12,Laboratory techniques
Human error,poor laboratory techniques and misuse of
equipment cause the majority
of laboratory injuries and work-related infections,This
chapter provides a compendium
of technical methods that are designed to avoid or
minimize the most commonly
reported problems of this nature.
Safe handling of specimens in the laboratory
Improper collection,transport and handling of specimens
in the laboratory carry a
risk of infection to the personnel involved.
Specimen containers
Specimen containers may be of glass or preferably plastic,
They should be robust and
should not leak when the cap or stopper is correctly
applied,No material should remain
on the outside of the container,Containers should be
correctly labelled to facilitate
identification,Specimen request or specification forms
should not be wrapped around
the containers but placed in separate,preferably
waterproof envelopes.
Transport of specimens within the facility
To avoid accidental leakage or spillage,secondary
containers,such as boxes,should be
used,fitted with racks so that the specimen containers
remain upright,The secondary
containers may be of metal or plastic,should be
autoclavable or resistant to the action
of chemical disinfectants,and the seal should preferably
have a gasket,They should be
regularly decontaminated.
Receipt of specimens
Laboratories that receive large numbers of specimens
should designate a particular
room or area for this purpose.
Opening packages
Personnel who receive and unpack specimens should be
aware of the potential health
hazards involved,and should be trained to adopt standard
precautions (2),particularly
when dealing with broken or leaking containers,Primary
specimen containers should
be opened in a biological safety cabinet,Disinfectants
should be available.
Use of pipettes and pipetting aids
1,A pipetting aid must always be used,Pipetting by mouth
must be prohibited.
2,All pipettes should have cotton plugs to reduce
contamination of pipetting devices.
3,Air should never be blown through a liquid containing
infectious agents.
4,Infectious materials should not be mixed by alternate
suction and expulsion through
5,Liquids should not be forcibly expelled from pipettes.
6,Mark-to-mark pipettes are preferable to other types as
they do not require expulsion
of the last drop.
7,Contaminated pipettes should be completely submerged
in a suitable disinfectant
contained in an unbreakable container,They should be left
in the disinfectant for
the appropriate length of time before disposal.
8,A discard container for pipettes should be placed within
the biological safety cabinet,
not outside it.
9,Syringes fitted with hypodermic needles must not be
used for pipetting.
10,Devices for opening septum-capped bottles that allow
pipettes to be used and avoid
the use of hypodermic needles and syringes should be used.
11,To avoid dispersion of infectious material dropped
from a pipette,an absorbent
material should be placed on the working surface; this
should be disposed of as
infectious waste after use.
Avoiding the dispersal of infectious materials
1,In order to avoid the premature shedding of their loads,
microbiological transfer
loops should have a diameter of 2–3 mm and be completely
closed,The shanks
should be not more than 6 cm in length to minimize
vibration.
2,The risk of spatter of infectious material in an open
Bunsen burner flame should
be avoided by using an enclosed electric microincinerator
to sterilize transfer loops.
Disposable transfer loops,which do not need to be
resterilized,are preferable.
3,Care should be taken when drying sputum samples,to
avoid creating aerosols.
4,Discarded specimens and cultures for autoclaving
and/or disposal should be placed
in leakproof containers,e.g,laboratory discard bags,Tops
should be secured (e.g.
with autoclave tape) prior to disposal into waste containers.
5,Working areas must be decontaminated with a suitable
disinfectant at the end of
each work period.
Use of biological safety cabinets
1,The use and limitations of biological safety cabinets
should be explained to all
potential users (see Chapter 10),with reference to national
standards and relevant
literature,Written protocols or safety or operations
manuals should be issued to
staff,In particular,it must be made clear that the cabinet
will not protect the
operator from spillage,breakage or poor technique.
2,The cabinet must not be used unless it is working
properly.
3,The glass viewing panel must not be opened when the
cabinet is in use.
4,Apparatus and materials in the cabinet must be kept to
a minimum,Air circulation
at the rear plenum must not be blocked.
5,Bunsen burners must not be used in the cabinet,The
heat produced will distort
the airflow and may damage the filters,An electric
microincinerator is permissible
but sterile disposable transfer loops are better.
6,All work must be carried out in the middle or rear part
of the working surface and
be visible through the viewing panel.
7,Traffic behind the operator should be minimized.
8,The operator should not disturb the airflow by repeated
removal and reintroduction
of his or her arms.
9,Air grills must not be blocked with notes,pipettes or
other materials,as this will
disrupt the airflow causing potential contamination of the
material and exposure
of the operator.
10,The surface of the biological safety cabinet should be
wiped using an appropriate
disinfectant after work is completed and at the end of the
day.
11,The cabinet fan should be run for at least 5 min before
beginning work and after
completion of work in the cabinet.
