Module 2
Safety precautions for AFB culture
and DST of Mycobacterium tuberculosis
Purpose
To provide you with an understanding of safe handling
techniques and precautions while performing AFB culture and
DST of Mycobacterium tuberculosis.
Prerequisite
modules
None
Module time
1 hour
Learning
objectives
At the end of this module, you will be able to:
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explain airborne transmission of TB;
adhere to safe practice;
explain the biosafety levels of laboratories for TB activities;
work safely in biological safety cabinets;
use adequate personal protective equipment;
use appropriate disinfectants in any circumstances;
cope with accidents in the TB laboratory;
handle chemicals safely in the laboratory.
Content outline
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How is TB transmitted?
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What are the biohazards in a TB laboratory?
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Minimum WHO recommendations for TB culture/DST
facilities
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Standard safe practices in a TB laboratory
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Personal protective equipment
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Disinfectants active on M. tuberculosis
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Safe disposal of infectious waste
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Coping with spills of infectious material
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Chemical safety
Exercises
Safety precautions for TB culture and DST
Reference
Laboratory biosafety manual, 3rd ed. Geneva, World Health
Organization, 2004 (WHO/CDS/CSR/LYO/2004.11).
HOW IS TB TRANSMITTED?
The TB bacilli are almost always transmitted by patients with active pulmonary disease. A patient
expels TB bacilli in small droplets of respiratory secretions, which quickly evaporate leaving
“droplet nuclei”, less than 5 μm in diameter. Droplet nuclei of this size, containing 1–3 bacilli, can
remain suspended in the air indefinitely and, following inhalation, are able to reach deep into the
lungs to produce infection. Larger particles do not remain airborne for as long and do not transmit
TB as efficiently.
The risk of infection depends on:
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the infectiousness of the source;
the environment (e.g. overcrowding and inadequate ventilation promote transmission of droplet
nuclei);
the duration and intensity of exposure; and
the susceptibility of the recipient.
Smear-positive patients who expel tubercle bacilli into the air, especially when they cough (also
when they sneeze, speak or sing), are the most infectious sources.
PROPER COLLECTION OF SPUTUM
Exposure to patients collecting sputum is one of the greatest hazards to a laboratory technician – if
the patient is an undiagnosed smear-positive case, he or she will produce infectious aerosols
during cough.
Any coughing patient who has to come to the laboratory should be asked to cover his or her mouth
with hand or tissue.
Always collect sputum specimens outdoors, where the movement of the air will rapidly dilute
infectious droplets and UV rays will inactivate TB bacilli. NEVER collect sputum specimens in
laboratories, toilets, waiting rooms, reception rooms, or other enclosed space.
BIOHAZARDS IN THE TB LABORATORY
Infectious aerosols are produced in the TB laboratory whenever a liquid suspension containing
tubercle bacilli is handled. Biosafety measures in the laboratory are essential to protect workers
against exposure to infectious aerosols
Because of their viscosity, sputa are a minimal source of infectious aerosols. By contrast, aerosols
produced during processing (especially during centrifugation steps) of homogenized sputa and
during culture handling must be minimized and contained in a biological safety cabinet (BSC).
Laboratory workers should be educated about the symptoms of TB and provided with ready access
to free medical care if symptoms arise.
Confidential HIV counselling and testing should be offered to laboratory workers. Options for
reassignment of HIV-positive workers away from the high-risk areas should be considered.
MINIMUM WHO RECOMMENDATIONS FOR TB CULTURE / DST FACILITIES
WHO recommends that all specimen processing procedures are carried out in a laboratory built
and equipped for biosafety level 2 (BSL2). Minimum requirements for BSL2 are that there is a BSC
in the laboratory and an autoclave or other means of decontamination available in the same
building.
Identification and DST should be performed in a BSL2 containment room with a directional airflow
from functionally clean to dirty areas, with 6 - 12 air exchanges per hour.
The containment room may consist of the blind end of a corridor, or be formed by constructing a
partition and door so that access to the laboratory is through an anteroom (e.g. double-door entry)
or through the basic BSL2 laboratory. The autoclave should be in the vicinity of the laboratory so
that the movement of contaminated materials is minimized.
BSCs have to be ducted to the outside or vented through a thimble. Recirculation from BSCs into
the laboratory room and recirculation to other areas within the building are not permitted.
SAFETY PRACTICES IN THE TB CULTURE AND DST LABORATORY
Good microbiological techniques (GMT) – working methods designed to eliminate or minimize
exposure to pathogens via, for example, aerosols, splashes, accidental inoculation – are essential
for minimizing biohazards. GMT are fundamental to laboratory safety: specialized equipment is an
aid to , NOT a replacement for, GMT.
