Procedure - World Health Organization

Procedure - World Health Organization
WHO/TB/98.258
WHO/TB/98.258
Original: English
Distr.: General
LABORATORY SERVICES
IN TUBERCULOSIS CONTROL
CULTURE
PART III
Writing committee:
ISABEL NARVAIZ DE KANTOR
SANG JAE KIM
THOMAS FRIEDEN
ADALBERT LASZLO
FABIO LUELMO
PIERRE-YVES NORVAL
HANS RIEDER
PEDRO VALENZUELA
KARIN WEYER
On a draft document prepared by:
KARIN WEYER
For the Global Tuberculosis Programme,
World Health Organization,
Geneva, Switzerland
World Health Organization
1998
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
CONTENTS
Preface ........................................................................................................................................................7
1. Introduction........................................................................................................................................9
2. Laboratory layout and equipment .....................................................................................11
Plan of a culture laboratory ........................................................................................................11
Arranging equipment and materials .......................................................................................13
Care and maintenance of essential equipment ..................................................................14
3. Specimen collection .....................................................................................................................25
Containers ...........................................................................................................................................25
Collection procedures ...................................................................................................................25
Sputum specimens ...........................................................................................................................25
Other specimens...............................................................................................................................26
4. Specimen storage and transport ..........................................................................................29
5. Specimen handling.......................................................................................................................31
Receipt of incoming specimens................................................................................................31
Safe handling of specimens ........................................................................................................31
6. Homogenisation and decontamination ............................................................................37
Digestion and decontamination procedures ........................................................................38
Sputum specimens ...........................................................................................................................38
Other specimens...............................................................................................................................42
7. Culture media .................................................................................................................................47
Advantages and disadvantages of egg-based media .......................................................47
Precautions during media preparation ...................................................................................48
Preparation of egg-based media ...............................................................................................48
Löwenstein-Jensen medium ........................................................................................................48
Ogawa medium.................................................................................................................................50
Acid-buffered Ogawa medium ...................................................................................................52
8. Inoculation and incubation procedures...........................................................................55
Inoculation procedures .................................................................................................................55
Incubation of cultures....................................................................................................................55
9. Culture examination and identification ..........................................................................57
Examination schedule ...................................................................................................................57
Reading of cultures.........................................................................................................................57
Differentiation of M. tuberculosis ...........................................................................................58
Niacin test ...........................................................................................................................................59
Nitrate reduction test .....................................................................................................................64
Catalase test .......................................................................................................................................71
Growth on medium containing p-Nitrobenzoic acid (PNB) .......................................74
3
CONTENTS
10. Recording and reporting of laboratory results ........................................................77
11. Quality control.............................................................................................................................79
12. Selected references ....................................................................................................................85
ANNEXES
Annex 1
Essential equipment and supplies for a culture laboratory
using modified petroff decontamination and Löwenstein-Jensen
culture medium (6 000 specimens per year) ................................................87
Annex 2
Sputum collection procedure ..............................................................................90
Annex 3
Laboratory request form .......................................................................................91
Annex 4
Culture report forms ...............................................................................................92
4a. Preliminary report ............................................................................................92
4b. Final report ..........................................................................................................93
Annex 5
Laboratory register ..................................................................................................94
Annex 6
Unit conversion factors..........................................................................................95
FIGURES
Figure 1
Plan of a containment laboratory for tuberculosis culture
Figure 2
Arranging equipment and materials in the biological safety
cabinet to ensure safe and logical flow of work .........................................14
Figure 3
Class I biosafety cabinet .......................................................................................15
Figure 4
Class II biosafety cabinet .....................................................................................16
Figure 5
Thimble system recommended
for air extraction in class II biosafety cabinet .............................................16
Figure 6
Pressure cooker laboratory autoclave .............................................................20
Figure 7
Autoclave with air discharge by gravity displacement ...........................21
4
...................12
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
DIAGRAMS
Diagram 1 Sodium hydroxide (NaOH)
(Modified Petroff) decontamination method...............................................40
Diagram 2 Niacin test for identification of M. tuberculosis ........................................61
Diagram 3 Niacin paper strip test for the identification of M. tuberculosis .........63
Diagram 4 Nitrate reduction test for identification of M. tuberculosis
classical method........................................................................................................65
Diagram 5 Nitrate reduction test for identification of M. tuberculosis
crystalline reagent method ...................................................................................68
Diagram 6 Nitrate paper strip pest for confirmation of M. tuberculosis ................68
Diagram 7 Heat labile catalase test (68°C, pH 7.0)
for identification of M. tuberculosis ................................................................74
5
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
PREFACE
Within the framework of National Tuberculosis Programmes the first purpose of
bacteriological services is to detect infectious cases of pulmonary tuberculosis,
monitor treatment progress and document cure at the end of treatment by means of
microscopic examination. The second purpose of bacteriological services is to
contribute to the diagnosis of cases of pulmonary and extra-pulmonary tuberculosis.
Standardisation of the basic techniques for tuberculosis bacteriology has so many
advantages that it has become an unavoidable need. The absence of standardised
techniques complicates the activities of new laboratory services as well as the
organisation of existing laboratories into an inter-related network. Standardisation
makes it possible to obtain comparable results throughout a country; it facilitates
staff training, delegation of responsibilities and the selection of equipment,
materials and reagents to be purchased; it also facilitates the evaluation of
performance and the establishment of suitable supervision in order to increase
efficiency and reduce operational costs.
Standardised techniques and procedures are useful if they meet the needs of - and
are prepared in accordance with - prevailing epidemiological conditions and
different laboratory levels. These techniques should be simple (to obtain the widest
coverage) and should be applicable by auxiliary laboratory workers. At the same
time, their sensitivity and specificity must guarantee the reliability of results
obtained.
While tuberculosis laboratory services form an essential component of the DOTS
strategy for National Tuberculosis Programmes, it is often the most neglected
component of these programmes. Furthermore, the escalation of tuberculosis
world-wide, driven by the HIV epidemic and aggravated by the emergence of
multidrug-resistance, has resulted in renewed concern about safety and quality
assurance in tuberculosis laboratories.
The above considerations have led to the preparation of guidelines for laboratory
services for the framework of National Tuberculosis Programmes. These
guidelines are contained in a series of three manuals, two of which are focused on
the technical aspects of tuberculosis microscopy and culture and a third which
deals with laboratory management, including aspects such as laboratory safety and
proficiency testing. These manuals have been developed for use in low-and
middle-income countries with high tuberculosis prevalence and incidence rates.
Not only are they targeted to everyday laboratory use, but also for incorporation in
teaching and training of laboratory and other health care staff.
Finally, in order to adapt the functioning of bacteriological laboratories to the
needs of integrated tuberculosis control programmes, information on control
programme activities has been included. It is hoped that the series on laboratory
services will enable National Tuberculosis Programmes to draw up national
laboratory guidelines as one of their essential components.
7
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
1
INTRODUCTION
Tuberculosis is a disease of global importance. One-third of the world population
is estimated to have been infected with Mycobacterium tuberculosis and eight
million new cases of tuberculosis arise each year. The tuberculosis crisis is likely
to escalate since the human immunodeficiency (HIV) epidemic has triggered an
even greater increase in the number of tuberculosis cases. The majority of
tuberculosis patients are 15 to 45 years of age, persons in their most productive
years of life. Tuberculosis kills over two million people world-wide each year,
more than any other single infectious disease, including AIDS and malaria.
Transmission of tuberculosis is virtually entirely by droplet nucleii created through
coughing by untreated persons suffering from pulmonary tuberculosis (the most
common form) in a confined environment. Infected droplets remain airborne for a
considerable time, and may be inhaled by susceptible persons.
Pulmonary tuberculosis usually occurs in the apex of the lungs. These develop
cavities which contain large populations of tubercle bacilli that can be detected in a
sputum specimen. Pulmonary tuberculosis is suggested by persistent productive
cough for three weeks or longer, weight loss, night sweats and chest pain. The
diagnosis can only be made reliably on demonstrating the presence of tubercle
bacilli in the sputum by means of microscopy and/or culture in the laboratory.
The cornerstone of the laboratory diagnosis of tuberculosis is direct microscopic
examination of appropriately stained sputum specimens for tubercle bacilli.
Between 5 000 and 10 000 tubercle bacilli per millilitre of sputum are required for
direct microscopy to be positive and only a proportion of tuberculosis patients
harbour large enough numbers of organisms to be detected in this way. It is also
virtually impossible to distinguish different mycobacterial species by microscopy.
Patients who have positive smears carry the greatest number of tubercle bacilli, are
the most infectious and are therefore the most important patients to detect early
because they are responsible for spreading tuberculosis disease.
Sputum examination by microscopy is relatively quick, easy and inexpensive and
must be performed on cases suspected of having tuberculosis. Smear microscopy
is also used to monitor treatment progress and control programme outcome.
Examination by bacteriological culture provides the definitive diagnosis of
tuberculosis. Depending on the decontamination method and the type of culture
medium used, as few as ten viable tubercle bacilli can be detected. However, the
usual microbiological techniques of plating clinical material on selective or
differential culture media and sub-culturing to obtain pure cultures cannot be
applied to tuberculosis bacteriology. Compared to other bacteria which typically
reproduce within minutes, M. tuberculosis proliferate extremely slowly
(generation time 18-24 hours). Furthermore, growth requirements are such that it
will not grow on primary isolation on simple chemically defined media. The only
media which allow abundant growth of M. tuberculosis are egg-enriched media
containing glycerol and asparagine, and agar or liquid medium supplemented with
serum or bovine albumin.
Culture increases the number of tuberculosis cases found, often by 30-50%, and
9
INTRODUCTION
detects cases earlier, often before they become infectious. Since culture techniques
can detect few bacilli, the efficiency of diagnosing failures at the end of treatment
can be improved considerably. Culture also provides the necessary material for
drug susceptibility testing. Culture of specimens is, however, much more costly
than microscopy and requires facilities for media preparation as well as skilled
staff.
Culture should be used selectively, in the following order of priority:
Selective use of culture
1. Surveillance of tuberculosis drug resistance as an integral part of the
evaluation of control programme performance
2. Diagnosis of cases with clinical and radiological signs of pulmonary
tuberculosis where smears are repeatedly negative
3. Diagnosis of extra-pulmonary and childhood tuberculosis
4. Follow-up of tuberculosis cases who fail a standardised course of treatment
and why may be at risk of harbouring drug resistant organisms
5. Investigation of high-risk individuals who are symptomatic, eg. Laboratory
workers, health care workers looking after multidrug resistant patients
10
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
2
LABORATORY LAYOUT AND EQUIPMENT
2.1
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.
11
Extraction fan
Window
(permanently closed)
12
Biological
safety cabine
Basin
Centrifuge
Refrigerator
Main working room
Reading area
Work bench with shelves
Window (permanently closed)
Cupboard
Work bench
Microscope
Specimen flow direction
35° - 37° C incubator
Sink
Basin
Inspissator
Kitchen
Window
(permanently
closed)
Work bench
Hatch for incoming
specimens
Office area
Basin
Basin
Autoclave
Work bench
Work bench
Balance
Sink with drying racks
Cupboar
d
Cupboard
with
shelves
Cupboard
Basin
Sink
Cupboard
Media
with
preparation room
shelves
Work bench
Refrigerator
Window
(permanently closed)
Extraction fan
Extraction fan
Shelves
Window
(permanently closed)
LABORATORY LAYOUT AND EQUIPMENT
Figure 1. Plan of a containment laboratory for tuberculosis culture
Window
(permanently
Extraction fan
closed)
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
2.2
Arranging equipment and materials
Entry to the laboratory is via the office which 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
laboratory area 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.
13
LABORATORY LAYOUT AND EQUIPMENT
Figure 2. Arranging equipment and materials in the biological safety cabinet to
ensure safe and logical flow of work.
Decontamination
Solution
2.3
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.
2.3.1
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 0.3µm (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).
14
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Figure 3. Class I biological safety cabinet
Exhaust filter cover
Exhaust HEPA filter
Control panel
Work surface
Front grill of work surface
Hinged window
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 hardducted 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).
15
LABORATORY LAYOUT AND EQUIPMENT
Figure 4. Class II biological safety cabinet
Exhaust filter cover
Exhaust HEPA filter
Primary pre-filter
Main HEPA filter
Control panel
Work surface
Front grill of work surface
Hinged window
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.
Figure 5. Thimble system recommended for air extraction
in Class II biosafety cabinets.
(Adapted from: Collins CH, Lyne PM. Microbiological Methods. 5th ed. Butterworths, London, 1984)
To extraction fan
Room air
From safety cabinet fan
16
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
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.
2.3.2
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
RCF
1.12 Rmax
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
17
LABORATORY LAYOUT AND EQUIPMENT
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 4°C to 18°C. 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
40°C 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 20°C to
30°C to 40°C. 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.
2.3.3
35°-37°C incubator
Cultures are incubated at 35°-37°C 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
18
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
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 37°C. 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 37°C and the other set to turn off
heating at around 39°C 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.
2.3.4
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 80°-85°C 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 (5°-10°)
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.
19
LABORATORY LAYOUT AND EQUIPMENT
2.3.5
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 121°C, 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 112°C. 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.
Figure 6. Pressure cooker laboratory autoclave
(Adapted from: Collins CH, Lyne PM. Microbiological Methods. 5th ed. Butterworths, London, 1984)
Pressure guage
Safety valve
Air/steam discharge valve
Lid
Chamber
Trivet
Water
Heater
20
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
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.
Figure 7. Autoclave with air discharge by gravity displacement
(Adapted from: Collins CH, Lyne PM. Microbiological Methods. 5th ed. Butterworths, London, 1984)
Pressure gauge
Jacket
Safety valve
Combined
pressure and
vacuum gauge
Safety valve
Valve
Cotton
wool filter
To jacket
Main
Valve
steam
supply To chamber
Door
Valve
To vacuum pump
or steam ejector
Strainers
Near-to-steam
traps
Thermometer
Valve
Non-return
valve
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.
21
LABORATORY LAYOUT AND EQUIPMENT
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 2°C
below that of saturated steam and closes within 2°C 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.
2.3.6
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.
2.3.7
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 microincinerators in which the loop or wire is inserted, and is recommended for use in
BSCs.
2.3.8
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.
22
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
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 1015ml.
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 presterilised.
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.
23
LABORATORY LAYOUT AND EQUIPMENT
•
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.
24
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
3
SPECIMEN COLLECTION
The presence of acid-fast bacilli in a clinical specimen may be confirmed either by
microscopy or by culture. However, since individual mycobacterial species cannot
be identified by smear examination, the definitive diagnosis of tuberculosis can
only be made if M. tuberculosis is isolated from the clinical specimen.
In tuberculosis bacteriology attention tends to be focused on the problems of
microscopy, culture and identification systems, while an often overlooked problem
is that of obtaining adequate specimens. The advantages of subtle decontamination
techniques, sensitive culture media and simple identification schemes will not be
fully realised unless specimens are collected with the utmost care and promptly
transported to the laboratory.
