14,Disinfection and sterilization
A basic knowledge of disinfection and sterilization is
crucial for biosafety in the laboratory,Since heavily soiled
items cannot promptly be disinfected or sterilized,it is
equally important to understand the fundamentals of
cleaning prior to disinfection (precleaning),In this regard,
the following general principles apply to all known classes
of microbial pathogens.
Specific decontamination requirements will depend on the
type of experimental work and the nature of the infectious
agent(s) being handled,The generic information given
here can be used to develop both standardized and more
specific procedures to deal with biohazard(s) involved in a
particular laboratory.
Contact times for disinfectants are specific for each
material and manufacturer,Therefore,all
recommendations for use of disinfectants should follow
manufacturers’ specifications.
Definitions
Many different terms are used for disinfection and
sterilization,The following are among the more common
in biosafety:
Antimicrobial – An agent that kills microorganisms or
suppresses their growth and multiplication.
Antiseptic – A substance that inhibits the growth and
development of microorganisms without necessarily killing
them,Antiseptics are usually applied to body surfaces.
Biocide – A general term for any agent that kills organis
ms.
Chemical germicide – A chemical or a mixture of
chemicals used to kill micro-organisms.
Decontamination – Any process for removing and/or
killing microorganisms,The same term is also used for
removing or neutralizing hazardous chemicals and radio-
active materials.
Disinfectant – A chemical or mixture of chemicals used to
kill microorganisms,but not necessarily spores,
Disinfectants are usually applied to inanimate surfaces or
objects.
Disinfection – A physical or chemical means of killing
microorganisms,but not necessarily spores.
Microbicide – A chemical or mixture of chemicals that
kills microorganisms,The term is often used in place of
“biocide”,“chemical germicide” or,antimicrobial”.
Sporocide – A chemical or mixture of chemicals used to
kill microorganisms and spores.
Sterilization – A process that kills and/or removes all
classes of microorganisms and
spores.
Cleaning laboratory materials
Cleaning is the removal of dirt,organic matter and stains,
Cleaning includes brushing,vacuuming,dry dusting,
washing or damp mopping with water containing a soap or
detergent,Dirt,soil and organic matter can shield
microorganisms and can interfere with the killing action of
decontaminants (antiseptics,chemical germicides and
disinfectants).
Precleaning is essential to achieve proper disinfection or
sterilization,Many germi- cidal products claim activity
only on precleaned items,Precleaning must be carried
out with care to avoid exposure to infectious agents.
Materials chemically compatible with the germicides to be
applied later must be used,It is quite common to use the
same chemical germicide for precleaning and
disinfection.
Chemical germicides
Many types of chemicals can be used as disinfectants
and/or antiseptics,As there is an ever-increasing number
and variety of commercial products,formulations must be
carefully selected for specific needs.
The germicidal activity of many chemicals is faster and
better at higher temperatures.
At the same time,higher temperatures can accelerate their
evaporation and also degrade them,
Particular care is needed in the use and storage of such
chemicals in tropical
regions,where their shelf-life may be reduced because of
high ambient temperatures.
Many germicides can be harmful to humans or the
environment,They should be selected,stored,handled,
used and disposed of with care,following manufacturers’
instructions,
For personal safety,gloves,aprons and eye protection are
recommended when preparing dilutions of chemical
germicides.
Chemical germicides are generally not required for
regular cleaning of floors,walls,equipment and furniture,
However,their use may be appropriate in certain cases of
outbreak control.
Proper use of chemical germicides will contribute to
workplace safety while reducing the risk from infectious
agents,As far as possible,the number of germicidal
chemicals to be used should be limited for economic
reasons,inventory control and to limit environmental
pollution.
Commonly used classes of chemical germicides are
described below,with generic information on their
applications and safety profiles,Unless otherwise indicated,
the germicide concentrations are given in weight/volume
(w/v),Table 12 summarizes the recommended dilutions of
chlorine-releasing compounds.
Chlorine,a fast-acting oxidant,is a widely available and
broad-spectrum chemical germicide,It is normally sold as
bleach,an aqueous solution of sodium hypochlorite
(NaOCl),which can be diluted with water to provide
various concentrations of available chlorine.
Chlorine,especially as bleach,is highly alkaline and can be
corrosive to metal,Its activity is considerably reduced by
organic matter (protein),
Storage of stock or working solutions of bleach in open
containers,particularly at high temperatures,releases
chlorine gas thus weakening their germicidal potential,
The frequency with which working solutions of bleach
should be changed depends on their starting strength,the
type (e.g,with or without a lid) and size of their containers,
the frequency and nature of use,and ambient conditions,
As a general guide,solutions receiving materials with high
levels of organic matter several times a day should be
changed at least daily,while those with less frequent use
may last for as long as a week.
A general all-purpose laboratory disinfectant should have
a concentration of 1 g/l available chlorine,
A stronger solution,containing 5 g/l available chlorine,is
recommended for dealing with biohazardous spillage and
in the presence of large amounts of organic matter,
Sodium hypochlorite solutions,as domestic bleach,contain
50 g/l available chlorine and should therefore be diluted
1:50 or 1:10 to obtain final concentrations of 1 g/l and 5 g/l,
respectively,Industrial solutions of bleach have a sodium
hypochlorite concentration of nearly 120 g/l and must be
diluted accordingly to obtain the levels indicated above.
Granules or tablets of calcium hypochlorite (Ca(ClO)2)
generally contain about 70% available chlorine,Solutions
prepared with granules or tablets,containing 1.4 g/l and
7.0 g/l,will then contain 1.0 g/l and 5 g/l available chlorine,
respectively.
Bleach is not recommended as an antiseptic,but may be
used as a general-purpose disinfectant and for soaking
contaminated metal-free materials,
In emergencies,bleach can also be used to disinfect water
for drinking,with a final concentration of 1–2 mg/l
available chlorine.
Chlorine gas is highly toxic,Bleach must therefore be
stored and used in well-ventilated areas only,Also,bleach
must not be mixed with acids to prevent the rapid
release of chlorine gas,
Many by-products of chlorine can be harmful to humans
and the environment,so that indiscriminate use of
chlorine-based disinfectants,in particular bleach,should
be avoided.
