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THE APPLICATION OF DISINFECTION AND STERILIZATION TO INFECTIOUS

WASTE MANAGEMENT
Eugene C. Cole, Dr.P.H.
School of Medicine
University of North Carolina, Chapel Hill, NC

The application of the principles of disinfection and sterilization

to effective infectious waste (IW) management must be viewed carefully.

While in general, both processes involve the inactivation of microbial

forms, the methods for achieving suitable disinfection and sterilization

for the on-site treatment of infectious wastes are very limited. I

might mention that on-site treatment has 3 potential advantages: (1)
.assurance that wastes are properly treated, (2) minimization of

potential risk to personnel as material moves through the waste stream,

and (3) cost-effectiveness.

Disinfection

A disinfectant can be described as an agent, usually chemical, which

destroys disease or other harmful microorganisms except, ordinarily,

bacterial spores. It refers to substances applied to inanimate objects.

Disinfectants may inactivate cells in a variety of ways including cell

wall and cytoplasmic membrane damage, electron transport interference,

and the coagulation of proteins and nucleic acids. While indeed,

disinfection is normally a chemical process, it is not the only one.

Ultraviolet (W)radiation has long been popular for the inactivation of

airborne and surface microbes within the close vicinity of the

-. generating lamp. UV radiation however, provides poor penetrability and

is therefore not effective as a means of IW treatment.

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Chemical disinfection is appropriate for the inactivation of liquid

wastes, such as cultures of etiologic agents, associated biologicals,
and human blood and blood products. It can also serve to decontaminate
some solid infectious wastes in a small clinic or office laboratory
where contaminated swabs, disposable culture loops, etc., are placed in
jars of disinfectant when steam sterilization or incineration are
unavailable.

The ideal disinfectant, in addition to being microbicidal, should

possess the characteristics listed in Table 1. Obviously, there is no
ideal disinfectant, so decisions must be made as to which factors are
most important in regard to the environment in question. Additionally,
in assessing the efficacy of a chemical disinfectant, one must consider
the important factors listed in Table 2.

When selecting a suitable disinfectant, consider first the type or

types or infectious agents that are of concern. Next, consider those
products with demonstrated efficacy against those agents. This warrants
becoming knowledgeable by reading and understanding product labels and
literature and consulting other appropriate references.

Normally it is inadequate to pour liquid waste (other than very

small amounts) into a disinfectant solution. Preferably, an amount of
concentrated disinfectant is placed into an appropriate container so

that when the liquid waste is added, the final use-dilution will be that
which is recommended. Mixing may be required. Following approximate
“inactivation, or at the end of the day, the container is emptied into
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Table 1. Characteristics of an Ideal Disinfectant

~~

Microbicidal

Easy to use

Detergent activity

Non - toxic

Non-irritating

Harmless to surfaces

Rapid action

Activity in presence of organic matter

Activity in presence of hard water

Stability

Residual activity

Inexpensive
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Table 2. Factors Affecting Disinfectant Efficacy

~ ~~~~

Hydrogen in concentration

Concentration

Exposure time

Presence of interfering substances

Temperature

Numbers of microorganisms

Types of microorganisms
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the sanitary sewer system (check local codes), and the system is flushed
with tap water to dilute the disinfectant and avoid damage to plumbing.
Solid waste items that have been decontaminated may then be regarded as
non-infectious trash and disposed of accordingly. One should always
remember that chemical disinfectants are toxic, and the use of proper
personal protective equipment is recommended.

Classes of Disinfectants

The following are the most commonly used classes of chemical

disinfectants :

A. Alcohols. (60-90%)
Advantages - bactericidal, tuberculocidal, virucidal (except
isopropanol and against hydrophilic viruses), non-staining,
non-irritating, rapid action.
Disadvantanes - non-sporicidal, organic matter interference,
incompatible with rubber and some plastics, highly flammable,
relatively expensive.
B. Quaternary Ammonium Compounds.
Advantanea - bactericidal (especially against gram-positive
organisms), virucidal (against lipophilic viruses), fungicidal,
pleasant odor, inexpensive.
pis a d v a n t u - non-tuberculocidal, non-sporicidal, organic
matter interference, non-virucidal (against hydrophilic
viruses).
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C. Phenolics.
Advantapes - bactericidal, fungicidal, tuberculocidal,
inexpensive.
Disadvantapes - questionable virucidal activity, non-
sporicidal, toxic, skin irritant, unpleasant odor,
corrosiveness.
D. Iodophors.
Advantapes - bactericidal, virucidal, fungicidal, detergent
action, storage stability.
Disadvantanes - prolonged exposure for tuberculocidal and
sporicidal activity, corrosiveness, inactivation by organic
matter, relatively expensive.
E. Gluteraldehydes.
Advantaees - bactericidal, virucidal, fungicidal,
tuberculocidal, sporicidal, lack or organic matter
interference, generally non-corrosive.
Disadvantaeeg - irritant, limited shelf life, expensive.
F. Hypochlorites. (2 500 ppm free available chlorine)
Advantaees - bactericidal, virucidal, tuberculocidal,
fungicidal, inexpensive.
Disadvantanes - non-sporicidal, toxic, corrosive, bleaching
agents.
G. Hydrogen Peroxide. (2 3%)
Advantages - bactericidal, virucidal, tuberculocidal,

fungicidal, sporicidal.
Disadvantaees - corrosive, expensive.
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Chemical Inactivation of HIV (AIDS virus)

