INTERNATIONAL UNION AGAINST Si £ TUBERCULOSIS AND F LUNG DISEASES
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INTERNATIONAL
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Bulletin of the INTERNATIONAL
UNION AGAINST
Si £
TUBERCULOSIS AND
F
LUNG DISEASES
tuberculosis / respiratory disease / community health
TECHNICAL
I
IDE
FOR
SPUTUM EXAMINATION
FOR TUBERCULOSIS
DIRECT MICROSCOPY
WHO EXPERT COMMITTEE OS El BTRl I LOSES
..■'The object of tuberculosis control is to break the chain
of transmission of infection. This can be achieved bv detecting
the sources of infection as early as possible and rendering them
tiori-infeetious by chemotherapy. Transmission is maintained in the
community particularly by subjects whose sputum is so heavily positive
That tubercle bacilli can be detected by smear microscopv "
Abstract from pax< I4"l'the \mth report
of the WHO Expt n < I'inmittion Tub' o n/inn
(Technical Report Senes 10’4 Ao GJ )
Requests for additional copies of the Guide should be put to
NT rlR NATION AL UNION AGAINST TUBERCULOSIS AND Lt NG DIM ASf S
199-207 rue des Pyrenees
75020 Paris, France
Tel 43 66 04 22
VOt R COMMENTS AND SUGGESTIONS ARt IMPOR 1 ANT
fO IMPROX E THE QUALITY AND VALUE OF Fl 11 R I Fill tltlAs
OF fHISGl IDE. PLEASE SENDTHEM IO IIII ABOX I \DHRIss
1978
3rd edition (revtsed >
Reprinted 1980
BULLETIN OF THE INTERNATIONAL UNION
AGAINST TUBERCULOSIS AND LUNG DISEASES
... to disseminate knowledge and promote the continuing education
ofphysicians and health personnel...
-'/•'-A i\>«' >» <tv
uca . -v-jq
ppp
SUPPLEMENT No 2
DECEMBER 1978
TECHNICAL
GUIDE
FOR
SPUTUM EXAMINATION
FOR TUBERCULOSIS
BY
DIRECT MICROSCOPY
1978 - 3rd edition (revised)
Reprinied 1986
PREFACE
This Guide is based upon one initiated as early as 1969 by Dr. J. Holm
(then Executive Director of the International Union AgainstTuberculosis). It was
felt that the auxiliary personnel, specially in developing countries, needed a
simple guide for collection, storage and transport of sputum specimens and for
examination for tuberculosis by direct microscopy.
This document, the third edition, has been carefully examined and revised
by the members of the two IUAT Scientific Committees on Bacteriology/
Immunology and Diagnostic Methods ; account was also taken of suggestions
made by other experienced authorities, as well as those of workers who have
been using the Guide in the field.
The Guide is intended for field laboratories which may often have very
limited facilities and personnel. It presents the basic general principles for
collection, transportation, and examination by smear of sputum possibly contai
ning tubercle bacilli.
While the Guide provides basic procedures for the detection of infectious
tuberculous patients, it is recognized that local modifications of methods may
be both desirable and appropriate.
To clarify all points of procedure or detail all possible modifications of
methods would be prohibitive. It is anticipated that some users of this Guide,
in consultation with colleagues, supervisors, and central laboratory personnel,
may modify certain procedures to accommodate local facilities and equipment.
Prof. V. Farga
Dr. Annik Rouillon
December 1 978
O
COMMUNITY HEALTH CELL
326. V Main, I Block
Koramongala
Bangalors-560034
India
CONTENTS
I
—Collection of sputum specimens...................................................... 4
A.
B.
C.
D.
II
— Storage and transport of sputum specimens............................ 4
A.
B.
III
Number of specimens requested ................................................. 4
Recording of sputum examinations............................................... 4
Place for collecting the specimens............................................... 4
Technique for collection................................................................ 4
Storage.............................................................................................. 5
Dispatch............................................................................................ 5
5
Safety................................................................................................ 5
Laboratory arrangement ................................................................. 5
— The laboratory..........................................................................................
A.
B.
IV
— Reception and registration of sputum specimens.................. 7
V
— Preparation of smears.........................................................................
A.
B.
C.
D.
E.
Engraving slides for smears.............................................................
Smear preparation............................................................................
Drying................................................................................................
Fixation..............................................................................................
Disinfection and sterilization of contaminated material..............
7
7
7
9
9
9
10
Staining.............................................................................................. 11
Decolorization................................................................................... 11
Counter-staining.............................................................................. 12
VI— Staining technique.....................................................................................
A.
B.
C.
12
Arranging the working table............................................................ 12
Use of the microscope...................................................................... 13
Technique of reading........................................................................ 13
VII — Examination by microscopy...................................................................
A.
B.
C.
VIII— Results of examination............................................................................. 13
IX — Recording at a microscopy centre.......................................................
14
X—
Disposal of examined slides.................................................................
14
XI—
Dispatch of results of examination...................................................