The following precautions are essential to protect both you and all other laboratory personnel.
Standard practices
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Access to the laboratory must be limited.
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Assume that all specimens are potentially infectious.
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Never smoke, eat, or drink in the laboratory.
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Wash hands with soap and water before and after performing any procedures.
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No mouth pipetting, no sucking pencils or pens.
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Do not perform work for which you are not trained.
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Always follow safety procedures.
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Perform all procedures carefully, following GMT, to minimize the creation of splashes or
aerosols.
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Decontaminate work surfaces and equipment at least once a day and after any spill of viable
material.
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Decontaminate all cultures, stocks, and other regulated wastes before disposal by autoclaving.
Materials to be decontaminated outside the immediate laboratory must be placed in durable,
leakproof containers, closed for transport from the laboratory. Containers must be packaged in
accordance with applicable local, state, and federal regulations before removal from the facility.
Work in a BSC
The BSC is the most important safety equipment in a TB laboratory performing culture and DST.
A Class I BSC is an open-fronted, ventilated cabinet with a unidirectional inward airflow away from
the operator. Exhaust air is hard-ducted through HEPA filters which remove particles of diameter
0.3 µm and above with an efficiency of at least 99.95%. The Class I BSC provides protection for
the worker and the environment but does not protect products (specimens, strain cultures) against
contamination.
A Class II BSC additionally provides protection against contamination of the product (specimens,
strain cultures) by recirculating part of the HEPA-filtered air in a laminar vertical flow inside the
cabinet so that a “curtain” of clean air descends across the whole working surface. Class II BSCs
should be vented to the outside through a thimble or with hard ducting.
Switch on the BSC 15 minutes before use to implement the air flow. Assemble all items needed for
work in the BSC to avoid frequent displacements, and organize the work to minimize arm
movements.
Do not overcrowd the working area as this disturbs the air flow. Conduct all manipulations within
the BSC as far as possible towards the back of the unit. In Class II BSCs, work in the BSC away
from the grille; never allow the grille to be covered by anything.
Do not use large open flames in the BSC: they create turbulence which disrupts the pattern of air
supplied to the work surface. Special Bunsen burners for use in BSCs are recommended.
The BSC should be switched off 15 minutes after the end of work to eliminate all infectious
aerosols.
PERSONAL PROTECTIVE EQUIPMENT
The decision of use of additional PPE should be based on risk assessment (e.g.. workload,
prevalence of tuberculosis, prevalence of multi-drug resistant TB, rate of failure/relapse). Risk
assessment should be reviewed routinely and revised when necessary.
Masks and respirators
One of the commonest misconceptions is that a standard surgical mask will protect the wearer
from becoming infected with TB. Surgical masks are made from porous material that will not trap
droplets nuclei, and they fit poorly, leaving large gaps between the face and mask. They are
designed to prevent the spread of microorganisms from the wearer to others by capturing the large
wet particles in the exhaled air. They are not designed to protect the wearer from inhaling small
infectious aerosols.
Although BSC and air flow in the laboratory are the main means of protection against exposure to
contaminated aerosols generated during culture and DST activities, additional personal protection
must be risk-assesses and e.g. considered in settings where MDR-TB and/or HIV are prevalent.
Staff may be HIV-infected and highly susceptible to contaminated aerosols.
Protection against inhalation of infectious aerosols can be provided by respirators, which are
devices with the capacity to filter particles of 0.3–0.4 µm diameter and which fit closely to the face
to prevent leakage around the edges. The N95 (or FFP2) respirator is a lightweight, disposable
nose and mouth respirator; it effectively filters out more than 95% of the particles of diameter 0.3
µm and above. The FFP3 respirator removes more than 98% of such particles.
Each user should be instructed in the proper use of the respirator and informed about its limitations.
Respirators should be correctly fitted to the face to prevent leakage around the face seal. This is done
by placing the mask over the nose and mouth with the top elastic band over the crown of the head and
the bottom elastic band over the back of the neck. The metal strip covering the nose should be firmly
moulded over the bridge of the nose. Facial hair between the wearer's skin and the sealing surfaces of
the respirator will prevent a good seal.
Respirators should also be worn during emergency cleaning of spillages that involve the release of
viable organisms in the work area.
Respirators should be stored in a convenient, clean, and sanitary location and should be discarded
after 8 hours (cumulative) of use and not be kept for more than one week.
Gloves
In accordance to universal precautions, appropriate gloves should be worn for all procedures that
involve handling of body fluids. Gloves must be worn in case of hand injury/skin disease or when
the risk of exposure to blood-borne pathogens is high; specimens resulting from invasive clinical
investigation must be handled with gloves.
