Controlling Microbial Growth

Chapter 7

Objectives
Learn some terminologies
Know the factors that affect the
effectiveness of anti-microbials
Know some physical and chemical methods
of controlling microbes

Microbial Control Methods

Figure 7.1

Terminologies
Sterilization:

Destruction/removal of all forms of microbial

life from an object
Physical processes

Filtration
High temperature
Incineration
Irradiation by ultraviolet light or gamma () rays

Chemical processes

Ethylene oxide (used in hospitals)

Chlorine dioxide (decontamination of buildings)
Steam sterilization (microbiology)

Mechanical processes
Filtration

Terminologies

Disinfection:

Use of chemicals (disinfectants) to kill or

inhibit microorganisms that cause disease
Does not kill all microorganisms
Usually toxic and injurious to human tissues

Widely use Chemicals disinfectants:

Chlorine (water supplies)

Phenols,
Biphenols (chlorhexidine),
Alcohols
Iodine
Aldehydes
Quats (quartenary ammonium salts)
Ozone and UV light (newer means)

Terminologies
Sanitization:

Related to disinfection, however sanitization only

reduces the microbial population to acceptable
standards.
Restaurants/cafeterias sanitize eating utensils
to acceptable public health standards.

Antisepsis - Related to disinfection:

Chemicals used to treat or prevent infection of living
tissues (still toxic)
Common antiseptics
Alcohol
Iodine
Chlorhexidine
Heavy metals (silver, mercury)

Terminologies
Antimicrobial agents

Agents that kill or inhibit the growth of

microorganisms (cidal or static)
cide: suffix indicating agent kills organisms
Biocide or germicide: kills microorganisms
Virocide: inactivates viruses
e.g. microbiocides for HIV used in
spermacides
Fungicide: kills fungi
static: agent that inhibit growth
Bacteriostatic agent: (inhibits) growth of
bacteria

Terminologies
Sepsis:
bacterial contamination

Asepsis:
absence of significant contamination

Aseptic technique
minimizes contamination

Factors Affecting Antimicrobial

Effectiveness

Population size (larger populations longer to kill)

Population composition: different sensitivity to
antimicrobial agents
Concentration or intensity of an antimicrobial agent
Time of exposure longer exposure more
organisms killed
Environment conditions
High temperatures (more killing)
Organic material (decreases killing)
Sewage, biofilms

Microbial characteristics

Spores more resistant than vegetative cells

Microbial Characteristics
Gram (-) vs Gram (+) bacteria.
Pseudomonas aeruginosa - most resistant gram (-)
Acid-fast vs non-acid fast
Mycobacterium tuberculosis- resistant to
aqueous bactericides (why?).
Vegetative vs Endospores most resistant bacteria forms
Viruses
Naked viruses more resistant than enveloped.
Protozoan
cysts vs trophozoite stage

Cryptosporidium cysts are resistant to chlorine

Microbial
Sensitivity
to Chemical
Biocides (Fig
7.11)

Concentration and
Duration of exposure

12

Figure 7.1

Physical Control Methods

Heat

Common method to destroy microorganisms

Moist Vs Dry
Steam at 100oC more dangerous that an oven
at 325oC (Why?)
Moist heat
Kills by coagulation of protein
Boiling water not as effective as steam
Boiling water not as effective as steam
(endospores resistant to boiling)
Dry heat
kills by oxidation (higher temps and longer
time required)

Moist Heat
Autoclaves (steam sterilizers)- Routinely
used in Microbiology lab
Operates like a pressure cooker
Sterilize by moist heat under pressure
15 psi for 15 minutes at 121C
Most effective
when organisms in small volume or in
direct contact with steam

Autoclave or Steam Sterilizer

Operates in a cycle
Saturated steam enters chamber
Air evacuated and temperature
raised to 121oC

Pressure applied (15 psi) 15

min
Steam condenses on objects
Pressure lowered to normal
atmospheric pressure before
opening the chamber
Why is this important for
liquids? Lowering pressure so
liquids will change to steam
Chamber opened before pressure
falls for solid objects (surgical
instruments)- Why?