Sodium dichloroisocyanurate
Sodium dichloroisocyanurate (NaDCC) in powder form
contains 60% available chlorine,Solutions prepared with
NaDCC powder at 1.7 g/l and 8.5 g/l will contain 1 g/l or 5
g/l available chlorine,respectively,Tablets of NaDCC
generally contain the equivalent of 1.5 g available chlorine
per tablet,
One or four tablets dissolved in 1 l of water will give
approximately the required concentrations of 1 g/l or 5 g/l,
respectively,NaDCC as powder or tablets is easy and safe
to store,Solid NaDCC can be applied on spills of blood or
other biohazardous liquids and left for at least 10 min
before removal,Further cleaning of the affected area can
then take place.
Chloramines
Chloramines are available as powders containing about
25% available chlorine,Chloramines release chlorine at a
slower rate than hypochlorites,Higher initial
concentrations are therefore required for efficiencies
equivalent to those of hypochlorites,
On the other hand,chloramine solutions are not
inactivated by organic matter to the same extent as
hypochlorite solutions,and concentrations of 20 g/l are
recommended for both,clean” and,dirty” situations.
Chloramine solutions are virtually odour-free,However,
items soaked in them must be thoroughly rinsed to remove
any residue of the bulking agents added to chloramine-
T (sodium tosylchloramide) powders.
Chlorine dioxide
Chlorine dioxide (ClO2) is a strong and fast-acting
germicide,disinfectant agent and oxidizer,often reported
to be active at concentrations levels lower than those
needed by chlorine as bleach,Chlorine dioxide is unstable
as a gas and will undergo decomposition into chlorine gas
(Cl ),oxygen gas (O ),giving off heat.
However,chlorine dioxide is soluble in water and stable in
an aqueous solution,Chlorine dioxide can be obtained in
two ways,(1) on-site generation by mixing of two separate
components,hydrochloric acid (HCl) and sodium chlorite
(NaClO2); and (2) ordering its stabilized form,which is
then activated on-site when required.
Of the oxidizing biocides,chlorine dioxide is the most
selective oxidant,Ozone and chlorine are much more
reactive than chlorine dioxide,and they will be consumed
by most organic compounds,Chlorine dioxide,however,
reacts only with reduced sulfur compounds,secondary and
tertiary amines,and some other highly reduced and
reactive organic compounds.
A more stable residue can therefore be achieved with
chlorine dioxide at much lower doses than when using
either chlorine or ozone,Generated properly,chlorine
dioxide can be used more effectively than ozone or chlorine
in cases of higher organic loading because of its selectivity.
Formaldehyde
Formaldehyde (HCHO) is a gas that kills all
microorganisms and spores at temperatures above 20 ° C,
However,it is not active against prions.
Formaldehyde is relatively slow-acting and needs a relative
humidity level of about 70%,It is marketed as the solid
polymer,paraformaldehyde,in flakes or tablets,or as
formalin,a solution of the gas in water of about 370 g/l
(37%),containing methanol (100 ml/l) as a stabilizer,Both
formulations are heated to liberate the gas,which is used
for decontamination and disinfection of enclosed volumes
such as safety cabinets and rooms.
Formaldehyde (5% formalin in water) may be used as a
liquid disinfectant.
Formaldehyde is a suspected carcinogen,It is a dangerous,
irritant gas that has a pungent smell and its fumes can
irritate eyes and mucous membranes,It must therefore
be stored and used in a fume-hood or well-ventilated area,
National chemical safety regulations must be followed.
Glutaraldehyde
Like formaldehyde,glutaraldehyde (OHC(CH2)3CHO) is
also active against vegetative bacteria,spores,fungi and
lipid- and nonlipid-containing viruses,It is non-corrosive
and faster acting than formaldehyde,However,it takes
several hours to kill bacterial spores.
Glutaraldehyde is generally supplied as a solution with a
concentration of about 20 g/l (2%) and some products may
need to be,activated” (made alkaline) before use by the
addition of a bicarbonate compound supplied with the
product,The activated solution can be reused for 1–4
weeks depending on the formulation and type and
frequency of its use,
Dipsticks supplied with some products give only a rough
indication of the levels of active glutaraldehyde available
in solutions under use,Glutaraldehyde solutions should be
discarded if they become turbid.
Glutaraldehyde is toxic and an irritant to skin and mucous
membranes,and contact with it must be avoided,It must
be used in a fume-hood or in well-ventilated areas,It
is not recommended as a spray or solution for the
decontamination of environmental surfaces,National
chemical safety regulations must be followed.
Phenolic compounds
Phenolic compounds,a broad group of agents,were among
the earliest germicides,However,more recent safety
concerns restrict their use,They are active against
vegetative bacteria and lipid-containing viruses and,when
properly formulated,also show activity against
mycobacteria,They are not active against spores and their
activity against nonlipid viruses is variable,Many phenolic
products are used for the decontamination of
environmental surfaces,and some (e.g,triclosan and
chloroxylenol) are among the more commonly used
antiseptics.
Triclosan is common in products for hand-washing,It is
active mainly against vegetative bacteria and safe for skin
and mucous membranes,However,in laboratory-based
studies,bacteria made resistant to low concentrations of
triclosan also show resistance to certain types of antibiotics,
The significance of this finding in the field remains
unknown.
Some phenolic compounds are sensitive to and may be
inactivated by water hardness and therefore must be
diluted with distilled or deionized water.
Phenolic compounds are not recommended for use on food
contact surfaces and in areas with young children,They
may be absorbed by rubber and can also penetrate the
skin,National chemical safety regulations must be
followed.
Quaternary ammonium compounds
Many types of quaternary ammonium compounds are
used as mixtures and often in combination with other
germicides,such as alcohols,They have good activity
against some vegetative bacteria and lipid-containing
viruses,Certain types (e.g,benzalkonium chloride) are
used as antiseptics.
The germicidal activity of certain types of quaternary
ammonium compounds is considerably reduced by organic
matter,water hardness and anionic detergents,Care
is therefore needed in selecting agents for precleaning
when quaternary ammonium compounds are to be used
for disinfection,Potentially harmful bacteria can grow in
quaternary ammonium compound solutions,Owing to low
biodegradability,these compounds may also accumulate in
the environment.