14
XII—
Formulation of reagents......................................................................... 14
ANNEXES......................................................................................................................
15-16
I. COLLECTION OF SPUTUM SPECIMENS
Collection of sputum specimens will be made
in peripheral health centres and in various types of
outpatient clinics. The specimens will be sent to
the laboratory for examination.
Special sputum containers are used. They
must be rigid to avoid crushing in transit and
possess a wide-mouthed screw top hermetically
sealable to prevent dessication and to minimize
contamination by leakage.
There are two acceptable types of container.
One. available from UNICEF, is plastic with a black
bottom, a translucent lid, and is readily destroyed
by burning ; the patient's identification must be
made on the container (not on the lid). The other
is a heavy glass, screw-capped jar that may be
re-used after disinfection by boiling (10 min.) and
thorough cleaning.
A. Number of sputum specimens requested
from each patient suspect of tuberculosis
Three specimens should be requested from
each suspect patient; for instance, one spot speci
men when the patient presents himself at the clinic;
an early morning specimen consisting of all sputum
raised within one or two hours after rising and a
second spot specimen collected at the time the
early morning sample is brought to the clinic.
B.
Recording of sputum examinations
It is very important to keep a full and accurate
register of all sputum examinations performed by
the laboratory.
The information necessary for the precise
identification of each sputum specimen studied
must be accurately recorded. The system of regis
tering may be, for instance, the following : on one
line of the sputum register corresponding to the
specimen, write the health centre's code letter and
accession number, the patient's surname, given
name, age, sex and address and the date the spu
tum specimen was collected (Annex I).
The health centre's code letter and the acces
sion number are those listed on sputum container.
It should be noted that the three specimens from
the same patient will have different identification
numbers.
C. Place for collecting the specimen at the
health centre
The risk of infection is very great when the
patient coughs, therefore specimens should be
collected in the open air and as far away as possible
from other people.
If conditions do not permit procuring the spu
tum out of doors, it is best to use a separate, wellventilated room.
D.
Technique for collection
Attained person must:
1) Give the patient confidence by explaining to him
the reason forthe examination, and also explain to
him how to cough so that the expectoration will
come from as deep down in the chest as possible.
2) Open the sputum container, keep the lid and give
only the bottom part to the patient.
3) Stand behind the patient, and ask him to hold the
sputum container close to his lips and spit into it.
4) Check the quality and the quantity of the spu
tum ; a specimen of sufficient volume (3 to 5 ml)
containing solid or purulent particles and not just
saliva, should be obtained. If the expectoration is
insufficient, the technician should encourage the
patient to cough again until a satisfactory result is
obtained. It must be realized that many patients
cannot produce sputum from deep in the respira
tory tract in a few minutes, consequently sufficient
time should be given the patient to produce expec
toration which he himself feels is produced by a
deep cough.
If there is no expectoration, the sputum contai
ner must be considered as used, and must be
properly disposed of.
5) Close the sputum container securely and if it
must be sent to-the laboratory, put it into a special
box fortransport.
6) Wash hands with soap and water.
7) Give the patient a new sputum container and
- make quite sure that the patient has understood
that he must spit into this container as soon as he
coughs up sputum in the morning;
- demonstrate how the container should be secu
rely closed ;
- instruct him to bring it back to the health centre.
II. STORAGE AND TRANSPORT OF SPUTUM SPECIMENS
If the health centre does not make its own
examinations for acid-fast bacilli, the collected
sputum specimens must be brought to the labora
4
tory where they will be examined. This transport
should normally take place once or twice a week.
Consequently, the specimens collected over a
period of a few days must be kept at the health
centre and transported all together in one batch to
the laboratory.
For this storage and transport, special trans
port boxes are used, each holding 10-20 sputum
containers.
A. Storage (if transport is needed)
1) The special box with the sputum containers
should be kept in as cool a place as possible until
it is dispatched.
2) Sputum can also be processed in the health
centre and the fixed smears be sent to the labora
tory (see section V).
Storage and transport of fixed smears is easier than
storage of sputum samples. When it is intended
to make cultures from the same sample, the spu
tum specimen must be kept under refrigeration.
B. Dispatch
With each transport box, an accompanying
list must be prepared which identifies the sputum
specimens it contains and the data forthe patients
from whom the specimens were collected. When
fixed smears are sent they should be accompanied
by the same list. This list is prepared by copying
from the health centre’s sputum register (as in
Annex II).
Before the dispatch from the health centre, the
trained person must verify for each transport box :
1) that the total number of sputum containers in
the box corresponds to that on the accompanying
list;
2) that the identification number on each sputum
container corresponds to the identification number
on the accompanying list;
3) that the accompanying list contains the neces
sary data for each patient.
When this check has been made, the trained
person :
- marks the date of dispatch on the accompanying
list,
- puts the list in an envelope which he attaches to
the outside of the transport box,
- closes the transport box carefully.