12,Paperwork should never be placed inside biological
safety cabinets.
Avoiding ingestion of infectious materials and contact with
skin and eyes
1,Large particles and droplets (> 5 Hm in diameter)
released during microbiological manipulations settle
rapidly on bench surfaces and on the hands of the operator.
Disposable gloves should be worn,Laboratory workers
should avoid touching their mouth,eyes and face.
2,Food and drink must not be consumed or stored in the
laboratory.
3,No articles should be placed in the mouth – pens,pencils,
chewing gum – in the laboratory.
4,Cosmetics should not be applied in the laboratory.
5,The face,eyes and mouth should be shielded or
otherwise protected during any operation that may result
in the splashing of potentially infectious materials.
Avoiding injection of infectious materials
1,Accidental inoculation resulting from injury with
broken or chipped glassware can be avoided through
careful practices and procedures,Glassware should be
replaced with plastic ware whenever possible.
2,Accidental injection may result from sharps injuries e.g,
with hypodermic needles (needle-sticks),glass Pasteur
pipettes,or broken glass.
3,Needle-stick injuries can be reduced by,(a) minimizing
the use of syringes and needles (e.g,simple devices are
available for opening septum-stoppered bottles so
that pipettes can be used instead of syringes and needles;
or (b) using engineered sharp safety devices when syringes
and needles are necessary.
4,Needles should never be recapped,Disposable articles
should be discarded into puncture-proof/puncture-
resistant containers fitted with covers.
5,Plastic Pasteur pipettes should replace those made of
glass.
Separation of serum
1,Only properly trained staff should be employed for this
work.
2,Gloves and eye and mucous membrane protection
should be worn.
3,Splashes and aerosols can only be avoided or minimized
by good laboratory technique,Blood and serum should be
pipetted carefully,not poured,Pipetting by mouth must be
forbidden.
4,After use,pipettes should be completely submerged in
suitable disinfectant,They should remain in the
disinfectant for the appropriate time before disposal or
washing and sterilization for reuse.
5,Discarded specimen tubes containing blood clots,etc,
(with caps replaced) should be placed in suitable leakproof
containers for autoclaving and/or incineration.
6,Suitable disinfectants should be available for clean-up of
splashes and spillages
Use of centrifuges
1,Satisfactory mechanical performance is a prerequisite of
microbiological safety in the use of laboratory centrifuges.
2,Centrifuges should be operated according to the
manufacturer’s instructions.
3,Centrifuges should be placed at such a level that
workers can see into the bowl to place trunnions and
buckets correctly.
4,Centrifuge tubes and specimen containers for use in the
centrifuge should be made of thick-walled glass or
preferably of plastic and should be inspected for defects
before use.
5,Tubes and specimen containers should always be
securely capped (screw-capped if possible) for
centrifugation.
6,The buckets must be loaded,equilibrated,sealed and
opened in a biological safety cabinet.
7,Buckets and trunnions should be paired by weight and,
with tubes in place,correctly balanced.
8,The amount of space that should be left between the
level of the fluid and the rim of the centrifuge tube should
be given in manufacturer’s instructions.
9,Distilled water or alcohol (propanol,70%) should be
used for balancing empty buckets,Saline or hypochlorite
solutions should not be used as they corrode metals.
10,Sealable centrifuge buckets (safety cups) must be used
for microorganisms in Risk Groups 3 and 4.
11,When using angle-head centrifuge rotors,care must be
taken to ensure that the tube is not overloaded as it might
leak.
12,The interior of the centrifuge bowl should be inspected
daily for staining or soiling at the level of the rotor,If
staining or soiling are evident then the centrifugation
protocols should be re-evaluated.
13,Centrifuge rotors and buckets should be inspected
daily for signs of corrosion and for hair-line cracks.
14,Buckets,rotors and centrifuge bowls should be
decontaminated after each use.
15,After use,buckets should be stored in an inverted
position to drain the balancing fluid.
16,Infectious airborne particles may be ejected when
centrifuges are used,These particles travel at speeds too
high to be retained by the cabinet airflow if the
centrifuge is placed in a traditional open-fronted Class I or
Class II biological safety cabinet,Enclosing centrifuges in
Class III safety cabinets prevents emitted aerosols
from dispersing widely,However,good centrifuge
technique and securely capped tubes offer adequate
protection against infectious aerosols and dispersed
particles.
Use of homogenizers,shakers,blenders and sonicators
1,Domestic (kitchen) homogenizers should not be used in
laboratories as they may leak or release aerosols,
Laboratory blenders and stomachers are safer.
2,Caps and cups or bottles should be in good condition
and free from flaws or distortion,Caps should be well-
fitting and gaskets should be in good condition.