Alcohols
Ethanol (ethyl alcohol,C2H5 OH) and 2-propanol
(isopropyl alcohol,(CH3)2CHOH) have similar disinfectant
properties,They are active against vegetative bacteria,
fungi and lipid-containing viruses but not against spores,
Their action on nonlipid viruses is variable,For highest
effectiveness they should be used at concentrations of
approximately 70% (v/v) in water,higher or lower
concentrations may not be as germicidal,A major
advantage of aqueous solutions of alcohols is that they do
not leave any residue on treated items.
Mixtures with other agents are more effective than alcohol
alone,e.g,70% (v/v) alcohol with 100 g/l formaldehyde,
and alcohol containing 2 g/l available chlorine,A 70% (v/v)
aqueous solution of ethanol can be used on skin,work
surfaces of laboratory benches and biosafety cabinets,and
to soak small pieces of surgical instruments,Since ethanol
can dry the skin,it is often mixed with emollients.
Alcohol-based hand-rubs are recommended for the
decontamination of lightly soiled hands in situations where
proper hand-washing is inconvenient or not possible,
However,it must be remembered that ethanol is ineffective
against spores and may not kill all types of nonlipid viruses.
Alcohols are volatile and flammable and must not be used
near open flames,Working solutions should be stored in
proper containers to avoid the evaporation of alcohols.
Alcohols may harden rubber and dissolve certain types of
glue,Proper inventory and storage of ethanol in the
laboratory is very important to avoid its use for purposes
other than disinfection,Bottles with alcohol-containing
solutions must be clearly labelled to avoid autoclaving.
Iodine and iodophors
The action of these disinfectants is similar to that of
chlorine,although they may be slightly less inhibited by
organic matter,Iodine can stain fabrics and environmental
surfaces and is generally unsuitable for use as a
disinfectant,
On the other hand,iodophors and tinctures of iodine are
good antiseptics,Polyvidone-iodine is a reliable
and safe surgical scrub and preoperative skin antiseptic,
Antiseptics based on iodine are generally unsuitable for
use on medical/dental devices,Iodine should not be used
on aluminium or copper.
Iodine can be toxic,Organic iodine-based products must
be stored at 4–10 ° C to avoid the growth of potentially
harmful bacteria in them.
Hydrogen peroxide and peracids
Like chlorine,hydrogen peroxide (H2O2) and peracids are
strong oxidants and can bepotent broad-spectrum
germicides,They are also safer than chlorine to humans
andthe environment.
Hydrogen peroxide is supplied either as a ready-to-use 3%
solution or as a 30% aqueous solution to be diluted to 5–10
times its volume with sterilized water,However,such 3–
6% solutions of hydrogen peroxide alone are relatively
slow and limited as germicides,Products now available
have other ingredients to stabilize the hydrogen peroxide
content,to accelerate its germicidal action and to make it
less corrosive.
Hydrogen peroxide can be used for the decontamination of
work surfaces of laboratory benches and biosafety cabinets,
and stronger solutions may be suitable for disinfecting
heat-sensitive medical/dental devices,The use of vaporized
hydrogen peroxide or peracetic acid (CH3COOOH) for
the decontamination of heat-sensitive medical/surgical
devices requires specialized equipment.
Hydrogen peroxide and peracids can be corrosive to
metals such as aluminium,copper,brass and zinc,and can
also decolorize fabrics,hair,skin and mucous membranes,
Articles treated with them must be thoroughly rinsed
before contact with eyes and mucous membranes,They
should always be stored away from heat and protected
from light.
Local environmental decontamination
Decontamination of the laboratory space,its furniture and
its equipment requires a combination of liquid and gaseous
disinfectants,Surfaces can be decontaminated using a
solution of sodium hypochlorite (NaOCl); a solution
containing 1 g/l available chlorine may be suitable for
general environmental sanitation,but stronger solutions (5
g/l) are recommended when dealing with high-risk
situations,For environmental decontamination,
formulated solutions containing 3% hydrogen peroxide
(H2O2) make suitable substitutes for bleach solutions.
Rooms and equipment can be decontaminated by
fumigation with formaldehyde gas generated by heating
paraformaldehyde or boiling formalin,This is a highly
dangerous process that requires specially trained
personnel,All openings in the room (i.e,windows,doors,
etc.) should be sealed with masking tape or similar before
the gas is generated,Fumigation should be conducted at an
ambient temperature of at least 21 ° C and a relative
humidity of 70%,
After fumigation the area must be ventilated thoroughly
before personnel are allowed to enter,Appropriate
respirators must be worn by anyone entering the room
before it has been ventilated,Gaseous ammonium
bicarbonate can be used to neutralize the formaldehyde.
Fumigation of smaller spaces with hydrogen peroxide
vapour is also effective but requires specialized equipment
to generate the vapour.
Decontamination of biological safety cabinets
To decontaminate Class I and Class II cabinets,equipment
that independently generates,circulates and neutralizes
formaldehyde gas is available,Alternatively,the
appropriate amount of paraformaldehyde (final
concentration of 0.8% paraformaldehyde in air) should be
placed in a frying pan on an electric hot plate,
Another frying pan,containing 10% more ammonium
bicarbonate than paraformaldehyde,on a second hot plate
is also placed inside the cabinet,
The hot plate leads are plugged in outside the cabinet,so
that operation of the pans can be controlled from the
outside by plugging and unplugging the hot plates as
necessary,
If the relative humidity is below 70%,an open container of
hot water should also be placed inside the cabinet before
the front closure is sealed in place with strong tape (e.g,
duct tape).
Heavy gauge plastic sheeting is taped over the front
opening and exhaust port to make sure that the gas cannot
seep into the room,Penetration of the electric leads
passing through the front closure must also be sealed with
duct tape.
The plate for the paraformaldehyde pan is plugged in,It is
unplugged when all the paraformaldehyde has vaporized,
The cabinet is left undisturbed for at least 6 h,The plate
for the second pan is then plugged in and the ammonium
bicarbonate is allowed to vaporize.
This plate is then unplugged and the cabinet blower is
switched on for two intervals of approximately 2 s each to
allow the ammonium bicarbonate gas to circulate,The
cabinet should be left undisturbed for 30 min before the
front closure (or plastic sheeting) and the exhaust port
sheeting are removed,The cabinet surfaces should be
wiped down to remove residues before use.
