Controlling Micriobial Growth

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

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).

• 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.

• 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

• 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.

• 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); if it
does burst, the cytoplasm escapes – this process is
known as plasmoptysis.
• 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. Those that do not
prefer to live in salty environments, but which are
capable of surviving there (e.g., Staphylococcus
aureus) are called haloduric organisms.
• Barometric Pressure
– Microbes that can survive in high atmospheric
pressure (> 14.7 psi) are know as piezophiles.
Changes in Osmotic Pressure

Factors That Affect Microbial Growth cont.
• Gaseous Atmosphere
– 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 (~5% O2).
– Obligate anaerobes are killed by the presence of
oxygen.
– Capnophiles require increased concentrations of CO2
(5-10% CO2).
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) 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).

Binary fission of
staphylococci.

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.
Culture Media, cont.
• A differential medium permits the differentiation of

organisms that grow on the medium; example =
MacConkey agar.
• 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 anaerobes.
– How is that possible? 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.

A Thioglycollate (THIO)
Broth Tube

Bacterial colonies on S. aureus on mannitol-
MacConkey agar (a salt agar (a selective &
selective & differential differential medium)
medium)

Colonies of a β-hemolytic Streptococcus
species on a blood agar plate (in this case, the
blood agar is both enriched and differential)

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.

Importance of Using “Aseptic Technique”
• Aseptic technique is practiced when it is necessary to

exclude microbes from a particular area (e.g., when
inoculating culture media).
• 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!
• Inoculating media within a biologic safety cabinet
minimizes contamination and protects the laboratorian.

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.
• 3 types of incubators are used in clinical microbiology
laboratories:
– A CO2 incubator (contains 5-10% CO2)
– A non-CO2 incubator (contains room air)
– An anaerobic incubator (the atmosphere is devoid of
oxygen)
Bacterial Population Counts
• Microbiologists sometimes need to know how many

bacteria are present in a particular liquid at a given time
(e.g., to determine bacterial contamination of drinking
water).
– Can determine either the total number of bacterial
cells or the number of viable (living) cells
• A spectrophotometer can be used to determine growth by
measuring the turbidity of the medium.
• A viable plate count is used to determine the number of
viable bacteria in a liquid sample by making serial
dilutions of the liquid and inoculating onto nutrient agar;
after overnight incubation, the number of colonies is
counted.
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).

A population growth curve of living organisms.
Stationary phase
Death phase
Logarithmic growth phase
Lag phase

A Chemostat is used
for continuous
cultures.

Culturing Obligate Intracellular Pathogens
in the Laboratory
• Obligate intracellular pathogens are microbes that can

only survive and multiply within living cells (called host
cells).
• Obligate intracellular pathogens include viruses and 2
groups of Gram-negative bacteria – rickettsias and
chlamydias.
• Culturing these organisms in the laboratory is a
challenge; they must be grown in embryonated chicken
eggs, lab animals, or cell cultures.

Culturing Fungi in the Laboratory
• Fungi (including yeasts, moulds and dimorphic fungi)

grow on and in a variety of solid and liquid culture media.
• There is no single medium that is best for all medically
important fungi.
• Examples of culture media for fungi include brain heart
infusion (BHI) agar, BHI with blood, and Sabouraud
dextrose agar (SDA); due to its low pH, SDA is selective
for fungi.
• Caution must be exercised when culturing fungi – some
are highly infectious!

Culturing Protozoa in the Laboratory
• Most microbiology laboratories do not culture protozoa;

some research and reference labs do, however.
• Examples of protozoa that can be cultured in vitro are

amebae, Giardia lamblia, Leishmania spp., Toxoplasma
gondii, Trichomonas vaginalis and Trypanosoma cruzi.
• Due to the severity of diseases that they cause, it is of

greatest importance to culture amebae: Acanthamoeba
spp., Balamuthia spp. and Naegleria fowleri.

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 a Bunsen Using an electrical

burner flame heating device

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.
A large, built-in autoclave.

Pressure-sensitive autoclave tape showing dark
stripes after sterilization.

Biological Indicators for Monitoring the
Effectiveness of Steam Sterilization

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
Growth, cont.
Characteristics of an ideal chemical antimicrobial agent:
• Should have a broad • Soluble in water and easy to
antimicrobial spectrum apply
• Fast acting • Inexpensive and easy to
prepare
• Not affected by the presence
of organic matter • Stable as both a concentrate
and a working solution
• Nontoxic to human tissues
and noncorrosive • Odorless
• Should leave a residual
antimicrobial film on surface

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?

Controlling Micriobial Growth

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Burton's Microbiology

for the Health Sciences

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• A differential medium permits the differentiation of

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Culture media are inoculated

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Aseptic technique is practiced when it is necessary to

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• After media are inoculated, they must be placed into an

• Microbiologists sometimes need to know how many

• A population growth curve for any particular species of

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Logarithmic growth phase

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Obligate intracellular pathogens are microbes that can

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Fungi (including yeasts, moulds and dimorphic fungi)

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Most microbiology laboratories do not culture protozoa;

• Examples of protozoa that can be cultured in vitro are

• Due to the severity of diseases that they cause, it is of

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• The suffix –cide or –cidal refers to “killing.”

• A microbistatic agent is a drug or chemical that inhibits

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Using a Bunsen Using an electrical

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Many foods brought into our kitchens are contaminated

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

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