Chapter 8.
Controlling Microbial Growth in Vitro
• Chapter 8 Outline
• Introduction
• Factors that Affect Microbial Growth
• Encouraging the Growth of Microbes in Vitro
• 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.
• Moisture
– 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 (dessication).
• Factors That Affect Microbial Growth, cont.
• Temperature
– Every organism has an optimum growth
temperature.
– The temperature (and pH) ranges over
which an organism grows best are largely
determined by its enzymes.
– Thermophiles are microorganisms that
grow best at high temperatures.
– Mesophiles are microbes that grow best at
moderate temperatures (e.g., 37o C).
• Factors That Affect Microbial Growth, cont.
• Temperature, cont.
– Psychrophiles prefer cold temperatures
(like deep ocean water).
• Psychrotrophs, a particular group
of psychrophiles, prefer
refrigerator temperature (4oC).
– Psychroduric organisms prefer warm
temperatures, but can endure very cold or
even freezing temperatures.
• Factors That Affect Microbial Growth, cont.
• pH
– “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
• Factors That Affect Microbial Growth, cont.
• 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 of
solutes.
– 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.
• Factors That Affect Microbial Growth, cont.
• Osmotic Pressure and Salinity, cont.
– Plasmolysis is a condition in which the cell
membrane and cytoplasm of a cell shrink
away from the cell wall; occurs when
bacteria with rigid cell walls are placed into
a hypertonic solution.
– 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.
– If a bacterial cell is placed into a hypotonic
solution, it may not burst (because of the
rigid cell wall.
• Factors That Affect Microbial Growth, cont.
• Osmotic Pressure and Salinity, cont.
– A solution is said to be isotonic when the
concentration of solutes outside a cell
equals the concentration of solutes inside
the cell.
– Organisms that prefer to live in salty
environments are called halophilic
organisms.
• Barometric Pressure
– Microbes that can survive in high
atmospheric pressure (> 14.7 psi) are know
as piezophiles.
• Changes in Osmotic Pressure
• Encouraging the Growth of Microbes in Vitro
• Culturing Bacteria in the Laboratory
Bacterial Growth
• Think of 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). http://youtu.be/J6akNYlkehY
• Binary fission continues until a colony is produced.
• 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).
• Culturing Bacteria in the Laboratory
Culture Media
• Media (sing., medium) are used in microbiology
labs to culture (i.e., grow) bacteria; media
prepared in the lab are referred to as artificial
media or synthetic media.
• A chemically defined medium is one in which all
ingredients are known.
• 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; example =
chocolate agar.
• A selective medium has added inhibitors that
discourage growth of certain organisms while
allowing the growth of a desired organism;
example = PEA agar.
• Differential Medium
• Culturing Bacteria in the Laboratory
Inoculation of Culture Media
• Culture media are inoculated with clinical
specimens (i.e., specimens collected from patients
with a suspected infectious disease).
• Inoculation involves adding a portion of a
specimen to the medium.
• Inoculation is accomplished using a sterile
inoculating loop.
• Culturing Bacteria in the Laboratory
Importance of Using “Aseptic Technique”
• Aseptic technique is practiced when it is necessary
to exclude microbes when inoculating culture
media.
• http://youtu.be/0odxJy0nR9s
• Unwanted organisms are referred to as
contaminants; the growth medium or plate is said
to be contaminated.
• The sterility of the media must be maintained
before inoculation.
– Avoid touching the surface of the agar!
• Culturing Bacteria in the Laboratory
Incubation
• 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.
• http://youtu.be/FaNvh3HlxQU
• Culturing Bacteria in the Laboratory
Bacterial Population Growth Curve
• A population growth curve for any particular
species of bacterium may be determined by
growing a pure culture of the organism in a liquid
medium at a constant temperature.
– Samples of the culture are collected at
fixed intervals to determine the number of
viable organisms.
– A graph is prepared by plotting the
logarithmic number of viable organisms
(on the vertical or Y- axis) against the
incubation time (on the horizontal or X-
axis).