Hand-washing/hand decontamination
Whenever possible,suitable gloves should be worn when
handling biohazardous materials,However,this does not
replace the need for regular and proper hand-washing
by laboratory personnel,Hands must be washed after
handling biohazardous materials and animals,and before
leaving the laboratory.
In most situations,thorough washing of hands with
ordinary soap and water is sufficient to decontaminate
them,but the use of germicidal soaps is recommended in
high-risk situations,Hands should be thoroughly lathered
with soap,using friction,for at least 10 s,rinsed in clean
water and dried using a clean paper or cloth towel (if
available,warm-air hand-dryers may be used).
Foot- or elbow-operated faucets are recommended,Where
not fitted,a paper/cloth towel should be used to turn off
the faucet handles to avoid recontaminating washed hands.
As mentioned above,alcohol-based hand-rubs may be used
to decontaminate lightly soiled hands when proper hand-
washing is not available.
Heat disinfection and sterilization
Heat is the most common among the physical agents used
for the decontamination of pathogens.,Dry” heat,which is
totally non-corrosive,is used to process many items of
laboratory ware which can withstand temperatures of 160
° C or higher for 2–4 h.
Burning or incineration (see below) is also a form of dry
heat.,Moist” heat is most effective when used in the form
of autoclaving.
Boiling does not necessarily kill all microorganisms and/or
pathogens,but it may be used as the minimum processing
for disinfection where other methods (chemical
disinfection or decontamination,autoclaving) are not
applicable or available.
Sterilized items must be handled and stored such that they
remain uncontaminated until used.
Autoclaving
Saturated steam under pressure (autoclaving) is the most
effective and reliable means of sterilizing laboratory
materials,For most purposes,the following cycles will
ensure sterilization of correctly loaded autoclaves:
1,3 min holding time at 134° C
2,10 min holding time at 126° C
3,15 min holding time at 121° C
4,25 min holding time at 115° C.
Examples of different autoclaves include the following.
Gravity displacement autoclaves,Figure 10 shows the
general construction of a gravity-displacement autoclave,
Steam enters the chamber under pressure and displaces
the heavier air downwards and through the valve in the
chamber drain,fitted with a HEPA filter.
Pre-vacuum autoclaves,These machines allow the removal
of air from the chamber before steam is admitted,The
exhaust air is evacuated through a valve fitted with a
HEPA filter,
At the end of the cycle,the steam is automatically
exhausted,These autoclaves can operate at 134° C and
the sterilization cycle can therefore be reduced to 3 min,
They are ideal for porous loads,but cannot be used to
process liquids because of the vacuum.
Fuel-heated pressure cooker autoclaves,These should be
used only if a gravity displacement autoclave is not
available,They are loaded from the top and heated by gas,
electricity or other types of fuels,
Steam is generated by heating water in the base of the
vessel,and air is displaced upwards through a relief vent,
When all the air has been removed,the valve on the relief
vent is closed and the heat reduced,The pressure and
temperature rise until the safety valve operates at a preset
level,
This is the start of the holding time,At the end of the cycle
the heat is turned off and the temperature allowed
to fall to 80° C or below before the lid is opened.
Loading autoclaves
Materials should be loosely packed in the chamber for easy
steam penetration and air removal,Bags should allow the
steam to reach their contents.
Precautions in the use of autoclaves
The following rules can minimize the hazards inherent in
operating pressurized vessels.
1,Responsibility for operation and routine care should be
assigned to trained individuals.
2,A preventive maintenance programme should include
regular inspection of the chamber,door seals and all
gauges and controls by qualified personnel.
3,The steam should be saturated and free from chemicals
(e.g,corrosion inhibitors) that could contaminate the items
being sterilized.
4,All materials to be autoclaved should be in containers
that allow ready removal of air and permit good heat
penetration; the chamber should be loosely packed so that
steam will reach the load evenly.
5,For autoclaves without an interlocking safety device that
prevents the door being opened when the chamber is
pressurized,the main steam valve should be closed and the
temperature allowed to fall below 80° C before the door is
opened.
6,Slow exhaust settings should be used when autoclaving
liquids,as they may boil over when removed due to
superheating.
7,Operators should wear suitable gloves and visors for
protection when opening the autoclave,even when the
temperature has fallen below 80 ° C.
8,In any routine monitoring of autoclave performance,
biological indicators or thermocouples should be placed at
the centre of each load,Regular monitoring
with thermocouples and recording devices in a,worst case”
load is highly desirable to determine proper operating
cycles.
9,The drain screen filter of the chamber (if available)
should be removed and cleaned daily.
10,Care should be taken to ensure that the relief valves of
pressure cooker autoclaves do not become blocked by
paper,etc,in the load.
Incineration
Incineration is useful for disposing of animal carcasses as
well as anatomical and other laboratory waste,with or
without prior decontamination,
Incineration of infectious materials is an alternative to
autoclaving only if the incinerator is under
laboratory control.
Proper incineration requires an efficient means of
temperature control and a secondary burning chamber,
Many incinerators,especially those with a single
combustion chamber,are unsatisfactory for dealing with
infectious materials,animal carcasses and plastics.
Such materials may not be completely destroyed and the
effluent
from the chimney may pollute the atmosphere with
microorganisms,toxic chemicals
and smoke,However,there are many satisfactory
configurations for combustion
chambers,Ideally the temperature in the primary
chamber should be at least 800 ° C
and that in the secondary chamber at least 1000 ° C.
Materials for incineration,even with prior
decontamination,should be transportedto the incinerator
in bags,preferably plastic,Incinerator attendants should
receive proper instructions about loading and temperature
control,
It should also be noted that the efficient operation of an
incinerator depends heavily on the right mix of materials
in the waste being treated.
There are ongoing concerns regarding the possible
negative environmental effects of existing or proposed
incinerators,and efforts continue to make incinerators
more environmentally friendly and energy-efficient.
Disposal
The disposal of laboratory and medical waste is subject to
various regional,national and international regulations,
and the latest versions of such relevant documents must be
consulted before designing and implementing a
programme for handling,transportation and disposal of
biohazardous waste,
In general,ash from incinerators may be handled as
normal domestic waste and removed by local authorities,
Autoclaved waste may be disposed of by off-site
incineration or in licensed landfill sites.
