for the Health Sciences
اعداد : أ. فريد ابو العمرين
Controlling Microbial Growth
Controlling Microbial Growth in Vitro
Chapter 8 Outline
2. Factors that Affect Microbial Growth
3. Encouraging the Growth of Microbes in Vitro
4. Inhibiting the Growth of Microbes in Vitro
Factors That Affect Microbial Growth
• Availability of Nutrients
All living organisms require nutrients to sustain life.
Nutrients are energy sources.
Organisms obtain energy by breaking chemical bonds.
Water is essential for life.
It is needed to carry out normal metabolic processes.
Certain microbial stages (e.g., bacterial endospores and
protozoal cysts) can survive a drying process.
Every organism has an optimum growth temperature.
Thermophiles are microorganisms that grow best at
Mesophiles are microbes that grow best at moderate
temperatures (e.g., 37o C).
Psychrophiles prefer cold temperatures (like deep
• “pH” refers to the acidity or alkalinity of a solution.
• Most microorganisms prefer a neutral or slightly alkaline
growth medium (pH 7.0 - 7.4)
• Acidophiles prefer a pH of 2 to 5
• Alkaliphiles prefer a pH > 8.5
Osmotic Pressure and Salinity
Osmotic pressure is the pressure that is exerted on a cell
membrane by solutions both inside and outside the cell.
Osmosis is the movement of a solvent, through a
permeable membrane, from a lower concentration of
solutes (dissolved substances) to a higher concentration
Osmotic Pressure and Salinity
When the concentration of solutes in the external
environment of a cell is greater than that of solutes inside
the cell, the solution in which the cell is suspended is said
to be hypertonic.
When the concentration of solutes outside a cell is less than
that of solutes inside a cell, the solution in which the cell is
suspended is said to be hypotonic.
A solution is said to be isotonic when the concentration of
solutes outside a cell equals the concentration of solutes
inside the cell.
Factors That Affect Microbial Growth,
In hypertonic solution a cell shrink
If a bacterial cell is placed into a hypotonic solution, Cells
swell up, and sometimes burst.
In isotonic soln. the cell has normal turgor.
In the case of erythrocytes, this bursting is called hemolysis
Factors That Affect Microbial Growth,
Organisms that prefer to live in salty environments
are called halophilic organisms.
Microbes that can survive in high atmospheric
pressure are know as piezophiles.
Microorganisms vary with respect to the type of gaseous
atmosphere that they require.
Obligate aerobes prefer the same atmosphere that humans do
(~20-21% O2 and 78-79% N2, other gases < 1%).
Microaerophiles require reduced concentrations of oxygen
Obligate anaerobes are killed by the presence of oxygen.
Facultative grow in presence or absence of oxygen
Capnophiles require increased concentrations of CO2 (5-10%
Encouraging the Growth
of Microbes in Vitro
• Bacterial growth as an increase in the number of organisms
rather than an increase in their size.
• Bacteria divide by binary fission (one cell divides to become
two cells) when they reach their optimum size.
• Binary fission continues through many generations until a
colony is produced on solid culture medium.
• Binary fission continues for as long as there is a sufficient
supply of nutrients, water, and space.
• The time it takes for one cell to become two cells is called the
generation time (e.g., E. coli = 20 minutes).
• Media are used in microbiology labs to culture (i.e.,
• Culture media can be liquid or solid.
• An enriched medium is a broth or solid containing a
rich supply of special nutrients that promote the growth
of fastidious organisms.
• A selective medium has added inhibitors that
discourage growth of certain organisms while allowing
the growth of a desired organism.
• A differential medium permits the differentiation of
organisms that grow on the medium.
• The various categories of media are not mutually
exclusive; e.g., blood agar is enriched and differential.
• Thioglycollate broth (THIO) is a popular liquid medium
in bacteriology labs; it supports the growth of all
categories of bacteria from obligate aerobes to obligate
– There is a concentration gradient of dissolved
oxygen in the tube; organisms grow only in that
part of the broth where the oxygen concentration
meets their needs.
Inoculation of Culture Media
• Culture media are inoculated
with clinical specimens (i.e.,
specimens collected from
patients with a suspected
• Inoculation involves adding a
portion of a specimen to the
• Inoculation is accomplished
using a sterile inoculating
Importance of Using “Aseptic Technique”
Aseptic technique is practiced to prevent
• (a) microbiology professionals from becoming infected,
• (b) contamination of their work environment.
• (c) contamination of clinical specimens, cultures, and
• After media are inoculated, they must be placed into an incubator
which will maintain the appropriate atmosphere, temperature, and
moisture level; the process is known as incubation.
• 3 types of incubators are used in clinical microbiology
A CO2 incubator (contains 5-10% CO2)
A non-CO2 incubator (contains room air)
An anaerobic incubator (the atmosphere is devoid of oxygen)
A population growth curve of living organisms.
lag phase during which the bacteria absorb nutrients,
synthesize enzymes, and prepare for cell division.
• The bacteria do not increase in number during
Logarithmic growth phase (log phase)
• the bacteria multiply so rapidly that the number of organisms
doubles with each generation time.
• Growth rate is the greatest during the log phase.
A population growth curve of living
Death phase or decline phase
The microorganisms then die at a rapid rate.
As the nutrients are used up and the concentration of toxic
waste products build up, the rate of division slows, such that
the number of bacteria that are dividing equals the number
that are dying.
As overcrowding occurs, the concentration of toxic waste
products continues to increase and the nutrient supply
Definition of Terms
In Vitro: In an artificial environment, as in a laboratory
setting; used in reference to what occurs outside an
In Vivo: Used in reference to what occurs within a living
• Sterilization is the complete destruction of all microbes,
including cells, spores, and viruses.
• Disinfection is the destruction or removal of pathogens
from nonliving objects.
Definition of Terms, cont.
• The suffix –cide or –cidal refers to “killing.”
Germicidal agents, biocidal agents, and microbicidal
agents are chemicals that kill microbes.
• Bactericidal agents are chemicals that specifically
• Sporicidal agents kill bacterial endospores.
Fungicidal agents kill fungi, including fungal spores.
Algicidal agents kill algae.
Viricidal agents destroy viruses.
Definition of Terms (cont.)
• A static agent is a drug or chemical that inhibits growth
and reproduction of microbes.
• A bacteriostatic agent is one that specifically inhibits the
metabolism and reproduction of bacteria.
• Lyophilization is a process that combines dehydration
(drying) and freezing.
• This process is widely used in industry to preserve foods,
antibiotics, microorganisms, and other biologic materials.
• Sepsis refers to the presence of pathogens in blood or
tissues, whereas asepsis means the absence of
Antisepsis is the prevention of infection.
Physical Methods to Inhibit
Heat is the most practical, efficient, and inexpensive method of
sterilization of those inanimate objects and materials that can
withstand high temperatures.
Because of these advantages, it is the means most frequently
• Types of Heat
– Dry heat – e.g., oven, electrical incinerator, or flame
– Moist heat – boiling or use of an autoclave
Dry Heat Sterilization
Dry Heat. Dry-heat oven provides effective sterilization of metals,
glassware, some powders, oils, and waxes.
These items must be at:
1. 160°C to 165°C for 2 hours
2. 170°C to 180°C for 1 hour.
The effectiveness of dry-heat sterilization depends on how deeply
the heat penetrates throughout the material, and the items to be
baked must be positioned so that the hot air circulates freely among
Incineration (burning) is an effective means of destroying
contaminated disposable materials.
Increased pressure raises the temperature above the
temperature of boiling water (above 100oC) and forces
steam into materials being sterilized.
1. a pressure of 15 psi
2. 121 oC
3. for 20 minutes.
A large metal pressure machine that uses steam under
pressure to completely destroy all microbial life.
To destroys vegetative microorganisms, bacterial
endospores, and viruses.
The autoclave quality-control
Pressure-sensitive autoclave tape and commercially
available strips or solutions containing bacterial spores
can be used as quality-control measures to ensure that
autoclaves are functioning properly.
After autoclaving, the spores are tested to see whether
they were killed.