3.1
Containers
An essential prerequisite for the safe collection of a satisfactory specimen is a
robust, leakproof and clean container. Containers must be rigid to avoid crushing
in transit and must possess a water-tight wide-mouthed screw top to prevent
leakage and contamination.
To facilitate the choice of a container the following specifications are
recommended:
•
Wide-mouthed (at least 35mm in diameter) so that the patient can expectorate
easily inside the container without contaminating the outside
•
•
Volume capacity of 50ml
•
•
•
Made of translucent material in order to observe specimen volume and quality
without opening the container
Made of single-use combustible material to facilitate disposal
Screw-capped to obtain an airtight seal and to reduce the risk of leakage
during transport
Easily-labelled walls that will allow permanent identification
An alternative container is the 28ml Universal bottle, which is a heavy glass,
screw-capped bottle with a wide neck. This container is reusable after thorough
cleaning and sterilisation in boiling water for at least 30 minutes.
3.2
Collection procedures
3.2.1
Sputum specimens
Although M. tuberculosis is capable of causing disease in almost any organ of the
body, more than 85% of tuberculosis disease in high prevalence countries is
pulmonary. Therefore, sputum is the specimen of choice in the investigation of
tuberculosis and should always be collected. If extra-pulmonary disease is
25
SPECIMEN COLLECTION
suspected, sputum should be collected in addition to any extra-pulmonary
specimens.
A good sputum specimen consists of recently-discharged material from the
bronchial tree, with minimum amounts of oral or nasal material. Satisfactory
quality implies the presence of mucoid or mucopurulent material and is of greater
significance than volume. Ideally, a sputum specimen should have a volume of 35ml, although smaller quantities are acceptable if the quality is satisfactory.
Collecting a good sputum specimen requires that the patient be given clear
instructions. Aerosols containing tubercle bacilli may be formed when the patient
produces a sputum specimen. Patients should, therefore, produce specimens either
outside in the open air or away from other people and not in confined spaces such
as toilets.
In some countries, patients may present first to the laboratory for diagnosis. It is
therefore appropriate that laboratory staff know the correct way of collecting
sputum specimens. This procedure is described in Annex 2. It is best to obtain
sputum early in the morning before the patient has eaten or taken medication
(which may interfere with the growth of tubercle bacilli). If sputum specimens are
collected for diagnostic purposes, tuberculosis chemotherapy should not be started
until the specimens have been collected.
Because of the increased sensitivity of culture, a single good-quality sputum
specimen may suffice. Some patients shed mycobacteria irregularly and in small
numbers; for these patients the chance of obtaining a positive culture result will be
improved if more specimens are cultured.
Specimens should be transported to the laboratory as soon as possible after
collection. If a delay is unavoidable the specimens should be refrigerated to inhibit
the growth of unwanted micro-organisms.
3.2.2
Other specimens
If a patient has a productive cough, obtaining a sputum specimen is a fairly
straightforward procedure. However, if a patient finds it difficult to produce
sputum, other methods may be used to obtain pulmonary secretions for diagnosis.
Collection techniques fall outside the scope of this document and will not be
discussed. However, induced sputum resemble saliva and it is important that these
specimens be marked “induced” in order not to be discarded as unsuitable.
Because M. tuberculosis may infect almost any organ in the body, the laboratory
should expect to receive a variety of extra-pulmonary specimens, eg. body fluids,
tissues, pus and urine. These specimens may be divided into two groups, namely:
•
•
aseptically collected specimens, usually free from other micro-organisms
specimens known to contain contaminating normal flora or specimens not
collected aseptically
26
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Aseptically collected fluids
Body fluids (spinal, pleural, pericardial, synovial, ascitic, blood, pus, bonemarrow) should be aseptically collected in a sterile container by the physician
using aspiration techniques or surgical procedures. For fluids that may clot, sterile
potassium oxalate (0.01-0.02ml of 10% neutral oxalate per ml fluid) or heparin
(0.2mg per ml) should be added. Specimens should be transported to the
laboratory as quickly as possible.
Aseptically collected tissues
Aseptically collected tissue specimens should be placed in sterile containers
without fixatives or preservatives. If the specimen is to be sent by mail it should be
protected from drying by adding sterile saline and packing the container in dry ice
or maintaining a temperature of 4-15°C. Specimens should be transported to the
laboratory as quickly as possible.
Specimens expected to be contaminated
Urine is the most commonly encountered extra-pulmonary specimen that requires
processing before culture. To minimise excessive contamination of urine
specimens the external genitalia should be washed before the specimens are
collected and the urine should be immediately processed or refrigerated. Three
early morning, voided midstream specimen should be collected.
27
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
4
SPECIMEN STORAGE AND TRANSPORT
For successful culture of specimens the time between specimen collection and the
culturing process should be kept to a minimum. Specimens should therefore be
dispatched with the least possible delay. If sputum specimens can be kept
refrigerated they could be sent to the laboratory once a week; extra-pulmonary
specimens, however, should be submitted as soon as possible after collection.
If specimens have to be transported at ambient temperatures, chemical
preservation may be used. Three methods provide reasonable results, viz:
•
Mixing the fresh specimen with an equal volume of 1% cetyl pyridinium
chloride in 2% sodium chloride. Tubercle bacilli will survive for up to a week,
while the growth of unwanted organisms will be restricted
•
Mixing the fresh specimen with anhydrous sodium carbonate in the
proportion of 50mg reagent to 2ml specimen
•
If the delay before cultural examination is to be less than 24 hours the
specimens may be mixed with an equal volume of 23% trisodium phosphate
However, none of the abovementioned preservation methods is optimal and speedy
transportation is essential for good results.
Requirements and recommendations for the safe transport of pathological
specimens are given in various national and international codes of practice and
guidelines. In addition, the postal and transport authorities of most countries as
well as the International Air Transport Association (IATA) have regulations about
conveying such materials.
As a general rule, diagnostic specimens must be packaged to withstand leakage of
contents, shocks, pressure changes and other conditions incident to ordinary
handling practices. Pathological material intended for postal or air transport should
be in approved, robust, leak-proof primary containers which are packed into
secondary containers made of metal, wood or strong cardboard with enough
absorbent material so that if they are damaged or leak the fluids will be absorbed.
For sending material across international or state boundaries this container may
have to be packed in the same way in an outer container and special administrative
arrangements with the postal authorities and airlines may be necessary.
Sputum specimens comprise the majority of specimens submitted to tuberculosis
culture laboratories and special transport boxes of metal or wood should be
provided. They should be made to hold between 20 and 30 specimen containers
packed vertically to avoid leaking. The lid should be securely fastened and the box
should preferably contain a locking mechanism. During transport it must be kept
as cool as possible and protected from sunlight.
Request forms should be located separately from specimen containers. With each
transport box an accompanying list must be prepared which identifies the
specimens and the patients from whom the specimens were collected. Before
29
SPECIMEN STORAGE AND TRANSPORT
dispatch from the health centre the following must be verified:
•
that the number of specimen containers in the box corresponds to that on the
accompanying list
•
that the identification number on each specimen container corresponds to the
identification number on the accompanying list
•
•
that the accompanying list contains the necessary data for each patient
that the date of dispatch and the particulars of the health centre are on the
accompanying list
A model laboratory request form is presented in Annex 3.
30
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
5
SPECIMEN HANDLING
5.1
Receipt of incoming specimens
Specimens should be received in the office area of the laboratory, preferably at a
separate specimen delivery counter. Delivery boxes should be opened in the
biosafety cabinet and the following procedures applied:
•
•
Wear disposable gloves during receipt and inspection of incoming specimens
•
Disinfect the outside of the delivery box using cotton wool or paper towels
saturated with a suitable disinfectant (eg. 5% phenol)
•
Open the delivery box carefully and check for cracked or broken specimen
containers. Autoclave or burn these without processing and request another
specimen
•
Check that specimens have been adequately labelled with individual
identification numbers and that these correspond with the numbers on the
accompanying list
•
Disinfect the inside of the delivery box, discard gloves and wash hands after
handling specimen containers (see part I, page 30) .
Inspect the delivery box for signs of leakage. If mass leakage is evident
discard the box by autoclaving or burning
5.2
Safe handling of specimens
5.2.1
Working within a BSC
•
Because of the increased risks of aerosol production during culture
procedures, all manipulations should be carried out within the BSC. The
cabinets are intended to protect the worker from airborne infection. They will
not, however, protect him/her from spillage and the consequences of poor
techniques
•
BSCs are designed to be operated 24 hours per day and in order to maintain
room air balance they should not be switched off. If electrical power has been
interrupted or the cabinet has been switched off (eg. following replacement of
filters), the cabinet blower should be operated for a least five minutes before
work is started. The work surface, interior walls and interior window surface
should be wiped with an appropriate disinfectant (eg. methylated spirits, see
part I, page 30). This should be followed by a second wiping with sterile water
•
Prepare a written checklist of materials necessary for tuberculosis culture.
This will minimise the number of arm-movement disruptions across the
fragile air barrier of the BSC, which may disrupt the air curtain and
compromise the partial barrier
•
Place only the materials and equipment required for immediate work in the
BSC and store extra supplies (eg. additional culture media) outside the
cabinet. Materials and equipment placed inside the BSC may cause
31
SPECIMEN HANDLING
disruption to the airflow resulting in turbulence, cross-contamination or
breach of containment
•
Allow a delay of 60 seconds after placing hands/arms inside the cabinet,
before manipulation of materials. This allows the BSC to stabilise and to
remove surface microbial contaminants
•
Ensure that the front grille is not blocked with laboratory notes, discarded
plastic wrappers, pipetting devices, etc
•
Perform all operations at least four inches from the front grille on the work
surface. Raise arms slightly to allow room air to be drawn through the front grille
•
Place absorbent paper towelling on the work surface (but not on the front or
rear grille openings). This will facilitate routine cleanup and will reduce
splatter and aerosol formation during an overt spill. Towelling can then be
folded and placed in an autoclavable bag when work is completed
•
Place all materials and aerosol-generating equipment (eg. vortex mixers) as
far back in the cabinet as practical, towards the rear edge of the work surface
and away from the front grille
•
Place bulky items such as autoclavable bags, pipette trays and collection
flasks to one side in the cabinet
•
Arrange materials and equipment to allow work to flow from a clean to a
contaminated area across the work surface. Place materials and supplies in
such a way as to limit the movement of dirty items over clean ones
•
Avoid the following common practices which may interfere with the
operation of the BSC:
• taping autoclavable disposal bags to the outside of the cabinet
• placing pipette collection containers upright in the BSC or on the floor
outside the cabinet
The frequent inward/outward movement needed to place objects in these
containers disrupts the integrity of the cabinet air barrier and can compromise
both staff and product protection
•
Use only horizontal pipette discard trays containing an appropriate
disinfectant (eg. 5% phenol, see part I, page 30)
•
Use proper microbiological techniques to avoid splatter and aerosols. This
will minimise the potential for staff exposure to infectious materials
manipulated within the cabinet. As a general rule, keeping clean materials at
least 12cm away from aerosol-generating activities will minimise the
potential for cross-contamination
•
Do not hold opened tubes or bottles in a vertical position and recap or cover
them as soon as possible. This will reduce the chance for cross-contamination
•
Do not use large open flames in the BSC. This creates turbulence which
disrupts the pattern of air supplied to the work surface. Special Bunsen
burners for use in BSC’s are recommended
32
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
5.2.2
5.2.3
•
Use an appropriate liquid disinfectant (eg. 5% phenol, see part I, page 30) in a
discard pan to decontaminate materials before removal from the BSC.
Introduce items into the pan with the minimal splatter and allow sufficient
contact time before removal. Alternatively, contaminated items may be placed
into an autoclavable disposal bag within the BSC. Water should be added to the
bag prior to autoclaving to ensure steam generation during the autoclave cycle
•
Surface-decontaminate all containers and equipment before removal from the BSC
(see part I, page 30)
•
At the end of the work day, surface-decontaminate the work surface of the
BSC, the sides and back and the interior of the glass window
•
Handle small spills within the BSC immediately by removing the contaminated
absorbent paper towelling and placing it into the autoclavable disposable bag.
Wipe any splatter onto items within the cabinet or on its interior immediately
with a paper towel saturated with a disinfectant solution (eg. 5% phenol)
•
Spills large enough to result in liquids flowing through the front or rear grilles
require more extensive decontamination. Surface-decontaminate and remove
all items from the BSC. Ensure that the drain valve is closed and pour
appropriate disinfectant (eg. 5% phenol) onto the work surface and through
the grille(s) into the drain pan. Allow at least 30 minutes for
decontamination. Empty the drain pan into a collection vessel containing
disinfectant by attaching a flexible tube to the drain valve with the open end
submerged in the disinfectant within the collection vessel. After
decontamination, flush the drain pan with water and remove the drain tube
•
Always decontaminate the BSC before HEPA filters are changed or internal
repair work is done. The most common decontamination method uses
formaldehyde gas and is described in the Management Series
Using the centrifuge
•
Select two centrifuge tubes of identical length and thickness. Place the
specimen to be centrifuged in one tube and an equal amount of 70% ethanol
in the other. Ensure that the tubes are balanced
•
Place the tubes in paired centrifuge buckets and place the paired buckets in
diametrically opposite positions in the centrifuge head
•
Close the centrifuge lid and ensure that the speed control is at zero before
switching on the current. (Many centrifuges are fitted with a “no volt” release
to prevent the machine starting unless this is done)
•
Move the speed control slowly until the speed indicator shows the required
rpm or g
Precautions
•
Make sure that the rubber buffers are in the buckets, otherwise tubes will
break
33
SPECIMEN HANDLING
5.2.4
•
Check the balancing carefully. Improperly balanced tubes will cause “head
wobble”, spin-off accidents and wear out bearings
•
Check that the balanced tubes are opposite one another in multi-bucket
centrifuges
•
•
•
Never start or stop the centrifuge with a jerk
Open sealed centrifuge buckets in the BSC
Using a pressure cooker autoclave
•
•
5.2.5
Observe the manufacturer instruction about the speed limits for various loads
There must be sufficient water inside the chamber
The autoclave is loaded and the lid is fastened down with the discharge tap
open. The safety valve is then adjusted to the required temperature and the
heat is turned on
•
When the water boils, the steam will issue from the discharge tap and carry
the air from the chamber with it. The steam and air should be allowed to
escape freely until all of the air has been removed. This may be tested by
attaching one end of a length of rubber tubing to the discharge tap and
inserting the other end into a bucket or similar large container of water.
Steam condenses in the water and the air rises as bubbles to the surface.