A basic knowledge of disinfection and sterilization is
crucial for biosafety in the laboratory,Since heavily soiled
items cannot promptly be disinfected or sterilized,it is
equally important to understand the fundamentals of
cleaning prior to disinfection (precleaning),In this regard,
the following general principles apply to all known classes
of microbial pathogens.
Specific decontamination requirements will depend on the
type of experimental work and the nature of the infectious
agent(s) being handled,The generic information given
here can be used to develop both standardized and more
specific procedures to deal with biohazard(s) involved in a
particular laboratory.
Contact times for disinfectants are specific for each
material and manufacturer,Therefore,all
recommendations for use of disinfectants should follow
manufacturers’ specifications.
Definitions
Many different terms are used for disinfection and
sterilization,The following are among the more common
in biosafety:
Antimicrobial – An agent that kills microorganisms or
suppresses their growth and multiplication.
Antiseptic – A substance that inhibits the growth and
development of microorganisms without necessarily killing
them,Antiseptics are usually applied to body surfaces.
Biocide – A general term for any agent that kills organis
ms.
Chemical germicide – A chemical or a mixture of
chemicals used to kill micro-organisms.
Decontamination – Any process for removing and/or
killing microorganisms,The same term is also used for
removing or neutralizing hazardous chemicals and radio-
active materials.
Disinfectant – A chemical or mixture of chemicals used to
kill microorganisms,but not necessarily spores,
Disinfectants are usually applied to inanimate surfaces or
objects.
Disinfection – A physical or chemical means of killing
microorganisms,but not necessarily spores.
Microbicide – A chemical or mixture of chemicals that
kills microorganisms,The term is often used in place of
“biocide”,“chemical germicide” or,antimicrobial”.
Sporocide – A chemical or mixture of chemicals used to
kill microorganisms and spores.
Sterilization – A process that kills and/or removes all
classes of microorganisms and
spores.
Cleaning laboratory materials
Cleaning is the removal of dirt,organic matter and stains,
Cleaning includes brushing,vacuuming,dry dusting,
washing or damp mopping with water containing a soap or
detergent,Dirt,soil and organic matter can shield
microorganisms and can interfere with the killing action of
decontaminants (antiseptics,chemical germicides and
disinfectants).
Precleaning is essential to achieve proper disinfection or
sterilization,Many germi- cidal products claim activity
only on precleaned items,Precleaning must be carried
out with care to avoid exposure to infectious agents.
Materials chemically compatible with the germicides to be
applied later must be used,It is quite common to use the
same chemical germicide for precleaning and
disinfection.
Chemical germicides
Many types of chemicals can be used as disinfectants
and/or antiseptics,As there is an ever-increasing number
and variety of commercial products,formulations must be
carefully selected for specific needs.
The germicidal activity of many chemicals is faster and
better at higher temperatures.
At the same time,higher temperatures can accelerate their
evaporation and also degrade them,
Particular care is needed in the use and storage of such
chemicals in tropical
regions,where their shelf-life may be reduced because of
high ambient temperatures.
Many germicides can be harmful to humans or the
environment,They should be selected,stored,handled,
used and disposed of with care,following manufacturers’
instructions,
For personal safety,gloves,aprons and eye protection are
recommended when preparing dilutions of chemical
germicides.
Chemical germicides are generally not required for
regular cleaning of floors,walls,equipment and furniture,
However,their use may be appropriate in certain cases of
outbreak control.
Proper use of chemical germicides will contribute to
workplace safety while reducing the risk from infectious
agents,As far as possible,the number of germicidal
chemicals to be used should be limited for economic
reasons,inventory control and to limit environmental
pollution.
Commonly used classes of chemical germicides are
described below,with generic information on their
applications and safety profiles,Unless otherwise indicated,
the germicide concentrations are given in weight/volume
(w/v),Table 12 summarizes the recommended dilutions of
chlorine-releasing compounds.
Chlorine,a fast-acting oxidant,is a widely available and
broad-spectrum chemical germicide,It is normally sold as
bleach,an aqueous solution of sodium hypochlorite
(NaOCl),which can be diluted with water to provide
various concentrations of available chlorine.
Chlorine,especially as bleach,is highly alkaline and can be
corrosive to metal,Its activity is considerably reduced by
organic matter (protein),
Storage of stock or working solutions of bleach in open
containers,particularly at high temperatures,releases
chlorine gas thus weakening their germicidal potential,
The frequency with which working solutions of bleach
should be changed depends on their starting strength,the
type (e.g,with or without a lid) and size of their containers,
the frequency and nature of use,and ambient conditions,
As a general guide,solutions receiving materials with high
levels of organic matter several times a day should be
changed at least daily,while those with less frequent use
may last for as long as a week.
A general all-purpose laboratory disinfectant should have
a concentration of 1 g/l available chlorine,
A stronger solution,containing 5 g/l available chlorine,is
recommended for dealing with biohazardous spillage and
in the presence of large amounts of organic matter,
Sodium hypochlorite solutions,as domestic bleach,contain
50 g/l available chlorine and should therefore be diluted
1:50 or 1:10 to obtain final concentrations of 1 g/l and 5 g/l,
respectively,Industrial solutions of bleach have a sodium
hypochlorite concentration of nearly 120 g/l and must be
diluted accordingly to obtain the levels indicated above.
Granules or tablets of calcium hypochlorite (Ca(ClO)2)
generally contain about 70% available chlorine,Solutions
prepared with granules or tablets,containing 1.4 g/l and
7.0 g/l,will then contain 1.0 g/l and 5 g/l available chlorine,
respectively.
Bleach is not recommended as an antiseptic,but may be
used as a general-purpose disinfectant and for soaking
contaminated metal-free materials,
In emergencies,bleach can also be used to disinfect water
for drinking,with a final concentration of 1–2 mg/l
available chlorine.
Chlorine gas is highly toxic,Bleach must therefore be
stored and used in well-ventilated areas only,Also,bleach
must not be mixed with acids to prevent the rapid
release of chlorine gas,
Many by-products of chlorine can be harmful to humans
and the environment,so that indiscriminate use of
chlorine-based disinfectants,in particular bleach,should
be avoided.