Results of examination will be reported from
the laboratory to the health centre on the same
accompanying list which the health centre sent
with the transport box (cf. Annex III). Details for
recording results of examination are presented in
Section VIII.
III. THE LABORATORY
A.
Safety
Each laboratory worker is responsible for his
own safety and that of his co-workers. Transmis
sion of tuberculosis results essentially from micro
aerosols - droplet nuclei of 5 microns diameter containing tubercle bacilli, the inhalation of which
produces a focus of infection in the alveoli of the
lung. Efforts should be made to avoid, minimize or
control those laboratory operations which create
potentially infectious aerosols.
Some common sources of aerosol production
in the laboratory are :
- opening of specimen containers ; this is espe
cially dangerous if sputum has dried between the
cover and the side of the sputum container, or if
thecontainerhasbeenshakenjustpriorto opening,
- preparing smears on microscope slides,
- flaming of transfer loops.
Equipment : For maximum protection of labo
ratory workers, it is highly desirable to have a
laboratory biological safety cabinet for use in
preparation and fixing of smears or for processing
specimens for culture (see Fig. 21). In the absence
of a safety cabinet, extreme care must be taken to
protect workers from infection.
B.
Laboratory arrangement
The detailed arrangement for the laboratory
will vary greatly as to whether other work is also
done and what size and shape of room is available
and also whether electricity or day light is used for
microscopic examination. Forexample, the labora
torycan be arranged so as to include three separate
sections (Fig. 1) :
- one well-lighted area for (A) preparing and (B)
staining smears,
- one area for microscopy,
- one area for the register and storage space.
It should contain at least three tables :
- one table (1), divided into two parts : one for
smear-preparation within the safety cabinet, if
available (A), the other for staining (B),
- one table (2) for microscopic examination. If
there is no electricity, this table should be placed
directly before a window (3),
- one table (6) for register books and slide storage.
The laboratory should also contain :
- a sink or basin (4), if possible with running water,
- a chair, or preferably a stool, the height of which
can be adjusted,
- a closed wardrobe (5) or locker.
5
Figure 1
Each time he enters, the technician must put on
his laboratory coat and wash his hands with soap
and water (Basin 4, Fig. 1). Similarly, each time he
goes out, he must wash his hands and leave his
coat hanging in the wardrobe.
It is forbidden to smoke or eat in the laboratory,
or to sit on the tables.
Before the preparation of smears is started, the
material on the table should have been arranged in
accordance with that shown in Figure 2.
6
All the manipulations for preparing a smear
should be completely standardized ; the arrange
ments of the material on the table should always
be the same, to ensure maximum safety, and to
achieve a satisfactory standardization for the ma
nipulations.
Details of material shown in Fig. 2
A. A non-porous surface plate, e.g. formica, gal
vanized metal or aluminium : this plate must be
about 80 cm wide, and its borders must be 5 cm
high. The front edge must be bent down at an angle
of 90° to meet the edge of the table, thus facilita
ting manipulations. These must be made strictly
over the surface plate, which must be sterilized
every day after use, either by flaming or by soaking
with aTB-germicide (e.g. 5°/o phenol, 3%cresol, or
other phenolic germicide). See Fig. 13 for details
of surface plate.
B. Box of engraved slides for the smears. (Slides
must have no scratches on the area to be smeared.
If the slides are greasy, they must be cleaned with
methylated spirit and then carefully wiped with a
fine cloth).
C. Special box fortransporting with specimens to
be examined.
D. Diamond-pointed stylus.
E. Slide-holder for the preparation of smears (see
also Fig. 4, 5 and 10).
F. Sputum container placed as near as possible to
the slide-holder on the right.
G. A wire loop (which may be flame-sterilized
between specimens).
H. Wooden applicators (if used) (see later).
I. Alcohol sand flask.
J. Bunsen-burner or spirit lamp.
K. Dryer on which to place the finished smears.
(This dryer must be as far as possible from the place
where the smears are prepared so as to avoid any
contamination from other specimens when they
are being handled.)
L. Forceps.
M. Metal waste receptacle and lid to receive waste
septic material.
NOTE : If the technician is left-handed, it may be
more convenient to arrange all (or most) items in
Fig. 2 in exactly the opposite position on the table
(i.e. in a mirror-image location).
IV. RECEPTION AND REGISTRATION OF SPUTUM SPECIMENS
The sputum specimens or fixed smears to
gether with identification data (see dispatch list.
Annex II) are delivered to the laboratory.
It is necessary to make sure that the request
for examination on the list and the indications on
the sputum container (or fixed smears) are the
same. These written indications as well as any other
coded information must be carefully recorded on
the examination register. This must also include
the date of collection of the sputum, the date of
arrival at the laboratory and the identification data
of the patient and of his health centre.
V. PREPARATION OF SMEARS
3) He takes the diamond-pointed stylus and engravesthe sputum specimen identification number
on the end of a slide. Using the list, he numbers a
slide for each specimen.
He must avoid making fingerprints on the rest of
the slide.