When all of the air has been removed from the chamber, bubbling in the
bucket will cease. When this stage has been reached, the air-steam discharge
tap is closed and the rubber tubing removed. The steam pressure then rises in
the chamber until the desired pressure, usually 15lb/in2, is reached and steam
issues from the safety valve
•
When the load has reached the required temperature, the pressure is held for
30 minutes
•
At the end of the sterilising period, the heater is turned off and the autoclave
allowed to cool
•
The air and steam discharge tap is opened very slowly after the pressure
gauge has reached zero (atmospheric pressure). If the tap is opened too soon,
while the autoclave is still under pressure, any fluid inside will boil
explosively and bottles containing liquids may even burst
•
The contents are allowed to cool. Depending on the nature of the materials being
sterilised, the cooling (or “run-down”) period needed may be several hours
Using an autoclave with air discharge by gravity displacement
•
If the autoclave is jacketed, the jacket must first be brought to the operating
temperature
•
The chamber is loaded, the door is closed and the steam-valve is opened,
allowing steam to enter the top of the chamber. Air and condense flow out
through the drain at the bottom
34
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
When the drain thermometer reaches the required temperature a further
period must be allowed for the load to reach that temperature. This should be
determined initially and periodically for each autoclave. Unless this is done
the load is unlikely to be sterilised
•
The autoclave cycle is then continued for the holding time. When it is
completed the steam valves are closed and the autoclave allowed to cool until
the temperature dial reads less than 80°C. Not until then is the autoclave safe
to open
•
The autoclave door should first be “cracked” or opened very slightly and left
in that position for several minutes to allow steam to escape and the load to
cool further
Serious accidents, including burns and scalds to the face and hands have
occurred when autoclaves have been opened, even when the temperature
gauge reads below 80°C and the doors have been “cracked”.
Liquids in bottles may still be over 100°C and under considerable pressure.
The bottles may explode on contact with air at room temperature.
When autoclaves are being unloaded operators should
wear full-face visors of the kind that cover the chin and throat.
They should also wear thermal-protective gloves.
35
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
6
HOMOGENISATION AND DECONTAMINATION
The usual microbiological techniques of plating clinical material on selective or
differential culture media and subculturing to obtain pure cultures cannot be
applied to tuberculosis bacteriology. M. tuberculosis requires special media not
used for other organisms and grows slowly, taking three to six weeks or longer to
give visible colonies. Cultures are usually made in bottles rather than in petri
dishes because tubercle bacilli are present in relatively small numbers in most
specimens; this necessitates large inocula which are spread out over the surface of
the media. Because of the long incubation time required, the bottles are tightly
stoppered to prevent drying of the cultures (which would occur in petri dishes).
The majority of clinical specimens submitted to the tuberculosis culture laboratory
are contaminated to varying degrees by more rapidly growing normal flora
organisms. These would rapidly overgrow the entire surface of the medium and
digest it before the tubercle bacilli start to grow. Most specimens must, therefore,
be subjected to a harsh digestion and decontamination procedure that liquefies the
organic debris and eliminates the unwanted normal flora.
All currently available digesting/decontaminating agents are to some extent toxic
to tubercle bacilli; therefore, to ensure the survival of the maximum number of
bacilli in the specimen, the digestion/decontamination procedure must be precisely
followed. In order for enough tubercle bacilli to survive to give a confirmatory
diagnosis, it is inevitable that a proportion of cultures will be contaminated by
other organisms. As a general rule, a contamination rate of 2%-3% is acceptable in
laboratories that receive fresh specimens; if specimens (especially sputum) take
several days to reach the laboratory then losses due to contamination may be as
high as 5%-10%. It is also important to note that a laboratory which experiences
no contamination is probably using a method that kills too many of the tubercle
bacilli.
When culturing tubercle bacilli, three important aspects should be borne in mind:
•
Specimens must be homogenised to free the bacilli from the mucus, cells or
tissue in which they may be embedded. The milder this homogenisation the
better the results
•
Neither homogenisation nor decontamination should unnecessarily diminish
the viability of tubercle bacilli
•
The success of homogenisation and decontamination depends on:
•
the greater resistance of tubercle bacilli to strongly alkaline or acidic
digesting solutions
• the length of exposure time to these agents
• the temperature build-up in the specimen during centrifugation
• the efficiency of the centrifuge used to sediment the tubercle bacilli
Many different methods of homogenisation and decontamination of sputum
specimens for culturing have been described but there is no universally recognised
37
HOMOGENISATION AND DECONTAMINATION
best technique. The choice of a suitable method is to a large extent determined by
the technical capability and the availability of staff in a laboratory, as well as the
quality and type of equipment available. Each method has its limitations and
advantages and it is recommended that regional/central laboratories standardise on
one method only. Methods which consistently yield the highest percentage of
positive cultures are those which require:
•
•
•
well trained staff
relatively expensive equipment (eg. centrifuges) and related supplies
continued maintenance of equipment and of good staff performance
Any method which require the use of a centrifuge present some problems which
must be considered:
•
The centrifuge must be fast enough to attain a relative centrifugal force (RCF)
of 3 000 x g. If the RCF is not high enough, many tubercle bacilli remain in
suspension following centrifugation and are poured off with the discarded
supernatant fluid. Recent studies have shown that 3 000 x g for 15 minutes
would sediment 95% of mycobacteria in a digested sputum specimen. The
specific gravity of tubercle bacilli ranges from 1.07 to 0.79, making
centrifugal concentration of specimens ineffective if the RCF is not 3 000 x g
•
Precautions must be taken to minimise the potential for staff infection in the
event of tube breakage during centrifugation. These include:
•
using a floor model centrifuge with lid and a fixed angle rotor. The mass of
the fixed angle rotor permits centrifugation of tubes with small weight
differences without causing vibration and possible tube breakage
•
always ensuring that tubes in the centrifuge are balanced. The weight of
centrifuge tubes can be balanced by adding sterile saline to specimens or by
inserting tubes with sterile water or 70% ethanol among the tubes
containing sputum (using 70% ethanol in stead of water in the tubes used as
balances will reduce the risk in the event of breakage)
•
using aerosol-free safety cups if available
•
enclosing the centrifuge in a specially ventilated cabinet if possible
6.1
Digestion and decontamination procedures
6.1.1
Sputum specimens
Sputum specimens should not be pooled because of the risk of crosscontamination. Since the exposure time to digestants/decontaminants has to be
strictly controlled it is best to work in sets equivalent to one centrifuge load (eg.
eight specimens at a time).
Always digest/decontaminate the whole specimen, ie. do not attempt to select
portions of the specimen as is done for direct microscopy. If the sputum will pour,
it should be gently decanted from the specimen container into the centrifuge tube.
38
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
If the specimen is too viscuous to pour, an equal volume of
digestant/decontaminant could be added to the sputum in the specimen container
and the mixture poured carefully into the appropriate centrifuge tube.
Since sputum specimens are the most common clinical specimens submitted for
tuberculosis culture, homogenisation and decontamination procedures have been
largely targeted towards their processing. Specimens other than sputum demand
even more care during processing because of the low numbers of tubercle bacilli
present in positive specimens.
6.2
Sodium hydroxide (Modified Petroff) method
This method is used widely in developing countries because of its relative
simplicity and the fact that the reagents are easy to obtain.
,NaOH is toxic, both for contaminants and for tubercle bacilli; therefore, strict
adherence to the indicated timing is required
•
Reagents
4% sodium hydroxide (NaOH) solution
Sodium hydroxide pellets (analytical grade)
Distilled water
4g
100ml
Dissolve NaOH in distilled water and sterilise by autoclaving
at 121°C for 15 minutes.
Sterile saline
Sodium chloride pellets (analytical grade)
Distilled water
0.85g
100ml
Dissolve NaCI in distilled water and sterilise by autoclaving
at 121°C for 15 minutes.
An alternative method for preparing 4% NaOH is as follows:
Add the contents of a 250g bottle NaOH pellets to 500ml distilled water and fill
water to the 625ml mark. Be careful since heat is released in this reaction.
When needed, prepare a fresh 4% NaOH solution by adding 40% NaOH to
sterile distilled water in the proportion 1:10.
•
Procedure
Refer to Diagram 1 on page 40.
39
HOMOGENISATION AND DECONTAMINATION
Diagram 1. Sodium hydroxide (NaOH) (Modified Petroff) method
PROCEDURE
To xml of sputum,
add 2xml of 4% NaOH
➜
Tighten cap of container
and shake to digest
➜
Let stand for 15 minutes
at room temperature with
occasional shaking
➜
2xml - 4% NaOH
Centrifuge at
3 000 x g for 15 minutes
➜
xml Sputum
Pour off supernatant
➜
Add 15ml sterile saline or distilled water and resuspend sediment
➜
Centrifuge at 3 000 x g for 15 minutes
➜
Decant supernatant and inoculate onto culture medium immediately
6.2.1
Advantages
•
The NaOH method is simple and inexpensive and provides fairly effective
control of contaminants
•
The time needed to process a single specimen is approximately one hour; 20
specimens would take approximately two hours, with centrifuge capacity
being the limiting factor
•
Sterilised NaOH solution will keep for several weeks. Store in plastic bottles
40
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
6.2.2
Limitations
•
The specimen exposure times must be strictly followed to prevent over-kill of
tubercle bacilli
•
The NaOH procedure is very robust and may kill up to 60% of tubercle bacilli
in clinical specimens. This initial kill is independent of additional
contributory factors such as heat build-up in the centrifuge and centrifugal
efficiency
A variety of more expensive and labour intensive homogenisation and
decontamination methods1 are available to countries with the required financial
and human resources. Some of these will be discussed briefly.
OPTION
N-ACETYL-L-CYSTEINE-SODIUM HYDROXIDE
(NALC-NaOH) METHOD
The mucolytic agent NALC (used for rapid digestion of sputum) enables
the decontaminating agent (NaOH) to be used at a lower final concentration
of 1%. Sodium citrate is included in the digestant mixture to bind the heavy
metal ions which may be present in the specimen and could inactivate the
acetyl-cysteine.
1
•
Properly performed, this method provides more positive cultures than
other methods, resulting in the killing of approximately 30% of tubercle
bacilli
•
The time needed to process a single specimen is approximately 40
minutes; 20 specimens would take approximately 60 minutes
•
Acetyl-cysteine loses activity rapidly in solution, so the digestant should
be made fresh daily
•
The indicated specimen exposure time must be strictly adhered to and a
1:10 dilution of resuspended sediment must be made to decrease the
concentration of any toxic components that may inhibit growth of tubercle
bacilli
•
Reagents such as bovine albumin and the required filters are expensive
Kent PT, Kubica GP. Public health mycobacteriology: Guide for the Level III Laboratory. US
Department of Health and Human Services, Centres for Disease Control, USA, 1985.
41
HOMOGENISATION AND DECONTAMINATION
OPTION
ZEPHIRAN-TRISODIUM PHOSPHATE (Z-TSP) METHOD
The use of trisodium phosphate and Zephiran (benzalkonium chloride) to
homogenise and decontaminate specimens results in a more gentle
digestion procedure.
•
The procedure need not be as critically timed as NaOH digestion
procedures
•
•
The method results in the killing of approximately 30% of tubercle bacilli
The time required for one specimen is nearly two hours; 20 specimens
would require four hours
Excessive contamination is sometimes encountered in clinical material from
certain patients, from certain areas or at certain times, and may present a difficult
problem. For these problem specimens alternative decontamination
methods such
1
as 5% oxalic acid or 4% sulphuric acid may be used.
OPTIONS
OXALIC ACID METHOD
This method is often helpful for specimens consistently contaminated with
Pseudomonas species.
SULPHURIC ACID METHOD
This method is sometimes helpful for urine and other thin watery body
fluids that consistently yield contaminated cultures when processed with
one of the alkaline digestants.
6.2.3
Other specimens
Gastric lavage
These specimens should be processed within four hours of collection since their
acidity is damaging to tubercle bacilli. Usually, gastric lavage does not need to be
decontaminated, provided it has been collected aseptically in a sterile container.
Centrifuge the total volume at 3 000 x g for 30 minutes. If contamination is suspected
the sediment should be mixed with 2ml of 4% sulphuric acid and allowed to stand for
15 minutes, after which 15ml sterile saline is added. Centrifuge this mixture at 3 000
x g for 15 minutes and neutralise the sediment with 4% NaOH containing a phenol
red indicator. Inoculate the sediment immediately onto culture medium.
1
Kent PT, Kubica GP. Public health mycobacteriology: Guide for the Level III Laboratory. US Department
of Health and Human Services, Centres for Disease Control, USA, 1985.
42
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Urine
Centrifuge the total volume at 3 000 x g for 15 minutes. Discard the supernatant
fluid and add 2ml of 4% sulphuric acid to the sediment. Let stand for 15 minutes,
add 15ml sterile saline and centrifuge at 3 000 x g for 15 minutes. Neutralise the
sediment with 4% NaOH containing a phenol red indicator. Inoculate the sediment
immediately onto culture medium.
Laryngeal swabs
Cover the swab (in its original tube) with 5% oxalic acid and allow to act for 15
minutes. Remove the swab to another tube containing sterile saline. Lift after a few
minutes, allow to drain and use to inoculate culture media.
For optimal results the oxalic acid (which might contain tubercle bacilli washed
off from the swab) should be transferred to a centrifuge tube and centrifuged at
3 000 x g for 15 minutes. Wash the sediment once with sterile saline, centrifuge at
3 000 x g for 15 minutes and inoculate immediately onto culture medium.
Tissue
Lymph nodes, biopsies and other surgically resected tissue should be cut into small
pieces with a sterile scalpel or scissors. Homogenise the specimen in a sterile
porcelain mortar or tissue grinder, using 0.5-1ml sterile saline and a small quantity
of sterilised sand (if necessary in mortar). This suspension can be directly
inoculated onto culture media if the sterility measurements described before have
been met; if not, decontaminate using 4% sulphuric acid as described for urine.
Mortars, pestles and tissue grinders must be cleaned and sterilised thoroughly to
prevent false positive results or contamination due to organisms left over from
previous specimens
Pus
This may be treated in the same way as aspirated fluids. If the material is very
thick, it should be treated in the same way as sputum.
Cerebrospinal fluid
Cerebrospinal fluid should be concentrated by membrane filtration, by high speed
centrifugation or by precipitation methods. Precipitation can be achieved by
adding 0.1ml sterile rabbit serum or an equivalent albumin solution for every 10ml
of cerebrospinal fluid. Mix until uniformly cloudy, centrifuge at 3 000 x g for 15
minutes and culture the sediment if contamination is not suspected.
If no sediment can be obtained by centrifugation, a sterile 20% solution of sulphosalicylic acid may be added drop by drop until turbidity sets in. This precipitate is
then more easily spun down to form a sediment.
43
HOMOGENISATION AND DECONTAMINATION
If contamination of cerebrospinal fluid is likely, the sediment is mixed with 2ml of
4% sulphuric acid and allowed to stand for 15 minutes. Add 15 ml of sterile saline
and centrifuge at 3 000 x g for 15 minutes. Inoculate sediment onto culture media.
Clots
In the case of specimens that form large clots, eg. pleural and ascitic fluids, it is
recommended that clot formation be avoided by the addition of sodium citrate at
the time of specimen collection. Add two drops of 20% sodium citrate for every
10ml fluid collected.
When clots are present, they can be digested with Petroff’s NaOH method after
homogenisation as described for tissue.
Other body fluids (including pleural fluid)
Mucopurulent fluid : Treat as for sputum when volume is 10ml or less.
Clear fluid
: If collected aseptically centrifuge at 3 000 x g for 15
minutes and inoculate sediment directly onto culture
media. If volume is more than 10ml treat as for gastric lavage.
