances. For example, for years, compounds con-
taining mercury were used in the pulping industry and
other industrial processes to control microbial contami-
nation. These mercury compounds ultimately entered
streams and rivers, resulting in considerable harm to
fish and other wildlife. Stringent regulations are now in
place for all potentially toxic compounds that are artifi-
cially introduced into the environment. They must be
tested for safety, biodegradability, carcinogenicity, or
other undesired effects on humans.
Another example of a carelessly applied agent is
DDT, which was once applied widely as an insecticide.
DDT was very effective against insects, but it accumu-
lated in the natural environment and was very harmful
to birds and other wildlife. Accumulation resulted from
the inability of microbes to mineralize DDT at rates com-
mensurate with application rates. DDT had a half-life of
about 30 years in most environments, and its use has
now been curtailed in the United States.
Acompound that kills a target group is identified by
combining the designated organisms name with the
suffix -cide, from the Latin cida, meaning having power
to kill (Table 7.3). Other agents are available that do not
kill but rather prevent or inhibit growth. These are
referred to as bacteriostatic agents (for bacteria) or
fungistatic agents (for fungi). Germicide is a general
term that describes substances that kill or inhibit
microbes (germs). Some antibacterial substances bring
about lysis, or disintegration, of the cell and are thus
CONTROL OF MICROBIAL GROWTH 153
Common antimicrobial agents and their uses
Compound Use
Organic compounds
Cresols Disinfectant in laboratories
O-Phenylphenol Disinfectant in laboratories
Phenol Disinfectant in laboratories
Formaldehyde Disinfect instruments
Quaternary ammonium Skin antiseptic
compounds
Ethanol and isopropanol Disinfect instruments
Ethylene oxide Sterilize instruments
Propylene oxide Sterilize instruments
Halogens
Chlorine gas Disinfect water
Iodine solution Skin antiseptic
Hexachlorophene Skin antiseptic
Chlorine bleach Domestic cleaning
Heavy metals
Mercury chloride Disinfectant
Silver nitrate In infant eyes to prevent
ophthalmic gonorrhea
Copper sulfate Algicide
Other
Hydrogen peroxide Skin antiseptic
Mercurochrome Skin antiseptic
Ozone Drinking water and fish tanks
Table 7.2
Categories of killing agents and their
target organisms
Agent Target Organism
Bactericide Bacteria
Fungicide Fungi
Algicide Algae
Pesticide Pests
Herbicide Plants
Insecticide Insects
Germicide Germs
Table 7.3
termed bacteriolytic agents. For example, lysozyme is
a lytic enzyme that destroys the cell wall of gram-posi-
tive bacteria. Loss of the cell wall results in water uptake
and subsequent bursting of the wall-less cell.
Adisinfectant can destroy microorganisms on con-
tact, including those that potentially cause disease.
Phenol and mercuric chloride are disinfectants. Disin-
fectants are indiscriminate destroyers that, if applied to
wounds, would also disrupt host tissue. An antiseptic
is a substance, such as Merthiolate or Mercurochrome,
that kills or prevents the growth of disease-causing
pathogens. An antiseptic should not do significant harm
to host tissue. As a rule, we use powerful disinfectants
such as phenol on inanimate objects such as laboratory
benches and less-potent antiseptics to treat wounds.
Achemotherapeutic agent is a chemical compound
that can be applied topically, taken orally, or injected
that will inhibit or kill microbes that cause infections.
The term is also applied to compounds that are effective
against viruses or tumor growth. Antibiotics are some-
times called chemotherapeutic agents and are widely
used as antimicrobials, but the term chemotherapeu-
tic is generally reserved for products of chemical syn-
thesis. The antibiotics will be discussed later.
Tests for Measuring Antimicrobial Activity
Robert Koch (18431910) first devised a laboratory pro-
cedure for quantifying the effectiveness of antimicrobial
compounds. Koch dried endospores of Bacillus anthracis
on threads and placed these in solutions of potential
antibacterial agents. Endospores were selected because
they are more resistant to harsh physical con-
ditions than other life forms.
