Part
III: Culture
laboratory layout and equipment
Plan of a culture
laboratory
Persons
who work with tubercle bacilli are at risk of laboratory-acquired
infection, mainly by the airborne route, and it is well-known
that sensible laboratory design may contribute much to the prevention
of such infections. The detailed arrangement of a tuberculosis
laboratory will vary according to the size and shape of the
available room, the type of laboratory activity and whether
other work is also done in the same room. Nevertheless, the
most important aspect in tuberculosis laboratory design is to
ensure a logical flow of specimens and activities, from clean
to less clean areas.
Mycobacterial
cultures should always be performed in containment laboratories,
physically separated from other laboratory areas. The objective
is to reduce the infection risk, not only to tuberculosis technologists,
but also to other individuals in the same building.
Contrary
to common belief containment laboratories need not be overly
sophisticated and expensive. Sophisticated and expensive air
conditioning is not an essential requirement in tuberculosis
culture laboratories. Rather, the principle should be that,
during working hours, air is continuously extracted to the outside
of the laboratory either through a biological safety cabinet
or through simple extraction fans in walls or windows. Ventilation
standards for air changes and pressure gradients should be considered
in relation to the number of specimens processed per year and
the prevalence of tuberculosis among these specimens. If bacteriological
methods are performed with strict adherence to safety standards
and high risk procedures are limited to the bio-safety cabinet,
air-borne contamination will be minimised. Six to twelve room
air changes per hour are sufficient to remove up to 99% of airborne
particles within 30 to 45 minutes.
Supply
and exhaust air devices should be located on opposite side walls,
with supply air provided from clean areas and exhaust air taken
from less clean areas. An excess of air supply of 50 cubic feet
per minute (23.6 litres per second) or a similar negative pressure
created by extracting air is sufficient to obtain the necessary
pressure gradient. Air should be exhausted directly to the outside.
Potentially contaminated air should be discharged at least 3m
above ground level.
Figure
1 illustrates a floor plan for a tuberculosis culture laboratory
with air flow in one direction, from clean to less clean areas.
Arranging
equipment and materials
Entry
to the laboratory is via the officewhich contains the
facilities necessary for administration and management of the
laboratory. These include storage space for laboratory registers,
laboratory reports, chemicals and reagents.
Specimens
arriving at the laboratory are presented through a window/hatch
to a separate reception counter. Here, specimen containers
are checked for leakage and their surfaces decontaminated. Cross-checking
of laboratory request forms against specimens is also done and
the relevant details are entered into the laboratory register.
On completion of these activities the specimens are passed into
the main laboratoryarea for further processing.
The
main laboratory area contains all the facilities necessary for
smear preparation, for specimen decontamination and digestion,
and for inoculation of media and incubation of cultures. This
area houses work benches, a pH meter, a large domestic refrigerator,
a wash basin with elbow-controlled taps and storage cabinets.
The isolation area is situated at the most extreme end
of the main laboratory and contains a biological safety cabinet
and a centrifuge.
The
reading room is reserved for performing microscopy on
smears prepared in the main laboratory and for reading of cultures.
This area contains work benches, a microscope and an elbow-operated
wash basin. Laboratory reports may be completed here and then
passed to the office for dissemination and entering of data
into the laboratory register.
The
kitchen functions as an area for the disposal of cultures
and for subsequent cleaning and sterilisation of glassware.
This area houses work benches, a large double-wash stainless
steel sink and an autoclave.
In
many countries, media preparation is performed at the central
level only. If, however, media is prepared as part of the activities
of the culture laboratory, a separate media preparation area
is recommended. This area houses workbenches, an inspissator,
a domestic refrigerator and a wash-basin with elbow-operated
taps.
Before
the processing of specimens and the preparation of cultures
are started, equipment and materials should be arranged to ensure
a logical and safe flow of work. All manipulations should be
standardised and the arrangement of materials should always
be the same to ensure maximum safety, as illustrated by Figure
2. For left-handed technologists it may be more convenient to
arrange all or most items in the opposite direction, ie. in
a mirror-image.