Tubercle bacilli may adhere to glass or plastic surfaces. To optimise recovery,
containers could be rinsed with sterile saline. Centrifuge the saline at 3 000 x g for
15 minutes and inoculate 2-3 drops onto culture media.
Specimens differ greatly in their degree of contamination and decontaminants
should be selected to suit the nature of the specimens. The need for
decontamination is also determined by the freshness of the specimen and by the
efficiency of refrigeration before processing.
The following specimens usually do not need decontamination when aseptically
collected into sterile containers:
•
•
•
•
•
Spinal, sinovial or other internal body fluids
Bone marrow
Pus from cold abscesses
Surgically resected specimens (excluding autopsy material)
Material obtained from pleural, liver and lymph nodes as well as biopsies (if
not fistulised)
Whenever doubt exists about the contamination of specimens, an untreated portion
may be inoculated onto nonselective bacteriological media, eg. nutrient agar, and
incubated for 24 hours to check for the presence of fast-growing nonmycobacterial
organisms. The remaining portion of the specimen is kept untreated and
refrigerated until the absence of contaminants is confirmed. Should this not be the
case the remaining specimen can then be appropriately decontaminated.
44
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Culture of Mycobacterium tuberculosis on Löwenstein-Jensen medium. Organisms show typical
cream-coloured, buff and rough colonies against the green egg-based medium.
(Courtesy of the South Africa Institute for Medical Research, Jahannesburg, South Africa).
Niacin production test to differentiate Mycobaterium tuberculosis from other mycobacterial
species, with the formation of a yellow colour ondicating a positive reaction
(Courtesy of the National Tuberculosis Research Programme of the Medical Research Council,
Pretoria, South Africa).
45
HOMOGENISATION AND DECONTAMINATION
Fluorescent microscopy of a culture of Mycobaterium tuberculosis showing typical cord formation
(Courtesy of the National Tuberculosis Research Programme of the Medical Research Council,
Pretoria, South Africa).
Nitrate reduction test to differentiate Mycobaterium tuberculosis from other mycobacterial species,
with the formation of an intense purple colour indicating a positive reaction.
46
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
7
CULTURE MEDIA
The definitive diagnosis of tuberculosis demands that M. tuberculosis be recovered
on culture media and identified using differential in vitro tests. Many different
media have been devised for cultivating tubercle bacilli and three main groups can
be identified, viz egg-based media, agar-based media and liquid media.
The ideal medium for isolation of tubercle bacilli should (a) be economical and
simple to prepare from readily available ingredients, (b) inhibit the growth of
contaminants, (c) support luxuriant growth of small numbers of bacilli and (d)
permit preliminary differentiation of isolates on the basis of colony morphology.
For the culture of sputum specimens, egg-based media should be the first choice,
since they meet all these requirements. There is increasing evidence that liquid
media may give better results with other specimens. While cost prevents their
routine use with sputum specimens, it is recommended that both egg-based and
liquid medium be used for non-repeatable specimens, eg. cerebrospinal fluid and
biopsy material.
It is recommended that all sputum specimens submitted for culture also undergo
microscopic examination as outlined in the Technical Series on Microscopy.
7.1
Advantages and disadvantages of egg-based media
7.1.1
Advantages
7.1.2
•
•
it is easy to prepare
•
it may be stored in the refrigerator for several weeks provided it was made
from fresh eggs and culture bottle caps are tightly closed to minimise drying
by evaporation
•
contamination during preparation is limited because it is inspissated after
being placed in bottles. In addition, the malachite green added to the media
suppresses the growth of nonmycobacterial organisms
it is the least expensive of all media available and supports good growth of
tubercle bacilli
Disadvantages
•
it may take as long as eight weeks before cultures become positive, especially
if specimens contain few bacilli or if decontamination procedures have been
overly harsh
•
when contamination does occur, it often involves the total surface of the
medium and the culture is usually lost
47
CULTURE MEDIA
7.2
Precautions during media preparation
For media of the best quality, chemicals of certified purity, clean glassware and
freshly distilled and sterilised water should be used. Directions for preparing
media must be followed precisely and without modification. A few general points
to obtain good quality media and avoid contamination of reagents and media are as
follows:
7.3
•
Keep the environment as clean as possible. Swab the work surface with a
suitable disinfectant (eg. 5% methylated spirits) before dispensing sterile
reagents and media. Clean the floor with a wet mop to limit dust
•
•
•
•
Use sterile glassware and equipment
Use reagent grade chemicals and reagents unless otherwise specified
Check the temperature of inspissators and hot air ovens
Follow strict aseptic techniques when preparing media, eg. flaming flasks and
tubes
•
•
•
When preparing egg-based media, carefully clean egg shells before breaking
•
Do not skimp on the volume of medium. Place 6-8ml of egg medium in each
bottle or 20ml into each test tube
Do not overheat medium during inspissation
Do not leave prepared media exposed to light (including ultra-violet light),
but store in the refrigerator in the dark when not in use
Preparation of egg-based media
1 LÖWENSTEIN-JENSEN MEDIUM
Löwenstein-Jensen (LJ) medium is most widely used for tuberculosis culture. The
modification of the International Union Against Tuberculosis and Lung Disease
(IUATLD) is recommended and will be described in detail. LJ medium containing
glycerol favours the growth of M. tuberculosis while LJ medium without glycerol
but containing pyruvate encourages the growth of M. bovis. Both should be used in
countries or regions where patients may be infected with either organism.
•
Ingredients
Mineral salt solution
Potassium dihydrogen phosphate anhydrous (KH2PO4)
Magnesium sulphate (MgSO4. 7H2O)
Magnesium citrate
Asparagine
Glycerol (reagent grade)
Distilled water
2.4g
0.24g
0.6g
3.6g
12ml
600ml
Dissolve the ingredients in order in the distilled water by heating. Autoclave at
48
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
121°C for 30 minutes to sterilise. Cool to room temperature. This solution
keeps indefinitely and may be stored in suitable amounts in the refrigerator.
Malachite green solution, 2%
Malachite green dye
Sterile distilled water
2.0g
100ml
Using aseptic techniques dissolve the dye in sterile distilled water by placing
the solution in the incubator for 1-2 hours. This solution will not store
indefinitely and may precipitate or change to a less-deeply coloured solution.
In either case discard and prepare a fresh solution.
Homogenised whole eggs
Fresh hens’ eggs, not more than seven days old, are cleaned by scrubbing
thoroughly with a hand brush in warm water and a plain alkaline soap. Let the
eggs soak for 30 minutes in the soap solution. Rinse eggs thoroughly in running
water and soak them in 70% ethanol for 15 minutes. Before handling the clean dry
eggs scrub the hands and wash them. Crack the eggs with a sterile knife into a
sterile flask and beat them with a sterile egg whisk or in a sterile blender.
•
Preparation of complete medium
The following ingredients are aseptically pooled in a large, sterile flask and
mixed well:
Mineral salt solution
Malachite green solution
Homogenised eggs (20-25 eggs, depending on size)
600ml
20ml
1000ml
The complete egg medium is distributed in 6-8ml volumes in sterile 14ml or
28ml McCartney bottles or in 20ml volumes in 20 x 150mm screw-capped test
tubes, and the tops are securely fastened.
Inspissate the medium within 15 minutes of distribution to prevent
sedimentation of the heavier ingredients.
•
Coagulation of medium
Before loading, heat the inspissator to 80°C to quicken the build-up of the
temperature. Place the bottles in a slanted position in the inspissator and coagulate
the medium for 45 minutes at 80°-85°C (since the medium has been prepared with
sterile precautions this heating is to solidify the medium, not to sterilise it). Heating
for a second or third time has a detrimental effect on the quality of the medium.
The quality of egg media deteriorates when coagulation is done at too high a
temperature or for too long. Discolouration of the coagulated medium may be
due to excessive temperature. The appearance of little holes or bubbles on the
surface of the medium also indicates faulty coagulation procedures.
Poor quality media should be discarded.
49
CULTURE MEDIA
•
Sterility check
After inspissation, the whole media batch or a representative sample of culture
bottles should be incubated at 35°-37°C for 24 hours as a check of sterility.
•
Storage
The LJ medium should be dated and stored in the refrigerator and can keep for
several weeks if the caps are tightly closed to prevent drying out of the
medium. For optimal isolation from specimens, LJ medium should not be older
than 4 weeks.
,
For the cultivation of M. bovis, LJ medium is enriched with 0,5% sodium
pyruvate. Glycerol is omitted and 8.0g sodium pyruvate is added to the mineral
solution.
2 OGAWA MEDIUM
This medium is cheaper than Löwenstein-Jensen because it is made without
asparagine.
•
Ingredients
Mineral salt solution
Potassium dihydrogen phosphate anhydrous (KH2PO4)
Sodium glutamate
Distilled water
3.0g
3.0g
300ml
Dissolve the ingredients in distilled water by heating. Autoclave at 121°C for
30 minutes to sterilise. Cool to room temperature. This solution keeps
indefinitely and may be stored in suitable amounts in the refrigerator.
Malachite green solution, 2%
Malachite green dye
Sterile distilled water
2.0g
100ml
Using aseptic techniques dissolve the dye in sterile distilled water by placing
the solution in the incubator for 1-2 hours. This solution will not store
indefinitely and may precipitate or change to a less-deeply coloured solution.
In either case discard and prepare a fresh solution.
Homogenised whole eggs
Fresh hens’ eggs, not more than seven days old, are cleaned by scrubbing
thoroughly with a hand brush in warm water and a plain alkaline soap. Let the
eggs soak for 30 minutes in the soap solution. Rinse eggs thoroughly in running
water and soak them in 70% ethanol for 15 minutes. Before handling the clean
dry eggs scrub the hands and wash them. Crack the eggs with a sterile knife
into a sterile flask and beat them with a sterile egg whisk or in a sterile blender.
50
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
Preparation of complete medium
The following ingredients are aseptically pooled in a large, sterile flask and
mixed well:
Mineral salt solution
Malachite green solution
Whole hens’ eggs (12-16 eggs, depending on size)
Glycerol
300ml
18ml
600ml
18ml
The resulting pH of the medium is 6.8. The medium is mixed well and
distributed in 6-8ml volumes in sterile 14ml or 28ml McCartney bottles or in
20ml volumes in 20x150mm screw-capped test tubes.
•
Coagulation of medium
Before loading, heat the inspissator to 80°C to quicken the build-up of the
temperature. Place the bottles in a slanted position in the inspissator and
coagulate the medium for 45 minutes at 80°-85°C (since the medium has been
prepared with sterile precautions this heating is to solidify the medium, not to
sterilise it). Heating for a second or third time has a detrimental effect on the
quality of the medium.
The quality of egg media deteriorates when coagulation is done at too high a
temperature or for too long. Discolouration of the coagulated medium may be
due to excessive temperature. The appearance of little holes or bubbles on the
surface of the medium also indicates faulty coagulation procedures.
Poor quality media should be discarded.
•
Sterility check
After inspissation, the whole media batch or a representative sample of culture
bottles should be incubated at 37°C for 24 hours as a check of sterility.
•
Storage
The medium should be dated and stored in the refrigerator and can keep for
several weeks if the caps are tightly closed to prevent drying out.
In laboratories where centrifuges are not available, a simple culture technique
could be employed as follows: Sputum specimens are decontaminated with
equal volumes of 4% NaOH and inoculated directly onto modified or acidbuffered Ogawa medium. This technique shows a fairly comparable case yield
when compared with concentrated culture techniques.
51
CULTURE MEDIA
3 ACID-BUFFERED OGAWA MEDIUM
•
Ingredients
Modified Ogawa
Potassium dihydrogen phosphate
Magnesium citrate (KH2PO4)
Sodium glutamate
Glycerol
Distilled water
2g
0.1g
0.5g
4ml
100ml
3g
1.0g
6ml
100ml
Homogenised whole eggs
2% Malachite green solution
200ml
4ml
200ml
6ml
6.4
6.2
Final pH
•
Acid-buffered Ogawa
Preparation
Dissolve the ingredients in the distilled water and boil for 30 minutes. Cool to
room temperature and add the homogenised eggs and malachite green solution.
Transfer 6-8ml volumes to suitable bottles and inspissate at 85°C for 45-60
minutes.
A variety of more expensive and labour intensive culture methods1 are available
to countries with the required financial and human resources. Some of these
will be discussed briefly:
OPTIONS
HERMAN KIRCHNER LIQUID MEDIUM
This medium is most useful and least expensive of the liquid media for
culture and tubercle bacilli. It has the additional advantage that it can
support a large inoculum.
DUBOS OLEIC ACID-ALBUMIN LIQUID MEDIUM
This medium is recommended for the cultivation of tubercle bacilli from
cerebrospinal, pleural and peritoneal fluid. It may be prepared from basic
ingredients or may be obtained commercially as a ready-to-use base to
which sterile albumin or serum is added.
1 Kent PT, Kubica GP. Public health mycobacteriology: Guide for the Level III Laboratory. US Department
of Health and Human Services, Centres for Disease Control, USA, 1985.
52
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
MIDDLEBROOK 7H-10 AND 7H-11 AGAR MEDIUM
Middlebrook 7H-10 may be made from basic ingredients or may be
prepared from commercially available 7H-10 agar-powdered base and
Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment.
7H-11 is a 7H-10 agar enriched by the addition of enzymatic digest of
casein. It is best to prepare 7H-10 and 7H-11 medium in small quantities of
200 to 400ml to minimise the amount of heat needed to melt the agar.
Boiling the basal medium before autoclaving (either to solubilise the agar or
to provide stocks of prepared base that may be stored and boiled for later
use) should be avoided because the repeat heating produces medium of
inferior quality.
When Middlebrook 7H-10 or 7H-11 medium is used for isolation cultures
must be incubated in an atmosphere of 10% CO2. Exposure of Middlebrook
7H-10 or 7H-11 agar to either daylight of heat results in the release of
formaldehyde in sufficient concentration to inhibit the growth of
mycobacteria.
OPTION
SELECTIVE MEDIUM
Specimens which are excessively contaminated may be inoculated onto
selective antibiotic-containing media. Use may be made of antibiotics to
which mycobacteria are not sensitive but which are capable of destroying
the contaminants, eg. penicillin (50-100 units/ml), nalidixic acid (35µg/ml)
or polymyxin (20-25µg/ml).
The antibiotics may be:
•
•
•
Added to egg medium before inspissation
Added to the surface of the medium slant or
Mixed with the inoculum
Mycobactosel medium contains several antibiotics, eg. cycloheximide
(0.4mg/ml), lincomycin (0.002mg/ml) and nalidixic acid (0.035mg/ml),
while mycobactosel agar is commercially available. Antibiotic enriched
medium should be stored in the refrigerator in the dark for a maximum of
four weeks.