To make engraving easier, he may place the slide
on a piece of black paper (Fig. 3).
4) After engraving, he checks that the identifi
cation on the slide is the same as that on the list,
and puts it in the slide box reserved for engraved
slides for this batch of specimens (See B, Fig. 2).
A. Engraving slides for smears
This is done at the microscope table.
1) The technician washes his hands.
2) He takes a new box of slides.
B. Smear preparation
The maximum chance of finding bacilli is in the
solid particles of the sputum. The result of the
examination depends to a great extent on the
choice of these particles. Do not attempt pro
Figure 3
cessing at onetime more than 10 or 1 2 specimens.
7
(Fig. 6). (If the sand and alcohol or lysol are placed
in a screw top 300-500 ml Erlenmeyer flask, this
may be used for long periods merely by refreshing
the alcohol or lysol to maintain the level at least
3 cm above the sand.)
Figure 4
This stage of the procedure (the opening of
the sputum containers and the preparation
of the smears) is most dangerous ; therefore, it
must be done carefully to prevent the formation
of infectious aerosols.
Here is an example of a suitable smear prepa
ration :
1) Take a slide from the slide box, holding it by the
part on which the number is engraved, place it
across the slide-holder (E) with the engraved side
uppermost and turned towards the operator (see
Fig-4).
Figure 6
4) When using wooden applicators :
- a) take a wooden applicator between the thumb
and index finger of each hand about three centi
metres from its centre and break it (Fig. 7);
2) Take the sputum container corresponding to the
number on the slide. Check that the number engra
ved on the slide corresponds to that on the sputum
container. Open the container, deposit the lid in the - b) choose yellowish, opaque, purulent par
waste receptacle (M, Fig. 2) and place the sputum ticles which will be placed on the slide. In order
container to the right of the slide-holder (Fig. 4). to do this, without changing position of the hands.
3) When using the loop :
- a) flame it and allow to cool ;
- b) pick a small portion of sputum selecting
purulent particles if present. A second loop may
facilitate selection of particles from very viscous
specimens;
- c) spread the sputum sample as thinly as pos
sible over two thirds of the slide (Fig. 5);
- d) sterilize the loop between successive speci
mens by holding in the flame of a Bunsen-burner
or spirit lamp until the wire is red hot. Before steri
lizing, larger particles of adherent sputum may be
removed from the wire by moving it up and down
through a flask containing sand and alcohol orlysol
8
Figure 8
use the broken ends of the two pieces of the a ppi icatorto break up the larger particles (Fig. 8);
Neither fixed nor unfixed slides should be left
exposed overnight. Not only is there a chance of
accidental breakage and infection, but insects and
rodents may enter the laboratory and eat the
smears off the slides. The technician must arrange
his working day so that slide preparation, staining
and reading are completed ; if reading cannot be
completed, slides should be placed in a covered
box for overnight storage.
D.
Fixation
The dryer with slides and the Bunsen-burner
or spirit lamp are placed :n the work area.
Figure 9
- c) if the particles are difficult to detach from the
bottom of the sputum container, turn it with one
of the sticks with a rapid circular movement (Fig. 9);
- Using forceps, take a slide from the dryer at the
engraved end, with the smear uppermost.
- Pass it three times through the flame of the
Bunsen-burner or the spirit lamp (Fig. 11) ; this
should take 3-5 seconds.
- Place it on the clean dryer.
When all the slides have been treated in this
way, flame the empty dryer.
- d) finally, using the two sticks in a pincer move
The dryer with the smeared and fixed slides is
ment, raise the particle and place it on the slide
(Fig. 10) (very often the particle is not homogenous brought to the staining part of the working table
(area
B, Fig. 1).
and one must, with the end of one stick, roll the
dependent part around the other stick);
- e) with one of the sticks, mix the particles placed
on the slide ; hold the stick firmly, and while
E. Disinfection and sterilization
applying downward pressure to the purulent par
of contaminated material
ticles, spread them evenly to cover 2/3 of the slide
(as indicated in Fig. 5).
When the manipulations are finished, infected
5) The wire loop is especially helpful in transferring materials and sputum containers must be thrown
into the waste receptacle.
excessively fluid specimens onto the slide. If no
acid fast bacilli are seen in "fluid" sputum samples,
1) Thewastereceptacle (M, Fig. 2)containing used
the report should indicate that the specimen was
materials and plastic sputum containers is half
unsatisfactory and that another should be sub
filled with water containing disinfectant (see E. 3)
mitted.
covered with its lid and brought to the boil and
6) After the smear is prepared, place the smear boiled for 10 minutes (Fig. 12). The contents are
then collected for later burning.
on the dryer.
2) In the event both burnable materials and glass
C. Drying
sputum jars are used, the latter should be discarded
into a separate container so that they may be boiled
The prepared slides must dry in the air for
and washed for re-use.
about 1 5-30,minutes. Do not use flame for drying.