Gloves must be worn while handling cultures.
Gloves may give a false sense of protection. Contaminated gloves may in fact be the source of
hazards for other staff members if used to handle or operate equipment in the laboratory. Change
gloves after every session that requires their use and after every interruption of the activity. Never
wear gloves outside the laboratory.
Change gloves after every activity session that requires their use and after every interruption of the
activity. Every time hands are removed from the BSC, gloves must be pulled out (as indicated
below) and discarded in a waste container in the BSC.
Disposable latex, vinyl or nitrile gloves can be used, and the correct size (small, medium or large)
should be available for all individuals. Hypoallergenic gloves should be provided in case of allergy
to latex proteins and/or to the cornstarch used for powder.
Reusing single-use gloves is not advised, discard used gloves as contaminated material.
Following the safe removal of gloves, wash hands immediately with water and liquid soap. Proper
hand-washing with soap and adequate care in the handling of contaminated materials are critical
elements of safe laboratory practice.
Gowns
Always use a gown inside the laboratory (never outside) and change at least weekly.
Long-sleeved with narrow cuffs, back-opening gowns or overalls give better protection than
laboratory coats and are preferred in microbiology laboratories. When worn, laboratory coats
should be fully buttoned.
An area of the laboratory must be designated for storage of used and new clothing. Laboratory
gowns must be stored apart from personnel clothing. Laundering services should be provided
at/near the facility.
Consideration should be given to having extra clothing suitable for visitors, maintenance, and
emergency response personnel.
Removing personal protective equipment
Always remove in the following order:
1. Respirator/mask
2. Gown/coat/suit/overalls
3. Disposable gloves
Personal protective equipment – summary
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Surgical masks do NOT protect against TB infection. TB bacilli pass through them, and they
may give a false sense of security.
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Effective respiratory protection is provided by an N95 respirator.
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Hand-washing and careful technique are acceptable practices for most countries.
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The additional protection conferred by respirators must be assessed for each setting.
DISINFECTANTS
Phenol should be used at a concentration of 2–5%, with a contact time of 15–30 minutes,
depending on the type and volume of material to be disinfected. It is useful in soaked paper towels
to cover working surfaces: this minimizes spatter and aerosol formation in the event of spillage.
Phenol is highly irritating to the skin, eyes and mucous membranes. It is also considered to be very
toxic through oral exposure. Because of this toxicity and the odour, phenol derivatives are now
generally used in its place. Many phenolic compounds are used for the decontamination of
surfaces and some (e.g. triclosan, chloroxylenol, orthophenylphenol) are among the more
commonly used antiseptics. Commercially available solutions should be used according to the
manufacturer's instructions for "dirty or worst-possible situations".
Hypochlorite is a widely available disinfectant. 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. It should be used at concentrations of 0.1% and 0.5% (1 and 5 g/l); the lower
concentration is recommended for “clean conditions” and the higher for “dirty conditions”, with a
contact time of 15–30 minutes, depending on the type and volume of material to be disinfected.
Bleach can be used as a general-purpose disinfectant and for soaking contaminated metal-free
materials (it is highly alkaline and can be corrosive to metal).
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.
Bleach, whether stock or in working solutions, must be stored in a well-ventilated, cool, dark area.
In good storage conditions, the 50 g/l solution may last as long as 3 months; diluted solutions
should be prepared daily. Storage of stock or working solutions in open containers releases
chlorine gas, thus weakening the germicidal potential. Moreover, chlorine gas is highly toxic.
Glutaraldehyde does not require dilution but an activator (provided separately by the
manufacturer) must be added. Glutaraldehyde is usually supplied as a 2% solution; the activator is
a bicarbonate compound. The activated solution should be used within 2 weeks and discarded if
turbid. Dipsticks supplied with some products give a rough indication of the levels of active
glutaraldehyde available in solutions under use.
Glutaraldehyde is useful for decontaminating bench surfaces and glassware.
Glutaraldehyde is toxic and an irritant to skin and mucous membranes, and direct contact with it
must be avoided. It must be used in a fume-hood or in well-ventilated areas. National chemical
safety regulations must be followed.
Alcohol, usually 70% ethanol (denatured ethanol, methylated spirits) or iso-propanol, can be used
on skin, on work surfaces of laboratory benches and BSCs, and to soak small surgical instruments.
A major advantage of aqueous solutions of alcohols is that they leave no residue on treated items.
When hands become contaminated, a rinse with 70% ethanol or isopropanol followed by thorough
washing with soap and water is effective.