53
CULTURE MEDIA
OPTION
RADIOMETRIC METHOD FOR TUBERCULOSIS CULTURE
Recent development in the diagnosis of tuberculosis include an automated
system for detecting early growth of mycobacteria by a radiometric method
(BACTEC: Beckton Dickinson). Sputum or other homogenates are
decontaminated as necessary and added to vials containing Middlebrook
7H12 medium, an antibiotic mixture (to avoid the growth of other
organisms) and 14C-labelled palmitic acid. The medium is prepared
commercially (BACTEC 12B: Beckton Dickinson) in rubber-sealed bottles
and inoculated with a syringe and hypodermic needle. If mycobacterial
growth occurs, 14C palmitic acid is utilised and 14CO2 is produced. The air
space above the medium in each bottle is sampled automatically by the
BACTEC machine at fixed intervals and the amount of radioactive gas is
estimated and recorded. Infectious aerosols are contained in the apparatus
and captured in HEPA filters before the air is exhausted.
Growth of mycobacteria may be detected within 5-7 days, but positive
results require further testing to distinguish between tubercle bacilli and
other mycobacteria. In the BACTEC machine, p-nitro-a-acetylamino-bpriophenone (NAP) is used and tubercle bacilli can be differentiated within
five days. NAP inhibits the growth of M. tuberculosis and usually does not
affect the growth of MOTT bacilli.
Comparative tests have shown that the method is very successful and
reliable and that confirmatory results for M. tuberculosis can be obtained
within two weeks. However, the BACTEC machine is very expensive to
purchase and to operate. In addition, two hazards must be considered if the
machine is to be used for routine tuberculosis bacteriology: the use of
hypodermic needles for the inoculation of media carries the risk of needlestick injury, while the culture media is radioactive and presents a problem in
terms of waste disposal.
In summary, the BACTEC method is invaluable for the detection of tubercle
bacilli in material such as cerebrospinal fluid where rapid results are crucial
in the management of the patient. However, the high cost of both the
apparatus and the radio-labelled medium prohibits its routine use in most
high tuberculosis prevalence countries.
54
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
8
INOCULATION AND INCUBATION PROCEDURES
8.1
Inoculation procedures
Condensed moisture is frequently observed at the bottom of culture medium
slants. This should be removed before inoculation is attempted.
A common fault in inoculation is the use of too small an inoculum. Either loops
(wire or disposable) or pipettes can be used for primary cultivation, although
plastic Pasteur pipettes are recommended. Each slope should be inoculated with
0.2-0.4ml (2-4 drops or 2-4 loopfuls) of the centrifuged sediment, distributed over
the surface. Fluid media can accommodate up to 1ml used for each specimen.
Two slopes of LJ medium should be inoculated per specimen. In areas where M.
bovis may be a problem, an additional slope containing pyruvate should be added.
8.2
Incubation of cultures
All cultures should be incubated at 35°-37°C until growth is observed or discarded
as negative after eight weeks.
Inoculated media should preferably be incubated in a slanted position for at least
24 hours to ensure even distribution of inoculum. Thereafter, if incubator space is
needed, bottles could be placed upright. Tops should be tightened to minimise
evaporation and drying of media.
The various Middlebrook agars require an atmosphere of 10% CO2 and 90% air to
ensure growth. CO2 is not essential to initiate growth on egg-based medium but
does stimulate earlier and more luxuriant growth. A separate CO2 incubator is not
necessary. Inlet and outlet petcocks can be attached to an airtight metal or plastic
box, built to fit on a shelf of the incubator. This box, which contains the incubating
cultures, should be flushed daily with a compressed mixture of 10% CO2 and 90%
air. Alternatively, agar plates can be placed in impermeable Mylar plastic bags and
these charged three times a week with CO2.
55
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
9
CULTURE EXAMINATION AND IDENTIFICATION
9.1
Examination schedule
All cultures should be examined 72 hours after inoculation to check that liquid has
completely evaporated, to tighten caps in order to prevent drying out of media and
to detect contaminants. Thereafter, cultures are examined weekly, or if this is not
operationally feasible, on at least three occasions, viz
•
after one week to detect rapidly growing mycobacteria which may be
mistaken for M. tuberculosis
•
after three to four weeks to detect positive cultures of M. tuberculosis as well
as other slow-growing mycobacteria which may be either harmless
saprophytes or potential pathogens
•
after eight weeks to detect very slow-growing mycobacteria, including
M. tuberculosis, before judging the culture to be negative
It is useful to label containers with cultures with the dates necessary for
examination and to place containers in the incubator in chronological order.
Should contaminated cultures be found during the examination, those where the
surface has been completely contaminated or where medium has been liquefied or
discoloured should be sterilised and discarded. Certain contaminating organisms
produce acid from constituents of the medium and the lowering of pH unbinds
some of the malachite green from the egg (indicated by the medium changing to
dark green). Tubercle bacilli will not grow under these conditions and cultures
should be discarded. Cultures with partial contamination should be retained until
the eighth week. Late contamination does not exclude the presence of
M. tuberculosis; it is therefore advisable to prepare a smear from the surface of the
medium. Should microscopy indicate the presence of acid-fast bacilli, an attempt
could be made to re-decontaminate and re-inoculate the culture.
9.2
Reading of cultures
Typical colonies of M. tuberculosis are rough, crumbly, waxy, non-pigmented
(cream coloured) and slow- growers, ie. only appearing three weeks after
inoculation.
With doubtful cultures or when less experienced staff read cultures, the acidfastness should be confirmed by Ziehl-Neelsen (ZN) staining. A very small
amount of growth is removed from the culture using a loop and gently rubbed into
one drop of sterile saline on a slide. At this point the ease with which the
organisms emulsify in the liquid should be noted: Tubercle bacilli do not form
smooth suspensions, unlike some other mycobacteria. The smear is allowed to dry,
fixed by heat and stained by the ZN method.
For preliminary identification of tubercle bacilli the following characteristics
apply:
57
CULTURE EXAMINATION AND IDENTIFICATION
9.3
•
Tubercle bacilli do not grow in primary culture in less than one week and
usually take three to four weeks to give visible growth
•
The colonies are buff coloured (never yellow) and rough, having the
appearance of bread crumbs or cauliflower
•
They do not emulsify in the saline used for making smears but give a granular
suspension
•
Microscopically they are frequently arranged in serpentine cords of varying
length or show district linear clumping. Individual cells are between 3µm and
4µm in length
Differentiation of M. tuberculosis
Although a presumptive diagnosis of tuberculosis may be made by an experienced
laboratory technologist on the basis of the characteristics of tubercle bacilli
described before, it is best to do confirmatory tests. Unfortunately there is no
completely reliable single test that will differentiate M. tuberculosis from other
mycobacteria. Nevertheless, the following tests, when used in combination with
the characteristics described before will enable the precise identification of >95%
of M. tuberculosis strains.
1 NIACIN TEST
Niacin (nicotinic acid) plays a vital role in the oxidation-reduction reactions that
occur during metabolic processes in all mycobacteria. Although all mycobacteria
produce niacin, comparative studies have shown that, because of a blocked
metabolic pathway, M. tuberculosis accumulates the largest amount of nicotinic
acid and its detection is useful for its definitive diagnosis. Niacin negative
M. tuberculosis strains are very rare, while very few other mycobacterial species
yield positive niacin tests.
Cultures grown on egg medium yield the most consistent results in the niacin test
and LJ medium is therefore recommended. A culture must be at least three to four
weeks old and must have sufficient growth of more than 50 colonies. Because
M. tuberculosis excretes niacin into the growth medium, cultures with confluent
growth may give a false-negative niacin reaction because the extracting fluid
cannot come in contact with the culture medium. When this occurs, expose the
underlying medium surface by either scraping away or puncturing through some of
the culture growth.
Aeration of cultures intended for niacin testing is very important. Caps should be
loose on slants throughout the entire incubation period and special Cap-o-Test
stoppers are recommended.
58
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
9.3.1
Niacin test with chemical reagents
Controls
Control the reagents by testing the extract from an uninoculated tube of medium
(negative control) and use an extract from a culture of M. tuberculosis H37Rv as
positive control.
•
Reagents
Aniline solution, 4%
•
Aniline is oncogenic and penetrates through the skin. Work with gloves and
be very careful
•
Aniline may change colour on exposure to air and light; prepare a fresh
solution when necessary
Fresh, clear colourless aniline
Ethanol 95%
4ml
96ml
Mix aniline with ethanol in an amber bottle and store in the dark in the
refrigerator. Discard if solution turns yellow.
Cyanogen bromide solution, 10%
•
Cyanogen bromide is a severe lacrimator and toxic if inhaled; work in a
well-ventilated fume hood when preparing the solution and in a biological
safety cabinet when testing cultures
•
Cyanogen bromide is oncogenic and penetrates through the skin. Work with
gloves and be very careful
•
In acid solution, cyanogen bromide hydrolyses to hydrocyanic acid, which
is extremely toxic. Discard all reaction tubes into a disinfectant solution
made alkaline by the addition of sodium hydroxide
Cyanogen bromide crystals
Distilled water
5g
50ml
•
Add cyanogen bromide crystals to distilled water in a glass beaker
•
Cover the beaker with foil and leave in the fume cupboard at room
temperature. The crystals take approximately 24 hours to dissolve at room
temperature
•
Do not heat the solution over a Bunsen flame
•
Pour into a tightly capped amber bottle and store in the refrigerator
•
Warm to room temperature to dissolve any precipitate formed upon cooling
59
CULTURE EXAMINATION AND IDENTIFICATION
• Prepare small amounts because cyanogen bromide is volatile and loses
strength on storage. Weak solutions give false-negative results
To avoid unnecessary prolonged exposure to the cyanogen bromide while
weighing the following procedure may be followed:
• Write down the weight of an empty beaker closed with a piece of
aluminium foil
• Remove the approximate quantity (eg. approximately 1/10 of the contents
of a 100g bottle for a 10% solution) of the white cyanogen bromide crystals
into the beaker, cover it and record the weight
• Calculate the difference between the two readings to obtain the exact
weight of crystals in the beaker
• Add the required amount of distilled water to give a final concentration of
10%
In some countries a 4% aqueous potassium cyanide solution containing
bromide is used and is prepared as follows:
•
Bromine water is highly corrosive and volatile and should be stored away
from other chemical reagents
•
KCN is very poisonous and should be handled in a fume hood
• Break an ampoule of bromine (50ml) in a 1 000ml capacity dark glass flask
(with glass stopper) containing 150ml cold distilled water
• Prepare a 4% aqueous potassium cyanide solution by dissolving 4g KCN in
100ml distilled water. The KCN must be pure and not hydrated
• With a pipette, remove 1ml of the bromine layer beneath the surface of the
bromine water and transfer it to the bottom of a 250ml Erleynmeyer flask.
Rapidly add, drop by drop, the potassium cyanide solution, shaking by
rotation until total decolorisation is obtained
•
Procedure
Refer to Diagram 2 on page 61.
60
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Diagram 2. Niacin test for identification of M. tuberculosis
PROCEDURE
Add 1 ml of sterile water to the culture slant. If growth is confluent, puncture the
medium with a Pasteur pipette to allow contact of the water with the medium
➜
Place the tube horizontally so the fluid covers the entire surface of the medium
➜
Allow 30 minutes for the extraction of niacin. The extraction time
may be longer if the culture has few colonies
➜
Raise the slants upright for 5 minutes to allow the fluid to drain to the bottom
➜
Remove 0.5ml of the fluid extract to a clean screwcap tube
➜
Sequentially add 0.5ml
of the 4% aniline solution
and 0.5ml of 10%
cyanogen bromide
➜
Close the tubes and observe the solution for the formation of a
yellow colour (= positive result) within 5 minutes. The yellow colour appears
as a ring at the interface of the two reagents, or if the tube is shaken,
as a yellow column of liquid
➜
Add 2-3 ml of 4% NaOH to each tube and discard
61
CULTURE EXAMINATION AND IDENTIFICATION
•
Results and interpretation
•
Negative :
No colour
•
Positive
Yellow colour appearing within 5 minutes. The colour
appears as a ring at the interface of the two reagents, or if
the tube is shaken, as a yellow column of liquid.
:
Niacin test with paper strips
Paper test strips for the detection of niacin are commercially available. They
compare well to the chemical reagents in detecting niacin production. A paperstrip method obviates the need to prepare and store the unstable and toxic
chemicals used to demonstrate the presence of niacin, but is much more
expensive.
•
Procedure
Refer to Diagram 3 on page 63.
•
•
Results and interpretation
•
Negative :
No colour
•
Positive
Yellow liquid in the bottom of the tube. Discard any colour
on the stip itself; this may occur because of oxidation of
chemicals, especially at the top of the strip
:
Precautions
•
Always check the expiry date of commercial test strips
•
To prevent false-negative results promptly reseal tubes after inserting
paper strip; if tubes are left unsealed the gas evolved as chemical mix on
the strip may escape into the atmosphere
62
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Diagram 3. Niacin paper strip test for the identification of M. tuberculosis
PROCEDURE
Add 1ml of sterile saline to the culture slant. If growth is confluent, puncture the
medium with a Pasteur pipette to allow contact of the saline with the medium
➜
Place the tube horizontally so the fluid covers the entire surface of the medium
➜
Allow 30 minutes for the extraction of niacin. The extraction time
may be longer if the culture has few colonies
➜
Raise the slant upright for 5 minutes to allow the fluid to drain to the bottom.
Remove 0.5ml of the fluid extract to a clean screwcap tube
➜
Insert the strip with the
identification end up
(an arrow may indicate
which end to insert first)
and immediately seal the tube.
Do not let the middle
of the strip get wet
➜
Leave at room temperature for 15-20 minutes.
Occasionally agitate the tube without tilting it
➜
Observe the colour of the liquid in the bottom of the tube against a white
background (yellow = positive). Discard any colour on the strip itself; this may
occur because of oxidation of chemicals, especially at the top of the strip
➜
Neutralise the strips with 10% sodium hydroxide
or discard them into alkaline disinfectant
63
CULTURE EXAMINATION AND IDENTIFICATION
2 NITRATE REDUCTION TEST
M. tuberculosis is one of the strongest reducers of nitrate among the mycobacteria,
which allows for this test to be used in combination with the niacin test in
differentiating M. tuberculosis from the other mycobacteria.
Cultures to be tested for nitrate reduction should be four weeks old and have
abundant growth Löwenstein Jensen egg medium is recommended.
Classical method with liquid reagents
•
Reagents
Sodium nitrate substrate in buffer
Prepare 0.01M sodium nitrate in 0.022M phosphate buffer, pH 7.0 as follows:
KH2PO4
Distilled water
3.02g
1000ml
Dissolve potassium phosphate in distilled water
to provide an 0.022M solution ...........................................................Solution 1
Na2HPO4
Distilled water
3.16g
1000ml
Dissolve sodium phosphate in distilled water
to provide an 0.022M solution ..........................................................Solution 2
Add 611ml of solution 2 to 389ml of solution 1 ‚ and mix well.
Check pH to be 7.0 ............................................................................Solution 3
Complete sodium nitrate substrate buffer
NaNO3
Solution 3
0.85g
1000ml
Dissolve the sodium nitrate in the buffer and dispense in 100ml aliquots.
Sterilise by autoclaving at 121°C for 15 minutes. When needed, aliquots of the
substrate solution are aseptically dispensed into sterile screw-capped tubes in
2ml quantities.
