Chapter 9:

Controlling Microbial Growth in the Environment

• Damaged to protein and nucleic acids

- Protein function depends on 3D shape.
- Extreme heat or certain chemicals
denature proteins
- Chemicals, radiation, and heat can alter or
destroy nucleic acids.
- Produce fatal mutants
- Halt protein synthesis through action of
RNA

Selection of Microbial Control Methods

Basic Principles of Microbial Control
Ideally, agents should be:
Action of Antimicrobial Agents
• Inexpensive
• Alteration of cell walls and membranes • Fast-acting
- Cell wall maintains integrity of cell. • Stable during storage
- When damage, effects of osmosis cause • Capable of controlling microbial growth
cells to burst. while being harmless to humans, animals,
and objects
- Cytoplasmic membrane contains
cytoplasm and controls passage of
chemical into and out of cell. Factors Affecting the Efficacy of Antimicrobial
- When damaged, cellular contents leak Methods
out. Site to be treated
- Nonenveloped viruses can better tolerate
harsh condition.
 • Harsh chemicals and extreme heat cannot Biosafety Levels
be used on humans, animals, and fragile
4 levels of safety in labs dealing with pathogen
objects.
• Method of microbial control based on site 1. Biosafety Level 1 (BSL -1)
of medical procedure. - Handling microbes that do not cause
disease in humans.
2. Biosafety Level 1 (BSL -2)
- Handling moderately hazardous agents.
3. Biosafety Level 1 (BSL -3)
- All manipulations of microbes done in
safety cabinets.
4. Biosafety Level 1 (BSL -4)
- Handling microbes that cause severe or
fatal disease.
- Lab space is isolated, and personnel wear
protective suits.

Physical Methods of Microbial Control

Heat Related Methods

• Effects of high temperatures

- Denatures proteins
- Interfere with integrity of cytoplasmic
membrane and cell wall
Relative susceptibility of microorganism - Disrupt structure and function of nucleic
acids.
Germicide Classification • Thermal death point
• High-level germicides - Lowest temperature that kills all cells in
- Kills all pathogens, include endospores. broth in 10mins
• Intermediate-level germicides • Thermal death time
- Kill fungal spores, protozoan cysts, viruses, - Time to sterilize volume of liquid at set
and pathogenic bacteria. temperature
• Low-level germicides
- Kills vegetative bacteria, fungi, protozoa,
and some viruses.
Moist heat

• Used to disinfect, sanitize, sterilize, and

pasteurize.
• Denatures protein and destroy cytoplasmic
membrane.
• More effective than dry heat.
• Methods of microbial control using moist
heat
- Boiling
- Autoclaving
- Pasteurization
- Ultrahigh-temperature sterilization

Boiling
• Kills vegetative cells of bacteria and fungi,
protozoan trophozoites, and most viruses.
• Boiling time is critical
- Different elevations require different
boiling times.
• Endospores, protozoan cysts, and some
viruses can survive boiling.
Pasteurization
Autoclaving
• Used for milk, ice cream, yogurt, and fruit
• Pressure applied to boiling water prevents juices.
steam from escaping. • Not sterilization
• Boiling temperature increases as pressure - Heat-tolerant microbes survive
increases. • Pasteurization of milk
• Autoclave condition: 121°C, 15 psi, 15 - Batch method
mins. - Flash pasteurization
- Ultrahigh-temperature pasteurization

Moist Heat
• Ultrahigh-temperature sterilization
- 140°C for 1 to 3 sec, then rapid cooling
- Treated liquids can be stored at room
temperature.

Dry Heat

• Used for materials that cannot be sterilized

with moist heat.
 • Denatures protein and oxidizes metabolic
and structural chemicals.
• Requires higher temperatures for longer
time than moist heat.
• Incineration is ultimate means of
sterilization.

Refrigeration and Freezing

• Decrease microbial metabolism, growth,
and reproduction.
- Chemical reaction occurs more slowly at
low temperatures
- Liquid water not available
• Refrigeration halts growth of most
pathogen.
• Some microbes can multiply in refrigerated
foods.
• Slow freezing is more effective than quick
freezing.
• Organisms vary in susceptibility to
freezing.
Desiccation and Lyophilization

• Desiccation (drying) inhibits growth as a

result of removal of water.
• Lyophilization (freeze-drying) is used for
Osmotic Pressure
long-term preservation of microbial
cultures. • High concentrations of salt or sugar in foods to
- Prevents formation of damaging ice inhibit growth.
crystals. • Cells in hypertonic solution of salt or sugar lose
water.
• Fungi have greater ability than bacteria to
survive hypertonic environments.