Care and maintenance of essential equipment
Annex
1 contains a list of essential equipment and supplies for a
culture laboratory using egg-based Löwenstein-Jensen culture
medium, and 4% sodium hydroxide for specimen decontamination.
Before purchasing new equipment and supplies it is worthwhile
to obtain personal advice of laboratory persons who have had
experience in their use. Do not rely entirely on advertisements,
catalogues, extravagant claims of sales representatives and
the opinion of purchasing officers.
Biological
safety cabinet
Selecting
the right model
The
single most important piece of laboratory equipment needed in
a tuberculosis culture laboratory is a well-maintained, properly
functioning biological safety cabinet (BSC). These cabinets
have been designed to provide a combination of staff, environmental
or product protection when appropriate practices and procedures
are followed. BSCs use high efficiency particulate air (HEPA)
filters in their exhaust and/or air supply systems. HEPA filters
remove particles equal to and greater than 0.3Fm (which essentially
includes all bacteria, spores and viruses) with an efficiency
of 99.97%.
Microbiological
risks are assigned to biosafety levels I through IV.Mycobacterium
tuberculosis is classified under Risk Group III. This group
includes microorganisms that are particularly associated with
infection by the airborne route. Precautions therefore involve
measures to minimise the production and dispersal of aerosols
and infected airborne particles and to prevent the laboratory
worker from inhaling those that might be released, as well as
measures intended to prevent infection by accidental ingestion
and inoculation.
Of
the three classes of biological safety cabinets, Class I and
Class II are suitable for tuberculosis bacteriology. The Class
I BSC provides staff and environmental protection, but no product
protection (Figure 3).
Unfiltered
room air is drawn across the work surface. Staff protection
is provided by this inward air flow as long as a minimum velocity
of 75 linear feet per minute (22.8 meter per second) is maintained
through the front opening. Any airborne bacteria are entrained
and conveyed into the HEPA filter. The Class I BSC is hard-ducted
to the building exhaust system and the building exhaust fan
provides the negative pressure necessary to draw room air into
the cabinet. Modern cabinets have airflow indicators and warning
devices. The filters must be changed when the airflow falls
below the minimum velocity level.
Class
II BSCs provide staff, environmental and product protection.
Air flow is drawn around the operator into the front grille
of the cabinet, which provides staff protection. In addition,
the downward laminar flow of HEPA-filtered air provides product
protection by minimising the chance of cross-contamination along
the work surface of the cabinet (Figure 4). Because cabinet
air has passed through the exhaust HEPA filter, it is contaminant-free
and may be circulated back into the laboratory (Type A BSC)
or ducted out of the building (Type B BSC).
If
a Class II BSC is preferred for tuberculosis bacteriology, the
?thimble system? should be used in stead of an extract fan,
as indicated by Figure 5. The thimble allows air to be extracted
continuously from the room and from the safety cabinet when
it is in use.
Placement
of the BSC
The
ideal location for the BSC is remote from the entry to the laboratory
(eg. the rear of the laboratory away from traffic) since people
walking parallel to the BSC can disrupt the air curtain. This
curtain is quite fragile, amounting to a nominal inward and
downward velocity of 1 mile per hour (1.609 kilometer per hour).
Open windows or laboratory equipment that create air movement
(eg. centrifuges) should not be located near the BSC.
Centrifuge
Centrifuges
are essential in laboratories where tubercle bacilli are cultured.
Methods involving the use of a centrifuge are more efficient
than simple decontamination and culture of sputum directly onto
medium.
The
recommended centrifuge for use in tuberculosis culture laboratories
is a floor model with a lid and fixed angle rotor which contains
sealed centrifuge buckets. Because of its great mass in relation
to that of the centrifuge tubes, the fixed angle rotor permits
centrifugation of tubes with small weight differences without
causing vibration and possible tube breakage. For maximum safety,
sealed centrifuge buckets should be fitted. These are usually
made of stainless steel and are fitted with rubber buffers.