9
3) Other items such as the slide holder, the dryer
and the work surface should be flamed (Fig. 13)
or literally soaked in a TB germicidal solution. (It
should be noted that a spirit lamp has limited
usefulness for this particular application). TB ger
micides are only those that have been shown to kill
tubercle bacilli by approved tests ; 5°/o phenol or
one of the phenol-derivative soap mixtures may be
used.
VI. STAINING TECHNIQUE
Figure 14
10
Arranging the table is shown in Figure 14.
A. Slide-rack for staining (can be used for 1 2 or more slides)
B. Filter-paper (cut up beforehand) or
C. Funnel with filter paper
D. Forceps
E. Plastic flask with Ziehl's carbol fuchsin
F. Alarm clock
G. Plastic flask with spirit
H. Cotton holder (in metal or wood)
I. Waste receptacle for the used filter papers
J. Plastic flask with 25% sulphuric acid
K. Plastic flask with 0,3% methylene blue
L. Extra slide rack
M. Basin (with running water if possible ; otherwise have an additional plastic flask containing water)
N. Flask of Ziehl's carbol fuchsin )
O. Flask of sulphuric acid
> Stock solutions to refill bottles E, J, K
P. Flask of methylene blue
)
Formulations for the above reagents are to be
found in Section XII.
The smeared and fixed slides are stained in
batches of up to about 1 2.
A. Staining
wool in methylated spirit (G. Fig. 14) fixed on the
end of a metal rod or a fairly strong stick of wood
(Fig. 16).
In no case must the stain boil or dry on
the slide. If the stain accidentally runs away, add
more and heat again. Leave the warm stain for five
1) Place the slides on the slide-rack (A, Fig. 14) minutes.
with the smeared sides uppermost, their edges
separated and the numbers turned towards the
operator. The smeared part of each slide can be
covered with a piece of filter-paper (B, Fig. 14).
2) Coverthe whole surf ace of the slides with Ziehl's
carbol fuchsin (E, Fig. 14).
NOTE : If filter-paper strips are not used, the carbol
fuchsin should be filtered through filter-paper in
funnel C (Fig. 14) directly onto the slides (Fig. 1 5).
3) Heat very gently until vapour rises. For this, use
the flame of a Bunsen-burner orof a wad of cotton
B. Decolorization
1) With forceps, remove the filter-papers and
deposit them in the waste receptacle (I, Fig. 14).
Bangalor®-66003^
2) Rinse each slide individually in a gentle stream
of running water (tap water, or bottled water
(M, Fig. 14)) until all free stain is washed away
(Fig. 17).
NOTE: Bulk staining, rinsing, acid decolorizing and
counter staining must be avoided because of the
real possibility of cross contamination from one
slide to another.
3) Replace all slides on the slide-rack (A, Fig. 14)
and cover each one individually with 25% sulphu
ric acid (J, Fig. 14) for 3 minutes (Fig. 1 8).
4) Rinse as in B.2) above.
5) Decolorize again for 1-3 minutes (as in B.3)
above) until all color has practically disappeared.
6) Rinse as in B.2) above.
C.
Counter-staining
1) Replace decolorized, rinsed slides on slide-rack
(A, Fig. 14) and flood smear with 0,3% methylene
blue counterstain (K, Fig. 14) for 60 seconds.
2) Rinse as in B. 2) above and allow to dry in
open air.
VII. EXAMINATION BY MICROSCOPY
For the examination of stained specimens, a
binocular microscope is most convenient, with an
immersion objective (X 100) and eye pieces of
moderate magnification (X 6 or X 8). Never
theless, if there is no electricity, and in hot or humid
conditions, a monocular microscope might be
better, because there are fewer surfaces to be
attacked by fungi and fewer days will be lost be
cause of lack of light.
If no electricity is available, daylight must be
used as light source and the table with the micros-
12
A. Arranging the working table (Fig. 1 9)
In addition to the microscope (A), the micro
scopist must have on the table :
- a bottle with immersion oil (B)
- toluene and clean cotton (C)
- a note book and pencil (D)
- the stained slides to be examined (E)
- the dispatch list for these sputum specimens (F)
- a slide box for examined slides (G).
B. Use of the microscope
Before starting the actual examination of
smears, the technician must make sure that all
elements of his microscope are correctly set. He
should, in particular, check that the source of light
is well regulated and focused, that the condenser
is in the upper position, with the diaphram open
and that the immersion objective and the ocular
lenses are clean.
Put a drop of immersion oil on the left edge of
the stained smear (nearthe engraved number) and
place the slide on the microscope stage. To avoid
possible contamination of the immersion oil,
do not touch the slide with the oil applicator,
but permit the drop of oil to fall freely onto the slide.
With the macrometric screw, lower the immersion
lens, keeping continuous watch until ittouchesthe
drop of oil. Looking through the eye-piece(s) bring
the immersion lens slowly upwards, by means of
the macrometric screw, until the image of the
smear appears. Complete the focusing by means
of the micrometric screw. All during the reading,
the correct focusing is ensured by using the micrometric screw.