Sodium dichloroisocyanurate
Sodium dichloroisocyanurate (NaDCC) in powder form
contains 60% available chlorine,Solutions prepared with
NaDCC powder at 1.7 g/l and 8.5 g/l will contain 1 g/l or 5
g/l available chlorine,respectively,Tablets of NaDCC
generally contain the equivalent of 1.5 g available chlorine
per tablet,
One or four tablets dissolved in 1 l of water will give
approximately the required concentrations of 1 g/l or 5 g/l,
respectively,NaDCC as powder or tablets is easy and safe
to store,Solid NaDCC can be applied on spills of blood or
other biohazardous liquids and left for at least 10 min
before removal,Further cleaning of the affected area can
then take place.
Chloramines
Chloramines are available as powders containing about
25% available chlorine,Chloramines release chlorine at a
slower rate than hypochlorites,Higher initial
concentrations are therefore required for efficiencies
equivalent to those of hypochlorites,
On the other hand,chloramine solutions are not
inactivated by organic matter to the same extent as
hypochlorite solutions,and concentrations of 20 g/l are
recommended for both,clean” and,dirty” situations.
Chloramine solutions are virtually odour-free,However,
items soaked in them must be thoroughly rinsed to remove
any residue of the bulking agents added to chloramine-
T (sodium tosylchloramide) powders.
Chlorine dioxide
Chlorine dioxide (ClO2) is a strong and fast-acting
germicide,disinfectant agent and oxidizer,often reported
to be active at concentrations levels lower than those
needed by chlorine as bleach,Chlorine dioxide is unstable
as a gas and will undergo decomposition into chlorine gas
(Cl ),oxygen gas (O ),giving off heat.
However,chlorine dioxide is soluble in water and stable in
an aqueous solution,Chlorine dioxide can be obtained in
two ways,(1) on-site generation by mixing of two separate
components,hydrochloric acid (HCl) and sodium chlorite
(NaClO2); and (2) ordering its stabilized form,which is
then activated on-site when required.
Of the oxidizing biocides,chlorine dioxide is the most
selective oxidant,Ozone and chlorine are much more
reactive than chlorine dioxide,and they will be consumed
by most organic compounds,Chlorine dioxide,however,
reacts only with reduced sulfur compounds,secondary and
tertiary amines,and some other highly reduced and
reactive organic compounds.
A more stable residue can therefore be achieved with
chlorine dioxide at much lower doses than when using
either chlorine or ozone,Generated properly,chlorine
dioxide can be used more effectively than ozone or chlorine
in cases of higher organic loading because of its selectivity.
Formaldehyde
Formaldehyde (HCHO) is a gas that kills all
microorganisms and spores at temperatures above 20 ° C,
However,it is not active against prions.
Formaldehyde is relatively slow-acting and needs a relative
humidity level of about 70%,It is marketed as the solid
polymer,paraformaldehyde,in flakes or tablets,or as
formalin,a solution of the gas in water of about 370 g/l
(37%),containing methanol (100 ml/l) as a stabilizer,Both
formulations are heated to liberate the gas,which is used
for decontamination and disinfection of enclosed volumes
such as safety cabinets and rooms.
Formaldehyde (5% formalin in water) may be used as a
liquid disinfectant.
Formaldehyde is a suspected carcinogen,It is a dangerous,
irritant gas that has a pungent smell and its fumes can
irritate eyes and mucous membranes,It must therefore
be stored and used in a fume-hood or well-ventilated area,
National chemical safety regulations must be followed.
Glutaraldehyde
Like formaldehyde,glutaraldehyde (OHC(CH2)3CHO) is
also active against vegetative bacteria,spores,fungi and
lipid- and nonlipid-containing viruses,It is non-corrosive
and faster acting than formaldehyde,However,it takes
several hours to kill bacterial spores.
Glutaraldehyde is generally supplied as a solution with a
concentration of about 20 g/l (2%) and some products may
need to be,activated” (made alkaline) before use by the
addition of a bicarbonate compound supplied with the
product,The activated solution can be reused for 1–4
weeks depending on the formulation and type and
frequency of its use,
Dipsticks supplied with some products give only a rough
indication of the levels of active glutaraldehyde available
in solutions under use,Glutaraldehyde solutions should be
discarded if they become turbid.
Glutaraldehyde is toxic and an irritant to skin and mucous
membranes,and contact with it must be avoided,It must
be used in a fume-hood or in well-ventilated areas,It
is not recommended as a spray or solution for the
decontamination of environmental surfaces,National
chemical safety regulations must be followed.
Phenolic compounds
Phenolic compounds,a broad group of agents,were among
the earliest germicides,However,more recent safety
concerns restrict their use,They are active against
vegetative bacteria and lipid-containing viruses and,when
properly formulated,also show activity against
mycobacteria,They are not active against spores and their
activity against nonlipid viruses is variable,Many phenolic
products are used for the decontamination of
environmental surfaces,and some (e.g,triclosan and
chloroxylenol) are among the more commonly used
antiseptics.
Triclosan is common in products for hand-washing,It is
active mainly against vegetative bacteria and safe for skin
and mucous membranes,However,in laboratory-based
studies,bacteria made resistant to low concentrations of
triclosan also show resistance to certain types of antibiotics,
The significance of this finding in the field remains
unknown.
Some phenolic compounds are sensitive to and may be
inactivated by water hardness and therefore must be
diluted with distilled or deionized water.
Phenolic compounds are not recommended for use on food
contact surfaces and in areas with young children,They
may be absorbed by rubber and can also penetrate the
skin,National chemical safety regulations must be
followed.
Quaternary ammonium compounds
Many types of quaternary ammonium compounds are
used as mixtures and often in combination with other
germicides,such as alcohols,They have good activity
against some vegetative bacteria and lipid-containing
viruses,Certain types (e.g,benzalkonium chloride) are
used as antiseptics.
The germicidal activity of certain types of quaternary
ammonium compounds is considerably reduced by organic
matter,water hardness and anionic detergents,Care
is therefore needed in selecting agents for precleaning
when quaternary ammonium compounds are to be used
for disinfection,Potentially harmful bacteria can grow in
quaternary ammonium compound solutions,Owing to low
biodegradability,these compounds may also accumulate in
the environment.