Koch then estimated the effectiveness of a
given compound by determining how long
the endospores would survive exposure to the
agent. At selected time intervals, threads were
removed from the test solution and placed in
a nutrient medium. Growth indicated sur-
vival. The shorter the time required to kill all
of the endospores, the more effective the dis-
infectant. Koch discovered that mercuric chlo-
ride was one of the most effective of all avail-
able compounds. Mercuric chloride has been
employed as a disinfectant up to the present
day. Unfortunately, the use of large quantities
of this mercury compound is discouraged
because of the environmental problems it
causes (see Chapter 24).
The relative strength of an antimicrobial
can be determined by serial dilution of the
parent compound. The more a compound can
be diluted and still inhibit the growth of a test
organism, the greater its relative strength. The
lowest concentration that will inhibit growth
is designated as the minimum inhibitory concentra-
tion, or MIC (Figure 7.5). Many factors affect the MIC,
including the test organism selected, the inoculum size,
and the amount of organic material present. Astrong
germicide, such as ethylene oxide or formaldehyde (37%
solution), will kill endospores and virtually all forms of
life. Intermediate-strength antimicrobials such as phe-
nolics can kill viruses and most vegetative cells but are
less effective against endospores. Such agents are used
for research and in hospital laboratories. Antimicrobials
with less killing power are effective against vegetative
cells (fungal and bacterial) but are not very effective
against endospores, fungal spores, or some viruses.
These weaker agents have less toxicity to humans and
can be applied directly to the skin or can be included as
a component in mouthwash. The general mode of action
of various germicides is in the destruction of proteins
(Table 7.4).
One standard that has been employed to compare the
relative efficiency of germicides is the phenol coeffi-
cient. Aphenol coefficient is a measure of the effective-
ness of a test compound compared with the disinfecting
power of phenol. For example, if a new antimicrobial at
a dilution of 1:100 kills a standard population of
Salmonella typhi that is killed by a 1:50 dilution of phe-
nol, the phenol coefficient would be 100/50, or 2. The
test antimicrobial would thus be twice as effective as
phenol in destroying the test population.
However, the phenol coefficient has some limitations,
as many factors can influence the effectiveness of disin-
fectants. Some are more affected by the physical envi-
154 CHAPTER 7
Figure 7.5 Dilution series for estimating antibiotic sensitivity
Atube dilution series for estimating the sensitivity of a microorganism
(in this case E. coli) to antibiotic (here, chloramphenicol). The minimum
inhibitory concentration (MIC) is the least amount of antibiotic that com-
pletely inhibits growth. Courtesy of Jerome Perry.
ronment including light, oxygen, organic matter, and
solid substrates. Gram-positive organisms are more
readily killed than gram-negatives. Members of the
genus Pseudomonas are not only resistant to, but may
also grow on, selected disinfectants. The ability of
pseudomonads to thrive in the presence of antiseptics is
of particular concern for burn patients.
CHEMOTHERAPEUTIC AGENTS
The antimicrobial agents discussed
in the previous section may be
used to limit the growth of unde-
sirable organisms in the environ-
ment. They may be employed as
disinfectants on laboratory bench-
es or other solid material but are
not suitable for control of viral,
bacterial, or fungal infections with-
in the animal or human body. They
would either be too toxic or be
inactivated by the presence of
organic matter.
From the time of Paul Ehrlich
(18541915), scientists have sought
chemicals that selectively inhibit
the growth of infectious bacteria
without harm to the human host.
Ideally, such a chemotherapeutic
agent would be specific for the
microbe invading the body but
with little or no harmful effect on
normal body function. There is a
constant search for better chemo-
therapeutics, including antibacteri-
als, antivirals, and antitumor agents.
Following is a discussion of some
of the chemotherapeutics that have been syn-
thesized and are effective against disease-
causing microorganisms.