Commercial sterilization
Used in the canning Industry
Steam sterilization designed to kill heat
resistant spores of Clostridium botulinum

The heat treatment reduces the number of

spore by 1012

Pasteurization
Reduces numbers of pathogens
Used when we dont want to change taste
Milk pasteurization
Designed to kill Mycobacterium tuberculosis
(heat resistant pathogen in milk).
Also kills pathogenic and non-pathogenic
Streptococcus, Brucella and coliforms
Low-temperature hold (LTH)
62.8C for 30 minutes
High-temperature short-term (HTST):
72C for 15 seconds
Ultra-high temperature (UHT):
Process (5 sec cycle 74oC 140C 74oC)

Dry Heat
Dry heat kills microbes by oxidation.
Higher temperatures and longer periods required.
Incineration
Flaming
Oven (170C, 2 hours)

Mechanical Methods
Filtration

Can reduce microbial

population in liquids
Sterilize heat-sensitive
materials (heat labile)
Can also reduce microbial
populations in air
Two types
Depth filtering
Membrane filters
0.1 mm thick
Pore sizes from 0.2 m

Low Temperature
Refrigeration is
bacteriostatic
Most pathogens do not
grow
Listeria monocytogenes
grows in refrigerator
Freezing: slow freezing
creates ice crystals

Radiation
Non-ionizing (UV) and Ionizing
radiation
UV- (Non ionizing radiation)
Not penetrating
Can damage eyes
Damages DNA

Thymine dimers in DNA

Germicidal lamps

vaccine disinfection

Radiation

Ionizing radiation:

Gamma () rays, X-rays, or high energy electron

beams (most energetic)
Cause mutations and death
Low level ionizing rad. used on spices,
certain meats and vegetables
High energy rad. used to sterilize medical
supplies

Limiting Water
Desiccation : bacteriostatic
lyophilization used to preserve cultures

Osmotic pressure high concentrations

of salt or sugar
Molds and yeasts most resistant to
desiccation and high salt conc.

Chemical Methods of Microbial

Control
Chemicals can be used as:
Disinfectants,
Sterilants,
Antiseptics
Surface-active agents (surfactants)
Chemical food preservatives
[Antibiotics]

Evaluating Disinfectants

Susceptibility test

Make serial dilution of disinfectant made

Known amounts of Staphylococcus aureus and
Salmonella enterica added to each dilutions
Incubate at 20 or 37oC for 5 or 10 minutes.
Remove aliquots and inoculate growth media
Determine the lowest dilution of disinfectant that
kills bacteria at 10 min but not at 5 min exposure (D).
Compare to phenol dilution (P) that does the same
Phenol Coefficient value = D/P
Value greater than 1 Disinfectant more effective

Conc. of disinfectant

MIC: Minimal inhibition concentration

MBC: Minimal bactericidal concentration

Evaluating Disinfectants
Use-dilution test

Metal rings dipped in test bacteria and dried,

Dried cultures placed in disinfectant for 10 min at 20C.
Rings then transferred to culture media to determine
whether bacteria survived the treatment.
Use information obtained to determine lowest dilution
to be used for disinfection.

Evaluating Disinfectants

Disc Dilution Methods:

Inoculate bacteria on nutrient agar plates

Place filter discs impregnated with disinfectant on
plates and incubate
Measure zones of inhibition

Phenols and Phenolics:

Disinfectants
Disrupt cell membranes
Phenol used as standard
Measure effectiveness of disinfectants
(Staphylococcus aureus or Salmonella typhi)

Active in presence of organic matter

Stable
Persist for long periods after application
Active against M. tuberculosis

Phenols
Originally used by Joseph Lister
Lysol
mixture of phenols used as
general disinfectant
Hexachlorophene excellent skin disinfectant
Effective against S. aureus
But is neurotoxic in rats
Triclosan
used in antibacterial soaps
and toothpaste
Broad spectrum of activity

Chlorhexidine
Disinfectant (member of biguanides)
Widely used by dentists
Also used as skin antiseptic
Low toxicity
Used on skin and mucous membranes

Halogens
Disinfectants

Iodine (I2) used in solution

Tincture (iodine+alcohol)
Iodophors (iodine+organic carrier)
Betadine and Isodine, Wescodyne
Stain skin and garments
Effectiveness- all bacteria, spores, fungus and viruses
Chlorine (Cl2) used to disinfect water supplies
Effective against wide range of microbes
Disadvantage:
Corrosive
Inactivated by organic matter
Forms chlorine disinfection by-products (DBPs) linked
to bladder cancer
Chlorine gas forms bleach (hypochlorite) in water
Chloramines are chlorine and ammonia

Alcohols
Disinfectants
Ethanol or Isopropanol widely used
Denature proteins and damage cell membrane

