Download Factors That Affect Microbial Growth, cont.

Burton's Microbiology
for the Health Sciences
Section IV.
Controlling the Growth of Microbes
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Burton's Microbiology
for the Health Sciences
Chapter 8.
Controlling Microbial Growth in Vitro
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Chapter 8 Outline
• Introduction
• Factors that Affect Microbial Growth
• Encouraging the Growth of Microbes in Vitro
• Inhibiting the Growth of Microbes in Vitro
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
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).
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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).
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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
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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.
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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.
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
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.
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Changes in Osmotic Pressure
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Encouraging the Growth of
Microbes in Vitro
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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).
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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.
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Differential Medium
A Thioglycollate (THIO)
Broth Tube
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Colonies of a β-hemolytic Streptococcus species on a
blood agar plate (in this case, the blood agar is both
enriched and differential)
http://textbookofbacteriology.net/themicrobialworld/pathogenes
is.html
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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.
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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/0odxJy0nR
9s
• 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!
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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/FaNvh3Hl
xQU
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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).
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Inhibiting the Growth of
Microbes in Vitro
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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.
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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.
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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.
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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
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Dry Heat Sterilization
Using a Bunsen
burner flame
Using an electrical
heating device
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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/
articles/show/dpr0208_ic_sterlizationmonitoring-1?print=1
Spore strips results
show tube at left is
clear (spores are
killed). Cloudy tube
(right) shows spores
are not killed.
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Pressure-sensitive autoclave tape showing dark
stripes after sterilization.
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Biological Indicators for Monitoring the
Effectiveness of Steam Sterilization
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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.
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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
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Using Chemical Agents to Inhibit
Microbial Growth, cont.
Characteristics of an ideal chemical antimicrobial agent:
• Should have a broad
antimicrobial spectrum
• Soluble in water and easy to
apply
• Fast acting
• Inexpensive and easy to
prepare
• Not affected by the presence
of organic matter
• Nontoxic to human tissues
and noncorrosive
• Stable as both a concentrate
and a working solution
• Odorless
• Should leave a residual
antimicrobial film on surface
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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.
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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.
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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?
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