Chemically Synthesized
Chemotherapeutics
Paul Ehrlich is considered the father of
chemotherapy (see quote at the beginning of
the chapter). He originated the concept that
invading pathogenic organisms might be
destroyed by selective chemicals that would
not seriously harm the host (see Chapter 2).
Others, aware of his contributions, have
sought chemical compounds that might be
employed as chemotherapeutic agents.
The sulfa drugs are chemotherapeutic
agents that were discovered by Gerhard
Domagk in the 1930s. Domagk was assessing
the potential use of dye substances for antibacterial
activity following the approach pioneered by Paul
Ehrlich. Sulfanilamide was one of the compounds test-
ed, and Domagk found that it possessed antibacterial
activity both in vitro [Latinin glass (outside the body)]
and in vivo [Latinliving (inside the body)]. Sulfa-
nilamide is a structural analog of the p-aminobenzoic
acid, a component of the B vitamin folic acid (Figure
7.6). Folic acid is a coenzyme involved in nucleic acid
synthesis. It is synthesized by enzymatically joining a
CONTROL OF MICROBIAL GROWTH 155
Mode of action of some antimicrobial agents
Agent Mode of Action
Alcohols Denature proteins and dissolve membrane lipids.
Aldehydes Combine with proteins and denature them.
Halogens Iodine oxidizes cellular constituents and can
iodinate cell proteins. Chlorine and hypochlorite
oxidize cell constituents.
Heavy metals Combine with proteins generally through
sulfhydryl groups.
Phenolics Denature proteins and disrupt cell membranes.
Quaternary Disrupt cellular membranes and can denature
ammonium proteins.
compounds
Table 7.4
CH
2
CH
CH
2
CH
2
C
N
H
2
N S
O
O
N
C C
O
O O

O
O

N
H H
H
H
Sulfanilamide
has the structure:
H
2
N
O
6-Methylpterin PABA Glutamate
Folic acid
H
2
N C
O
OH
PABA
(p-aminobenzoic acid)
HN
N N
N
The end of this molecule may
have additional glutamates,
added as -glutamyl residues.
Sulfanilamide competes
with PABA for addition
to 6-methylpterin to
form folic acid.
Figure 7.6 Sulfa drug as structural analog
Sulfanilamide is a competitive inhibitor of metabolic function in microorganisms. It is
an analog of and competes with p-aminobenzoic acid in the synthesis of the vitamin
folic acid.
molecule of 6-methyl pterin with p-aminobenzoic acid
and a molecule of the amino acid glutamic acid. The
presence of sulfanilamide results in the displacement of
p-aminobenzoic acid in microorganisms that synthesize
folic acid. The product thus generated cannot form a
peptide bond with glutamic acid, as the inactive pteri-
dine-sulfa conjugate predominates. Because folic acid is
a mandatory catalyst in the synthesis of purine and
pyrimidines, a microorganism without functional folic
acid would not survive. Animals are unaffected by sul-
fanilamide because they obtain folic acid in their diet
and do not synthesize it from the base constituents.
An understanding of the selective toxicity of sul-
fanilamide led to a concerted search for other chemo-
therapeutic agents. Structural analogs of amino acids,
purines, pyrimidines, and vitamins have been syn-
thesized. Emphasis has been placed on synthesizing
analogs of the bases in DNAand RNA(adenine, thy-
mine, guanine, uracil, and cytosine) in an effort to find
chemical compounds that have antiviral or antitumor
activity. Thousands of compounds have been synthe-
sized, but a limited number have proven effective as
chemotherapeutics.
Some important antiviral agents that have been
developed in recent years, and these include acyclovir
and azidothymidine (AZT). Acyclovir is a structural
analog of deoxyguanosine (Figure 7.7) and can be used
in the treatment of herpes virus infections. When a her-
pes virus (a DNAvirus) multiplies in a cell, the enzyme
thymidine kinase is activated. When acyclovir is pres-
ent, this enzyme gratuitously phosphorylates, resulting
in the formation of an unnatural triphosphate, which
then blocks the DNApolymerase. As a consequence, the
assembly of DNAin viral particles is curtailed.