They are paired and their weight engraved on them. Sealed buckets
are always used in pairs, opposite one another and it is convenient
to paint each pair with different colour patches to facilitate
recognition. If the buckets fit in the centrifuge head on trunnions,
these are also paired.
Centrifuges
should preferably be fitted with an electrically operated safety
catch which prevents the lid from being opened while the rotor
is spinning.
Many
reports and manuals describing sputum processing in tuberculosis
laboratories record centrifuge speeds in revolutions per minute
(rpm). However, revolutions per minute is a measure of speed
for a particular centrifuge head and not a measure of sedimenting
efficiency or relative centrifugal force (RCF). The amount of
artificial gravity created by the spinning of a centrifuge is
determined by the rate of spin (revolutions per minute) and
by the distance from the centre of the spinning head to an outer
point where the force is to be measured. This relative centrifugal
force can be increased by either increasing the rate of spin
or the distance from the centre and is expressed in multiples
of g, eg. 3000 x g. The RCF may be calculated from the following
formula:
RCF
= 1.12Rmax (rpm/1000)2
where
Rmax = radius (mm) from the center of the rotating
head to the bottom of the spinning centrifuge tube
The required rpm to generate a desired RCF may be calculated
as follows:
rpm = 1000
If the RCF is not high enough,
many mycobacteria will remain in suspension following centrifugation
and will be poured off with the discarded supernatant. A 95%
sedimenting efficiency should be attained for optimal isolation
of mycobacteria. This requires a RCF of 3 000 x g. Many of the
old centrifuges still used in tuberculosis laboratories commonly
spin at 2 300 to 3 000 rpm (only 1 500-2 000 RCF); most users
of such equipment spin digested specimens for 15 minutes, thereby
achieving sedimenting efficiencies ranging from 75%-84% and
lower.
Depending
on the type of rotor in a non-refrigerated centrifuge and the
number of runs, the temperature in the specimen tube may increase
by 4EC to 18EC. Tubes spun in a streamlined angle head are least
affected by temperature rise even after several runs, but the
contents of tubes in unprotected horizontal rotors may exceed
40EC if the centrifuge has been used for five or more successive
runs. Even when centrifuge time remains consistent at 15 minutes,
the percentage of organisms killed increase from 13% to 22%
to 30% as the temperature rises from 20EC to 30EC to 40EC. It
is, therefore, important to keep the spinning time low (15 minutes)
and the RCF high (3 000 x g) to achieve 95% sedimentation. The
use of angle head rotors minimises heat build-up due to air
friction.
Glass
tubes may break under the stress of centrifugation. If a centrifuge
tube breaks, the liquid will splash or be blown out and aerosolised.
Screw top centrifuge tubes should therefore be used for potentially
infectious material.
The centrifuge head must
be in balance while spinning. An out-of-balance head vibrates
and may break. If a tube is added to one side of the head, an
equivalent weight must be added to the opposite side. Tubes
used in processing specimens will usually be balanced if matched
pairs have matched levels of liquid in them. As an added safety
precaution, matched tubes should contain 70% ethanol rather
than water, which may limit the risk of infection should breakage
occur.
Do
not touch any centrifuge head while it is spinning. Touching
it may not only cause injury, it may also cause rapid or erratic
stops which stir and resuspend the sediment. Some centrifuges
are equipped with a brake to gradually slow the spinning head.
35E-37EC
incubator
Cultures
are incubated at 35E-37EC for eight weeks. Incubators are available
in various sizes. In general, it is best to obtain the largest
possible model that can be accommodated and afforded. Small
incubators suffer wide fluctuations in temperature when the
doors are opened. Ensure a proper circulation of air by avoiding
overloading and by using perforated trays. Maintain a constant
temperature by not opening the incubator door unnecessarily.