C. Technique of reading
Examine at least 100 microscopic fields. For
a skilled microscopist, this will take 5 minutes.
The reading must be systematic and standar
dized. For instance, begin the reading of the slide
in the centre of the left end of the smear, by slight
adjustementsof the micrometricscrew, systemati
cally examine the field, beginning at the periphery
and ending at the centre.
After examining a microscopic field, move the
slide longitudinally so that the neighbouring field
to the right can be examined. In this manner, all
the microscopic fields from beginning to end of
this central length of the slide should be examined.
The number of microscopic fields in one length of
the slide corresponds to at least 100
When no Acid-Fast Bacilli (AFB) are found in
100 fields, a more thorough search should be
made in 100 new fields.
As shown in Fig. 20, move the slide a few milli
meters towards its back and read a second length
(from right to left).
Tubercle bacilli look like fine red rods, slightly
curved, more or less granular, isolated, in pairs, or
in groups, standing out clearly against the blue
background. Count the number of AFB and report
this number on the note book.
At the end of examination, take the slide from
the microscope stage, check the identification
engraved on it, and enter the result of the exami
nation in the last column of the dispatch list (see
Annex III). Dip the slide into toluene (or xylol) to
remove the immersion oil and place it in the box
for examined slides.
Examine the slides in the order given on the
dispatch list.
Before examining the next slide, wipe the
immersion lens with a piece of clean cotton.
VIII. RESULTS OF EXAMINATION
The number of bacilli found is a very important
piece of information because it relates to the
degree of infectivity of the patient, as well as
to the severity of the disease.
- No AFB1 to 9 AFB
10 to 99 AFB
1 to 10 AFB
more than 10 AFB
For this reason, the examination must be not
only qualitative, but also quantitative.
The following is an example of a method of
reporting which is sufficiently quantitative to be
valuable to the clinician :
per 100 immersion fields
per 100 immersion fields
per 100 immersion fields
per field
per field
0
record exact figure
+
+ +
+ + +
13
IX. RECORDING AT A MICROSCOPY CENTRE
The microscopy centre keeps the following
documentation :
1) Work-record book with daily information on the
total number of slides examined and the total
number of positive slides, by health centre
(Annex IV).
2) Positive sputum register with detailed infor
mation on all positive patients identified at the
microscopy centre (Annex V).
X. DISPOSAL OF EXAMINED SLIDES
A. Positive slides
A slide in which acid-fast bacilli have been
demonstrated is a document on which the dia
gnosis of pulmonary tuberculosis of a person
depends. It must be recorded and kept in the labo
ratory, and the reading should be confirmed by a
second reader. The record of all positive slides is
made in a special record book of the laboratory
(cf. Annex V), in the order in which the slides have
been examined.
The positive slides are removed to a special
box and kept for about one year. Before discarding,
they must be broken and buried to prevent their
re-use.
B. Negative slides
All the negative slides must be kept in the
laboratory for at least one week, in order to allow
for a control of the reading. After this time, they
may be discarded (e.g. buried).
XL DISPATCH OF RESULTS OF EXAMINATION
Upon completion of a series of microscopic
examinations, the date of examination is recorded
on the dispatch list which is returned to the health
centre as soon as possible. The transport box is
cleaned by swabbing with a cloth wet with a TBgermicide (e.g. 3°/o cresol or other phenol deri
vative) and also returned to the health centre.
XII. FORMULATION OF REAGENTS
A. Ziehl’s carbol fuchsin
FORMULA 1
To prepare 100 ml of stain, use the following
formula. Larger volumes may be made for stock
solutions, if desired :
1) Saturated alcoholic solution of fuchsin
- basic fuchsin .........................................
3 g
- 95% ethyl alcohol.................................. 100 ml
2) Working solution
- phenol crystals......................................
5 g
Heat gently in a flask to liquefy, and bring to
90 ml with water.
- add : saturated fuchsin solution.........
10 ml
FORMULA 2 (needs no scales)
- saturated solution of fuchsin, filtered . 100 ml
- aqueous phenol 5%............................. 900 ml
Saturated solution of fuchsin : the contents of
a 25 g flask of basic fuchsin is introduced into
a 250 ml bottle, which is then filled with methy
14
lated spirits. The bottle is shaken vigorously and is
shaken three times more on the same day It is
allowed to settle. The solution is ready for use the
following day.
Methylated spirits may be added until the
deposit is used up.
The 5% aqueous phenol is prepared by adding
5 ml of crystalised phenol melted at 45° C (over
100 ml of water).
B. Decolourization reagents
SULPHURIC ACID 25%
Empty 300 ml of water into a 1-litre flask.
Slowly add 100 ml of sulphuric acid, allowing it
to flow along the side of the flask. Mix. The contents
will heat up. Never empty the water into the
sulphuric acid.
Sulphuric acid 25% may be replaced by acid
alcohol, prepared as follows :
ACID ALCOHOL
- methylated spirits ............................... 970 ml
- hydrochloric acid.................................