Hydrochloric acid solution
Concentrated HCI
Distilled water
10ml
10ml
Slowly add concentrated HCI to distilled water (never the reverse) to obtain a
1:1 dilution. Store in an amber bottle in the dark in the refrigerator.
64
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Sulfanilamide solution, 0.2%
Sulfanilamide
Distilled water
0.2g
100ml
Dissolve sulfanilamide in distilled water and store in an amber bottle in the
dark in a refrigerator.
N-naphthylethylene-diamine solution, 0.1%
N-naphthylethylene-diamine
Distilled water
0.1g
100ml
Dissolve naphthylethylene-diamine in distilled water and store in an amber
bottle in the dark in a refrigerator.
•
Controls
Control the reagents by testing the extract from an uninoculated tube of
medium (negative control) and use an extract from a culture of M. tuberculosis
H37Rv as positive control.
•
Procedure
Refer to Diagram 4 .
Diagram 4.
Nitrate reduction test for identification of M. tuberculosis classical method
PROCEDURE
Add 0.2ml of sterile saline to a screw-cap tube
➜
Use a sterile loop/spade to emulsify two loopfuls/spadefuls of a
4-week old culture in the saline
➜
Add 2ml of the NaNo3 substrate
➜
Shake well and incubate upright in a 37°C water bath for 3 hours and remove
➜
65
CULTURE EXAMINATION AND IDENTIFICATION
2 DROPS
Sulfanilamide
1 DROP
HCI
2 DROPS
N-Napthtylethylene-diamine
➜
Add in the following order:
1 drop diluted HCI
(shake tube well)
2 drops 0.2% sulfanilamide
2 drops 01.%
N-napthtylethylene-diamine
➜
Examine immediately for a pink to red colour and compare to colour standard
•
Results and interpretation
•
Negative : No colour. If no colour develops, the test is either negative or
the reduction has proceeded beyond nitrite. Add a small amount of
powdered zinc to all negative tests by tipping the end of a slightly
moistened applicator stick into dry zinc and shaking into the liquid.
a) If nitrate is still present, it will be catalysed by the zinc and a red colour
will develop, indicating a true negative
b) If no colour develops the original reaction was positive but the nitrate
was reduced beyond nitrite. Repeat the test to confirm the observation
•
Positive
:
Faint pink
Clear pink
Deep pink
Red
Deep red
Purplish red
Red colour, which vary from pink to very deep red-crimson:
=
=
=
=
=
=
+/1+
2+
3+
4+
5+
Only 3+ to 5+ is considered positive.
Method with crystalline reagent
The dry crystalline reagent is easy to prepare, has a shelf-life of a least six months
and has the added advantage that only one reagent is needed to detect nitrate rather
than the three liquid reagents used in the conventional chemical test.
66
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
Reagents
Sodium nitrate substrate in buffer
Prepare as described in classical method, page 62.
Crystalline reagent
Sulfanilic acid
N-(1-naphthyl)-etylenediamine dihydrochloride
L(+) - tartaric acid
1 part
1 part
10 parts
Put the chemicals in an amber bottle and mix by vigorous manual shaking
about 30 times. (Tartaric acid is much more crystalline than the other two
chemicals and may have to be ground using a mortar and pestle to ensure good
mixture of the reagents). The dry mixture has a heterogeneous crystalline
appearance. Store in the amber bottle at room temperature.
•
Controls
Control the reagents by testing the extract from an uninoculated tube of
medium (negative control) and use an extract from a culture of M. tuberculosis
H37Rv as positive control.
•
Procedure
Refer to Diagram 5 on page 68.
•
Results and interpretation
•
Negative : No colour. If no colour develops, the test is either negative or
the reduction has proceeded beyond nitrite. Add a small amount of
powdered zinc to all negative tests by tipping the end of a slightly
moistened applicator stick into dry zinc and shaking into the liquid.
a) If nitrate is still present, it will be catalysed by the zinc and a red colour
will develop, indicating a true negative
b) If no colour develops the original reaction was positive but the nitrate
was reduced beyond nitrite. Repeat the test to confirm the observation
•
Positive
:
Faint pink
Clear pink
Deep pink
Red
Deep red
Purplish red
Red colour, which vary from pink to very deep red-crimson:
=
=
=
=
=
=
+/1+
2+
3+
4+
5+
Only 3+ to 5+ is considered positive.
67
CULTURE EXAMINATION AND IDENTIFICATION
Diagram 5. Nitrate reduction test for identification of M. tuberculosis
Crystalline reagent method
PROCEDURE
Add 0.2ml of sterile saline to a screw-cap tube
➜
Use a sterile loop/spade to emulsify two loopfuls/spadefuls of a
4-week old culture in the saline
➜
Add 2ml of the NaNo3 substrate
➜
Shake well and incubate upright in a 37°C water bath for 3 hours and remove
➜
Use the tip of a spatula to add a small amount of crystalline reagent to the test
solution. The quantity of reagent is not critical
➜
Examine immediately for
a pink to red colour and compare
to colour standard
Nitrate test with paper strips
Paper test strips for the detection of nitrate following nitrate reduction are
commercially available. The paper strip test method yields most consistent results
with mycobacteria that vigorously reduce nitrate, such as M. tuberculosis. It
therefore provides reliable results and is much less labour-intensive than the
chemical method, but is much more expensive.
68
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
Procedure
Refer to Diagram 6 on page 69.
•
•
Results and interpretation
•
Negative :
No colour change
•
Positive
Top portion of the strip changes to light or dark blue
:
Precautions
•
Always check the expiry date of commercial test strips
•
Because the strips are sensitive to sunlight, excess heat and moisture, they
should be stored between 2°C and 8°C in the original container, tightly
capped
•
Discard strips if they become discoloured, for this indicates deterioration
of the reagent
•
Do not rely on results of test strips if the positive control culture gives
weak or negative reactions
Diagram 6. Nitrate paper strip test for confirmation of M. tuberculosis
PROCEDURE
Add 1ml sterile saline to a sterile screwcap test tube
➜
Use a sterile spade/applicator stick to emulsify in the saline
two spadefuls of growth from a four-week-old culture
➜
Use sterilised forceps
and carefully insert a nitrate test
strip (arrow indicates which
end to insert first); do not let
the strip contact any fluid
on the side of the tube
➜
69
CULTURE EXAMINATION AND IDENTIFICATION
Cap the tube tightly and incubate in a vertical position at 37°C for two hours
➜
After one hour of incubation, shake the tube gently without tilting
➜
After two hours of incubation, tilt the tube six times to wet the entire strip
➜
Allow the tube to remain slanted for 10 minutes with the liquid covering the strip
➜
Examine the top portion of the strip for changes to light or dark blue (= positive)
9.4
Nitrate reduction standards
In order to ensure consistency in interpreting nitrate reduction reactions it is
recommended that a series of standards depicting the colour intensity from +/- to
5+ be prepared. These keep indefinitely and should be used whenever nitrate tests
are done.
•
Reagents
Stock solution
1 0.067M disodium phosphate (9,47g of anydrous Na2HPO4 per 1 000ml)
2 0.067M monopotassium phosphate (9.07g of KH2PO4 per 1 000ml)
3 0.067M trisodium phosphate (25.47g of Na3PO4C12H2O per 1 000ml)
4 1% phenolphthalein (1g in 100ml 95% ethyl alcohol)
5 1% bromthymol blue (1g in 100ml 95% ethyl alcohol)
6 0.01% bromthymol blue: prepare by mixing 1.0ml of no.“ above in 100ml
of distilled water.
Working buffer solution
Mix 35ml of stock solution 1, 5ml of stock solution 2 and 100ml of solution 3.
•
Procedure
•
Place eight clean test tubes (number 1-8) in a rack. Use the same size tubes
as used to perform the nitrate reduction test.
•
Put 2ml of working buffer solution into tubes 2 through 8.
70
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
To 10ml of working buffer solution,
add 0.1ml of 4 and 0.2ml of 6 ............................................................... Solution 7
•
Add 2ml of solution 7 to the tube numbered 1. This is the 5+ colour
standard.
•
To the tube number 2 in the series, add 2ml of solution 7. Mix well and
transfer 2ml to the next tube (number 3). Continue to make serial dilutions
of 2ml, discarding 2ml from the 8th tube.
•
The colour standards:
tube 1
tube 2
tube 3
tube 5
tube 6
tube 8
•
=
=
=
=
=
=
5+
4+
3+
2+
1+
+/-
Autoclave tubes, seal and store at 5°C.
3 CATALASE TEST
Catalase is an intracellular, soluble enzyme capable of splitting hydrogen peroxide
into water and oxygen, ie. 2H2O2 ➛ 2H2O + O2. The oxygen bubbles into the
reaction mixture to indicate catalase activity. Virtually all mycobacteria passes
catalase enzymes, except for certain isoniazid-resistant mutants of M. tuberculosis
and M. bovis.
Mycobacteria posses several kinds of catalase that vary in heat stability.
Quantitative differences in catalase activity can be demonstrated by one or more of
the following tests:
•
•
•
Room temperature or drop method (indicates the presence of catalyse)
Semiquantitative test (indicates level of catalyse production)
68°C test at pH7 (indicates loss of catalyse activity due to heat)
Drug susceptible strains of M. tuberculosis do form catalyse as indicated by the
drop method, produce less than 45mm of bubbles in the semiquantitative test and
lose catalase activity when heated to 68°C for 20 minutes. For these tests 14 dayold cultures on LJ butts should be used, ie. the media tubes should be inspissated
in an upright position to provide a butt and should not be slanted. The tubes must
have stoppers which permit exchange of air, eg. Cap-o-Test stoppers. The cultures
should be incubated in a well-humidified incubator at 35°-37°C, with loose caps,
for 14 days.
71
CULTURE EXAMINATION AND IDENTIFICATION
•
Reagents
0.067M phosphate buffer solution, pH 7.0
Na2HPO4 anhydrous
Distilled water
9.47g
1000ml
Dissolve disodium phosphate in distilled water
to provide an 0.067M solution ..........................................................Solution 1
KH2PO4
Distilled water
9.07g
1000ml
Dissolve monopotassium phosphate in distilled water
to provide an 0.067M solution ...........................................................Solution 2
Hydrogen peroxide, 30%
30% hydrogen peroxide (H2O2), also known as Superoxol
(Merck) is stored in the refrigerator.
•
Ensure that the H2O2 used is 30% and not the 3% kind obtained from
pharmacies
• Wear rubber or plastic gloves and a protective eye shield when handling
superoxol
Tween 80, 10%
Tween 80
Distilled water
10ml
90ml
Mix Tween 80 with distilled water and autoclave at 121°C for 10 minutes. The
Tween may settle during autoclaving and may be resuspended by swirling
immediately after autoclaving and during cooling. Store in the refrigerator.
Complete catalase reagent (Tween-peroxide mixture)
Immediately before use, mix equal parts of 10% Tween 80 and 30% hydrogen
peroxide. Allow 0.5ml reagent for each strain to be tested.
•
Controls
• Drop method
Use an uninoculated tube of medium as negative control and an LJ butt of M.
tuberculosis H37Rv as positive control
• Semiquantitative and 68°C tests
Use an uninoculated tube of medium as negative control and an LJ butt of M.
terrae as positive control.
72
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
Procedures
•
Drop method
Examine the 14 day-old LJ slant to ascertain that growth has occurred. Add one
to two drops of the freshly-prepared Tween-peroxide mixture to the slant with
the culture growth. Observe for a period of 5 minutes for the formation of
bubbles.
Results and interpretation
Negative
:
No bubbles formed
Positive (slow)
:
Few slowly forming bubbles
Positive (rapid)
:
Immediate copious formation of bubbles
•
Semiquantitative test
Examine the 14 day-old LJ butt to ascertain that growth has occurred. Add 1ml
of the freshly-prepared Tween-peroxide mixture, replace caps loosely and
allow to stand at room temperature for 5 minutes. A column of foam will form.
Measure the height of the foam column (ie. from the top of the liquid on the LJ
medium to the top of the foam).
Results and interpretation
Low or no catalase activity
Inconclusive result
High catalase activity
•
:
:
:
Less than 31mm of foam
Between 31 and 45mm of foam
More than 45mm of foam
68°C, pH7.0 test
Refer to Diagram 7 on page 74.
Results and interpretation
•
•
Positive
Negative
: Bubbles
: No bubbles
On rare occasions, bubbles may be seen rising from the sedimental cells in
such small quantity that foam does not form at the surface of the fluid. This is
still recorded as a positive reaction.
73
CULTURE EXAMINATION AND IDENTIFICATION
Diagram 7. Heat labile catalase test (68°C, pH 7.0) for identification of
M. tuberculosis
PROCEDURE
With a sterile pipette, aseptically add 0.5ml of 0.067M
phosphate buffer, pH 7.0 to 16x125mm screw-cap tubes
➜
Suspend several loopfulls of test cultures
in the buffer solution, using sterile loops
➜
Place the tubes containing the emulsified cultures in a previously heated
water bath at 68°C for 20 minutes. Time and temperature are critical
➜
Remove the tubes from heat and allow to cool to room temperature
➜
Add 0.5ml of the freshly-prepared Tween-peroxide mixture
to each tube and replace caps loosely
➜
Observe the formation of bubbles appearing on the surface of the liquid.
Do not shake the tubes because Tween 80 also may form bubbles
when shaken, resulting in false positive results
➜
Hold negative tubes for 20 minutes before discarding
4 GROWTH ON MEDIUM CONTAINING p-NITROBENZOIC ACID (PNB)
In laboratories where facilities and reagents for niacin and nitrate testing are not
available, identification of tubercle bacilli may be done by a combination of one or
more of the catalase tests described previously together with growth at 25°C on LJ
medium and growth on LJ medium containing p-nitrobenzoic acid at 37°C.
Problems with incubation at 25°C may be encountered in tropical regions. A
refrigerated incubator should be used where available; as an alternative, a water
bath within a refrigerator or cold room should be used.
74
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
•
Procedure
•
Inoculate two slopes of LJ medium containing glycerol and one tube of LJ
medium containing p-nitrobenzoic acid (PNB) at a concentration of
500mg/litre
•
Incubate one LJ slope and the PNB slope at 37°C in an internally
illuminated incubator and examine at 3, 7, 14 and 21 days. When growth is
evident on the LJ slope examine it for pigment. If an internally illuminated
incubator is not available, remove slopes from the dark incubator as soon
as growth is evident, loosen the caps to admit some oxygen and expose
them to daylight (but not direct sunlight) or place 1m from a laboratory
bench lamp for 1 hour. Reincubate and examine for pigment the following
day
•
Incubate the other LJ slope at 25°C and examine at 3, 7, 14 and 21 days
Results and interpretation
M. tuberculosis does not grow within three days at 37°C and does not grow at
all at 25°C or on PNB medium. It also does not produce yellow or orange
pigment in the dark or after exposure to light.