The AIDS virus has been found to be extremely susceptible to
chemical disinfection. Disinfectants used in lower than normal
concentrations and yet able to inactivate lo5 HIV during a 10 min
exposure at room temperature include: ethyl alcohol, isopropyl alcohol,
sodium hypochlorite ( 5 0 ppm), phenolics, and hydrogen peroxide.

Chemical Inactivation of HeDatitis B virus

High concentrations (106 ) of hepatitis B virus were found to be

inactivated within 10 min at 20C by sodium hypochlorite ( 5 5 0 ppm),

alkaline glutaralhdehyde ( 2 % ) , glutaraldehyde-phenate (0.13%/0.44%),
isopropyl alcohol (70%), and iodophor (80 ppm).
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Sterilization

Sterilization is the act or process, physical or chemical, which

destroys all forms of life, especially microorganisms. Common

sterilization techniques include steam heat, dry heat, ethylene oxide,

and ionizing radiation. Of all the methods, heat, and particularly

moist heat, is the most reliable and widely used.

Ethylene oxide may present a carcinogenic, mutagenic, genotoxic,

reproductive, neurologic, and sensitization hazard to personnel and is

not recommended for IW treatment. Drv heat inactivation may be applied

to solid infectious waste. As sterilization times are prolonged,

however, and energy requirements are extensive, dry heat treatment of IW

is not preferred. Ionizing radiation is an effective, low temperature

sterilization method that is used extensively for a wide range of

medical products. Because of its high cost it is only suitable for

large scale sterilization.

In considering all of the aforementioned sterilization methods,

steam sterilization is preferred. Moist heat destroys microorganisms by

the irreversible coagulation and denaturation of enzymes and structural

proteins. The basic principle of steam sterilization, as accomplished

in an autoclave, is to expose each item to direct steam contact at the

required temperature and pressure for the specified time. Thus, there

are four parameters of steam sterilization: pressure, temperature,

time, and steam. Recognized exposure periods for sterilization of clean

wrapped supplies (not infectious waste) are 30 min @ 121C in a gravity

displacement sterilizer, and 4 min @ 132C in a prevacuum unit. At

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constant temperatures, sterilization times vary depending on the size

and type of load as well as the sterilizer type.

In the gravity displacement unit, steam is admitted to the top of

the chamber and because steam is lighter than air it forces air out the

bottom of the chamber through the drain vent. Such autoclaves are

primarily used to process culture media, water, pharmaceutical products,

infectious waste, and non-porous articles whose surfaces have direct

steam contact. For gravity displacement units, the penetration time is

prolonged because of incomplete air elimination. High speed prevacuum

sterilizers are similar to the gravity displacement type, except they

are fitted with a vacuum pump to insure air removal from the sterilizing

chamber and load before the steam is admitted. The advantage is nearly

instantaneous steam penetration, even into porous loads.

Autoclave monitoring is an essential part of the steam sterilization

process. This includes in-use monitoring of temperature and pressure.

Periodic preventive maintenance should include calibration of gauges and

indicators. Biological indicators (using spores of Bacillus

stearothermoDhilus) should be run with actual loads on a daily or weekly

basis depending on frequency of use.

In 1982, Rutala et al. published data from a study of a gravity

displacement steam autoclave that was tested to determine the operating

parameters that affected sterilization of microbiological waste.

Commercially available 1.5 mil polyethylene biohazard bags were used.

They were tested in two modes: (1) in the open position, with the sides
of the bag folded down to expose the top layer of petri plates, and (2)
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with the opening in the bag loosely constricted with a twist tie. Four
holes were punched in the tips of all twist tied bags. Loads were
tested both with and without 500 ml of water added to the bags. 5, 10,
and 15 lb. loads of contaminated petri dishes were tested. They
contained 67, 136, and 205 plates, respectively. An average of 8 5 % of
the plates were contaminated with viable bacteria. The waste bags were
placed into shallow stainless steel (ss) or polypropylene (pp)
containers. The bags were monitored for time-temperature profiles by a
digital potentiometer, and for sterilization efficacy by a biological
indicator (spores of E. stearothermoDhilus) within the load. At the end
of the cycle, contents were sampled and cultured for viable microbes,
both aerobically and anaerobically. Bacteria included Escherichia coli,
StaDhvlococcus auxeus, StaDhvlococcus eDidermidis, Klebsiella
pneumoniae, and species of Acinetobacter, Enterobacter, Pseudomonas,
Proteus, Streptococcus, and Bacillus.