30 ml
C.
Counter-staining with methylene blue
- methylene blue chloride or
hydrosoluble methylene blue..................
- distilled water..........................................
Diagram of a transfer cabinet (Fig. 21)
0.3 g
100 g
to the fan
A transfer cabinet can be made of wood or
other material and placed on the laboratory bench
or ordinary table. The dimensions, which may be
varied, are as follows :
length : 1 m, height: 85 cm, width of base: 60 cm,
width of top : 33 cm.
X. outlet for contaminated air to be disposed of
safety. The contaminated air must be drawn by
exhaust fan through filter. If no ventilation is
possible, a transfer cabinet can be more dangerous
than no cabinet at all.
W. large window (thick glass) as from automobile.
May be framed and hinged for opening.
P. slit to allow the introduction of the technician's
hands and forehands (20 cm high).
ANNEXES
HEALTH CENTRE'S SPUTUM REGISTER
ANNEX I
No. sputum
container
At
At 4*2
/*?
At
M tfZi
H VZf
bl W
*7
M V3l
M VU
Name and surname
be^t Joints
FoJ-m t>e*f At*tr
i9n Ah'
Alkyds i*-*
Zhu. be-r ft/;!.
be-f bteh,~z
Ain*
S
beH' farkt- be*
Age
Sex
Address
Date of
’ collection
it
bl
F
p
K tSa/x.
KlS»b.
KtS^r
4z/Z4/b9
F
F
5/z/zu
f*
/<Sru^
a.
At
i)e<d-
38
At
Ib/ez A 9
i9p/t>8
J/.'M
Kt
F
P
F
F
bl
AFB results
/I
3>z
Z8
21
vc
3S
21
2V
V<2?
Zi
25
K>Sn.r
Date of
results
A
H/ze/6f
••
J/.M
'bLtrc/t.
Sh'Hbjerde
>•
zr/rr/*9
15
ANNEX II
DISPATCH LIST
(accompanying the transport box containing sputum containers
sent by the health centre to the laboratory)
Results on :
Technician :
No. sputum
container
(
Laboratory :
Sent on :
Health centre :
Name and surname
copy FROf1
ANNEX III
Results on :
No. sputum
container
Age
Sex
2/Xa.
Z2.
F
F
2-ie^-- iSoocf- *7lr*cA#i
44.
3f
2Z.
Name and surname
'frc+J-faok.
f'1'
finyor
FftyxOr^^J
H *£***».
/7Z/“X/^X
A*****
44
2^
26
2S
26
2>S
Address
Date of
collection
AFB results
!’/;/»
H,/«/(>•>
fl/2A
36/4 43/2 Ad/ii.
o/2i~
>So/L_
O/2.L.
0/2 a.
q/2 1So/2 a
o/a a
m/n/6^
It
//.'A
M
W'/t*
/?/*»/« 9
KS*-’-
WORK-RECORD
Date received
Health centre
ib/lilil
fi.
C
Z^/4/A?
/«/«>?
First and last
number
gz$H'S •2.^0^
lylS-
6S6
H-2V
"2.64
HiS
Total
number
examined
Total
number
positive
Date results
sent off
/Z
/o
2
/2/-H/6?
/i
o
'll
A
B
i
a
!
£
POSITIVE SPUTUM REGISTER
ANNEX V
Name and given name Age Sex
Address
A
Sl,K
Ks<^
a.K
b/ui<-
44
Af 4£4
W 42?
Microscopist : At*'
Received on :
Examined on :
Sent back on :
///'Ac.
/TSux
///'Ac
F
F
F
F
-2>
frZ^dic-
ANNEX IV
Number
container
results
R EG-/57
Laboratory :
Technician :
/■tQiz
433
« 434
AFB
DISPATCH LIST SENT BACK TO THE HEALTH CENTRE
Sent on :
/H
M VW
n>/io
Date of
collection
Address
5 PUl■un
Health centre :
A fZS
Microscopist:
Sex
Age
Received on :
Examined on :
Sent back on :
ft-bfltluu's if-
/V 4X
M
um
* By the sec ond reader.
Jf
44
26
p
J*
Date of
collection
/3/2/Zh
/V/-»//>♦
Date of
examination AFB results Confirmation*
/?Z///4e
/»?//*’
11/^/iy
l9/U/i9
54//A
?/2A
>Sefr
S»72L
P
5
3
( ONSTITl F.NI
AFGHANISTAN the Director Notional I uber ti s«
institute Kabul
ALGERIA Pr. i D LARBAtH I vr.
- lidneral
Alg<'ten de t uttc . ontre - Tuhricuh.x IGO rue Dkdcuche
Mourad Alger
ANGOLA Mr ( A HRNANDfcS MmwP f A-MSanie
Directum dEndermes 'Lai go Premier Mat) Luanda
ARGENTINA |k H RtJDRIGt FZ.t AS I F II S Prendenir
g.i Argentic
nt’. . i f.<
- Santa Fe 4292.