Alcohols
Ethanol (ethyl alcohol,C2H5 OH) and 2-propanol
(isopropyl alcohol,(CH3)2CHOH) have similar disinfectant
properties,They are active against vegetative bacteria,
fungi and lipid-containing viruses but not against spores,
Their action on nonlipid viruses is variable,For highest
effectiveness they should be used at concentrations of
approximately 70% (v/v) in water,higher or lower
concentrations may not be as germicidal,A major
advantage of aqueous solutions of alcohols is that they do
not leave any residue on treated items.
Mixtures with other agents are more effective than alcohol
alone,e.g,70% (v/v) alcohol with 100 g/l formaldehyde,
and alcohol containing 2 g/l available chlorine,A 70% (v/v)
aqueous solution of ethanol can be used on skin,work
surfaces of laboratory benches and biosafety cabinets,and
to soak small pieces of surgical instruments,Since ethanol
can dry the skin,it is often mixed with emollients.
Alcohol-based hand-rubs are recommended for the
decontamination of lightly soiled hands in situations where
proper hand-washing is inconvenient or not possible,
However,it must be remembered that ethanol is ineffective
against spores and may not kill all types of nonlipid viruses.
Alcohols are volatile and flammable and must not be used
near open flames,Working solutions should be stored in
proper containers to avoid the evaporation of alcohols.
Alcohols may harden rubber and dissolve certain types of
glue,Proper inventory and storage of ethanol in the
laboratory is very important to avoid its use for purposes
other than disinfection,Bottles with alcohol-containing
solutions must be clearly labelled to avoid autoclaving.
Iodine and iodophors
The action of these disinfectants is similar to that of
chlorine,although they may be slightly less inhibited by
organic matter,Iodine can stain fabrics and environmental
surfaces and is generally unsuitable for use as a
disinfectant,
On the other hand,iodophors and tinctures of iodine are
good antiseptics,Polyvidone-iodine is a reliable
and safe surgical scrub and preoperative skin antiseptic,
Antiseptics based on iodine are generally unsuitable for
use on medical/dental devices,Iodine should not be used
on aluminium or copper.
Iodine can be toxic,Organic iodine-based products must
be stored at 4–10 ° C to avoid the growth of potentially
harmful bacteria in them.
Hydrogen peroxide and peracids
Like chlorine,hydrogen peroxide (H2O2) and peracids are
strong oxidants and can bepotent broad-spectrum
germicides,They are also safer than chlorine to humans
andthe environment.
Hydrogen peroxide is supplied either as a ready-to-use 3%
solution or as a 30% aqueous solution to be diluted to 5–10
times its volume with sterilized water,However,such 3–
6% solutions of hydrogen peroxide alone are relatively
slow and limited as germicides,Products now available
have other ingredients to stabilize the hydrogen peroxide
content,to accelerate its germicidal action and to make it
less corrosive.
Hydrogen peroxide can be used for the decontamination of
work surfaces of laboratory benches and biosafety cabinets,
and stronger solutions may be suitable for disinfecting
heat-sensitive medical/dental devices,The use of vaporized
hydrogen peroxide or peracetic acid (CH3COOOH) for
the decontamination of heat-sensitive medical/surgical
devices requires specialized equipment.
Hydrogen peroxide and peracids can be corrosive to
metals such as aluminium,copper,brass and zinc,and can
also decolorize fabrics,hair,skin and mucous membranes,
Articles treated with them must be thoroughly rinsed
before contact with eyes and mucous membranes,They
should always be stored away from heat and protected
from light.
Local environmental decontamination
Decontamination of the laboratory space,its furniture and
its equipment requires a combination of liquid and gaseous
disinfectants,Surfaces can be decontaminated using a
solution of sodium hypochlorite (NaOCl); a solution
containing 1 g/l available chlorine may be suitable for
general environmental sanitation,but stronger solutions (5
g/l) are recommended when dealing with high-risk
situations,For environmental decontamination,
formulated solutions containing 3% hydrogen peroxide
(H2O2) make suitable substitutes for bleach solutions.
Rooms and equipment can be decontaminated by
fumigation with formaldehyde gas generated by heating
paraformaldehyde or boiling formalin,This is a highly
dangerous process that requires specially trained
personnel,All openings in the room (i.e,windows,doors,
etc.) should be sealed with masking tape or similar before
the gas is generated,Fumigation should be conducted at an
ambient temperature of at least 21 ° C and a relative
humidity of 70%,
After fumigation the area must be ventilated thoroughly
before personnel are allowed to enter,Appropriate
respirators must be worn by anyone entering the room
before it has been ventilated,Gaseous ammonium
bicarbonate can be used to neutralize the formaldehyde.
Fumigation of smaller spaces with hydrogen peroxide
vapour is also effective but requires specialized equipment
to generate the vapour.
Decontamination of biological safety cabinets
To decontaminate Class I and Class II cabinets,equipment
that independently generates,circulates and neutralizes
formaldehyde gas is available,Alternatively,the
appropriate amount of paraformaldehyde (final
concentration of 0.8% paraformaldehyde in air) should be
placed in a frying pan on an electric hot plate,
Another frying pan,containing 10% more ammonium
bicarbonate than paraformaldehyde,on a second hot plate
is also placed inside the cabinet,
The hot plate leads are plugged in outside the cabinet,so
that operation of the pans can be controlled from the
outside by plugging and unplugging the hot plates as
necessary,
If the relative humidity is below 70%,an open container of
hot water should also be placed inside the cabinet before
the front closure is sealed in place with strong tape (e.g,
duct tape).
Heavy gauge plastic sheeting is taped over the front
opening and exhaust port to make sure that the gas cannot
seep into the room,Penetration of the electric leads
passing through the front closure must also be sealed with
duct tape.
The plate for the paraformaldehyde pan is plugged in,It is
unplugged when all the paraformaldehyde has vaporized,
The cabinet is left undisturbed for at least 6 h,The plate
for the second pan is then plugged in and the ammonium
bicarbonate is allowed to vaporize.
This plate is then unplugged and the cabinet blower is
switched on for two intervals of approximately 2 s each to
allow the ammonium bicarbonate gas to circulate,The
cabinet should be left undisturbed for 30 min before the
front closure (or plastic sheeting) and the exhaust port
sheeting are removed,The cabinet surfaces should be
wiped down to remove residues before use.
