Lecture #6
Control of Microbial Growth
Important Terms
• Sterilization
• The killing or removal of all viable organisms within a growth medium
• Inhibition
• Effectively limiting microbial growth
• No killing taking place
• Decontamination
• The treatment of an object to make it safe to handle
• Disinfection
• Directly targets the removal of all pathogens
• Not necessarily all microorganisms
Heat Sterilization
• Heat sterilization is the most widely used
method of controlling microbial growth
• High temperatures denature macromolecules
• Amount of time required to reduce viability
tenfold is called the decimal reduction time
(D)
• Time it takes for a 10 fold reduction in
bacterial numbers
• Some bacteria produce resistant cells
called endospores
• Can survive heat that would rapidly kill
vegetative cells
Pasteurization
• Pasteurization: is the process of using precisely controlled heat to
reduce the microbial load in heat-sensitive liquids
• Does not kill all organisms-> not sterilization
• In the case of milk many different time and temperature combinations
can be used.
• LTLT (low-temperature/long-time)
• 63oC for 30 minutes
• HTST (high-temperature/short-time)
• 72oC for 15 seconds
• Both processes kill Coxiella burnetii, the causative agent of Q fever, which is
the most heat resistant pathogen found in milk
The Autoclave
• The autoclave is a sealed
Chamber
pressure
gauge
device that uses steam under
Steam exhaust
Steam
exhaust
valve
pressure
• Allows temperature of water to
get above 100oC
Door
Jacket chamber
Thermometer
and valve
• 121oC for 15 min at 15 pounds
Air exits through vent
Steam supply
valve
Steam enters here
per square inch of pressure is
typically used
Autoclave time
Stop
steam
• To ensure sterility this means
that the point that takes the
longest to heat must stay at 121
for 15 min
Begin
pressure
Flowing
steam
Sterilization time
Temperature
of object being
sterilized
Temperature
of autoclave
Radiation
• Physical method of growth control
• Microwaves, UV, X-rays, gamma rays, and electrons can reduce
microbial growth
• UV has sufficient energy to cause modifications and breaks in
DNA
• UV is useful for decontaminating surfaces
• Cannot penetrate solid, opaque, or light-absorbing surfaces
Radiation
• Ionizing radiation
•
Electromagnetic radiation that produces
ions and other reactive molecules
generates electrons, hydroxyl radicals,
and hydride radicals.
•
Some microorganisms are more
resistant to radiation than others.
•
amount of energy required to reduce
viability tenfold is analogous to D value
Radiation
• Sources of radiation include cathode ray tubes, X-rays, and radioactive
nuclides
• Radiation is used for sterilization in the medical field and food industry
•
Radiation is approved by the WHO and is used in the USA for
decontaminating foods particularly susceptible to microbial contamination
• Hamburger, chicken, spices may all be irradiated
Filtration
• Filtration avoids the use of heat on sensitive liquids and gases
•
Pores of filter are too small for organisms to pass through
• Allow liquid or gas to pass through
• Ex) HEPA filters, membrane filters
•
With membrane filters, filtration can be accomplished by:
• Syringe
• Pump
• Vacuum
Filtration
a) Pore size is 5 µm
•
Note the size of the pores relative
to the size of some of the bacteria
•
Is this an appropriate filter to
prevent bacteria from going
through?
b) Pore size 0.2 µm
• Note the pore size relative to
Leptospira (0.1 x 20 µm)
• This filter prevents most bacteria
from going through
Chemical Control of Microbial Growth
• Antimicrobial agents can be classified as:
•
•
•
Bacteriostatic: prevents cell growth as long as the antimicrobial agent is
present
Bacteriocidal: kills the cells (does not lyse them)
Bacteriolytic: kills and lyses the cells
Chemical Control of Microbial Growth
• Minimum inhibitory concentration (MIC)
• The smallest amount of an agent needed to inhibit growth of a microorganism
• Varies with the organism used, inoculum size, temperature, pH, etc.
• Increasing concentration of antibiotic from left to right.
• Lowest concentration with no growth = MIC
• May still have living (non-growing) organisms
• i.e. the antibiotic may be bacteriostatic
• Plate aliquots of broth from the MIC and from
tubes with higher concentrations of antibiotic,
on appropriate nutrient agar plates, without
antibiotic
• Do colonies form?
• No colonies = Minimal Lethal Concentration (MLC)
• MLC usually higher than MIC
Chemical Control of Microbial
Growth
• Disc diffusion assay
•
Uses solid media
•
Antimicrobial agent is added to filter paper disc
•
The MIC is reached at some distance
•
Forms a zone of inhibition
•
Area of no growth around disc
Chemical Control of Microbial Growth
These antimicrobial agents can be divided into two categories:
1.
Products used to control microorganisms in commercial and industrial applications
• Ex) chemicals in foods, air conditioning cooling towers, textile and paper
products, fuel tanks
2.
Products designed to prevent growth of human pathogens in inanimate
environments and on external body surfaces
• Sterilant: destroys all microorganisms including endospores
• Disinfectant: kills microorganisms but not all endospores.
• Used on inanimate objects
• Sanitizer: reduces the numbers of microorganisms on surfaces (e.g. hand
sanitizers)
• Antiseptic: kills or inhibits the growth of microorganisms (non-toxic enough to
be applied on living tissue (e.g. mouth wash))