AZT is an antiviral used in the treatment of AIDS
(acquired immunodeficiency syndrome). This com-
pound inhibits the multiplication of HIV, the retrovirus
that causes AIDS. Unfortunately, neither of these antivi-
ral agents can destroy the nonreplicating intracellular
virus. Both the herpes virus and HIV persist for life in
selected cells within the human host from the time of
infection (see Chapter 29).
Antibiotics as Chemotherapeutics
The term antibiotic, which is familiar to most of us,
was coined by Selman Waksman in 1953. Waksman was
the discoverer of the antibiotic streptomycin, which
played a significant role in the control of tuberculosis.
He defined an antibiotic as . . . a chemical substance,
produced by microorganisms, which has the capacity to
inhibit the growth and even to destroy bacteria and
other microorganisms, in dilute solutions. Antibiotics
that are effective in controlling disease are produced
during growth by certain bacteria, actinomycetes, and
fungi. In fact, the ability to produce antibiotic substances
is widespread in the microbial world, and the group of
bacteria termed the actinomycetes is particularly adept at
producing them. The antibiotics synthesized by actino-
mycetes have remarkable chemical diversity. However,
the first commercially successful antibiotic used in
chemotherapy was produced by a fungus. This antibi-
otic was penicillin and was produced by the fungus
Penicillium notatum (Box 7.1). The search for antibiotic
substances and their industrial production is presented
in Chapter 31. The medical aspects of antibiotics are cov-
ered in Chapters 28 and 29. The present section will pro-
vide a brief discussion of their basic mode of action and
the manner in which infectious organisms become
resistant to them.
Antibiotics are selectively toxic to certain types of liv-
ing cells and less so to others. This selectivity is due
to distinct differences in fundamental physiological
processes in affected cells and unaffected cells. Some
antibiotics are effective against a limited range of bacte-
ria and thus are considered to be narrow-spectrum
antibiotics. Penicillin is an example of this type because
it is mostly effective against gram-positive organisms. A
broad-spectrumantibiotic, such as tetracycline, inhibits
both gram-positive and gram-negative bacteria. Anti-
biotics that inhibit bacteria generally do not inhibit
eukaryotes. Conversely, antibiotics that inhibit eukary-
otes are generally not effective against bacteria. The
Archaea are generally less sensitive to antibacterial
antibiotics.
Antibiotic Actions: Sites and Modes
An antibiotic interferes in some manner with a normal
physiological function in a susceptible cell (Table 7.5).
156 CHAPTER 7
Perry / Staley Lory
Microbiology 2/e, Sinauer Associates
Figure 07.07 Date 12/11/01
CH
2
HO
H
2
N
HOCH
2
H
2
N
N
H
OH
H
H
N
N
C
O
HN
O
N
N
N
C
O
HN
O
Deoxyguanosine
(nucleoside)
Acyclovir
(antiviral)
Figure 7.7 Acyclovir as structural analog
The antiviral agent acyclovir is structurally related to the
nucleoside deoxyguanosine. Phosphorylation of acyclovir
yields an analog of deoxyguanosine triphosphate, which
inhibits the viral DNApolymerase.
The major actions of antibiotics include:
Disruption of cell wall synthesis
Destruction of cell membranes
Interference with some aspect of protein or nucleic
acid synthesis
The most useful antibiotics employed to control
human infections interfere with structural or physio-
logical reactions that are unique to the invading
pathogen. This is possible because the bacteria are phys-
iologically quite different from the eukaryotes.
Consequently, antibacterial antibiotics can exploit these
differences and destroy the invader without significant
harm to the human host.