SUMMARY
IDENTIFICATION OF M. tuberculosis
•
•
•
•
•
•
•
Growth rate slow
Growth temperature 35°-37°C only
No pigmentation
Niacin positive
Nitrate positive
Catalase negative at 68°C
No growth on LJ medium containing p-nitrobenzoic acid
75
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
10
RECORDING AND REPORTING OF LABORATORY RESULTS
Tuberculosis laboratories must establish a uniform procedure for reporting culture
results. If laboratory findings are to be useful, they must be communicated in ways
that make sense to the different authorities: Health care workers use the findings
for the diagnosis and management of tuberculosis; public health authorities use
them for statistical and epidemiological purposes, while tuberculosis managers use
the information to ensure that bacteriologically proven patients are receiving
appropriate chemotherapy.
Culture procedures for tuberculosis bacteriology are notoriously time-consuming,
often taking weeks or months to complete. For this reason, interim reports should
be issued. The following schedule is recommended:
•
If the cultures have been contaminated, a report should be sent out immediately
and a repeat specimen requested
•
If cultures are positive and growth has been identified as M. tuberculosis a
report should be sent out immediately
•
At four weeks an interim report (optional) could be sent out on all negative
specimens, stating that another report will be issued in the event of the
specimen becoming positive later on
•
At eight weeks a final report should be issued containing all the data previously
reported so that earlier interim reports can be destroyed and only the final
report retained in the patients’ file
Culture reports should be qualitative (ie. positive or negative) as well as
quantitative (ie. number of colonies isolated). The average number of colonies on
all the bottles/tubes per specimen should be reported. The following scheme is
recommended:
Reading
No growth
1-19 colonies
20-100 colonies
100-200 colonies
200-500 colonies (
almost confluent growth)
>500 colonies (confluent growth)
Contaminated
Report
Negative
Positive (number of colonies)
Positive (1+)
Positive (2 +)
Positive (3 +)
Positive (4 +)
Contaminated
77
RECORDING AND REPORTING OF LABORATORY RESULTS
In high tuberculosis prevalence countries more than 85% of disease is due to
M. tuberculosis and other mycobacteria rarely are responsible for clinical disease.
Also, the robust decontamination techniques used favour the isolation of
M. tuberculosis while killing some of the more fragile mycobacteria. A positive
culture with the characteristics as described before can, therefore, fairly safely be
labelled as “tubercle bacilli” and the recommended way of reporting is as follows:
“Cultivation yielded __________ growth of mycobacteria with the characteristics
of tubercle bacilli”
Model tuberculosis culture reports are presented in Annex 4.
Obviously, all the technical details of each laboratory test cannot be recorded on
the reports, but they should be precisely outlined in the laboratory manual and all
the results must be noted in the laboratory register. A copy of the completed final
report should be retained in the laboratory. When reading cultures the following
should be recorded in the laboratory register:
•
•
•
•
•
Growth rate (slow / rapid)
Number of colonies isolated
Pigment production in the colonies (none / present and colour)
Colony morphology (rough / smooth / shiny / flat)
Results of differential tests
A model laboratory register is presented in Annex 5.
78
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
11
QUALITY CONTROL (see part I, page 41)
Quality assurance with regard to tuberculosis culture is a system designed to
continuously improve the reliability, efficiency and use of culture as diagnostic
and monitoring option. The purpose of a quality assurance programme is to
improve the efficiency and reliability of culture services. The components of a
quality assurance programme are:
•
•
•
quality control (see part I, page 41),
quality improvement (see part I, page 41),
proficiency testing (see part I, page 43).
The following section will focus on aspects of quality control in the culture
laboratory. For a discussion on quality improvement and proficiency testing please
refer to the Management Series.
Quality control of culture is a process of effective and systematic internal
monitoring of the performance of bench work in the culture laboratory. Quality
control ensures that the information generated by the laboratory is accurate,
reliable and reproducible. This is accomplished by assessing - against acceptable
established limits - the quality of specimens, the performance of decontamination,
digestion and culture procedures, the quality of reagents, media and equipment, by
reviewing culture results and by documenting the validity of culture methods.
Quality control should be performed on a regular basis in the culture laboratory to
ensure reliability and reproducibility of laboratory results. For a quality control
programme to be of value, it must be practical and workable.
Quality control is the responsibility of all laboratory workers
Quality control must be applied to:
•
•
•
•
•
•
•
laboratory arrangement
equipment
collection and transport of specimens
handling of specimens
reagents and media
culture methods
reporting of results
79
QUALITY CONTROL
The keys to successful quality control are:
•
•
•
•
adequately trained, interested and committed staff
common-sense use of practical procedures
a willingness to admit and rectify mistakes
effective communication
Quality control measures which must be in place in all tuberculosis culture
laboratories include:
Laboratory arrangement and administration
•
Ensure that doors in the laboratory are always closed. Work areas, equipment
and supplies should be arranged for logical and efficient work flow. Work areas
should be kept free of dust. Benches should be swabbed at least once a day
with an appropriate disinfectant (eg. 5% phenol)
•
Every procedure performed in the laboratory must be written out exactly as
carried out and be kept in the laboratory for easy reference. Any changes must
be dated and initialised by the laboratory supervisor
•
•
All records should be retained for two years
Laboratory procedures used routinely should be those that have been published
in reputable microbiological books, manuals or journals
Laboratory equipment
•
•
•
•
Equipment should meet the manufacturers claims and specifications
Written operating and cleaning instructions must be kept in a file for all
equipment
Dated service records must be kept for all equipment
Equipment must be monitored regularly to ensure the constant accuracy and
precision necessary.
•
Biological safety cabinet: The BSC is the primary containment device that
protects the worker, product and/or environment from exposure to
tuberculosis and its performance needs to be verified at the time of
installation and annually thereafter. The purpose and acceptance level of the
performance tests are to ensure the balance of inflow and exhaust air, the
distribution of air onto the work surface and the integrity of the cabinet.
Other tests check electrical and physical features of the BSC.
Daily checks of the BSC include the following:
•
Ensure that the rate of airflow across the front opening is 75 linear
feet/minute (22.86 meter/second) for Class I and 75 to 100 linear
feet/minute (22.86 to 30.48 meter/second) for Class II cabinets.
80
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
Check the magnetic gauge in the exhaust duct for any pressure drop
across the filters and replace the filters when the gauge indicates that the
airflow across the front opening has dropped below optimal levels.
The following tests should be performed annually on Class I and Class II
cabinets:
• Downflow velocity and volume test
This test is performed to measure the velocity of air moving through the
cabinet workspace.
•
Inflow velocity test
This test is performed to determine the calculated or directly measured
velocity through the work access opening, to verify the nominal set point
average inflow velocity and to calculate the exhaust airflow volume rate.
An electronic vane type anemometer should be used to measure airflow.
The airflow into a Class I cabinet should be measured in at least five places
in the plane of the working face and an average calculated. At no place
should there be a reading that is 20 linear feet/minute (0.1meter/second)
more or less than any of the others. If there is such a difference there will be
turbulence within the cabinet.
In Class II cabinets the airflow is greater at the bottom than at the top of the
working face. The average inward flow is calculated by measuring the
velocity of air leaving the exhaust and the area of the exhaust vent. From
this the volume per minute is calculated, which is also the amount entering
the cabinet. Divided by the area of the working face it gives the average
velocity. The downward velocity of air should be measured at 18 points in
the horizontal place, 10cm above the top edge of the working face. No
reading should differ from the mean by more than 20%.
•
Airflow smoke patterns tests
This test is performed to determine if the airflow along the entire perimeter
of the work access opening is inward, if airflow within the work area is
downward with no dead spots or refluxing, if ambient air passes onto or
over the work surface, and if there is refluxing to the outside at the window
wiper gasket and side seals. The smoke test is an indicator of airflow
direction, not of velocity.
Commercial airflow testers are recommended. They are small glass tubes,
sealed at each end. Both ends are broken off with the gadget provided and a
rubber bulb fitted to one end. Pressing the bulb to pass air through the tube
causes it to emit white smoke.
81
QUALITY CONTROL
•
HEPA filter leak test
This test is performed to determine the integrity of supply and exhaust
HEPA filters, filter housing, and after-mounting frames while the cabinet is
operated at the nominal set point velocities. An aerosol in the form of
generated particulates of dioctylphthalate (DOP) or an accepted alternative
is required for leak-testing HEPA filters and their seals. Although DOP has
been identified as a potential carcinogen, competent service personnel are
trained to use this chemical in a safe manner. The aerosol is generated on
the intake side of the filter, and particles passing through the filter or around
the seal are measured with a photometer on the discharge side. This test is
suitable for ascertaining the integrity of all HEPA filters.
•
Cabinet leak test
The pressure holding test is performed to determine if exterior surfaces of
all plenums, welds, gaskets, and plenum penetrations or seals are free of
leaks. It is performed just prior to initial installation when the BSC is in a
free-standing position in the room in which it will be used, after a cabinet
has been relocated to a new location, and again after removal of access
panels to plenums for repairs or a filter change. This test may also be
performed on fully installed cabinets.
• Electrical leakage and ground circuit resistance and polarity tests
These safety tests are performed to determine if a potential shock hazard
exists by measuring the electrical leakage, polarity ground fault interrupter
function, and ground circuit resistance to the cabinet connection. The
polarity of electrical outlets are checked using a polarity tester. The ground
fault circuit interrupter should trip when approximately 5 milliampere (ma)
is applied.
•
Lighting intensity test
This test is performed to measure the light intensity on the work surface of
the cabinet as an aid in minimising cabinet operator’s fatigue.
• Vibration test
This test is performed to determine the amount of vibration in an operating
cabinet as a guide to satisfactory mechanical performance, as an aid in
minimising cabinet operator’s fatigue, and to prevent damage to delicate
tissue culture specimens.
• Noise level test
This test is performed to measure the noise levels produced by the cabinet,
as a guide to satisfactory mechanical performance and an aid in minimising
cabinet operator’s fatigue.
82
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
•
Centrifuge: Check brushes and bearings every 6 months
•
Incubator 35°-37°C: Record the temperature daily, preferably in the
morning. Test the temperature at several sites within the incubator by
placing a thermometer in a water reservoir (eg. Erlenmeyer flask). Control
the light within the incubator by covering the glass front of the incubator
door and by restricting the use of any lights inside the incubator
•
Inspissator: Check temperature daily. Clean after each batch of culture
media prepared
•
pH meter: Compensate for temperature with each run. Date buffer solutions
and discard when unsatisfactory. Standardise with pH 4.0 and 7.0 buffers
before each test or series of tests
•
Water baths: Check temperature before and during use. Clean monthly
•
Refrigerator 2°-8°C: Check temperature daily. Clean monthly. Defrost or
check refrigerator and freezer compartment every 3 months
• Freezers: Check daily. Clean every 6 months
•
Glassware: Discard chipped or etched glassware. Ensure that glassware are
free of detergents. Do not store sterile glassware for more than three weeks
before it is used
Specimens and request forms
•
Perform tests only upon written request of authorised persons and do not allow
oral requests without follow-up written instructions
•
Insist on specimen request forms being kept separate from the specimens
themselves. Forms that have been contaminated by specimens should be
sterilised by autoclaving
•
Insist on adequately completed request forms and proper labelling of
specimens to ensure positive identification of patients. Reject specimens that
cannot be properly identified
•
Evaluate the quality of sputum specimens and make a note if a specimen
resembles saliva. The report should state “specimen resembled saliva - treat a
negative result with caution” (to facilitate reporting a rubber stamp of the
comment can be made)
•
Discard leaking and broken specimen containers by autoclaving and request a
repeat specimen
•
Document the arrival time of specimens in the laboratory and note any delays
in delivery on the report form, particularly with negative/contaminated results
83
QUALITY CONTROL
Reagents and stains
•
All containers of stains and reagents should show the date received and the date first
opened. Any material found to be unsatisfactory should be recorded as such and
removed from the laboratory immediately. Stocks should be limited to six months’
supply and regular stock rotation should take place to avoid unnecessary expiry.
Digestion and decontamination
•
•
Process sputum specimens in batches according to centrifuge capacity
Keep a monthly record of the percentage of clinical specimens contaminated:
the acceptable range is 2-5%. Contamination rates <2% indicates overly harsh
decontamination, which means that too many tubercle bacilli are killed. If the
laboratory is experiencing delays in delivery of specimens the contamination rate
may be greater than 5%. If a rate of >5% persists, ensure that specimens are
completely digested, since partially digested specimens may not be completely
decontaminated. Thoroughly mix the contents of centrifuge tubes to ensure that the
inside surfaces have been well decontaminated
Culture media
•
•
•
•
Use fresh eggs (< seven days) for preparation of Löwenstein-Jensen media
Control coagulation time and temperature for egg-based medium. Discard
media that are discoloured or have bubbles following inspissation
Check all batches of media for sterility by incubation at 35°-37°C for 24 hours
Keep all media in the dark in the refrigerator and discard unused media after
four weeks
Culture procedures
•
Avoid cross-contamination of cultures by using individual pipettes or loops and
strict aseptic techniques
•
Be suspicious of several successively positive specimens or of cultures with
few colonies that follow a heavily positive culture
Biochemical tests
Prepare reagents as indicated and check the expected biochemical test response by
using appropriate positive and negative controls
Water
Check both distilled and tap water regularly for the presence of acid-fast
contaminants. If water appears cloudy or dirty, centrifuge 200-250ml in multiple
tubes and make a smear of the combined sediment. Alternatively, filter 1 000ml of
water through a sterile 0.22µm pore size membrane filter, cut the filter into strips
with a sterile scissor and place on Löwenstein-Jensen culture medium
84
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
12
SELECTED REFERENCES
1.
Collins CH, Lyne PM. Microbiological Methods. 5th ed. Butterworths,
London, 1984.
2.
Collins CH, Grange JM, Yates MD. Tuberculosis bacteriology. 2nd ed.
Organization and practice. Oxford: Butterworth-Heineman, 1997.
3.
Fadda G, Virgilio GD, Mantellini P, Piva P, Sertoli M. The organization of
laboratory services for a tuberculosis control programme. JPMA 1988; 38(7):
187-193.
4.
Grange JM, Yates MD. Guidelines for speciation within the Mycobacterium
tuberculosis complex. WHO/Zoon/94.174. WHO Veterinary Public Health
Unit, 1994.
5.
Kent PT, Kubica GP. Public health mycobacteriology: Guide for the Level III
Laboratory. US Department of Health and Human Services, Centres for
Disease Control, USA, 1985.
6.
Kleeberg HH, Koornhof HJ, Palmhert H. Laboratory Manual of Tuberculosis
Methods. 2nd. ed. revised by EE Nel, HH Kleeberg, EMS Gatner. MRC
Tuberculosis Research Institute Pretoria, South Africa, 1980.
7.
Kubica GP. Correlation of acid-fast staining methods with culture results for
mycobacteria. Bull Int Union Tuberc 1980; 55(3-4): 117-124.
8.
Mitchison DA. Examination of sputum by smear and culture in case-finding.
Bull Int Union Tuberc 1968; 41: 139-147.
9.
Mitchison DA, Keyes AB, Edwards EA, Ayuma P, Byfield SP, Nunn AJ.