Physical Methods to Inhibit
• Cold; most microorganisms are not killed, but their
metabolic activities are slowed.
• Desiccation; For many centuries, foods have been
preserved by drying. Many dried microorganisms remain
viable, but they cannot reproduce.
• Radiation; an ultra-violet (UV) lamp is useful for
reducing the number of microbes in the air.
• Ultrasonic waves; used in hospitals and medical and
dental clinics to clean equipment.
• Filters; used to separate cells/microbes from liquids or
Gaseous atmosphere; can be altered to inhibit growth.
UV lamp is useful for reducing the number of
microorganisms in the air.
Sterility may also be maintained by having a UV lamp
placed in a hood or cabinet containing instruments,
paper and cloth equipment, liquid, and other inanimate
Many biologic materials, such as sera, antisera, toxins,
and vaccines, are sterilized with UV rays.
X-rays and gamma and beta rays
Radiation may be lethal or cause mutations in microorganisms and
tissue cells because they damage DNA and proteins within those
Studies performed in radiation research laboratories have
demonstrated that these radiations can be used for the prevention
of food spoilage, sterilization of heat-sensitive surgical equipment,
preparation of vaccines, and treatment of some chronic diseases
such as cancer, all of which are very practical applications for
In hospitals, medical clinics, and dental clinics, ultrasonic
waves are a frequently used means of cleaning delicate
Ultrasonic cleaners consist of tanks filled with liquid
solvent (usually water); the short sound waves are then
passed through the liquid.
The sound waves mechanically dislodge organic debris
on instruments and glassware.
Filters of various pore sizes are used to filter or separate cells, larger
viruses, bacteria, and certain other microorganisms from the liquids
or gases in which they are suspended.
Filters with tiny pore sizes (called micropore filters) are used in
laboratories to filter bacteria and viruses out of liquids.
The variety of filters is large and includes sintered glass (in which
uniform particles of glass are fused), plastic films, unglazed porcelain,
asbestos, diatomaceous earth, and cellulose membrane filters.
Small quantities of liquid can be filtered through a filter-containing
syringe, but large quantities require larger apparatuses.
Microbes, even those as small as viruses, can be removed from
liquids using filters having appropriate pore sizes.
In limited situations, it is possible to inhibit growth of microorganisms by
altering the atmosphere in which they are located. Because aerobes and
microaerophiles require oxygen, they can be killed by placing them into an
atmosphere devoid of oxygen or by removing oxygen from the environment
in which they are living.
Conversely, obligate anaerobes can be killed by placing them into an
atmosphere containing oxygen or by adding oxygen to the environment in
which they are living.
For instance, wounds likely to contain anaerobes are lanced (opened) to
expose them to oxygen. Another example is gas gangrene, a deep wound
infection that causes rapid destruction of tissues.
Gas gangrene is caused by various anaerobes in the genus Clostridium.
• Chemical disinfection refers to the use of chemical
agents to inhibit the growth of pathogens, either
temporarily or permanently.
• Disinfectants are affected by:
Prior cleaning of the object or surface
The organic load (e.g., feces, blood, pus)
The bioburden; types and numbers of microbes
Concentration of the disinfectant
Physical nature of the object being disinfected
Temperature and pH
Using Chemical Agents to Inhibit
Microbial Growth, cont.
Characteristics of an ideal chemical antimicrobial agent:
Should have a broad
• Not affected by the presence
of organic matter
• Nontoxic to human tissues
• Should leave a residual
44 antimicrobial film on surface
• Soluble in water and easy to
• Inexpensive and easy to
• Stable as both a concentrate
and a working solution
Using Chemical Agents to Inhibit
Microbial Growth (cont.)
May safely be used on human tissues.
Reduce the number of organisms on the surface of the
skin; do not penetrate pores and hair follicles.
• Antiseptic soaps and scrubbing are used by healthcare
personnel to remove organisms lodged in pores or folds of
• Antimicrobial chemical agents that can safely be applied to
skin are called antiseptics.
Controlling Microbial Growth in Vivo
Using Antimicrobial Agents