Hand-washing/hand decontamination
Whenever possible,suitable gloves should be worn when
handling biohazardous materials,However,this does not
replace the need for regular and proper hand-washing
by laboratory personnel,Hands must be washed after
handling biohazardous materials and animals,and before
leaving the laboratory.
In most situations,thorough washing of hands with
ordinary soap and water is sufficient to decontaminate
them,but the use of germicidal soaps is recommended in
high-risk situations,Hands should be thoroughly lathered
with soap,using friction,for at least 10 s,rinsed in clean
water and dried using a clean paper or cloth towel (if
available,warm-air hand-dryers may be used).
Foot- or elbow-operated faucets are recommended,Where
not fitted,a paper/cloth towel should be used to turn off
the faucet handles to avoid recontaminating washed hands.
As mentioned above,alcohol-based hand-rubs may be used
to decontaminate lightly soiled hands when proper hand-
washing is not available.
Heat disinfection and sterilization
Heat is the most common among the physical agents used
for the decontamination of pathogens.,Dry” heat,which is
totally non-corrosive,is used to process many items of
laboratory ware which can withstand temperatures of 160
° C or higher for 2–4 h.
Burning or incineration (see below) is also a form of dry
heat.,Moist” heat is most effective when used in the form
of autoclaving.
Boiling does not necessarily kill all microorganisms and/or
pathogens,but it may be used as the minimum processing
for disinfection where other methods (chemical
disinfection or decontamination,autoclaving) are not
applicable or available.
Sterilized items must be handled and stored such that they
remain uncontaminated until used.
Autoclaving
Saturated steam under pressure (autoclaving) is the most
effective and reliable means of sterilizing laboratory
materials,For most purposes,the following cycles will
ensure sterilization of correctly loaded autoclaves:
1,3 min holding time at 134° C
2,10 min holding time at 126° C
3,15 min holding time at 121° C
4,25 min holding time at 115° C.
Examples of different autoclaves include the following.
Gravity displacement autoclaves,Figure 10 shows the
general construction of a gravity-displacement autoclave,
Steam enters the chamber under pressure and displaces
the heavier air downwards and through the valve in the
chamber drain,fitted with a HEPA filter.
Pre-vacuum autoclaves,These machines allow the removal
of air from the chamber before steam is admitted,The
exhaust air is evacuated through a valve fitted with a
HEPA filter,
At the end of the cycle,the steam is automatically
exhausted,These autoclaves can operate at 134° C and
the sterilization cycle can therefore be reduced to 3 min,
They are ideal for porous loads,but cannot be used to
process liquids because of the vacuum.
Fuel-heated pressure cooker autoclaves,These should be
used only if a gravity displacement autoclave is not
available,They are loaded from the top and heated by gas,
electricity or other types of fuels,
Steam is generated by heating water in the base of the
vessel,and air is displaced upwards through a relief vent,
When all the air has been removed,the valve on the relief
vent is closed and the heat reduced,The pressure and
temperature rise until the safety valve operates at a preset
level,
This is the start of the holding time,At the end of the cycle
the heat is turned off and the temperature allowed
to fall to 80° C or below before the lid is opened.
Loading autoclaves
Materials should be loosely packed in the chamber for easy
steam penetration and air removal,Bags should allow the
steam to reach their contents.
Precautions in the use of autoclaves
The following rules can minimize the hazards inherent in
operating pressurized vessels.
1,Responsibility for operation and routine care should be
assigned to trained individuals.
2,A preventive maintenance programme should include
regular inspection of the chamber,door seals and all
gauges and controls by qualified personnel.
3,The steam should be saturated and free from chemicals
(e.g,corrosion inhibitors) that could contaminate the items
being sterilized.
4,All materials to be autoclaved should be in containers
that allow ready removal of air and permit good heat
penetration; the chamber should be loosely packed so that
steam will reach the load evenly.
5,For autoclaves without an interlocking safety device that
prevents the door being opened when the chamber is
pressurized,the main steam valve should be closed and the
temperature allowed to fall below 80° C before the door is
opened.
6,Slow exhaust settings should be used when autoclaving
liquids,as they may boil over when removed due to
superheating.
7,Operators should wear suitable gloves and visors for
protection when opening the autoclave,even when the
temperature has fallen below 80 ° C.
8,In any routine monitoring of autoclave performance,
biological indicators or thermocouples should be placed at
the centre of each load,Regular monitoring
with thermocouples and recording devices in a,worst case”
load is highly desirable to determine proper operating
cycles.
9,The drain screen filter of the chamber (if available)
should be removed and cleaned daily.
10,Care should be taken to ensure that the relief valves of
pressure cooker autoclaves do not become blocked by
paper,etc,in the load.
Incineration
Incineration is useful for disposing of animal carcasses as
well as anatomical and other laboratory waste,with or
without prior decontamination,
Incineration of infectious materials is an alternative to
autoclaving only if the incinerator is under
laboratory control.
Proper incineration requires an efficient means of
temperature control and a secondary burning chamber,
Many incinerators,especially those with a single
combustion chamber,are unsatisfactory for dealing with
infectious materials,animal carcasses and plastics.
Such materials may not be completely destroyed and the
effluent
from the chimney may pollute the atmosphere with
microorganisms,toxic chemicals
and smoke,However,there are many satisfactory
configurations for combustion
chambers,Ideally the temperature in the primary
chamber should be at least 800 ° C
and that in the secondary chamber at least 1000 ° C.
Materials for incineration,even with prior
decontamination,should be transportedto the incinerator
in bags,preferably plastic,Incinerator attendants should
receive proper instructions about loading and temperature
control,
It should also be noted that the efficient operation of an
incinerator depends heavily on the right mix of materials
in the waste being treated.
There are ongoing concerns regarding the possible
negative environmental effects of existing or proposed
incinerators,and efforts continue to make incinerators
more environmentally friendly and energy-efficient.
Disposal
The disposal of laboratory and medical waste is subject to
various regional,national and international regulations,
and the latest versions of such relevant documents must be
consulted before designing and implementing a
programme for handling,transportation and disposal of
biohazardous waste,
In general,ash from incinerators may be handled as
normal domestic waste and removed by local authorities,
Autoclaved waste may be disposed of by off-site
incineration or in licensed landfill sites.