Control of fungal, protozoan, and viral invaders is
much more difficult because physiological reactions in
these pathogens are quite similar to that in all eukary-
otes including a human host. The physiological differ-
ences among the eukaryotes are simply not so distinct
that these differences can readily be exploited. Thus,
effective antifungal and antiprotozoan agents are gen-
erally toxic to humans. Because viral infections are intra-
cellular, they are also difficult to control with chemother-
apeutic agents. Avirus is synthesized, for the most part,
by the metabolic machinery of the host, and destruction
of this machinery can lead to the destruction of the host.
Several antibiotics and their modes of action are list-
ed in Table 7.5. Penicillin and chemically related antibi-
otics prevent the transpeptidation reaction, which is an
important step in the assembly of the cell wall polymer,
peptidoglycan (see Chapter 4). This results in a weak-
ened cell wall, especially in gram-positive organisms.
Because microorganisms generally live in osmotically
unfavorable environments, one having a weakened cell
wall will take up water and burst, or lyse. Gram-nega-
tive bacteria tend to be less sensitive to penicillin
because their outer envelope can prevent the antibiotic
from reaching the peptidoglycan layer of the cell. The
Archaea that do not have peptidoglycan in their cell wall
are generally unaffected by antibiotics such as penicillin
that interfere with the synthesis of this polymer. Some
of the Archaea are sensitive to selected ionophores that
disrupt membranes (see Table 7.5).
Tyrocidine and polymyxin are both polypeptide
antibiotics that disrupt cell membranes. Both are pro-
duced by bacteria of the genus Bacillus. Tyrocidine is an
ionophore, which destroys selective permeability by
forming channels across the cell membrane, resulting in
leakage of monovalent cations. As a consequence, the
organism cannot establish a proton motive force, and
transport into and out of the cell is impaired. Polymyxin
causes similar cytoplasmic membrane damage. Peptide
antibiotics are not taken internally but are applied topi-
cally to treat skin infections. Enzymes present in the intes-
tinal tract would digest an ingested peptide antibiotic.
Certain antibiotics interfere directly with bacterial
DNA replication. Among these is novobiocin, which
binds directly to the beta subunit of the DNAgyrase
responsible for unwinding supercoiled DNA during
replication. Other antibiotics, such as rifampicin, block
the RNApolymerase involved in transcribing the infor-
mation on the DNAmolecule to make messenger RNA
for protein synthesis (see Chapter 13). Most of these
antibiotics are ineffective in the Archaea.
Many of the clinically useful antibiotics inhibit pro-
tein synthesis in bacteria. Protein synthesis requires
ribosomes, which are structures made up of subunits of
unequal size. Ribosomes are involved in the synthesis
of protein. Bacterial and eukaryotic ribosomes differ in
size, protein content, and ability to bind antibiotics. On
the basis of sedimentation velocity during ultracen-
trifugation, the smaller bacterial ribosome is designated
70S and the larger eukaryotic ribosome size is 80S.
Antibacterial antibiotics that inhibit protein synthe-
sis do so by binding to the bacterial ribosome. This
occurs with the antibiotics tetracycline, chlorampheni-
CONTROL OF MICROBIAL GROWTH 157
The producing organisms and mode of action of several antibiotics
Antibiotic Producer Mode of Action
Penicillin Penicillium spp. Blocks transpeptidation involved in cell wall synthesis
Erythromycin Streptomyces erythreus Binds to 50S ribosomal subunit and stops peptidyltransferase
Chloramphenicol S. venezuelae Binds to ribosomes blocking peptidyltransferase
Rifampicin S. mediterranei Blocks transcribing enzyme RNApolymerase
Novobiocin S. spheroides Binds to a subunit of DNAgyrase
Tyrocidine Bacillus brevis Ionophore disrupts cell membrane integrity and function
Polymyxins B. polymyxa Disrupts membrane transport and function
Streptomycin S. griseus Inhibits 30S ribosomes, blocks amino acid incorporation into peptides
Tetracycline S. aureofaciens Inhibits binding of aminoacyl tRNAto ribosomes
Table 7.5
col, streptomycin, and erythromycin. The antifungal
antibiotic cycloheximide binds to eukaryotic ribosomes
(80S) but not the 70S ribosome that is present in bacte-
ria. Consequently, cycloheximide inhibits fungi but does
not inhibit the growth of bacteria. As humans have 80S
ribosomes, they too would be adversely affected by
cycloheximide. Afew antibiotics, including tetracycline,
can bind to both 70S and 80S ribosomes. However, tetra-
cycline can be used in humans because it does not inhib-
it the synthesis of protein in eukaryotic cells at the con-
centrations used chemotherapeutically. It does impede
wound healing in humans under some circumstances.
