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Control of Microbial Growth (page 1)
(This chapter has 6 pages)
© Kenneth Todar, PhD
In the 19th century, surgery was risky and
dangerous, and patients undergoing even the most routine operations
were at very high risk of infection. This was
so because surgery was not performed under aseptic
conditions. The operating room, the surgeon's hands, and the
surgical instruments were laden with microbes, which caused high levels
of infection and mortality.
Surgeons in the mid-1800s often operated wearing their street clothes,
without washing their hands. They frequently used ordinary sewing
thread to suture wounds, and stuck the needles in the lapels of their
frock coats in between patients. Surgical dressings were often
made up of surplus cotton or jute from the floors of cotton mills. It
was against this background that French scientist Louis Pasteur
demonstrated that invisible microbes caused disease.
Pasteur's work influenced the English surgeon Joseph
Lister, who applied Pasteur's germ theory of disease to surgery, thus
modern antiseptic surgery. To disinfect, Lister used a solution
of carbolic acid (phenol), which was sprayed around the operating room
surgery using Lister's carbolic acid
It was clear
that Lister's techniques were effective in increasing the rates of
surviving surgery, but his theories were controversial because many
surgeons were unwilling to accept something they could not see. Also,
perhaps another reason that
surgeons were slow to pick up on Lister's methods was the fact that
during surgery they were required
to breathe an irritating aerosol of phenol.
The control of microbial growth is necessary in many
practical situations, and significant advances in agriculture,
and food science have been made through study of this area of
"Control of microbial growth", as used here,
means to inhibit or prevent
growth of microorganisms. This control is affected in two basic ways:
by killing microorganisms or (2) by inhibiting the growth of
Control of growth usually involves the use of physical or chemical
which either kill or prevent the growth of microorganisms. Agents which
kill cells are called cidal agents; agents which inhibit the
of cells (without killing them) are referred to as static
Thus, the term bactericidal refers to killing bacteria, and bacteriostatic
refers to inhibiting the growth of bacterial cells. A bactericide
kills bacteria, a fungicide kills fungi, and so on.
In microbiology, sterilization refers to
the complete destruction
or elimination of all viable organisms in or on a substance being
There are no degrees of sterilization: an object or substance is either
sterile or not.
Sterilization procedures involve the use of heat, radiation or
or physical removal of cells.
Methods of Sterilization
Heat: most important
and widely used. For sterilization one must consider the type of
heat, and most importantly, the time
application and temperature
to ensure destruction of all microorganisms.
Endospores of bacteria are considered the most thermoduric of all cells
so their destruction guarantees sterility.
Incineration: burns organisms and
destroys them. Used for needles, inoculating wires, glassware, etc.
objects not destroyed in the incineration process.
Boiling: 100o for 30 minutes.
Kills everything except some endospores. To kill endospores, and
therefore sterilize a solution,
very long (>6 hours) boiling, or intermittent boiling is
required (See Table 1 below).
Autoclaving (steam under pressure or pressure
Autoclaving is the most effective and most
efficient means of sterilization. All autoclaves operate on a
time/temperature relationship. These two variables are extremely
important. Higher temperatures ensure more rapid killing. The usual
temperature/pressure employed is 121ºC/15 psi for 15 minutes.
Longer times are needed for larger loads, large
volumes of liquid, and more dense materials. Autoclaving is ideal for
sterilizing biohazardous waste, surgical dressings, glassware, many
types of microbiologic media, liquids, and many other things. However,
certain items, such as plastics and certain medical instruments (e.g.
fiber-optic endoscopes), cannot withstand
autoclaving and should be sterilized with chemical or gas sterilants.
When proper conditions and time are employed, no living organisms will
survive a trip through an autoclave.
Schematic diagram of a laboratory
autoclave in use
to sterilize microbiological culture medium. Sterilization
of microbiological culture media is is often carried out with the
microbiological media are prepared, they must be sterilized and
rendered free of microbial contamination from air, glassware, hands,
etc. The sterilization process is a 100% kill, and guarantees
medium will stay sterile unless exposed to contaminants.
An autoclave for
use in a laboratory or hospital
Why is an autoclave such an effective
sterilizer? The autoclave is a large pressure cooker; it operates by
using steam under pressure as the sterilizing agent. High pressures
enable steam to reach high temperatures, thus increasing its heat
content and killing power. Most of the heating power of steam comes
from its latent heat of vaporization. This is the amount of heat
required to convert boiling water to steam. This amount of heat is
large compared to that required to make water hot. For example, it
takes 80 calories to make 1 liter of water boil, but 540 calories to
convert that boiling water to steam. Therefore, steam at 100º C
has almost seven times more heat than boiling water.
Moist heat is thought to kill microorganisms by
causing denaturation of essential proteins. Death rate is directly
proportional to the concentration of microorganisms at any given time.
The time required to kill a known population of microorganisms in a
specific suspension at a particular temperature is referred to as thermal death time (TDT). Increasing
the temperature decreases TDT, and lowering the temperature increases
TDT. Processes conducted at high temperatures for short periods of time
are preferred over lower temperatures for longer times.
Environmental conditions also influence TDT. Increased heat causes
increased toxicity of metabolic products and toxins. TDT decreases with
pronounced acidic or basic pHs. However, fats and oils slow heat
penetration and increase TDT. It must be remembered that thermal death
times are not precise values; they measure the effectiveness and
rapidity of a sterilization process. Autoclaving 121ºC/15 psi for 15 minutes exceeds the thermal death time for most organisms except some
Dry heat (hot air oven): basically the cooking oven. The rules of
relating time and temperature apply, but dry heat is not as effective
moist heat (i.e., higher temperatures are needed for longer periods of
time). For example 160o/2hours
or 170o/1hour is necessary for sterilization. The dry heat
oven is used for
glassware, metal, and objects that won't