Transcript Chemical Methods for Controlling Microbial Growth

Controlling Microbial Growth
Vocabulary
• Bactericidal: Bacteria-killing
• Bacteriostasis: Bacterial growth inhibiting
• Disinfection: Destruction of vegetative pathogens
• Antisepsis: Destruction of vegetative pathogens
on living tissue
• Aseptic: Absence of a pathogen from an object or
an area
Vocabulary Cont.
• Sanitation: Reduction of pathogens on eating
utensils, etc. to public health standards
• Antiseptic: chemical used for disinfection of
skin or mucous membranes
• Disinfectant: Chemical used on inanimate
objects to kill or inhibit bacterial growth
• Sterile: Without life, free of microorganisms
Measuring the effectiveness of
bacterial control methods
• Not all genera of bacteria are equally
susceptible to a given method of control.
– Example:
• E. coli bacteria are highly susceptible to heat and die
quickly when they reach boiling temperatures.
• Pathogens that create endospores on the other hand,
can survive long periods of boiling.
• How do we determine how hot and how long
we need to keep a microbe at to kill it?
Three acceptable ways to measure
microbial tolerance to heat
1. Decimal reduction time (DRT): time in which
90% of the population of bacteria at a given
temperature will die.
2. Thermal death time (TDT): Time in which all
cells in a suspension are killed at a given
temperature.
3. Thermal death point (TDP): The lowest
temperature in which all microorganisms will
be killed in 10 minutes.
Death Curve
Microbes die at a constant, logarithmic rate when treated.
Death Curve
What is the DRT for the above graph?
What is the TDT?
Physical Methods of Controlling
Microbial Growth
Moist Heat Methods
• More effective than dry heat
• Kills by coagulation of proteins
• Three methods: boiling, autoclaving,
pasteurization
Boiling
• 100 C for 10 min: kills vegetative bacteria,
most viruses, and fungal spores.
– Endospores and some viruses (hepatitis) can
survive for long boiling periods.
• Good for making drinking water and food safe
for consumption.
– Unreliable for sterilization of surgical equipment
(endospores survive without stomach acid to kill
them). Free flowing unpressurized steam is
equivalent to boiling.
Autoclave
• Pressurizing of steam to make it hotter. 1 atm
temp = 121 C kills all organisms and their
endospores. Most effective and most
preferred method of sterilization. Steam must
contact all solid surfaces since solids don’t
have convection currents like liquids do.
Autoclave
Pasteurization
• Mildly heating food products to kill particular
spoilage microorganisms or pathogens. 72 C
for 15 seconds with refrigeration needed
afterwards. Many heat resistant bacteria
survive pasteurization but these are unlikely
to cause disease.
• Ultra high temperature pasteurization 140 C
for 3 seconds sterilizes milk.
Dry Heat
• Kills and sterilizes by oxidation effect.
• Direct flaming or incineration
• Hot air sterilization (170 C for 2 hours)
Filteration
• Filters are made of cellulose or plastic
polymers with pores of about .2 micrometers.
Viruses can still get through.
• There are filters with pore size as small as .01
micrometers for viruses but filtration is very
slow.
• Filters are important when you can’t heat
something up because you’ll destroy what
you’re trying to keep.
Cold
• Cold is bacteriostatic
• Slow freezing is more harmful as crystals can
set up better.
• Deep freezing is quick freezing at temp
between -50 and -95 degrees. This is usually
done to preserve the specimen.
Desiccation
• Desiccation is the absence of water. Microbes
need water in order to grow and reproduce.
However, microbes in a desiccated state can
be viable for years. When water is added, they
can resume growth.
• Desiccation is bacteriostatic, though the ability
to survive varies by species.
Lyophilization (freeze drying)
• A suspension of microbes is quickly frozen at
temp between -54 and -72 C and water is
removed by high vacuum. Microbes can be
stored for years in this state.
Chemical Methods for
Controlling Microbial Growth
Test Guide to Chemotherapy
• Different strains of microbes have different
levels of susceptibility to different
chemotherapeutic agents.
• Susceptibility of a microorganism can change
over the course of a treatment.
• Physicians need to know the sensitivities of
the pathogen before treatment can be
started. Various tests are employed to give
Physicians the knowledge they need.
Methods for Testing Disinfectants
• The American Official Analytical Chemist’s
Use-dilution method (Use-Dilution Method)
• The Disk-diffusion Method
• Three bacteria, Salmonella, Staphylococcus,
and Pseudomonas, are used as standards to
examine the effectiveness of a given
disinfectant.
Use-Dilution
• Small stainless steel cylinders are dipped and
coated with these bacteria and than allowed to
dry.
• The cylinder is than dipped into one of several
dilutions of the chemical agent to be tested for 10
minutes, removed, rinsed with water, and placed
into a tube of nutrient broth.
• Chemicals that prevent growth at the greatest
dilutions are considered to be the most effective.
Example Results
Disinfecting Agents
Dilution Ratio Agent 1
Agent 2
1:1
1:10
+
1:50
+
1:500
+
+ indicates growth
- indicates no growth
Agent 3
+
+
Disk-diffusion (Filter Paper) Method
• Most common testing method.
• Small disc of filter paper is soaked with
chemical and placed on surface of agar plate
that has been previously inoculated with the
test organism.
Disk-diffusion Cont.
• The chemical is concentrated in one area, and
as it diffuses out, it is less concentrated. The
effectiveness is based on how big of a circle
“kill-zone” or zone of inhibition is around
where the chemical is introduced.
• Multiple chemicals can be tested against one
bacteria at a time with this method.
Example Results
Chemical Disinfectants
• Can kill in three ways:
– Damage of Plasma Membrane
– Inactivate Enzymes
– Denature Proteins
Phenol and Phenolic
• Phenol: First used by Joseph Lister in an
operating room. It proved too toxic for skin
and had a terrible odor.
• Phenolic: Adapted version of phenol that is
less potent and more useful.
• Kills in all three ways.
Iodine (a halogen)
• Kills by inactivating enzymes by attaching to the
amino acid tyrosine.
• Effective against bacteria, endospore, fungi, and
some viruses.
• Iodine is available as tincture solution (in solution
with alcohol). Tincture solutions often stain and
patients may develop hypersensitivity to it.
• Iodine can come in Iodophor (combined with an
organic molecule for slower releasing).
Chlorine (a halogen)
• Chlorine is mostly just a disinfectant. Chlorine
turns to hyochlorous acid, its active form,
when it’s added to water.
• Kills by deactivating enzymes. The acid
oxidizes cellular molecules into inactive forms.
Alcohol (ethanol and isopropyl)
• Alcohols work in two ways: by denaturing
proteins and dissolving lipids (plasma
membrane).
• Effective against bacteria and fungi but not
endospores and non-enveloped viruses.
Alcohol is most effective when it is diluted by
water. (ethanol 70%, isopropyl 90%). Nonwatered alcohol doesn’t carry into cells very
well.
Don’t drink isopropyl alcohol.
That is all.
Heavy Metals
• Gold, silver, copper and zinc are germicidal.
Silver nitrate is used in infants’ eyes.
• Selenium and zinc in shampoos control fungi
that cause dandruff.
• Heavy metals combing with –SH groups to
denature proteins.