Mixtures of alcohol 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. The greater toxicity and
irritant capacity of such mixtures should be taken into consideration.
Alcohols are volatile and flammable and must not be used near open flames. Bottles of alcoholcontaining solutions must be clearly labelled to avoid autoclaving.
Iodophores are combinations of iodine and an inert polymer such as polyvinyl-pyrrolidone that
reduces surface tension and slowly releases the iodine. Iodophors are less irritating than iodine
and do not stain. Iodophor preparations should be used at concentrations of 3–5% and contact
time should be 15–30 minutes, depending on the type and volume of material to be disinfected.
The action of these disinfectants is similar to that of chlorine, although they may be slightly less
inhibited by organic matter. Iodophors are useful for mopping up spills and for hand-washing.
SAFE DISPOSAL OF INFECTIOUS WASTE
All infected materials, including closed specimen containers, should be placed in the BSC in
autoclavable bags.
Autoclave all cultures and related materials, placing the infected materials inside autoclavable bags
and following procedures for adequate decontamination (more detailed instructions are given in
Module 3, “Use, calibration and maintenance of laboratory equipment”).
COPING WITH AN ACCIDENT
Spills in the BSC
In the event of a spill within the BSC, laboratory workers are protected from inhalation of infectious
particles as any aerosols released from the contaminated liquids are trapped by the HEPA filter in
the BSC which retains virtually all of the aerosolized tubercle bacilli.
Spilled infectious substances should be covered with absorbent tissue. Disinfectant should then be
poured liberally over the tissue and left for the appropriate time (at least 2 hours). The absorbent
tissue and any broken material should then be cleared away and placed in a contaminated-waste
container.
Allow at least 2 hours for decontamination!
Spills outside the BSC
If spills occur outside the BSC, EVACUATE the laboratory. Stay outside the room with the door
closed for at least 30 minutes to let aerosols settle down. Place the “DO NOT ENTER” warning
sign on the door of the laboratory.
Notify the laboratory supervisor of the accident. Give as much information as possible on the
location, nature and extent of the accident, as well as any possible contamination of equipment.
Using appropriate respiratory protection devices, return to the accident area to clean the spill.
After the appropriate time (at least 2 hours), prepare to re-enter the room where the spill occurred
to disinfect the contaminated area. Assemble a clean-up team of at least two persons. Put on
protective clothing – gown, thick rubber gloves for protection from broken glass, overshoes,
goggles and a single-use disposable N95/FFP2 respirator. Re-enter the contaminated area and
assess the extent of contamination.
Spilled infectious substances and broken containers contaminated with infectious substances
should be covered with absorbent paper towel. Disinfectant should then be poured liberally over
the tissue. If walls have been splashed, disinfectant solution should be sprayed or poured out onto
the contaminated surfaces which are then covered with a layer of absorbent tissue.
Allow adequate time for disinfection.
Allow at least 2 hours for decontamination!
Once disinfection is completed, discard all waste into suitable waste containers and
autoclave.
CHEMICAL SAFETY
AFB microscopy requires the use of several hazardous chemicals, including concentrated acids,
alcohols and phenol. Take the following precautions when working with chemicals in the TB
microscopy laboratory:
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Take particular care in diluting concentrated acids. ALWAYS ADD THE CONCENTRATED
ACID TO WATER. This avoids splashes of acid which will burn the skin or eyes.
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Do not use alcohols near an open flame – they are flammable.
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Phenol is a corrosive chemical. Avoid direct contact with the skin or mucous membranes.
Reduce exposure to phenolic fumes by staining smears in a well-ventilated area and by limiting
the number of slides in each staining batch to a maximum of 12.
Table1 Summary of biosafety level requirements, from Laboratory biosafety manual, 3rd ed,
WHO, 2004
KEY MESSAGES
 TB bacilli are almost always transmitted by patients with active pulmonary
disease.
 Never smoke, eat, or drink in the laboratory.
 Wash your hands frequently with soap and water, and always before and
after performing any procedures.
 Avoid hazards that may occur in a TB laboratory by paying careful attention
to safety procedures.
 ALWAYS work carefully and in a safe manner following good microbiological
technique.
 The additional protection conferred by respirators must be assessed for
each setting.
Module 2: Review
Find out how much you have learned by answering these questions.
How is TB transmitted from person to person?
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What are the main biohazards in a TB laboratory?
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What precautions must be taken when collecting sputum specimens?
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What is the main protective equipment in laboratory for culture and DST activities?
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What precautions must you take when working in a BSC?
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What procedures must you follow when coping with biohazards occurring outside a
BSC ?
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What precautions must you take when handling acids?
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Do surgical masks confer protection against laboratory-acquired TB?
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