3,Pressure builds up in the vessel during the operation of
homogenizers,shakers and sonicators,Aerosols containing
infectious materials may escape from between the cap and
the vessel,Plastic,in particular,polytetrafluoroethylene
(PTFE) vessels are recommended because glass may break,
releasing infectious material and possibly wounding the
operator.
4,When in use,homogenizers,shakers and sonicators
should be covered by a strong transparent plastic casing,
This should be disinfected after use,Where possible,these
machines should be operated,under their plastic covers,in
a biological safety cabinet.
5,At the end of the operation the containers should be
opened in a biological safety cabinet.
6,Hearing protection should be provided for people using
sonicators.
Use of tissue grinders
1,Glass grinders should be held in absorbent material in a
gloved hand,Plastic (PTFE) grinders are safer.
2,Tissue grinders should be operated and opened in a
biological safety cabinet.
Care and use of refrigerators and freezers
1,Refrigerators,deep-freezers and solid carbon dioxide
(dry-ice) chests should be defrosted and cleaned
periodically,and any ampoules,tubes,etc,that have
broken during storage removed,Face protection and
heavy duty rubber gloves should be worn during cleaning,
After cleaning,the inner surfaces of the cabinet should be
disinfected.
2,All containers stored in refrigerators,etc,should be
clearly labelled with the scientific name of the contents,the
date stored and the name of the individual who stored
them,Unlabelled and obsolete materials should be
autoclaved and discarded.
3,An inventory must be maintained of the freezer’s
contents.
4,Flammable solutions must not be stored in a
refrigerator unless it is explosion-proof,Notices to this
effect should be placed on refrigerator doors.
Opening of ampoules containing lyophilized infectious
materials
Care should be taken when ampoules of freeze-dried
materials are opened,as the contents may be under
reduced pressure and the sudden inrush of air may
disperse some of the materials into the atmosphere,
Ampoules should always be opened in a biological safety
cabinet,The following procedures are recommended for
opening ampoules.
1,First decontaminate the outer surface of the ampoule.
2,Make a file mark on the tube near to the middle of the
cotton or cellulose plug,if present.
3,Hold the ampoule in alcohol-soaked cotton to protect
hands before breaking it at a file scratch.
4,Remove the top gently and treat as contaminated
material.
5,If the plug is still above the contents of the ampoule,
remove it with sterile forceps.
6,Add liquid for resuspension slowly to the ampoule to
avoid frothing.
Storage of ampoules containing infectious materials
Ampoules containing infectious materials should never be
immersed in liquid nitrogen because cracked or
imperfectly sealed ampoules may break or explode on
removal,If very low temperatures are required,ampoules
should be stored only in the gaseous phase above the liquid
nitrogen,Otherwise,infectious materials should be stored
in mechanical deep-freeze cabinets or on dry ice,
Laboratory workers should wear eye and hand protection
when removing ampoules from cold storage,The outer
surfaces of ampoules stored in these ways should be
disinfected when the ampoules are removed from storage.
Standard precautions with blood and other body fluids,
tissues and excreta
Standard precautions (which include,universal
precautions” (19)) are designed to reduce the risk of
transmission of microorganisms from both recognized and
unrecognized sources of infection.
Collection,labelling and transport of specimens
1,Standard precautions should always be followed; gloves
should be worn for all procedures.
2,Blood should be collected from patients and animals by
trained staff.
3,For phlebotomies,conventional needle and syringe
systems should be replaced by single-use safety vacuum
devices that allow the collection of blood directly into
stoppered transport and/or culture tubes,automatically
disabling the needle after use.
4,The tubes should be placed in adequate containers for
transport to the laboratory (see Chapter 15 for transport
requirements) and within the laboratory facility (see
section on Transport of specimens within the facility in
this chapter),Request forms should be placed in separate
waterproof bags or envelopes.
5,Reception staff should not open these bags.
Opening specimen tubes and sampling contents
1,Specimen tubes should be opened in a biological safety
cabinet.
2,Gloves must be worn,Eye and mucous membrane
protection is also recommended (goggles or face shields).
3,Protective clothing should be supplemented with a
plastic apron.
4,The stopper should be grasped through a piece of paper
or gauze to prevent splashing.
Glass and,sharps”
1,Plastics should replace glass wherever possible,Only
laboratory grade (borosilicate) glass should be used,and
any article that is chipped or cracked should be discarded.
2,Hypodermic needles must not be used as pipettes (see
also section on Avoiding injection of infectious materials in
this chapter).
Films and smears for microscopy
Fixing and staining of blood,sputum and faecal samples
for microscopy do not necessarily kill all organisms or
viruses on the smears,These items should be handled
with forceps,stored appropriately,and decontaminated
and/or autoclaved before disposal.
Automated equipment (sonicators,vortex mixers)
1,Equipment should be of the closed type to avoid
dispersion of droplets and aerosols.
2,Effluents should be collected in closed vessels for further
autoclaving and/or disposal.
3,Equipment should be disinfected at the end of each
session,following manu-facturers’ instructions.
Tissues
1,Formalin fixatives should be used.
2,Frozen sectioning should be avoided,When necessary,
the cryostat should be shielded and the operator should
wear a safety face shield,For decontamination,the
temperature of the instrument should be raised to at least
20 AC
Decontamination
Hypochlorites and high-level disinfectants are
recommended for decontamination,Freshly prepared
hypochlorite solutions should contain available chlorine at
1 g/l for general use and 5 g/l for blood spillages,
Glutaraldehyde may be used for decontaminating surfaces
Precautions with materials that may contain prions
Prions (also referred to as,slow viruses”) are associated
with the transmissible spongiform encephalopathies
(TSEs),notably Creutzfeldt-Jakob disease (CJD; including
thenew variant form),Gerstmann-Str?ussler-Scheinker
syndrome,fatal familial insomnia and kuru in humans;
scrapie in sheep and goats; bovine spongiform
encephalopathy (BSE) in cattle; and other transmissible
encephalopathies of deer,elk and mink.
Although CJD has been transmitted to humans,there
appear to be no proven cases of laboratory-associated
infections with any of these agents,Nevertheless,it is
prudent to observe certain precautions in the handling of
material from infected or potentially infected humans and
animals.
The selection of a biosafety level for work with materials
associated with TSEs will depend on the nature of the
agent and the samples to be studied,and should be
undertaken in consultation with national authorities,The
highest concentrations of prions are found in central
nervous system tissue,Animal studies suggest that it is
likely that high concentrations of prions are also found in
the spleen,thymus,lymph nodes and lung,Recent studies
indicate that prions in lingual and skeletal muscle
tissue may also present a potential infection risk.
As complete inactivation of prions is difficult to achieve,it
is important to stress the use of disposable instruments
whenever possible,and to use a disposable protective
covering for the work surface of the biological safety
cabinet.
The main precaution to be taken is to avoid ingestion of
contaminated materials or puncture of the laboratory
worker’s skin,The following additional precautions should
be taken,as the agents are not killed by the normal
processes of laboratory disinfection and sterilization.
1,The use of dedicated equipment,i.e,equipment not
shared with other laboratories,is highly recommended.
2,Disposable laboratory protective clothing (gowns and
aprons) and gloves must be worn (steel mesh gloves
between rubber gloves for pathologists).
3,Use of disposable plastic ware,which can be treated and
discarded as dry waste,is highly recommended.
4,Tissue processors should not be used because of the
problems of disinfection,Jars and beakers (plastic) should
be used instead.
5,All manipulations must be conducted in biological safety
cabinets.
6,Great care should be exercised to avoid aerosol
production,ingestion,and cuts and punctures of the skin.
7,Formalin-fixed tissues should be regarded as still
infectious,even after prolonged exposure to formalin.
8,Histological samples containing prions are substantially
inactivated after exposure to 96% formic acid for 1 h.
9,Bench waste,including disposable gloves,gowns and
aprons,should be autoclaved using a porous load steam
sterilizer at 134–137 AC for a single cycle of 18 min,or
six successive cycles of 3 min each,followed by
incineration.
10,Non-disposable instruments,including steel mesh
gloves,must be collected for decontamination.
11,Infectious liquid waste contaminated with prions
should be treated with sodium hypochlorite containing
available chlorine at 20 g/l (2%) (final concentration) for
1 h.
12,Paraformaldehyde vaporization procedures do not
diminish prion titres and prions are resistant to ultraviolet
irradiation,However,the cabinets must continue to be
decontaminated by standard methods (i.e,formaldehyde
gas) to inactivate other agents that may be present.
13,Prion-contaminated biological safety cabinets and
other surfaces can be decontaminated with sodium
hypochlorite containing available chlorine at 20 g/l
(2%) for 1 h.
14,High-efficiency particulate air (HEPA) filters should be
incinerated at a minimum temperature of 1000 AC after
removal,Recommended additional steps prior to
incineration include:
a,spraying of the exposed face of the filter with lacquer
hairspray prior to removal,
b.,bagging” of filters during removal,and
c,removal of the HEPA filter from the working
chamber so that the inaccessible
plenum of the cabinet is not contaminated.
15,Instruments should be soaked in sodium hypochlorite
containing available chlorine at 20 g/l (2%) for 1 h and
then rinsed well in water before autoclaving.
16,Instruments that cannot be autoclaved can be cleaned
by repeated wetting with sodium hypochlorite containing
available chlorine at 20 g/l (2%) over a 1-h period.
Appropriate washing to remove residual sodium
hypochlorite is required,
Human error,poor laboratory techniques and misuse of
equipment cause the majority
of laboratory injuries and work-related infections,This
chapter provides a compendium
of technical methods that are designed to avoid or
minimize the most commonly
reported problems of this nature.
Safe handling of specimens in the laboratory
Improper collection,transport and handling of specimens
in the laboratory carry a
risk of infection to the personnel involved.
Specimen containers
Specimen containers may be of glass or preferably plastic,
They should be robust and
should not leak when the cap or stopper is correctly
applied,No material should remain
on the outside of the container,Containers should be
correctly labelled to facilitate
identification,Specimen request or specification forms
should not be wrapped around
the containers but placed in separate,preferably
waterproof envelopes.
Transport of specimens within the facility
To avoid accidental leakage or spillage,secondary
containers,such as boxes,should be
used,fitted with racks so that the specimen containers
remain upright,The secondary
containers may be of metal or plastic,should be
autoclavable or resistant to the action
of chemical disinfectants,and the seal should preferably
have a gasket,They should be
regularly decontaminated.
Receipt of specimens
Laboratories that receive large numbers of specimens
should designate a particular
room or area for this purpose.
Opening packages
Personnel who receive and unpack specimens should be
aware of the potential health
hazards involved,and should be trained to adopt standard
precautions (2),particularly
when dealing with broken or leaking containers,Primary
specimen containers should
be opened in a biological safety cabinet,Disinfectants
should be available.
Use of pipettes and pipetting aids
1,A pipetting aid must always be used,Pipetting by mouth
must be prohibited.
2,All pipettes should have cotton plugs to reduce
contamination of pipetting devices.
3,Air should never be blown through a liquid containing
infectious agents.
4,Infectious materials should not be mixed by alternate
suction and expulsion through
5,Liquids should not be forcibly expelled from pipettes.
6,Mark-to-mark pipettes are preferable to other types as
they do not require expulsion
of the last drop.
7,Contaminated pipettes should be completely submerged
in a suitable disinfectant
contained in an unbreakable container,They should be left
in the disinfectant for
the appropriate length of time before disposal.
8,A discard container for pipettes should be placed within
the biological safety cabinet,
not outside it.
9,Syringes fitted with hypodermic needles must not be
used for pipetting.
10,Devices for opening septum-capped bottles that allow
pipettes to be used and avoid
the use of hypodermic needles and syringes should be used.
11,To avoid dispersion of infectious material dropped
from a pipette,an absorbent
material should be placed on the working surface; this
should be disposed of as
infectious waste after use.
Avoiding the dispersal of infectious materials
1,In order to avoid the premature shedding of their loads,
microbiological transfer
loops should have a diameter of 2–3 mm and be completely
closed,The shanks
should be not more than 6 cm in length to minimize
vibration.
2,The risk of spatter of infectious material in an open
Bunsen burner flame should
be avoided by using an enclosed electric microincinerator
to sterilize transfer loops.
Disposable transfer loops,which do not need to be
resterilized,are preferable.
3,Care should be taken when drying sputum samples,to
avoid creating aerosols.
4,Discarded specimens and cultures for autoclaving
and/or disposal should be placed
in leakproof containers,e.g,laboratory discard bags,Tops
should be secured (e.g.
with autoclave tape) prior to disposal into waste containers.
5,Working areas must be decontaminated with a suitable
disinfectant at the end of
each work period.
Use of biological safety cabinets
1,The use and limitations of biological safety cabinets
should be explained to all
potential users (see Chapter 10),with reference to national
standards and relevant
literature,Written protocols or safety or operations
manuals should be issued to
staff,In particular,it must be made clear that the cabinet
will not protect the
operator from spillage,breakage or poor technique.
2,The cabinet must not be used unless it is working
properly.
3,The glass viewing panel must not be opened when the
cabinet is in use.
4,Apparatus and materials in the cabinet must be kept to
a minimum,Air circulation
at the rear plenum must not be blocked.
5,Bunsen burners must not be used in the cabinet,The
heat produced will distort
the airflow and may damage the filters,An electric
microincinerator is permissible
but sterile disposable transfer loops are better.
6,All work must be carried out in the middle or rear part
of the working surface and
be visible through the viewing panel.
7,Traffic behind the operator should be minimized.
8,The operator should not disturb the airflow by repeated
removal and reintroduction
of his or her arms.
9,Air grills must not be blocked with notes,pipettes or
other materials,as this will
disrupt the airflow causing potential contamination of the
material and exposure
of the operator.
10,The surface of the biological safety cabinet should be
wiped using an appropriate
disinfectant after work is completed and at the end of the
day.
11,The cabinet fan should be run for at least 5 min before
beginning work and after
completion of work in the cabinet.
12,Paperwork should never be placed inside biological
safety cabinets.
Avoiding ingestion of infectious materials and contact with
skin and eyes
1,Large particles and droplets (> 5 Hm in diameter)
released during microbiological manipulations settle
rapidly on bench surfaces and on the hands of the operator.
Disposable gloves should be worn,Laboratory workers
should avoid touching their mouth,eyes and face.
2,Food and drink must not be consumed or stored in the
laboratory.
3,No articles should be placed in the mouth – pens,pencils,
chewing gum – in the laboratory.
4,Cosmetics should not be applied in the laboratory.
5,The face,eyes and mouth should be shielded or
otherwise protected during any operation that may result
in the splashing of potentially infectious materials.
Avoiding injection of infectious materials
1,Accidental inoculation resulting from injury with
broken or chipped glassware can be avoided through
careful practices and procedures,Glassware should be
replaced with plastic ware whenever possible.
2,Accidental injection may result from sharps injuries e.g,
with hypodermic needles (needle-sticks),glass Pasteur
pipettes,or broken glass.
3,Needle-stick injuries can be reduced by,(a) minimizing
the use of syringes and needles (e.g,simple devices are
available for opening septum-stoppered bottles so
that pipettes can be used instead of syringes and needles;
or (b) using engineered sharp safety devices when syringes
and needles are necessary.
4,Needles should never be recapped,Disposable articles
should be discarded into puncture-proof/puncture-
resistant containers fitted with covers.
5,Plastic Pasteur pipettes should replace those made of
glass.
Separation of serum
1,Only properly trained staff should be employed for this
work.
2,Gloves and eye and mucous membrane protection
should be worn.
3,Splashes and aerosols can only be avoided or minimized
by good laboratory technique,Blood and serum should be
pipetted carefully,not poured,Pipetting by mouth must be
forbidden.
4,After use,pipettes should be completely submerged in
suitable disinfectant,They should remain in the
disinfectant for the appropriate time before disposal or
washing and sterilization for reuse.
5,Discarded specimen tubes containing blood clots,etc,
(with caps replaced) should be placed in suitable leakproof
containers for autoclaving and/or incineration.
6,Suitable disinfectants should be available for clean-up of
splashes and spillages
Use of centrifuges
1,Satisfactory mechanical performance is a prerequisite of
microbiological safety in the use of laboratory centrifuges.
2,Centrifuges should be operated according to the
manufacturer’s instructions.
3,Centrifuges should be placed at such a level that
workers can see into the bowl to place trunnions and
buckets correctly.
4,Centrifuge tubes and specimen containers for use in the
centrifuge should be made of thick-walled glass or
preferably of plastic and should be inspected for defects
before use.
5,Tubes and specimen containers should always be
securely capped (screw-capped if possible) for
centrifugation.
6,The buckets must be loaded,equilibrated,sealed and
opened in a biological safety cabinet.
7,Buckets and trunnions should be paired by weight and,
with tubes in place,correctly balanced.
8,The amount of space that should be left between the
level of the fluid and the rim of the centrifuge tube should
be given in manufacturer’s instructions.
9,Distilled water or alcohol (propanol,70%) should be
used for balancing empty buckets,Saline or hypochlorite
solutions should not be used as they corrode metals.
10,Sealable centrifuge buckets (safety cups) must be used
for microorganisms in Risk Groups 3 and 4.
11,When using angle-head centrifuge rotors,care must be
taken to ensure that the tube is not overloaded as it might
leak.
12,The interior of the centrifuge bowl should be inspected
daily for staining or soiling at the level of the rotor,If
staining or soiling are evident then the centrifugation
protocols should be re-evaluated.
13,Centrifuge rotors and buckets should be inspected
daily for signs of corrosion and for hair-line cracks.
14,Buckets,rotors and centrifuge bowls should be
decontaminated after each use.
15,After use,buckets should be stored in an inverted
position to drain the balancing fluid.
16,Infectious airborne particles may be ejected when
centrifuges are used,These particles travel at speeds too
high to be retained by the cabinet airflow if the
centrifuge is placed in a traditional open-fronted Class I or
Class II biological safety cabinet,Enclosing centrifuges in
Class III safety cabinets prevents emitted aerosols
from dispersing widely,However,good centrifuge
technique and securely capped tubes offer adequate
protection against infectious aerosols and dispersed
particles.
Use of homogenizers,shakers,blenders and sonicators
1,Domestic (kitchen) homogenizers should not be used in
laboratories as they may leak or release aerosols,
Laboratory blenders and stomachers are safer.
2,Caps and cups or bottles should be in good condition
and free from flaws or distortion,Caps should be well-
fitting and gaskets should be in good condition.
3,Pressure builds up in the vessel during the operation of
homogenizers,shakers and sonicators,Aerosols containing
infectious materials may escape from between the cap and
the vessel,Plastic,in particular,polytetrafluoroethylene
(PTFE) vessels are recommended because glass may break,
releasing infectious material and possibly wounding the
operator.
4,When in use,homogenizers,shakers and sonicators
should be covered by a strong transparent plastic casing,
This should be disinfected after use,Where possible,these
machines should be operated,under their plastic covers,in
a biological safety cabinet.
5,At the end of the operation the containers should be
opened in a biological safety cabinet.
6,Hearing protection should be provided for people using
sonicators.
Use of tissue grinders
1,Glass grinders should be held in absorbent material in a
gloved hand,Plastic (PTFE) grinders are safer.
2,Tissue grinders should be operated and opened in a
biological safety cabinet.
Care and use of refrigerators and freezers
1,Refrigerators,deep-freezers and solid carbon dioxide
(dry-ice) chests should be defrosted and cleaned
periodically,and any ampoules,tubes,etc,that have
broken during storage removed,Face protection and
heavy duty rubber gloves should be worn during cleaning,
After cleaning,the inner surfaces of the cabinet should be
disinfected.
2,All containers stored in refrigerators,etc,should be
clearly labelled with the scientific name of the contents,the
date stored and the name of the individual who stored
them,Unlabelled and obsolete materials should be
autoclaved and discarded.
3,An inventory must be maintained of the freezer’s
contents.
4,Flammable solutions must not be stored in a
refrigerator unless it is explosion-proof,Notices to this
effect should be placed on refrigerator doors.
Opening of ampoules containing lyophilized infectious
materials
Care should be taken when ampoules of freeze-dried
materials are opened,as the contents may be under
reduced pressure and the sudden inrush of air may
disperse some of the materials into the atmosphere,
Ampoules should always be opened in a biological safety
cabinet,The following procedures are recommended for
opening ampoules.
1,First decontaminate the outer surface of the ampoule.
2,Make a file mark on the tube near to the middle of the
cotton or cellulose plug,if present.
3,Hold the ampoule in alcohol-soaked cotton to protect
hands before breaking it at a file scratch.
4,Remove the top gently and treat as contaminated
material.
5,If the plug is still above the contents of the ampoule,
remove it with sterile forceps.
6,Add liquid for resuspension slowly to the ampoule to
avoid frothing.
Storage of ampoules containing infectious materials
Ampoules containing infectious materials should never be
immersed in liquid nitrogen because cracked or
imperfectly sealed ampoules may break or explode on
removal,If very low temperatures are required,ampoules
should be stored only in the gaseous phase above the liquid
nitrogen,Otherwise,infectious materials should be stored
in mechanical deep-freeze cabinets or on dry ice,
Laboratory workers should wear eye and hand protection
when removing ampoules from cold storage,The outer
surfaces of ampoules stored in these ways should be
disinfected when the ampoules are removed from storage.
Standard precautions with blood and other body fluids,
tissues and excreta
Standard precautions (which include,universal
precautions” (19)) are designed to reduce the risk of
transmission of microorganisms from both recognized and
unrecognized sources of infection.
Collection,labelling and transport of specimens
1,Standard precautions should always be followed; gloves
should be worn for all procedures.
2,Blood should be collected from patients and animals by
trained staff.
3,For phlebotomies,conventional needle and syringe
systems should be replaced by single-use safety vacuum
devices that allow the collection of blood directly into
stoppered transport and/or culture tubes,automatically
disabling the needle after use.
4,The tubes should be placed in adequate containers for
transport to the laboratory (see Chapter 15 for transport
requirements) and within the laboratory facility (see
section on Transport of specimens within the facility in
this chapter),Request forms should be placed in separate
waterproof bags or envelopes.
5,Reception staff should not open these bags.
Opening specimen tubes and sampling contents
1,Specimen tubes should be opened in a biological safety
cabinet.
2,Gloves must be worn,Eye and mucous membrane
protection is also recommended (goggles or face shields).
3,Protective clothing should be supplemented with a
plastic apron.
4,The stopper should be grasped through a piece of paper
or gauze to prevent splashing.
Glass and,sharps”
1,Plastics should replace glass wherever possible,Only
laboratory grade (borosilicate) glass should be used,and
any article that is chipped or cracked should be discarded.
2,Hypodermic needles must not be used as pipettes (see
also section on Avoiding injection of infectious materials in
this chapter).
Films and smears for microscopy
Fixing and staining of blood,sputum and faecal samples
for microscopy do not necessarily kill all organisms or
viruses on the smears,These items should be handled
with forceps,stored appropriately,and decontaminated
and/or autoclaved before disposal.
Automated equipment (sonicators,vortex mixers)
1,Equipment should be of the closed type to avoid
dispersion of droplets and aerosols.
2,Effluents should be collected in closed vessels for further
autoclaving and/or disposal.
3,Equipment should be disinfected at the end of each
session,following manu-facturers’ instructions.
Tissues
1,Formalin fixatives should be used.
2,Frozen sectioning should be avoided,When necessary,
the cryostat should be shielded and the operator should
wear a safety face shield,For decontamination,the
temperature of the instrument should be raised to at least
20 AC
Decontamination
Hypochlorites and high-level disinfectants are
recommended for decontamination,Freshly prepared
hypochlorite solutions should contain available chlorine at
1 g/l for general use and 5 g/l for blood spillages,
Glutaraldehyde may be used for decontaminating surfaces
Precautions with materials that may contain prions
Prions (also referred to as,slow viruses”) are associated
with the transmissible spongiform encephalopathies
(TSEs),notably Creutzfeldt-Jakob disease (CJD; including
thenew variant form),Gerstmann-Str?ussler-Scheinker
syndrome,fatal familial insomnia and kuru in humans;
scrapie in sheep and goats; bovine spongiform
encephalopathy (BSE) in cattle; and other transmissible
encephalopathies of deer,elk and mink.
Although CJD has been transmitted to humans,there
appear to be no proven cases of laboratory-associated
infections with any of these agents,Nevertheless,it is
prudent to observe certain precautions in the handling of
material from infected or potentially infected humans and
animals.
The selection of a biosafety level for work with materials
associated with TSEs will depend on the nature of the
agent and the samples to be studied,and should be
undertaken in consultation with national authorities,The
highest concentrations of prions are found in central
nervous system tissue,Animal studies suggest that it is
likely that high concentrations of prions are also found in
the spleen,thymus,lymph nodes and lung,Recent studies
indicate that prions in lingual and skeletal muscle
tissue may also present a potential infection risk.
As complete inactivation of prions is difficult to achieve,it
is important to stress the use of disposable instruments
whenever possible,and to use a disposable protective
covering for the work surface of the biological safety
cabinet.
The main precaution to be taken is to avoid ingestion of
contaminated materials or puncture of the laboratory
worker’s skin,The following additional precautions should
be taken,as the agents are not killed by the normal
processes of laboratory disinfection and sterilization.
1,The use of dedicated equipment,i.e,equipment not
shared with other laboratories,is highly recommended.
2,Disposable laboratory protective clothing (gowns and
aprons) and gloves must be worn (steel mesh gloves
between rubber gloves for pathologists).
3,Use of disposable plastic ware,which can be treated and
discarded as dry waste,is highly recommended.
4,Tissue processors should not be used because of the
problems of disinfection,Jars and beakers (plastic) should
be used instead.
5,All manipulations must be conducted in biological safety
cabinets.
6,Great care should be exercised to avoid aerosol
production,ingestion,and cuts and punctures of the skin.
7,Formalin-fixed tissues should be regarded as still
infectious,even after prolonged exposure to formalin.
8,Histological samples containing prions are substantially
inactivated after exposure to 96% formic acid for 1 h.
9,Bench waste,including disposable gloves,gowns and
aprons,should be autoclaved using a porous load steam
sterilizer at 134–137 AC for a single cycle of 18 min,or
six successive cycles of 3 min each,followed by
incineration.
10,Non-disposable instruments,including steel mesh
gloves,must be collected for decontamination.
11,Infectious liquid waste contaminated with prions
should be treated with sodium hypochlorite containing
available chlorine at 20 g/l (2%) (final concentration) for
1 h.
12,Paraformaldehyde vaporization procedures do not
diminish prion titres and prions are resistant to ultraviolet
irradiation,However,the cabinets must continue to be
decontaminated by standard methods (i.e,formaldehyde
gas) to inactivate other agents that may be present.
13,Prion-contaminated biological safety cabinets and
other surfaces can be decontaminated with sodium
hypochlorite containing available chlorine at 20 g/l
(2%) for 1 h.
14,High-efficiency particulate air (HEPA) filters should be
incinerated at a minimum temperature of 1000 AC after
removal,Recommended additional steps prior to
incineration include:
a,spraying of the exposed face of the filter with lacquer
hairspray prior to removal,
b.,bagging” of filters during removal,and
c,removal of the HEPA filter from the working
chamber so that the inaccessible
plenum of the cabinet is not contaminated.
15,Instruments should be soaked in sodium hypochlorite
containing available chlorine at 20 g/l (2%) for 1 h and
then rinsed well in water before autoclaving.
16,Instruments that cannot be autoclaved can be cleaned
by repeated wetting with sodium hypochlorite containing
available chlorine at 20 g/l (2%) over a 1-h period.
Appropriate washing to remove residual sodium
hypochlorite is required,