Quality control in tuberculosis bacteriology: The origin of isolated positive
cultures from the sputum of patients in four studies of short course
chemotherapy in Africa. Tubercle 1980; 61: 135-144.
10.. PAHO / WHO Advisory Committee on Tuberculosis Bacteriology. Manual of
Technical Standards and Procedures for Tuberculosis Bacteriology. Part II:
The culture of Mycobacterium tuberculosis. Pan American Health
Organization, Martinez, Argentina, 1998.
11. PAHO / WHO Advisory Committee on Tuberculosis Bacteriology. Manual of
Technical Standards and Procedures for Tuberculosis Bacteriology. Part III:
Sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs. Pan
American Health Organization, Martinez, Argentina, 1998.
12. Salfinger M, Pfyffer GE. The new diagnostic mycobacteriology laboratory.
Eur J Clin Microbiol Infect Dis 1994; 961-979.
13. Strong B, Kubica GP. Isolation and identification of Mycobacterium
tuberculosis. US Department of Health and Human Services, Centres for
Disease Control, USA, 1981.
85
SELECTED REFERENCES
14. Urbanczik R. Present position of microscopy and a culture in diagnostic
mycobacteriology. Zbl Bakt Hyg A 1985; 260: 81-87.
15. US Department of Health and Human Services. Primary containment for
biohazards: selection, installation and use of biological safety cabinets.
Centres for Disease Control and Prevention and National Institutes of Health,
USA, 1995.
86
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
ANNEX 1
ESSENTIAL EQUIPMENT AND SUPPLIES FOR A CULTURE
LABORATORY USING MODIFIED PETROFF DECONTAMINATION
AND LÖWENSTEIN-JENSEN CULTURE MEDIUM
(6000 SPECIMENS PER YEAR)
Item
Quantity/volume required
Equipment
Autoclave, laboratory type, mixed load pressure cooker type or gravity
displacement model with automatic air and condensate discharge.........................1
Balance, top pan .....................................................................................................1
Biological safety cabinets, Class I or Class II, complying with international
standards .................................................................................................................1
Bunsen burner for use with butane gas ................................................................3
Centrifuge, with lid, 3 000 x g RCF capacity, fixed angle rotor, sealed buckets,
safety catch .............................................................................................................1
Culture bottle washer ...........................................................................................1
Fogging device .......................................................................................................1
Homogenizer, for eggs...........................................................................................1
Incinerator.............................................................................................................1
Inspissator 240 litre capacity, thermostatically controlled at 80-85°C ..................1
Microscope, binocular, oil immersion lens (100x), eye pieces (8x or 10 x),
spare bulbs ..............................................................................................................1
Refrigerator...........................................................................................................1
Steam pots .............................................................................................................3
Vortex mixer..........................................................................................................1
Walk-in incubator, walk-in or shelf, heater and circulating fan, steel or
aluminium racks .....................................................................................................1
Supplies
Aluminium foil, heavy duty...........................................................................8 rolls
Adhesive labels for sputum containers ............................................................7000
Aspirator flask, with outlet for distilled water 5 to 10 litre capacity.....................1
Autoclave tape, 1cm width ..........................................................................12 rolls
Beakers, glass, 100ml, 250ml, 500ml, 1 000ml .....................................................5
Bowl, plastic, 50x30cm ..........................................................................................4
Canisters for sterilising pipettes ............................................................................5
Centrifuge tubes, glass or plastic, screw cap, round bottom ...........................7000
Cotton wool, white absorbent ............................................................................2kg
Cultures boxes, 50 culture capacity.....................................................................10
Culture tube racks,48 tube capacity, polypropylene or metal covered with
polypropylene or nylon ...........................................................................................5
Diamond pens .......................................................................................................2
Discard bottles, splashproof .................................................................................2
Discard dishes, stainless steel or polypropylene ...................................................3
Disinfectant, bactericidal eg.5% phenol, 5% sodium hypochlorite ...........40 liters
Filter funnels, glass, 45mm or 60mm diameter /
90mm or 125mm diameter .............................................................................2 each
Filter paper, 15cm diameter, No .................................................................4 boxes
87
ANNEX 1
Forceps , stainless steel, 15cm ...............................................................................2
Glassware, 250ml and 500ml Erleynmeyer flasks, 250ml, 500ml ................2 each
Gloves, disposable ..............................................................................................400
Hand lens ..............................................................................................................1
Inoculation loops,25 SWG nichrome wire, 15cm long, 5mm diameter or .......500
Inoculation loops, disposable ..........................................................................7000
Laboratory registers for culture.......................................................................2-4
Laboratory request forms ..............................................................................7000
Laboratory report forms, preliminary and final ....................................7000 each
Lens tissue, for cleaning objectives and eye-pieces ........................................1 box
Loop holders........................................................................................................10
Masks, industrial, disposable .............................................................................400
McCartney bottles, 14ml or 28ml, wideneck.................................................9 000
Measuring cylinders, glass, 25ml, 100ml, 250ml, 1 000ml..........................2 each
Microscope slides, 25mmx75mm, 1.1-1.3mm thick..........................................600
Nichrome wire, for loops .........................................................................20 metres
Overalls, laboratory coats...................................................................2 per person
Paper towels, disposable..............................................................................2 boxes
Pasteur pipettes, low-density polypropylene, integral teats ..............................500
Pens, ball point, red ink ........................................................................................12
ball point, black or blue ........................................................................................24
Pipette washer .......................................................................................................1
Pipettes, blow-out 1ml, 5ml, 10ml.................................................................5 each
Racks, for inspissation of bottles containing media ............................................10
Reagent bottles, different capacities varying from 50ml to 1 000ml...................25
Rubber teats, for pipettes, 2ml, 5ml..............................................................5 each
Scissors, stainless steel, 25cm, ...............................................................................4
Self-filling syringes, 5ml and 10ml capacity .................................................1 each
Slide rack, plastic, 12-25 slide capacity.................................................................4
Slide storage box, cardboard or metal, 12-25 slide capacity ...............................20
Spatulas, stainless steel..........................................................................................4
Specimen containers, plastic, disposable, 50ml capacity, wide mouthed,
screw-capped, combustible .....................................................................................3
Stain bottles, amber glass, 100ml capacity ............................................................3
plastic, 10ml capacity .............................................................................................3
Staining racks .......................................................................................................2
Stainless steel buckets, large, with lids .................................................................4
Stirring rods ..........................................................................................................5
Test tubes, glass, rimless, 16mm x 152mm........................................................200
glass, rimless, 19mm x 152mm...........................................................................200
Test tube caps, aluminium, Cap-o-Tests............................................................600
Thermometers.......................................................................................................2
Timers, 0-60 minutes with alarm ...........................................................................2
Tripod stand with wire mat ...................................................................................2
Volumetric flasks, glass, 500ml capacity ..............................................................4
Wash bottles, plastic, 500ml capacity....................................................................2
Waterproof markers ...........................................................................................12
Waterbath, with lid, thermostat and heater............................................................2
88
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
Reagents
Acid-alcohol, for Ziehl-Neelsen staining ....................................................6 liters
Aniline ...........................................................................................................125ml
Aqueous methylene blue..............................................................................4 liters
Basic fuchsin....................................................................................................125g
Carbolfuchsin, for Ziehl-Neelsen staining ...............................................12 liters
Cyanogen bromide (BrCN) ............................................................................250g
Dry zink dust .......................................................................................................2g
Eggs ..............................................................................................10 doz per month
Ethyl alcohol (70%) ..............................................................................2.5 litre x 2
Glycerol ..................................................................................................2.5litre x 1
Hydrochloric acid concentrated (reagent grade) .................................2.5 litre x 1
Hydrogen peroxide 30% (superoxol) ..........................................................250ml
Immersion oil ................................................................................................100ml
L-asparagine ...................................................................................................250g
Magnesium sulphate MgSO4.7H2O ...............................................................250g
Magnesium citrate ..........................................................................................250g
Malachite green.................................................................................................25g
Methylated spirit.....................................................................................750ml x 5
N-naphthylethylene-diamine-dihydrochloride..............................................2.5g
Na2HPO4 (anhydrous) ....................................................................................250g
p-Nitrobenzoic acid (PNB) .............................................................................200g
Phenol crystals ................................................................................................125g
Potassium permanganate KMNO4 ................................................................250g
Potassium dihydrogen phosphate KH2PO4 (anhydrous) ..............................500g
Sodium hydroxide (NaOH) ............................................................................500g
Sodium chloride .............................................................................................250g
Sodium pyruvate.............................................................................................100g
Sodium nitrate.................................................................................................125g
Sulfanilamide ....................................................................................................15g
Sulfuric acid (reagent grade) ...............................................................2.5 litre x 1
Tween 80 ........................................................................................................100ml
Xylene ............................................................................................................400ml
89
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
ANNEX 2
SPUTUM COLLECTION
PROCEDURE
•
Give the patient confidence by explaining to him/her the reason for
sputum collection
•
Instruct the patient to rinse his/her mouth with water before producing the
specimen. This will help to remove food and any contaminating bacteria in
the mouth
•
Instruct the patient to take two deep breaths, holding the breath for a few
seconds after each inhalation and then exhaling slowly. Ask him/her to
breathe in a third time and then forcefully blow the air out. Ask him/her to
breathe in again and then cough. This should produce a specimen from
deep in the lungs. Ask the patient to hold the sputum container close to the
lips and to spit into it gently after a productive cough. Sputum is
frequently thick and mucoid, but it may be fluid, with chunks of dead
tissue from a lesion in the lung. The colour may be a dull white or a dull
light green. Bloody specimens will be red or brown. Thin, clear saliva or
nasopharyngeal discharge is not sputum and is of little diagnostic value for
tuberculosis
•
If the sputum is insufficient encourage the patient to cough again until a
satisfactory specimen is obtained. Remember that many patients cannot
produce sputum from deep in the respiratory track in a few minutes. Give
him/her sufficient time to produce an expectoration which s/he feels is
produced by a deep cough
•
If there is no expectoration, consider the container used and dispose of it
in the appropriate manner
•
Check that the container is securely closed and label the container (not the
lid) clearly
•
•
Wash hands with soap and water
•
•
Give the patient a new sputum container and make sure that s/he
understands that a specimen must be produced as soon as s/he wakes up in
the morning
Demonstrate to the patient how the container should be securely closed
Instruct the patient to bring the specimen back to the health centre or
laboratory
90
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
LABORATORY REQUEST FORM
ANNEX 3
Name of Health Centre __________________________ Date _____________
Name of patient _____________________________ Age _____ Sex M ❐ F ❐
Complete address:________________________________________________
_______________________________________________________________
Patient’s register number* _____________________
Source of specimen
❐ Pulmonary
❐ Extra-pulmonary
Site ____________________
Reason for examination ❐ Diagnosis
❐ Follow-up of chemotherapy
Specimen identification number ________________ Date ________________
Signature of person requesting examination ____________________________
* Be sure to enter the District TB Register number for the follow-up of patients on
chemotherapy
_________________________________________________________________
91
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
CULTURE RESULTS: PRELIMINARY REPORT
ANNEX 4a
_________________________________________________________________
Laboratory serial number ______________ Date specimen received ___________
Culture results
Culture method ______________________
No growth
❐
3+
❐
1-19 colonies
❐
4+
❐
1+
❐
Contaminated
❐
2+
❐
Cultivation yielded _____________ growth of mycobacteria with the characteristics of tubercle bacilli.
A final report will be issued within the next four weeks.
Date ___________________________
Signature ______________________
92
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
CULTURE RESULTS: FINAL REPORT
ANNEX 4b
_________________________________________________________________
Laboratory serial number _____________ Date specimen received ______________
Microscopy results
Staining method
❐
Negative
❐ Ziehl-Neelsen ❐ Fluorochrome
1+
❐
Not done
❐
2+
❐
1-9 AFB
❐
3+
❐
Culture results
Culture method ____________________
1-19 colonies
❐
Contaminated ❐
1+
❐
❐
2+
❐
3+
❐
4+
❐
No growth
Not done
❐
Culture identification
Growth rate_______________________ Colony morphology ________________
Niacin production
❐ positive
❐ negative
Nitrate production
❐ positive
❐ negative
Other, list__________
❐ positive
❐ negative
__________
❐ positive
❐ negative
Mycobacterium tuberculosis
MOTT
❐
❐
Culture identified as
Date __________________________ Signature __________________________
93
Lab
Serial no.
Name (in full)
Sex
M/F
Age
Complete adress
(for new patients)
Name of
referring Microscopy
Health Center
results
(for new
patients)
Culture
results
Colony
morphology
LABORATORY REGISTER FOR CULTURE
Growth
rate
Nitrate
Niacin
PNB
Catalase
ZiehlNeelsen
confirma- Signature
tion
ANNEX 5
Remarks
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
94
LABORATORY SERVICES IN TUBERCULOSIS CONTROL
UNIT CONVERSION FACTORS
ANNEX 6
LENGTH
1mm
=
1m
=
1m
=
1m
=
0.0394 inch
39.37 inches
3.28 feet
1.09 yard
1 inch
1 inch
1 foot
1 yard
=
=
=
=
25.4mm
0.0254m
0.305m
0.91m
0.035 ounce
2.2lb
1 ounce
1lb
=
=
28g
0.454kg
1m2 = 104 cm2 = 10.76ft2
TEMPERATURE
0°C
=
(°F-32) x 5/9
EF
=
(°C x 9/5) + 32
100°C
=
212°F
WEIGHT
1g
=
1kg
=
AREA
1cm2
=
0.155in2
1in
=
6.452cm
2
1ft2 = 144in2 = 0.0929m2
2
VOLUME
1 litre = 1000cm3 = 10-3 m3 = 0.0351ft3 = 61.02in3
1ft3 = 0.02832m3 = 28.32 liters = 7.477 gallons
1 US gallon = 3.78 liters
UK gallon = 0.26 US gallon
1ml = 1cc = 0.034fl oz
VELOCITY
1cm / s = 0.03281 ft/s
1km/h = 0.2778 m/s
500ml = 16fl oz = 1pint
1 000ml = 1 liter = 33.8fl oz = 1 quart
1UK gallon = 4.55 liters 1 000ml = 0.22
1ft/s = 30.48cm/s
PRESSURE
1Pa = 1N/m2 = 1.451 x 10-4 lb/in2 = 0.209 lb/ft2
1lb/in2 = 6 891Pa
1atm = 1.013 x 105Pa = 14.7lb/in2 = 2117 lb/ft2
95
1lb/ft2 = 47.85Pa
Copyright © World Health Organization (1998)
This document is not a formal publication of the World Health Organization (WHO)
and all rights are reserved by the Organization. The document may, however,
be freely reviewed, abstracted, reproduced, or translated in part, but not for sale
or for use in conjunction with commercial purposes.
For authorisation to reproduce or translate the work in full, and for any use by commercial entities,
applications and enquiries should be addressed to the Global Tuberculosis Programme, World Health
Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes in the
text, plans for new editions, and the reprints, regional adaptations and translations that are available.
The views expressed herein by named authors are solely the responsibility of those authors.
Printed in Italy
Design and printing: Jotto Associati s.a.s. - Biella - Italy
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement