Chapter 8 Control of Microorganisms

Fomites

inanimate objects that may be the source of microbial spread from their surfaces

Biocides

antimicrobial agents that control microbes

Gram negative bacteria and control

outer membrane is less susceptible to treatments

Mycobacterium and control

endospores and cysts give extra protection

Sterilization

remove viable microbes

Disinfection

killing microbes

Disinfectants

chemicals used on inanimate objects

ex: bleach, ammonia

Sanitization

cleanliness level considered safe by public health standards

ex: isopropyl alcohol

Antiseptics

chemical agents on tissue to prevent infection

ex: iodine, h2o2, antibiotics

Bactericides

kill bacteria

Bacteriostatic agents

prevent growth, do not kill

SporKlenz

h2o2, peracetic, acetic acid, filtered to 0.22 micrometers, low toxicity

D value

decimal reduction time measures agent’s killing efficiency

D value equation

D value = time required to kill 90% of microbes

Z value

temperature change that decreases microbial population by 90% at a given D value

Filtration

sterilize gas and liquids with a barrier for microbes

ex: depth and membrane filters

Depth filters

vacuum pump force liquid through filter

Membrane filter

0.2 micron pores to remove living cells, not viruses

Air filters

remove airborne pathogens from air

ex: N95

High efficiency particulate air filters

HEPA, keep microbes out of areas

N95 masks

remove 95% of particles bigger than 0.3 microns

HEPAs

remove 97% of particles greater than 0.3 microns and remove viruses 0.1 microns and smaller

Laminar flow hoods

force air through HEPA and send vertical curtain of sterile air through cabinet, contaminated air removed

Chemical fume hood protects

the user

Biosafety cabinet protects

the user, the environment, and the material

Classes of BSCs

Class 1, 2, and 3

Class 2 and 3 BSCs provide

product protection

How does moist heat kill cells and viruses?

degrades nucleic acids, denatures proteins, and disrupts cell membranes

To destroy bacterial endospores…

moist heat 121C with steam and pressure for 10-12 minutes

Charles Chamberland

created autoclave

How autoclave works

air forced out until chamber is filled with saturated steam, 121C, 5 lbs PSI

Pasteurization

treating liquids through controlled heating

VH Soxhlet and F Soxhlet

created pasteurization in 1886

Tyndallization

destroys heat resistant microbes in dust with steam exposure, created by John Tyndall

UV radiation 260 nm

causes thymine thymine dimerization that prevents DNA rep and transcription

Cold plasma

Antimicrobial agent with ionized gases that emits UV and reactive oxygen species to damage DNA, proteins, etc

Ionizing Radiation

excellent sterilizing that penetrates deeply to create free radicals

Joseph Lister

first used phenol in surgery

Alcohols

denature proteins and dissolve membranes, not sporicidal

Halogens

oxidize cell constituents, iodine

Metals as antimicrobials

arsenic/mercury for germicides, Ag/Zn/Cu for antimicrobials, CuSO4 for algicide

Quaternary Ammonium Compounds

broad spectrum detergents with polar and nonpolar parts to disrupt membranes and denature proteins

do not kill TB or endospores

Aldehydes

inactivate nucleic acids and proteins

Ethylene oxide

gas used to sterilize heat sensitive items, kills microbes by reacting with DNA and proteins

Chlorine dioxide

gaseous disinfectant used in humidified environment, no resistance yet

Vaporized H2O2

decontaminates BSCs, ORs, etc

Factors effecting antimircrobial agents

1) Population size

2) Population composition

3) Concentration of agent

4) Contact time

5) Temperature

6) Local environment

Phenol coefficient test

potency of a disinfectant compared to phenol

>1 = more efficient

<1 = less efficient

Use dilution test

contaminated stainless steel dipped into test agents and then cultured

Bdellovibrio

gram neg predator, eliminate human intestinal pathogens

Felix d’Herelle

isolated bacteriophages from dysentery and found the phage destroyed the bacteria

Enzybiotics

cell lysing proteins from bacteriophages that attack PG and sometimes hydrolyze biofilm

Youyou Tu

extract from wormwood tree with mefloquine treated malaria

Antibiotics

natural products that kill microbes

Antimicrobials

natural and chemically synthesized compounds that kill microbes

Describe how an autoclave works. What conditions are required for sterilization by moist heat? What three things must one do when operating an autoclave to help ensure success?

An autoclave uses moist heat in the form of pressurized steam. It continues to force out steam until it reaches the desired temperature and pressure to kill microorganisms. Usually it will reach 121 ℃ and 15 psi. Moist heat must reach a specific temperature for a certain amount of time to denature microorganism proteins. For example, vegetative bacterial cells need to wait for 10 minutes at 70 ℃ to be inactivated. The temperature of boiling water is not enough heat to destroy endospores so it cannot sterilize. For moist heat to sterilize, the temperature, pressure, and time specifications must be met. The autoclave chamber should not be packed with material so that steam can circulate more effectively. A biological indicator is used to determine if the autoclaving was successful in that no culture of endospores grows after the autoclaving. The right temperature and pressure must be set for effective autoclave use.

In the past, spoiled milk was responsible for a significant proportion of infant deaths. Why is untreated milk easily spoiled?

Milk has lactic acid bacteria that causes the milk to ferment if left out. It also contains many proteins and enough moisture for bacterial growth. Milk still has an expiration date after pasteurization because pasteurization does not sterilize the milk but merely kills pathogens and slows spoilage.

List the advantages and disadvantages of ultraviolet light and ionizing radiation as sterilizing agents. Provide a few examples of how each is used for this purpose.

UV light is effective in sterilizing air and exposed surfaces with overhead lamps. UV can also be used for water treatment, and the microbes are killed when water passes underneath the light. However it is unable to penetrate glass, dirt, water, and other substances so it is not a preferred sterilizing agent. Ionizing radiation is a better sterilizing agent as it can penetrate deeply through objects. It can destroy endospores and all microbial cells. However, ionizing radiation cannot kill viruses. Some radiation including gamma and beta are used to cold sterilize antibiotics, hormones, sutures, and syringes. Irradiation is also used for food safety in that it kills E. coli and other food borne infections.

What is the correlation between radiation energy and the mechanisms of sterilization?

Higher energy radiation is more effective in killing microorganisms. For example, UV radiation is lower energy and therefore cannot penetrate through substances to effectively kill microorganisms. Ionizing radiation, on the other hand, is high energy and is very effective in breaking DNA and denaturing proteins in that it can penetrate deeply into substances.