RESISTANCE TO ANTIBIOTICS
Antibiotics have been effective in controlling many of
the diseases that have been a scourge to humankind.
Tuberculosis, bacterial pneumonia, syphilis, and many
other human infectious diseases that were once fatal can
now generally be treated with antibiotics. Antibiotics
have been called wonder drugs because they effect a
dramatic cure for what had previously been incurable.
But there is a problem with wonder drugs that
became evident after widespread use.
The extensive use of penicillin as a chemotherapeu-
tic agent led to the evolution of pathogenic bacterial
strains that were unaffected by the drug. These resist-
ant organisms retained their potent pathogenicity but
were no longer controlled by the administration of
penicillin. For example, when penicillin G (see Figure
4.53) was first introduced, virtually all strains of
Staphylococcus aureus were sensitive to the drug. Within
a span of only 10 years, essentially all staph infections
acquired in hospitals were caused by strains that were
resistant to penicillin.
Why do microorganisms become resistant to chemo-
therapeutics? Abundant microbial populations have
existed throughout all of Earths history because
microbes are adaptable. For example, when oxygen
appeared in the atmosphere or the climate became cool-
158 CHAPTER 7
Fortune favors the
prepared mind.
LOUIS PASTEUR
The more I practice,
the luckier I get.
LEE TREVINO
These quotes are a brief summation
of the circumstances that led Sir
Alexander Fleming (18811955) to
the discovery of penicillin. All too
often, scientific advances are attrib-
uted to chance or serendipitous
good fortune, but usually they are
not. More often they are the product
of intuition and hard work. The dis-
covery of penicillin resulted from
Flemings long-held and firm belief
that naturally occurring substances
existed that had useful antimicrobial
properties.
During World War I Fleming
worked in a field hospital in France.
There he treated casualties of battle
and experimented with better ways
of treating deep wounds. The
accepted treatment at that time was
copious quantities of antiseptic
carbolic acid, boric acid, or peroxide.
Unfortunately, in deep wounds
these antiseptics were quickly neu-
tralized by tissue, and serious infec-
tions often followed. Fleming and
his colleague, Sir Almroth Wright
(18611947; a noted developer of
antityphoid vaccine), observed that
wounds carefully cleansed and
treated with minimal antiseptic
healed faster and with fewer serious
infections than wounds treated with
massive amounts of antiseptic. They
believed that stimulation of natural-
defense mechanisms by encourag-
ing the exudation of lymph into the
wound site was particularly effective
in promoting healing. They found
that bathing wounds with a saline
solution was effective in stimulating
the exuding of lymph. The success of
this experimentation convinced
Alexander Fleming that naturally
produced substances could be
employed successfully in the treat-
ment of infectious disease. Fleming
explained to a physician visiting his
laboratory at Boulogne,What we
are looking for is some chemical
substance which can be injected
without danger into the blood
stream for the purpose of destroy-
ing the bacilli of infection, as sal-
varsan destroys the spirochaetes.
Following the war, Fleming
returned to his position at St. Marys
The Antibiotic Age
Sir Alexander Fleming Photo by
Sydney R. Bayne/NLM.
Milestones Box 7.1
er, microbes evolved to survive these new environmen-
tal conditions. Environmental stresses and constraints
became natural selection processes for microbes. Those
that survived carried genes that better adapted them to
serve in the altered conditions. Human control methods
of all types create a challenge to bacteria that leads to
natural selection for those organisms in the environment
that can withstand the prevailing condition. Thus, the
widespread use of penicillin resulted in the selection of
strains that could survive in the presence of the drug.