Radiation

2 Types of radiations

1. Particulate Radiation
- High-speed subatomic particles freed from
their atoms.
2. Electromagnetic Radiation
- Energy without mass traveling in waves at the
speed of light.
- The shorter tha wavelenght, the more energy
tha wave carries.
• All types of radiation are described as their
ionizing or nonoinizing.
- Based on effects of chemicals within cells.
Ionizing Radiation Chemical Methods of Microbial Control
• Wavelenghts shorter than 1nm • Affects microbes cell wall, cytoplasmic
- Electron beams, gamma rays, some X rays
membrane, protein, or DNA.
• Ejects electron from atoms to create ions
• Effect varies with differing environmental
• Ions disrupt hydrogen bonding, oxidize double
conditions
covalent bonds, and create hydroxyl radicals.
- Ions denature other molecules (DNA) • Often more effective against enveloped
• Electron beam effective at killing microbes but viruses and vegetative cells of bacteria,
do not penetrate well fungi, and protozoa.
• Gamma rays penetrate well but require hours
Phenol and Phenolics
to kill microbes.
• X ray require long time to kill microbes. • Denature proteins and disrupt cell
- Not practical for microbial control membranes.
Nonionizing Radiation • Effective in presence of organic matter.
• Remains active for prolonged time.
• Wavelenght greater than 1nm
• Commonly used in health care settings,
• Excites electrons, causing than to make new
labs, and homes.
covalent bonds.
- Affects 3D structure of protein and nucleic • Have disagreeable odor and possible side
acids. effects.
• UV light causes pyrimidine dimers in DNA.
• UV light does not penetrate well.
• Suitable for disinfecting air, transparent fluids,
and surfaces of objects.
Alcohols - Ideal for many medical and industrial
applications
• Alcohols Intermediate-level disinfectants.
• Denature proteins and disrupt cytoplasmic
membranes.
• More effective than soap in removing
bacteria from hands.
• Swabbing skin with alcohol prior to
injection removes most microbes
Heavy Metals
Halogens
• Heavy-metal ions denature proteins
• Include iodine, chlorine, bromine, and • Low-level bacteriostatic and fungistatic
fluorine.
agents
• Intermediate-level antimicrobial chemicals • 1% silver nitrate once commonly used to
• Damage proteins by denaturation. prevent blindness caused by N.
• Widely used in numerous applications gonorrhoeae
- iodine tablets, iodophores, chlorine • Thimerosal used to preserve vaccines
treatment, bleach, chloramines, bromine
• Copper controls algal growth
disinfection, and the addition of fluoride to
water and toothpastes. Aldehydes

Oxidizing Agents • Compounds containing terminal -CHO

groups
• Peroxides, ozone, and peracetic acid.
• Cross-link functional groups to denature
• Kill by oxidation of microbial enzymes. proteins and inactivate nucleic acids
• High-level disinfectants and antiseptics.
• Glutaraldehyde disinfects and sterilizes
• Hydrogen peroxide can disinfect and
• Formalin used in embalming and in
sterilize surfaces.
disinfection of rooms and instruments
- Not useful for treating open wounds
because of catalase activity. Gaseous Agents
• Ozone treatment of drinking water.
• Microbicidal and sporicidal gases used in
• Peracetic acid is effective sporicide used to closed chambers t sterilize items
sterilize equipment.
• Denature proteins and DNA by cross-
Surfactants linking functional groups
• Used in hospitals and dental offices
• "Surface active" chemicals
• Disadvantages:
- Reduce surface tension of solvents
- Can be hazardous to people
• Soaps and detergents
- Often highly explosive
- Soaps have hydrophilic and hydrophobic - Extremely poisonous
ends
- Potentially carcinogenic
- Good degerming agents but not
antimicrobial Enzymes
• Detergents are positively charged organic
• Antimicrobial enzymes act against
surfactants
microorganisms
- Quaternary ammonium compounds
• Human tears contain lysozyme
(quats)
- Digests peptidoglycan cell wall of bacteria
- Low-level disinfectants
• Uses of enzymes to control microbes in the
- Disrupt cellular membranes
environment
 - Lysozyme is used to reduce the number of • Contaminated cylinder immersed into
bacteria in cheese dilution of disinfectant
- Prionzyme can remove prions on medical • Cylinders removed, washed, and placed
instruments into tube of medium
• Most effective agents entirely prevent
Antimicrobials
growth at highest dilution
• Antibiotics and semisynthetic and • Current standard test in the U.S.
synthetic chemical • New standard procedure being developed
• Typically, are used to treat disease
• Some are used for antimicrobial control
outside the body

Kelsey-Sykes capacity test

Methods for Evaluating Disinfectants and • Alternative assessment approved by the

Antiseptics European Union
• Bacterial suspensions added to the
Phenol coefficient chemical being tested
• Evaluates efficacy of disinfectants and • Samples removed at predetermined times
antiseptics and incubated
• Compares to phenol an agent's ability to • Lack of bacterial reproduction reveals
control microbes minimum time required for the
• Greater than 1.0 indicates agent is more disinfectant to be effective
effective than phenol In-use test
• Has been replaced by newer methods
• Swabs taken from objects before and after
Use-dilution test application of disinfectant or antiseptic
• Metal cylinders dipped into broth cultures • Swabs inoculated into growth medium and
of bacteria incubated
 • Medium monitored for growth
• Accurate determination of proper strength
and application procedure for each specific
situation

Development of Resistant Microbes

• Little evidence that products containing
antiseptic: disinfecting chemicals add to
human or animal health
• Use of such products promotes
development of resistant microbes