Although
incubators rarely develop faults, it is advisable, before choosing
one, to ascertain that service facilities are available. The
electrical circuits are not complex but require expert technical
knowledge to repair. Transporting incubators back to the manufacturers
is most inconvenient.
In
a laboratory with a large volume of cultures it is of great
advantage to incubate them in an incubator room. A hot room
or walk-in incubator is not difficult to adapt from a small
room or corner of a large room: Windows must be blocked up.
The walls need two layers of building paper on which is glued
48mm-thick slabs of expanded polystyrene or cork (more expensive)
between battens at 600mm centres. The inner lining can be ordinary
plaster or insulation board. The ceiling must be lagged in the
same way and in rooms lower false ceilings are preferable. The
floor can be covered with insulation board and hardboard and
the doors lagged in their inner surfaces in the same way, and
fitted in their jambs on sponge rubber draught-prevention strips.
Two
methods of heating are possible. Tubular heaters around the
walls are satisfactory and a power of 3kW is more than adequate
for a room of 5-7m3 to be maintained at 37EC. A large
circulating fan to avoid hot and cold spots must be fitted on
one wall and should operate constantly. An alternative arrangement
is the greenhouse-or space-heater in which a 2.5-3.0 kW heater
and a fan are built into a steel casing. The wiring must be
altered so that the fan is always on and the heater connected
to sensitive thermostats. (The thermostats build into ordinary
greenhouse- or space heaters are too coarse for this purpose).
Two thermostats are recommended, one normally operational at
37EC and the other set to turn off heating at around 39EC as
a precaution against failure of the first thermostat and consequent
over-heating of cultures. The two methods of heating may be
combined. Smaller tubular heaters, permanently switched on,
will supply background heat and the space heater will maintain
the required temperature.
Wooden
shelving and racks are undesirable. If a high humidity is maintained
fungi may grow on the wood. Steel or aluminium racks are preferable
and can be custom-made. Shelves should be free, ie. removed
easily for cleaning and there should be space between the shelves
and the walls to allow for circulation of air.
Inspissator
In
the preparation of slopes of egg-based medium the amount of
heating required to coagulate the protein must be carefully
controlled. A steamer heats the medium too rapidly and raises
the temperature too high.
Inspissators
for the preparation of egg-based culture medium should be able
to reach and maintain a constant temperature of 80E-85EC for
45 minutes. Modern inspissators are thermostatically controlled
and fitted with a large internal circulating fan. Inside shelves
on which the tubes are sloped should be made of wire mesh so
that circulation is not impeded. It is convenient to have wire
mesh or aluminium racks made which hold tubes or bottles at
the correct angle (5E-10E) and which slide onto the shelf brackets.
These facilitate rapid loading and unloading while the inspissator
is hot.
A
glass door that seals off the inside of the oven will contribute
to maintaining the required temperature and is recommended.It
is also recommended that the required temperature be raised
first before batches of media are loaded for inspissation.
Autoclave
Tubercle
bacilli are more readily killed by moist heat (saturated steam)
than by dry heat. Steam kills tubercle bacilli by denaturing
their protein. Air has an important influence on the efficiency
of steam sterilisation because its presence changes the pressure-temperature
relationship. For example, the temperature of saturated steam
at 15 lb/in2 is 121EC,
provided that all of the air is first removed from the vessel.
With only half of the air removed the temperature of the resulting
air-steam mixture at the same pressure is only 112EC.
In addition, the presence of air in mixed loads will prevent
penetration by steam.
All
of the air that surrounds and permeates the load must first
be removed before steam sterilisation can commence. Materials
to be sterilised should therefore be packed loosely. Contaminated
material (eg. discarded cultures) should be in solid bottomed
containers not more than 20cm deep. Large air spaces should
be left around each container and none should be covered.
Only
autoclaves designed for laboratory work and capable of dealing
with a mixed load should be used. ?Porous load? and ?bottled
fluid sterilisers? are not satisfactory for laboratory work.
Two varieties of laboratory autoclaves are suitable:
- pressure
cooker types
- gravity
displacement models with automatic air and condensable discharge
Pressure cooker autoclaves
The
most common type is a device for boiling water under pressure.
It has a vertical metal chamber with a strong metal lid which
can be fastened down and sealed with a rubber gasket. An air
and steam discharge tap, pressure gauge and safety valve are
fitted in the lid. Water in the bottom of the autoclave is heated
by external gas burners, an electric immersion heater or a steam
coil.
Figure
6 illustrates a typical pressure cooker autoclave.
Autoclaves
with air discharge by gravity displacement
These autoclaves are usually
arranged horizontally and are rectangular in shape, thus making
the chamber more convenient for loading. A palette and trolley
system can be used. Figure 7 shows in diagrammatic form a jacketed
gravity displacement type of autoclave. Similar autoclaves can
be constructed without jackets. The door should have a safety
device to ensure that it cannot be opened while the chamber
is under pressure.
The
jacket surrounding the autoclave consists of an outer wall enclosing
a narrow space around the chamber, which is filled with steam
under pressure to keep the chamber wall warm. The steam enters
the jacket from the mains supply, which is at high pressure,
through a valve that reduces this pressure to the working level.
The working pressure is measured on a separate pressure gauge
fitted to the jacket. This jacket also has a separate drain
for air and condensate to pass through.
The
steam enters the chamber from the same source which supplies
steam to the jacket. It is introduced in such a way that it
is deflected upwards and fills the chamber from the top downwards,
thus forcing the air and condensate to flow out of the drain
at the base of the chamber by gravity displacement. The drain
is fitted with strainers to prevent blockage by debris. The
drain is usually fitted with a thermometer for registering the
temperature of the issuing steam. The temperature recorded by
the drain thermometer is often lower than that in the chamber.
A ?near-to-steam? trap is also fitted.
The
automatic steam trap or 'near-to-steam' trap is designed to
ensure that only saturated steam is retained inside the chamber,
and that air and condensate, which are at a lower temperature
than saturated steam, are automatically discharged. It is called
a 'near-to-steam' trap because it opens if the temperature fall
to about 2EC below that of saturated steam and closes within
2EC to the saturated steam temperature.
The
trap operates by the expansion and contraction of a metal bellows,
which opens and closes a valve. The drain discharges into a
tundish in such a way that there is a complete airbreak between
the drain and the dish. This ensures that no contaminated water
can flow back from the waste-pipe into the chamber.
Water
bath
The contents of a test tube
placed in a water bath are raised to the required temperature
much more rapidly than in an incubator. Water baths are therefore
useful for short term incubation required, for example, in some
biochemical tests.
Modern
water baths are equipped with electrical stirrers and in some
the heater, thermometer and stirrer are in one unit, easily
detached from the batch for servicing. Water baths must also
be lagged so as to prevent heat loss through the walls. A bath
that has not been lagged by the manufacturers can be insulated
with slabs of expanded polystyrene.
Water
baths should be fitted with lids in order to prevent heat loss
and evaporation. These lids must slope so that condensation
water does not drip on the contents. To avoid chalky deposits
on tubes and internal surfaces only distilled water should be
used.
Bunsen
burners
For
material that may spatter or that is highly infectious a hooded
Bunsen burner should be used. Electric burners are also available.
These are tubular micro-incinerators in which the loop or wire
is inserted, and is recommended for use in BSCs.
Glassware
and plastics
Soda-glass
or pyrex are satisfactory for tuberculosis culture and the use
of more expensive resistance glass is not justified. New unwashed
soda-glass should be soaked in hydrochloric acid overnight to
partially neutralise the alkali content of the glass.
Culture
bottles
Several
sizes of culture bottles are useful for tuberculosis bacteriology.
