Continuous Culture of Microorganisms

Growth in an open system.

Maintains cells in log phase at a constant biomass

concentration for extended periods.

Achieved using a continuous culture system.

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 Chemostats and Turbidostats
Chemostat
Rate of incoming medium = rate of removal
of medium from vessel.
An essential nutrient is in limiting
quantities.
Most stable and effective at lower dilution
rates.
Turbidostat
Regulates flow rate of media through vessel
to maintain predetermined turbidity or cell
density.
Dilution rate varies.
Contains all nutrients in excess.
Operates best at high dilution rates.
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 Dilution Rate and Microbial Growth
Dilution rate—rate at
which medium flows
through vessel.
Note: cell density
maintained at wide
range of dilution rates.
Chemostat operates
best at low dilution
rate.

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 Microbial Growth Control

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 Common Microbial Control Methods

Disinfection: The destruction or removal of vegetative pathogens but not bacterial endospores.
Usually used only on inanimate objects.
Sterilization: The complete removal or destruction of all viable microorganisms. Used on inanimate
objects.
Antisepsis: Chemicals applied to body surface to destroy or inhibit vegetative pathogens.
Chemotherapy: Chemicals used internally to kill or inhibit growth of microorganisms within host
tissues.
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 Definition of Frequently Used Terms
Sterilization.
• Destruction or removal of all viable organisms.
Disinfection.
• Killing, inhibition, or removal of disease causing (pathogenic) organisms.
• Disinfectants—agents, usually chemical, used for disinfection, usually used
on inanimate objects.
Sanitization.
• Reduction of microbial population to levels deemed safe (based on public
health standards).
Antisepsis.
• Prevention of infection of living tissue by microorganisms.
• Antiseptics—chemical agents that kill or inhibit growth of microorganisms
when applied to tissue.
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 Impact of Biocide Exposure

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 Antimicrobial Agents
Chemotherapy.
• Use of chemicals to kill or inhibit growth of
microorganisms within host tissue.
Cidal agents kill (-cide suffix indicates agent that kills).
• Include bactericides, fungicides, and viricides.
Static agents inhibit growth (-static suffix indicates
growth inhibiting agent).
• Include bacteriostatic and fungistatic.

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 The Pattern of Microbial Death
Microorganisms are not killed instantly.
Population death usually occurs exponentially.
Measure of agent’s killing efficiency.
• Decimal reduction time—time to kill 90%.
Must be sure viable but nonculturable cells are dead.
• Once they recover, they may regain the ability to
reproduce and cause infection.

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 D and Z Values

Microbial Number at Microorganisms Killed in 1 Microorganisms at Log10 of

Minute Start of Minute Minute (90% of total) End of 1 Minute Survivors
1 106 9 x 105 105 5
2 105 9 x 104 104 4
3 104 9 x 103 103 3
4 103 9 x 102 102 2
5 102 9 x 101 10 1
6 101 9 1 0
7 1 0.9 0.1 -1
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 8.3 Mechanical Removal Methods
Rely on Barriers
=Filtration

Reduces microbial population or sterilizes

solutions of heat-sensitive materials by removing
microorganisms.
Also used to reduce microbial populations in air.

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 Filtering Liquids
Membrane filters.
• Porous membranes with defined pore
sizes that remove microorganisms
primarily by physical screening.

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 Filtering Air
Surgical masks.
High-efficiency
particulate air (HEPA)
filters.
• Used in laminar flow
biological safety cabinets.

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 8.4 Physical Control Methods Alter
Microorganisms to Make Them Nonviable
a. Describe the application of heat and radiation
to control microorganisms.
b. Explain the mechanisms by which heat and
radiation kill microbes.
c. Design novel antimicrobial control applications
using heat and radiation.

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 8.4 Physical Control Methods Alter Microorganisms to Make Them
Nonviable

Moist Heat
Destroys viruses, fungi, and bacteria.
Boiling will not destroy endospores and does not sterilize.
Degrades nucleic acids, denatures proteins, and disrupts
membranes.
Entity Vegetative Cells Spores
Yeasts 5 minutes at 50– 5 minutes at 70–80°C
60°C
Molds 30 minutes at 62°C 30 minutes at 80°C
Mesophilic 10 minutes at 60– 2 to over 800 minutes at 100°C
Bacteria 70°C 0.5–12 minutes at 121°C
Viruses 30 minutes at 60°C
Prions 90 minutes at 134°C
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 Steam Sterilization
Carried out above 100oC which
requires saturated steam
under pressure.
Uses an autoclave.
Effective against all types of
microorganisms (including
spores!).
Quality control—includes
strips with Geobacillus
stearothermophilus.
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 Dry Heat Incineration
Bench top
incinerators are used
to sterilize inoculating
loops used in
microbiology
laboratories.

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 Chemical Control Agents and Their
Requirements
Disinfection.
• Ideally is effective against wide variety of infectious
agents at low concentrations and in the presence of
organic matter.
Antisepsis.
• Overuse of antiseptics such as triclosan has selected
for triclosan-resistant bacteria.
Sterilization.

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 Effects of Glutaraldehyde

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 Sterilizing Gases
Used to sterilize heat-
sensitive materials.
Microbicidal and
sporicidal.
Ethylene oxide
sterilization is carried out
in equipment resembling
an autoclave.
Vaporized hydrogen
peroxide can also be
used.
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 8.6 Antimicrobial Agents Must Be
Evaluated for Effectiveness
a. Predict the effects of (1) microbial population
size and composition, (2) temperature, (3)
exposure time, and (4) local environmental
conditions on antimicrobial agent
effectiveness.
b. Describe the processes used to measure
microbial killing rates, dilution testing, and in-
use testing of antimicrobial agents.

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 Evaluation of Antimicrobial Agent
Effectiveness
Complex process regulated by U.S. federal
agencies:
• Environmental Protection Agency regulates
disinfectants.
• Food and Drug Administration regulates agents used
on humans and animals.

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Conditions Influencing the Effectiveness of
Antimicrobial Agent Activity 1

Population size.
• Larger populations take longer to kill than smaller
populations.
Population composition.
• Microorganisms differ markedly in their sensitivity to
antimicrobial agents.
Concentration or intensity of an antimicrobial agent.
• Usually higher concentrations kill more rapidly.
• Relationship is not linear.
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 Efficiency Evaluation of Chemical Agents
Phenol coefficient test.
• Potency of a disinfectant is compared to that of phenol.
• Useful for screening but may be misleading.
Use dilution test.
• Determines rate at which selected bacteria are destroyed
by various chemical agents.
Normal in-use testing.
• Testing done using conditions that approximate normal
use of disinfectant.
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 Biological Control of Microorganisms
Emerging field showing great promise.
Natural control mechanisms:
• Predation by Bdellovibrio.
• Viral-mediated lysis using pathogen specific
bacteriophage lysins (or bacteriophages themselves,
such as in a spray format recently approved by the
FDA for use on food products).
• Toxin-mediated killing using bacteriocins.

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