Used to clean skin prior to surgery

Evaporate quickly
More active as 70% aqueous solutions (instead
of 100%)

Heavy Metals
Disinfectants
Denature proteins
Silver nitrate (topical cream)
Mercury (phenylmercury) in eyedrops and
contact lenses preservatives
Copper sulfate (algicide)
Zinc (mouthwash, paints)

Surfactants
Decrease surface tension or can damage
lipid membranes
Soaps and detergents
Cleaning value (little value as disinfectants)

Acidianionic sanitizers
Used to clean dairy utensils and equipment

Quaternary ammonium compounds

Microbicidal
Fungicides, amoebicidal, and virucidal (enveloped viruses)

Quaternary Ammonium
Compounds (Quats)
Common quat (Zephiram, Cepacol)
Used in mouthwash
Pseudomonas auriginosa not affected, can grow in quats.
More effective against G(+)
Not effective against endospores or mycobacteria

Aldehydes
Formaldehyde (formalin) and
glutaraldehyde
Cross-link protein molecules
Disinfect instruments
Used to preserve tissues for pathology

Chemical Sterilization
Gas Sterilization
Ethylene oxide and Chlorine dioxide

Plasmas
Gas excited by an electromagnetic field to generate a mixture of nuclei
and free electrons
Form free radicles that can damage microbial structures. Use: surgical
devises with long hollow tubes, catheters, laparoscopic instruments.

Supercritical fluids
Compressed CO2 with both gas and liquid properties (use:
bone, tendons, ligaments etc)

Peroxygens
Hydrogen peroxide, peracetic acid, etc.

Gas Sterilization
Ethylene oxide

Denatures proteins
High penetration power
Kills all microbes and spores

But require lengthy exposure time

Used in hospitals as a chemical sterilant

(beddings, mattresses)
(chambers resemble autoclaves)

Chlorine dioxide

Short-lived
Used to sterilize buildings and contents
(anthrax endospores)
Initial treatment of water supplies before
chlorination

Oxidizing Agents
Interfere with metabolism (especially of
anaerobes)
Peroxygens
Hydrogen peroxide
Common antiseptic
Benzoyl peroxide
Peracetic acid (washing fruits and
vegetables)
Ozone
Used by the GVRD as primary water
disinfectant (allowing chlorine to be
used in lower conc.)

Microbial Characteristics & Control

Gram (-) vs Gram (+)
bacteria

Pseudomonas & Burkholderia spp. most resistant Gram ve (porins)

Acid-fast vs non-acid fast

Mycobacterium tuberculosisresistant to aqueous bactericides

(why?)

Vegetative vs endospores

most resistant bacteria forms

Viruses

Naked viruses more resistant than

enveloped (protein vs lipid coat)

Protozoan cysts vs vegetative

(trophozoite) protozoa.
Cryptosporidium cysts are
resistant to chlorine

Other Antimicrobials
Antibiotics
Examples discussed here are not useful for ingestion or
injection to treat disease
E.g. Nisin to inhibit endospore-forming spoilage bacteria
in cheese

Plasmas
Gas is excited by electromagnetic field to form plasma
state of matter
Free radicals in plasmas destroy microbes and spores
Used to sterilize long, narrow, hollow surgical
instruments

Other Antimicrobials
Supercritical fluids
When CO2 is compressed into supercritical
state it has both gas and liquid properties
Microbes are exposed to supercritical fluids
inactivated
Endospores inactivated at lower temperatures (45 oC)
Used to disinfect medical implants (bone, tendons, ligaments from
donors)

Chemical Food Preservatives

Sulfur dioxide
Sodium benzoate
Sorbic acid
Calcium propionate
Sodium nitrate and sodium nitrite

Heat Preservation
Must know temperature
and time needed to kill
critical bacteria
Thermal death time
(TDT)

Minimum time required to

kill all bacteria in a liquid
culture at a particular
temperature

Antiseptic Effectiveness

The steeper the downward slope of the killing curve, the more affective the
antiseptic is. Which antiseptic is most effective?

Commercial Sterilization
Used in the canning Industry
Steam sterilization designed to kill heat
resistant spores of Clostridium botulinum
More-resistant endospores of thermophilic
bacteria may survive

Limiting Water
Desiccation : bacteriostatic
lyophilization used to preserve cultures

Osmotic pressure high

concentrations of salt or sugar
Molds and yeasts most resistant to
desiccation and high salt conc