The most useful sizes are the small McCartney (14ml), the standard
McCartney (28ml) and the Universal container (28ml), which has
a larger neck than the others and is also used as a specimen
container. These bottles usually have aluminium screw caps with
rubber liners. The liners should be made of black rubber; some
red rubbers are thought to give off bactericidal substances.
Test
tubes
Rimless
test tubes of heavy quality are most suitable for tuberculosis
bacteriology. Thin glass, lipped chemical tubes should not be
used. The most frequently used sizes of test tubes are 152x16mm,
holding 5-10ml and 152x19mm, holding 10-15ml.
Cotton
wool plugs have been used for many years to stopper test tubes
but have largely been replaced by metal caps. Aluminium caps
(Cap-O-Test) closures are recommended. They have a wide tolerance
and fit most tubes, being held in place with a small spring.
These caps are inexpensive, last a long time, are available
in many colours and save a great deal of time and labour.
Temporary
closures for bottles, flasks and tubes can be made from kitchen
aluminium foil.
Pasteur
pipettes
Pasteur
pipettes are probably the most dangerous pieces of laboratory
equipment in unskilled hands. Safer pasteur pipettes with integral
teats and made of low density polypropylene (rather than glass)
are available and are supplied pre-sterilised.
Pasteur pipettes are used
once only.
Graduated
pipettes
Straight
side blow out pipettes, 1-10ml capacity are often used. They
must be plugged with non-absorbent cotton wool at the suction
end to prevent bacteria from entering from the teat and contaminating
the material in the pipette. These plugs must be tight enough
to stay in place during pipetting but not so tight that they
cannot be removed during cleaning. About 25mm of non-absorbent
cotton wool is pushed into the end with a piece of wire. The
ends are then passed through a Bunsen flame to tidy them. (Wisps
of cotton wool which get between the glass and the teat may
permit air to enter and the contents to leak).
Rubber
teats
Rubber
teats provide a safe alternative to the highly dangerous practice
of mouth pipetting. Teats with a capacity greater than that
of the pipettes for which they are intended should be used,
eg. a 1ml teat for pasteur pipettes, a 2ml teat for a 1ml pipette
etc. (otherwise the teat must be fully compressed, which is
tiring). Most novice laboratory workers compress the teat completely,
then suck up the liquid and try to hold it at the mark while
transferring it. This is unsatisfactory and leads to spilling
and inaccuracy. Compress the teat just enough to suck the liquid
a little way pass the mark of the pipette. Withdraw the pipette
from the liquid, press the teat lightly to bring the fluid to
the mark and then release it. The correct volume is now held
in the pipette without tiring the thumb and without risking
loss. To discharge the pipette, press the teat slowly and gently
and then release it in the same way. Violent operation usually
fails to eject all the liquid; bubbles are sucked back and aerosols
are formed.
Inoculating
loops and wires
These are usually made of
25 SWG Nicrome wire. They should be short (not more than 15cm
long) in order to minimise vibration and therefore involuntary
discharge of contents. Loops should be small (not more than
5mm in diameter). Large loops are inclined to empty spontaneously
and scatter infected airborne particles. Loops should be completely
closed. This can be achieved by twisting the end of the wire
round the shank, or by taking a piece of wire 15cm long, bending
the centre round a nail or rod of appropriate diameter and twisting
the ends together in a drill chuck.
Loops
and wires should not be fused into glass rods. Aluminium holders
are available from most laboratory suppliers.
Disposable
loops are excellent, albeit more expensive.
Racks
and baskets
Test
tube and culture bottle racks should preferably be made of polypropylene
or nylon so that they can be autoclaved. This also minimises
breakage, which is not uncommon when metal racks are used. Wooden
racks are unhygienic.
Traditional wire baskets
are unsafe for holding test tubes. They contribute to breakage
hazards and do not retain spilled fluids. Autoclavable plastic
boxes of various sizes are safer for use with cultures.
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