Controlling Microorganisms
Physical and Chemical
• The methods of
Control of Microbes microbial control used
outside of the body are
designed to result in
four possible outcomes
– sterilization
– disinfection
– decontamination (also
called sanitization)
– antisepsis
Controlling Microorganisms Controlling Microorganisms
• Sterilization • Decontamination (sanitization)
– The killing or removal of all – Cleansing technique that mechanically
microorganisms in a removes microorganisms as well as other
material or on an object debris to reduce contamination to safe
including endospores. levels
– Ex: autoclave or chemical – Ex. Soaps, detergents
sterilants
• Antisepsis
• Disinfection – Reduces the number of microbes on the
– Removes vegetative bacteria human skin. A form of decontamination
but not endospores but on living tissues.
– Ex: Bleach, iodine, boiling – Ex: Alcohol, surgical hand scrubs
water
Controlling Microorganisms Controlling Microorganisms
• Antiseptics: chemical agents
• Sepsis: the growth of applied directly to exposed
microorganisms in the blood body surfaces (skin and
and other tissues
mucous membranes),
• Asepsis: wounds, and surgical
– any practice that prevents the incisions to prevent
entry of infectious agents into vegetative pathogens
sterile tissues and thus prevents
infection – preparing the skin before surgical incisions
– aseptic techniques: practiced in
healthcare; range from sterile – swabbing an open sore
methods to antisepsis – ordinary hand washing with a germicidal soap
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More microbial control terminology 3 Major Principles of Microbial Control
• Chemicals can be used to kill (‐cide) or inhibit (‐ 1. A definite proportion of the organisms die in a
stat) microbial growth given time interval.
• Not all microbes die immediately.
2. The fewer organisms present, the shorter the
– Chemical agents are used on living tissues (as time needed to achieve sterility.
antiseptics) and on inanimate objects (as • Think about cleaning up a mess. The bigger the mess,
disinfectants). the more time it will take.
– Few chemicals achieve sterility. 3. Microbes differ in their susceptibility to
– Why can’t we always use disinfectants on our skin or antimicrobial agents.
mucous membranes? • Need to match antimicrobial agents appropriately
Relative Resistance of Different Microbial Types to
Microbial Control Agents Effectiveness of Control Depends On:
More resistant
Prions
• Number of microorganisms
Bacterial endospores
Mycobacterium
• Target population (bacteria, fungi, spores,
viruses)
Staphylococcus and Pseudomonas
Protozoan cysts
• Temperature
Protozoan trophozoites
• pH
Most gram-negative bacteria • Concentration of agent
Fungi and fungal spores • Mode of action
Nonenveloped viruses • Interfering agents (solvents, debris, saliva,
Most gram-positive bacteria blood, feces)
Enveloped viruses
Less resistant
Microbial Death How do we control microbial growth?
• Death: permanent termination of
an organism’s vital processes
– microbes have no conspicuous vital
processes, therefore death is difficult
to determine
– permanent loss of reproductive
capability, even under optimum
growth conditions has become the
accepted microbiological definition
of death
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Targets to Control Microbial Presence
Cell Wall
• Bacteria and fungi
– Injure cell wall – Block synthesis
– Injure cell membranes – Degrade cellular components
– Interfere with nucleic acid synthesis – Destroy or reduce stability
– Interfere with protein synthesis • Agent
– Interfere with protein function – Chemical agent – Penicillin, detergents, alcohols
– Remove microbes – Physical agent –
• Heat, radiation
Which of the above would effect our cells too?
Cell Membrane Nucleic Acid Synthesis
• All microbes and enveloped
viruses • Irreversibly bind to DNA
– Bind and penetrate lipids – Stop transcription and
– Lose selective permeability translation
(leakage) – Cause mutations
• Agent • Agent
– Chemical agent – Surfactants – Chemical agent –
– Physical agent – formaldehyde
• Heat, radiation – Physical agent –
radiation, heat
Protein Synthesis Protein Function
• Block protein
• Binds to ribosomes active sites
– Stops translation
• Prevent binding
– Prevents peptide bonds
to substrate
• Agent • Denature protein
– Chemical agent – • Agent
• chloramphenicol – Chemical –
– Physical agent – alcohols, acids,
• radiation, heat phenolics,
metallic ions
– Physical – Heat
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Physical Control Methods Killing with Heat
• Temperature
• Moist heat
• The most common method of sterilization.