• Inhibiting the Growth of Microbes in Vitro
• Definition of Terms
• Sterilization is the complete destruction of all
microbes, including cells, spores, and viruses.
– Accomplished by dry heat, autoclaving
(steam under pressure), gas, various
chemicals, and certain types of radiation.
• Disinfection is the destruction or removal of
pathogens from nonliving objects by physical or
chemical methods; pasteurization is an example of
a disinfection technique.
– Disinfectants are chemical substances that
eliminate pathogens on inanimate objects.
– Antiseptics are solutions used to disinfect
skin and other living tissues.
• 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
kill bacteria, but not necessarily bacterial
endospores.
– 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 microbistatic 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
pathogens.
• Antisepsis is the prevention of infection.
• Using Physical Methods to Inhibit Microbial
Growth
• Heat
– 2 factors – temperature and time -
determine the effectiveness of heat for
sterilization.
– The thermal death point (TDP) of any
species is the lowest temperature that will
kill all of the organisms in a standardized
pure culture within a specified time.
• Types of Heat
– Dry heat – e.g., oven, electrical incinerator,
or flame
– Moist heat – boiling or use of an autoclave
• Dry Heat Sterilization
• Using Physical Methods to Inhibit Microbial
Growth, cont.
• The autoclave
– A large metal pressure cooker that uses
steam under pressure to completely
destroy all microbial life.
– Increased pressure raises the temperature
above the temperature of boiling water
(above 100oC) and forces steam into
materials being sterilized.
– Autoclaving at a pressure of 15 psi at
121.5oC for 20 minutes destroys vegetative
microorganisms, bacterial endospores, and
viruses.
– Can use pressure-sensitive tape or spore
strips or solutions as a quality control
measure to ensure proper autoclaving.
– http://www.dentalproductsreport.com/art
icles/show/dpr0208_ic_sterlization-
monitoring-1?print=1
• Biological Indicators for Monitoring the
Effectiveness of Steam Sterilization
• Using Physical Methods to Inhibit Microbial
Growth, cont.
• Cold; most microorganisms are not killed, but their
metabolic activities are slowed.
• Desiccation; 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 gases.
• Gaseous atmosphere; can be altered to inhibit
growth.
• Using Chemical Agents to Inhibit Microbial Growth
• 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
– Contact time
– Physical nature of the object being
disinfected
– Temperature and pH
• Using Chemical Agents to Inhibit Microbial Growth,
cont.
• Should have a broad antimicrobial spectrum
• Fast acting
• Not affected by the presence of organic matter
• Nontoxic to human tissues and noncorrosive
• Should leave a residual antimicrobial film on
surface
• Soluble in water and easy to apply
• Inexpensive and easy to prepare
• Stable as both a concentrate and a working
solution
• Odorless
• Using Chemical Agents to Inhibit Microbial Growth
(cont.)
• Antiseptics
– 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 the skin.
• Inhibiting the Growth of Pathogens in Our Kitchens
(from the CD-ROM)
• Many foods brought into our kitchens are
contaminated with pathogens; examples = E. coli
O157:H7, Salmonella and Campylobacter spp. on
poultry and ground beef.
• Problems arise when handling foods before
cooking.
• Remain aware of pathogens when preparing foods.
• Wash hands frequently.
• Thoroughly clean plates and counter tops that
have had poultry or meat on them with hot soapy
water
• The use of antibacterial kitchen sprays is
controversial.
• Controversies Relating to the Use of Antimicrobial
Agents in Animal Feed and Household Products
• 40% of the antibiotics manufactured in the U.S. are
used in animal feed; microorganisms resistant to
these antibiotics survive!
– Drug resistant organisms are transmitted
in animal feces and in food products.
– Efforts are underway to eliminate or
reduce the practice of adding antibiotics to
animal feed.
• Use of antimicrobial agents is widespread in toys,
cutting boards, in hand soaps, and many other
household products; resistant microorganisms
survive!
• Controversy: Should children be exposed to all
sorts of microorganisms for their immune systems
to develop properly?