When 5 lbs of microbiological waste in ss containers with or without

water, or pp containers with water was exposed to a steam sterilizing
cycle of 30 min, no growth of vegetative or sporeforming bacteria

occurred. In a pp container without water, all organisms were killed

after a 45 min cycle. When 10 lbs of microbiological waste was tested
in ss containers with water, 121C was reached in 45 min, and all
organisms except the indicator spores were killed. Without water at 45
min, all organisms with the exception of the indicator spores were
killed, but the temperature within the load did not reach 121C.
Utilizing the ss containers, either with or without water, the indicator
spores were not killed until a 90 min cycle was used. When the pp
containers were used, either with or without water, 121C could not be
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reached and indicator spores survived even when a 90 min cycle was used.
All other organisms were killed after 45 min in the presence of water,
and after 60 min without water. The 15 lb load data were essentially

the same as for the 10 lb loads.

The investigators thus concluded that factors that facilitated heat

transfer and the sterilization of microbiological waste included the
type of container in which the waste was placed, the physical
characteristics of the load, and the autoclave bag. They also noted
that the bag closest to the door heated more slowly than the middle and
Pack bags and that the tops of bags must be adjusted to allow for the
free passage of air and steam. The question was also raised as to the
necessity of using a cycle (90 min) that will kill all the spores of the
indicator, &. stearothermoDhilus. Since those spores are much more heat
resistant than the average organism it is unrealistic to require the
elimination of all spores in order to render waste "non-infectious".
Depending on the characteristics of the load, as already stated, spore
forming bacteria other then 8 . stearothermoDhiluS will be killed after
45 or 60 min.

The use of microwave oven irradiation as a method for sterilizing

bacterial waste was reported by Latimer and Matsen. They undertook a

quantitative study to determine the effect of timed microwave
irradiation on commonly encountered laboratory bacteria, using an oven

operating at 2 , 4 5 0 MHz. Organisms grown in broth culture and exposed to

microwaves for 5 min included $. coli, E. plirabilis, E. aerueinosa, S .

marcescenq, S. Bureus , S. eDidermidig, and enterococcus. All organisms

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were killed within the 5 min period. Spore strips containing viable E.
stearothermoDhilus spores were likewise exposed, with none surviving
after a 5 min exposure. Loads of contaminated plastic petri dishes
(about 100/load) exposed to the microwaves were rendered sterile within
5 min. The authors conclude that the utilization of microwave ovens for
bacterial decontamination in laboratories is entirely feasible. It
appears to be a practical time and energy saving method for the
treatment of bacterial waste. The treatment of fungal, viral, and
mycobacterial waste however, warrants additional investigation.

Lastly, concern exists over the proper treatment of combined

'infectious/radioactive waste, Normally, the component representing the
greatest hazard is addressed first, with the final disposal of the
material subject to the regulations of the Nuclear Regulatory Commission
(NRC). If the waste is considered "highly infectious" and is
contaminated with low level radioisotopes, then extended autoclaving
followed by storage for decay, or approved incineration (for solids), or
autoclaving with release to the sanitary sewer (for liquids) may be
utilized. In general, however, the time of storage for decay will
result in the death of the infectious agent. The Centers for Disease
Control (CDC) recommends treating radioactive blood and urine by
chemical disinfection using sodium hypochlorite or hydrogen peroxide to
inactivate the biological component prior to approved disposal.
However, if chemical inactivation is not feasible, the waste should be
steam-sterilized,tagged non-infectious, and disposed of according to
the NRC.
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References
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Decontamination of laboratory microbiological waste by steam
sterilization. Appl Envir Microbiol 43:1311-1316.
2. Latimer, J.M., and J.M. Matsen. 1977. Microwave oven irradiation
as a method for bacterial decontamination in a clinical
microbiology laboratory. J Clin Microbiol 6:340-342.
3. Gardner, J.F., and M.M. Peel. 1985. Introduction to sterilization
and disinfection. Churchill Livingstone Inc., New York.
4. National Committee for Clinical Laboratory Standards. Clinical
laboratory hazardous waste; proposed guideline. NCCLS document
GP5-P. Villanova, Pennsylvania.
5. Martin, L.S., J . S . McDougal, and S.L. Loskoski. 1985.
Disinfection and inactivation of the human T lymphotropic virus
type III/lymphadenopathy-associated virus. J Infect Dis 152:400-
403.
6. Kobayashi, H., M. Tsuzuki, K. Koshimizu, H. Toyarma, N. Yoshihara,
T. ShiKata, K. Abe, K. Mizuno, N. Otomo, and T. Oda. 1984.
Susceptibility of hepatitis B virus to disinfectants or heat. J
Clin Microbiol 20:214-216.
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Inactivation of hepatitis B virus by intermediate-to-highlevel
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13. Rutala, W.A., 1987. Disinfection, sterilization, and waste

disposal. In Wenzel, R.P., Prevention and control of nosocomial
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