• Dry heat • Modes of action:
• Cold – Oxidizes proteins and nucleic acids
• Radiation – Denatures proteins/enzymes
• Ionizing • Effectiveness varies with: kinds of microbes,
• Ultraviolet their number, intensity, length of exposure,
pH, moisture, nature of product
Moist Heat Sterilization Boiling
• Effective on glassware and
instruments
• Most common and • Kills fungi, protozoans, bacteria,
efficient method used viruses in 10‐30 minutes
• Requires 3, separate, boilings to
• Two kinds: kill endospores
– boiling • Can use at home
– steam sterilization • Messy, time consuming,
(autoclave) materials may require drying;
endospores may require longer
time
• Can you boil plastics items?
Steam Sterilization (Autoclaving) Dry Heat
• Uses: liquids, glassware,
instruments, bandages, • Types:
contaminated material – Oven (hot air) sterilization
• Steam must reach all – Flaming inoculating loops
surfaces to be effective – Incineration/burning
• Most efficient and • Temperature and time of
convenient. Kills all exposure is greater than
microbes in 15‐20 moist heat. Why would
minutes. Materials may this be?
require drying
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Dry Heat ‐ Ovens Dry Heat ‐ Incineration
• Heat at 160‐170 oC for 2 hrs. • Destroys
contaminated
• Kills microbes and endospores materials:
by oxidation or denaturation – Blood soaked swabs,
• Used on glassware and bandages
instruments – Contaminated objects
and materials
• Can’t be used on liquid media, – Entire, diseased
cloth, plastics, or articles animal carcasses infrared incinerator
wrapped in paper • Safe and effective
Thermal Death Measurements Radiation
• Movement of energy in waves
• Thermal death time (TDT): shortest length of through space and materials
time required to kill all test microbes at a • High frequency waves have the
specified temperature greatest penetrability
– Waves strike molecules and knock
out electrons
• Thermal death point (TDP): the lowest – Releases ions and creates free
radicals in cells
temperature required to kill all microbes in a – Ions attach to proteins and nucleic
sample in 10 minutes acids, damage cell structures, cause
cell death
• Kills microbes on surfaces and
within materials
• Good for heat‐sensitive items.
A UV treatment system for disinfection of water
Ultraviolet (UV) Radiation
• Moderate wavelengths, low
penetrability. Won’t penetrate
paper, glass or skin.
• Kills microbes on surfaces
• Cross‐links DNA, inhibits replication,
not safe to use on skin, causes
burns, cancer
• Uses: sterilize surfaces (floors ,walls
etc) in labs and operating rooms.
Also vaccines, serum, toxins,
drinking water and waste water
– Germicidal lamp in hospitals,
schools, food preparation areas
(inanimate objects, air, water)
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High Energy
Ionizing Mechanical Control Methods
Radiation
• Filtration
• Gamma rays, X‐rays,
Electron beams • Liquid
• Gamma rays used to • Gas
sterilize glassware,
surgical instruments,
sterile drapes
• Electron beams used
to sterilize
pharmaceuticals,
disposable plastic
syringes, surgical
gloves, etc.
The effects of ionizing and nonionizing radiation on DNA.
Filtration Filtration and Filters
• The passage of liquids and
gases through screen‐like • Gases are forced through
material with pore sizes small under positive pressure.
enough to retain microbes. • Liquids are either forced
• Removes microbes. Doesn’t through under pressure or
kill or inhibit. pulled through under
vacuum.
• Used to sterilize air and heat
sensitive material. • Fluids are collected in
sterile vessels
Uses of Filtration Air Filtration
• Used in operating rooms, burn units, laminar
• Heat sensitive material. flow hoods in high security pathogen
i.e., plasma, sugar research. Also in rooms housing TB patients
solutions, intravenous
• Use High Efficiency Particulate Air (HEPA)
solutions, vaccines,
antibiotic solutions
• Removes bacteria, but not
viruses, from plasma. Slow
because of plasma’s high
viscosity
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Chemical Agents in Microbial Control Chemical Agents in Microbial Control:
• Range from disinfectants and antiseptics to Principles of Effective Disinfection
sterilants and preservatives
• Careful attention should be paid to the
properties and concentration of the
• Aqueous solutions: chemicals dissolved in pure
disinfectant to be used.
water as the solvent
• Tinctures: chemicals dissolved in pure alcohol • The presence of organic matter, degree of
or water‐alcohol mixtures contact with microorganisms, and
temperature should also be considered.
Chemical Agents in Microbial Control:
Chemical Agents in Microbial Control:
Selecting a Disinfectant How do you know if a disinfectant works?
• Weigh the risks and
benefits for each Test it!
situation
• An ideal disinfectant – Serial‐dilution test
should have: – Calculates # of surviving microbes after 10 minutes in
disinfectant solution
• Antimicrobial activity
• Solubility
• Stability
• Lack of toxicity for humans and animals
– Disk‐diffusion test
• Minimum activity by extraneous material – Filter paper disk is soaked with disinfectant/antibiotic and
applied to inoculated plate. Look for inhibited growth.
• Activity at ordinary temperatures
• Ability to penetrate
• Material safety
• Availability and low cost
Serial‐Dilution Test Disk‐Diffusion Test
• The minimum inhibitory concentration (MIC) is the
concentration required to inhibit growth of a specific
isolate in vitro under standardized conditions. Which is the
• It is determined by finding the lowest dilution without best control
visible growth during serial dilution testing. This will vary agent?
for individual isolates.
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Chemical Agents in Microbial Control: Phenolics
Types of Chemical Control Agents • Vary based on
• You won’t need to know specifics about any of functional groups
the chemical control agents. This is more for attached to the
your information aromatic ring
• Phenols • Examples:
• Dr. Lister used phenol to control surgical infections Hexachlorophene,
• Rarely used today because it is a skin irritant Triclosan
– Microbicidal
• Phenolics – Ingredient in soaps
• Often used because they are stable and persist for long to kitty litter
periods • Disrupts cell walls
• Example=Lysol and membranes,
Types of Chemical Control Agents
Types of Chemical Control Agents • Alcohols
• Disinfect and then evaporate
• Halogens • Used to degerm (remove microbes by swab)
– Iodine • Tinctures = solutions of disinfectant in alcohol
– Betadine
• Alcohol based hand sanitizer rubs—effective alternative
– Used for skin disinfection and wound treatment
to hand washing
– Also used for water treatment
• Heavy Metals
– Chlorine
• Silver nitrate‐‐used in many applications,
– HOCl‐‐hypochlorous acid bleach (calcium hypochlorite)
– Used to disinfect instruments and water for instance, in eye‐drops applied to
– 10% bleach in water‐‐good disinfectant, but needs to be fresh infants to protect against gonococcal
infections which may cause blindness
• Copper sulfate‐‐destroys algae in
ponds/pools
Demonstration of the Action of Heavy Metals
Types of Chemical Control Agents
• Quaternary Ammonium Compounds
(Quats)
• Most widely used surface‐active
agents (foamy)
• Bind and disrupt cell membrane
• Zephiran
– Disinfectant when concentrated, but can
be used in dilute form as an antiseptic
• Cepacol
– Disinfectant and antiseptic
– Mouthwash
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Types of Chemical Control Agents Types of Chemical Control Agents
• Chemical Food Preservatives • Aldehydes
• sulfur dioxide‐‐wine‐making • Very effective
• sodium benzoate‐‐prevent molds in acidic foods • Formaldehyde & glutaraldehyde‐‐used to disinfect
• sodium nitrate‐‐meat product additive hospital instruments, however carcinogenic
– link between increased levels of nitrates and increased deaths • Used by morticians for embalming
from certain diseases including Alzheimer's, diabetes mellitus,
and Parkinson's; possibly through the damaging effect of
nitrosamines on DNA (De La Monte, SM; Neusner, A; Chu, J; Lawton, M (2009). "Epidemilogical trends strongly suggest
exposures as etiologic agents in the pathogenesis of sporadic Alzheimer's disease, diabetes mellitus, and non‐alcoholic steatohepatitis". Journal of
Alzheimer's disease : JAD 17 (3): 519–29)
Controlling Microbes in Food and the
Types of Chemical Control Agents
Lab: Refrigeration and Freezing
• Gaseous Chemosterilizers (good for
heat‐sensitive items) • Bacteriostatic: Inhibits growth and toxin
• Chemicals that sterilize in a closed chamber production
• Ethylene oxide‐‐used on hospital equipment • Slow freezing kills many microbes, but not all.