Some penicillin-resistant strains produce an enzyme,
penicillinase, that destroys the antibiotic.
Microbial resistance to an antibiotic, a chemothera-
peutic agent, or other chemicals such as heavy metals
can occur for several of reasons:
Natural resistance
The organism may lack the structure that the antibi-
otic inhibits, as occurs with mycoplasma, which lacks
cell walls and is thus unaffected by penicillin.
The cell wall structure or cytoplasmic membrane of
an organism may be impermeable to an antibiotic or
other chemical.
Acquired resistance
Aresistant microorganism may produce a substance
that inactivates the antibiotic, as occurs in strains of
Staphylococcus aureus in producing the enzyme peni-
cillinase, which disrupts the penicillin molecule.
Agradual accumulation of mutations in chromosomal
DNAmay result in cellular structures that will not
bind the antibiotic or other chemical. For example, the
gene for transpeptidase synthesis in staphylococci can
mutate so that the enzyme does not bind penicillin.
Another significant problem in antibiotic and other
forms of resistance is the acquisition of bits of extra chro-
mosomal DNAthat carry the information that renders
a microorganism resistant. These bits of DNA are
termed plasmids and can be transferred from cell to
cell (see Chapter 15).
CONTROL OF MICROBIAL GROWTH 159
Hospital in London. As time permit-
ted, he tested various naturally
occurring substances for antibacter-
ial activity. Among the materials he
tested was nasal mucus, and he
observed that addition of mucus to
a suspension of Staphylococcus
aureus led to a rapid clearing of the
turbidity. He discovered that this
clearing resulted from lysis of the
bacterial cells. The factor involved
was an enzyme termed lysozyme
(see Chapter 4). This discovery, made
in 1921, was important scientifically,
but lysozyme was not useful as a
chemotherapeutic agent. It did,
however, offer confirmation to
Fleming that his belief in natural
antibacterials was warranted.
One of Flemings research inter-
ests was Staphylococcus aureus, and
he often had cultures growing on
Petri dishes lying about the lab. One
day while examining some of these
old cultures, he observed that one
was contaminated with a mold and
that bacterial colonies did not devel-
op in the area adjacent to the mold
growth. Fleming was intrigued and
likened the phenomenon to the
action of lysozyme on S. aureus. He
then took a picture of the plate
(shown in the drawing) and wrote
up the observation in his research
notebook. He showed the plate to a
colleague with the remarkTake a
look at that, its interestingthe
kind of thing I like; it may well turn
out to be important.Important,
indeed; the antibacterial substance
produced by the mold (Penicillium
notatum) was penicillin. This ush-
ered in the antibiotic age.Flemings
monumental discovery led eventu-
ally to the conquest of such ancient
scourges as pneumonia, tuberculo-
sis, syphilis, staphylococcal infec-
tions, typhus, and a host of other
human ills.
(continued)
The original Penicilliumplate From
Maurois, Andre. 1959. Life of Sir
Alexander Fleming, E. P. Dutton Co.
Milestones Box 7.1
Flemings drawings and notes
SUMMARY
The role of microorganisms in the cycles of nature is
essentially destructive; human survival often depends
on counteracting their activities.
An understanding of the nature of microorganisms led
to more direct means for controlling them.
Sterilization is the destruction of all viable life forms.
It can be attained by fire, moist heat, dry heat, radia-
tion, filtering, or toxic chemicals.
Microbial death by heating occurs at an exponential
rate. The length of time required to reduce a popula-
tion tenfold is termed the decimal reduction time.
The most effective means for routine sterilization of
media and food is steam under pressure. The common
laboratory apparatus employed for this is the autoclave.
Dry heat at 160C is effective in sterilizing glassware,
pipettes, and other heat-stable material.
Pasteurization is a method for reducing the number
of microorganisms present in milk or other products.
It can destroy selected disease or spoilage-causing
microorganisms.