• Reacts with functional groups of DNA and
proteins • Survivors multiply when returned to growth
• Sterilizes and disinfects plastic materials temperatures. Toxins produced previously are
not affected.
• Peroxygens • Not a Sterilant
• Hydrogen peroxide‐‐better used on inanimate • In the lab it is used
objects vs. open wounds
• Benzoyl peroxide‐‐treat acne by killing to store microbes
anaerobic bacteria in hair follicles
• Peracetic acid‐‐effective, considered a sterilant
– used in food processing and medical equipment
Controlling Microbes in Food and the Controlling Microbes in Food and the Lab:
Lab: Desiccation Freeze Drying ‐ Lyophilization
• At ambient temperatures • Slow freezing under vacuum
• Essentially bacteriostatic removes water without ice
– Kills many microbes (species crystal formation in cells.
sensitive)
– Used to preserve foods, • Avoids cell damage.
meats
– Not a reliable sterilant!! Does • Used to store bacteria and
not kill endospores and viruses as powders. Lyophilized
protozoan cysts. microbes can be rehydrated and
• In the lab it is used to store grown in culture.
microbes
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Controlling Microbes in Food: Microwave Controlling Microbes in Food:
Radiation Osmotic Pressure
• Kills bacteria by heating. • Adding large amounts of salt or
• Unreliable sterilant!! sugar to foods creates a
– Ovens have “cold spots” hypertonic environment for
– Materials must be rotated to bacteria, causing plasmolysis
achieve even temperature
distribution.
– Won’t kill Trichinella cysts. • Pickling, smoking, and drying
foods have been used for
• A new version for lab use centuries to preserve foods
sterilizes media in 10 min.
• Osmotic pressure is never a
sterilizing technique
Controlling Microbes in Food:
Controlling Microbes in Food:
Pasteurization
Pasteurization
• Disinfection of beverages • Need to maintain taste and
appearance
• Exposes beverages to 71.6
• Mild heat is used to kill
˚C for 15 seconds pathogens and reduce microbe
– Stops fermentation populations in liquid food and
beverages.
• Prevents the transmission
• Standard method: Heat
of milk‐borne diseases beverages to 60‐66oC for 30
– Salmonella, minutes. Cool rapidly and store in
Campylobacter, sterile containers in cold.
Listeria, Mycobacteria • Flash pasteurization: Heat milk to
71.7oC for 15sec
• Examples: Milk industry,
• Ultra High Temperature
wineries, breweries Sterilization: Heat milk to 140oC
for 3 sec. Store in sterile
containers. Long shelf life
without refrigeration.
Controlling Microbes in Food:
Gamma Rays
• Used on poultry, pork, fresh
fruits, white potatoes, spices.
• Kills bacteria in food
• Eliminates insects
• Prevents premature sprouting
of seeds
• Extends shelf life of foods
• May discolor food and/or alter
taste
• Animals fed irradiated feed
loss weight
• No demonstrated risk from
residual radiation
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