The wavelengths of light that can cause the death of
microorganisms are ultraviolet light (UV), which dis-
rupts RNA/DNA, and ionizing radiation, which can
potentially harm any constituent of a cell.
Microorganisms have DNArepair systems that can
correct damage caused by radiation.
Filters with pores small enough to retain bacteria can
be employed to sterilize liquids. Anumber of types are
available, but membrane filters are the most widely
used.
Toxic gases such as propylene oxide is used to steril-
ize plastic Petri dishes, filters, and other disposable
material.
Antimicrobial agents may be products of chemical
synthesis, natural products, or a combination of the
two. The favored agent has selective toxicity in that it
destroys the target microbe but produces limited activ-
ity toward other cells.
Adisinfectant is an agent such as phenol that destroys
all living cells on contact. An antiseptic is less potent
and prevents the growth of disease-causing organ-
isms. Merthiolate that is applied to superficial wounds
is an antiseptic.
The minimum inhibitory concentration (MIC) is the
lowest concentration of a germicide that will inhibit a
test organism.
The phenol coefficient is a measure of the effective-
ness of a test compound as compared with phenol.
The first effective chemically synthesized chemother-
apeutic agent was sulfanilamide. An understanding
of the role of this compound in blocking vitamin syn-
thesis in bacteria encouraged the scientific world to
search for other chemical agents that would be effec-
tive against viruses, protozoa, fungi, and cancer.
By definition an antibiotic is a product of microor-
ganisms. Antibiotics have proven to be effective in
controlling human and animal diseases caused by bac-
teria. They generally interfere with metabolic activi-
ties or structures in disease-causing bacteria but do not
interfere with them in a host eukaryote.
The major modes of action of antibiotics include: dis-
rupt cell wall synthesis, destroy cytoplasmic mem-
branes, and disrupt nucleic acid or protein synthesis.
REVIEW QUESTIONS
1. What is the difference between sterilization and pas-
teurization? What agent is most often used in steril-
ization? Why are we so concerned with sterilizing
things?
2. Dry heat is employed to sterilize selected materials.
Name some of the materials best sterilized by dry
heat.
3. What agent is most often used to sterilize air in a
closed room? List what concerns there would be
with other methods.
4. Give some advantages of sterilization by filtration.
How is filtration applied in the laboratory? In
industry?
5. How did Robert Koch determine the effectiveness of
disinfectants? What compound did he find most
effective with this method?
6. How would one calculate a phenol coefficient? Is
this a useful number? List the pros and cons.
7. Sulfanilamide is a classical example of a competitive
inhibitor. How does it function?
8. What are the basic shortcomings of antiviral agents
such as AZT and acyclovir?
9. Why is it so difficult to find effective antiviral and anti-
fungal agents when antibacterials are quite plentiful?
10. Discuss the mode of actions of some common
antibacterials.
160 CHAPTER 7
SUGGESTED READING
Block, S. S., ed. 1991. Disinfection, Sterilization, and
Preservation. 4th ed. Philadelphia: Lea and Febiger.
Favero, M. S. and W. W. Bond. 1991. Sterilization,
Disinfection, and Antisepsis in the Hospital. In A.
Ballows, W. J. Hausler, K. L. Herrmann, H. O. Isenberg
and H. J. Shadomy, eds. Manual of Clinical Microbiology.
5th ed. (pp. 183200). Washington, DC: American Society
for Microbiology.
Hardman, J. G. and L. E. Limbird, eds. 1995. The
Pharmacological Basis of Therapeutics. 9th ed. New York:
Pergamon Press.
Harte, J., C. Holdren, R. Schneider and C. Shirley. 1991.
Toxics Ato Z: AGuide to Everyday Pollution Hazards. Los
Angeles: University of California Press.
Levy, S. B. 1992. The Antibiotic Paradox, How Miracle Drugs
Are Destroying the Miracle. New York: Plenum Press.
CONTROL OF MICROBIAL GROWTH 161