Hurni» Aire*
AlsTRAIIA D' KWH HARRIS Finn. ,
*<-•Austrulwn 7ubc«uto-v»* and < nest AvMMjauon Ini
I Wandeen Road < larrHIle NSW 2IU7
M STRIA (V \ KH v .st . V «
A ..IkgcsundbctisneMiiDn BundesmtnisierHim flit Gesuudbett
.nd I m<*cHs*huU siybonrint I llllOWienl
RAM.IADF.SH Dr
HOSSAIN " •
Secrviatv National Ann Tubervuiuna A s%. ma l ion of
Bangladesh 24 Rarrtabandhu \»enut ! 1st and Ind Floor* 1
Dacca
HI I (.11 M P
I PRK.N. ■ p.
Belga de Defen-H.- Hiirr i.i lubcnuh
Sa. rue de la ( onmrdr
10541 Bruxelles A
BENIN M i v1>' i
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Idbercvi..*.: Mmi* . ic k i. tak PuHiyue < monou
BOLIVIA
l N.H nji
Tuber*
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de PrcvLstonSraial y SaTud Publua
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BOTSWANA I) t
M Au A Nt. I
’ B I mt Minin . 4 |> ,Uh Gaborone
BRAZH o
VI XIMIMS NE
..... i n
Stu.ldudcB ..tlL-n l<
IO.I.I..K-. V I > •' a SI. AS
(Viat.ZSuP Quadra 616 Laie IS 7Q20B Hrastlia D F
B< 1 G ARI A
A Al ■ IU 1
Pneumo*, g* A PhthmK.it^* MatJital Vitiemt
Dim Nr*iero» Sir 19 Sofia
HIRKINAFaSO M I, Pr Ol.n, . - • x.. ub»
...
s
B P 3423, Ouagadougou
Hl RM A Burma Medi* . A ’
.
I '■
- Sc )O
249 Iheinbvu Road Kanaa»»ela» P o Rangoon
I A.NADA M 8 MKKl N/H f - ui, . On .
C jn*<Lun t«ng Awn-uilicti
‘ Albert. Suite
Ull»aahiP<f’
« F NIR Al AFRH AN RFPl Bi It M te Pies.deni t arrutd
National Ant»iuteriuku» Dinxhon Genetic de i- Santc
Puhkqi « i h AH n
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Baugu
(HID l.'iv <fa. Program., "lutn.i t .' ual Jv [□<* .uihms,
MWHBerK.aeiali.it Pubitcu Siers tcin Nacfonaldr Salad.
Data* oGemr . t Mac l»er *41 ( asilia J*O9 Santiago
I HINA Uaipcb' Or C F HS!*M . *
Njiwnul Tuberculosis A*m-» mitnn
IA4 Min < huan West Road I aipeh
COI OMBI A
•
HiftF s iti sNi
i
I iga Ant. ■•« . uh . C aJornhi.H
Atentda Jtmenez No I US.
A part ado Aereo 41414. Bogina
(OSTA Rll A Dr J M\H(N MON< f hek de P> »rimj
IhsuMunde Fubercult'sm Mmrsicrm dr Sulud san Imt
< l B» l>. k « . H .
<
\............ . .
.
Ci-eupu Natirunal de T utx rcutemis Minufferm de Vahid Publics
I alle 25. I sq AN I a Habana
< /b( HOSI.OA AM A h
h x, n Ik . • .
liapnunicni institute <*( Postgraduate Lducauon A Imfitute
of F B and Rcipualury D«c«m«
H2A M 8rattsia»a Pod Biskupmr
riOMINIl AN RtP( Bl It
DFMOt RAID PFUPi F S RF Pl Hi.K OF KOREA
Dr U Zl»V SEL<N Prtsittent Association amine ta rubercuh sc
de la Repubiique Popu...ire Demm -Hqur d <
•
lapxc-Dong laedongang Pyongyang
DEMtMRAlit YEMEN r ub< - |F»S * I h
n.r
Al
ot'H atilh Aden
DENMARK m kv
xs. Man.gmgDThe Naluma) Abtix-Olum against 18 A L ung Dim aset
57 ileiafchulmvej 2 700 \ an lose
MFMBI Rs • IMX5>
DIIBOl Tl
, .f m .
i. .
<■
Djlboati
Kt A DOR ;>
A t REIRE Rer-^pVn.r t,
-ai
Suctedbd EcuAionana de TunnlOgia * tn/crmen*ars det Forav
( aaitia 4^99, Gtra*aoutl
EGYPT D» FQ DCS. A DtlWCtor t.r neral i.er.- «j
Assonatruo Againv Tubercukmm 19 Amia Sa«i> Street
Mantra. I aire
El SALVADOR • Il M '.R A. F ' s * Sq» F Z
Secretsctn General Pairomuo NacKitut Antitutercuioso
! I ( P 111, Sen saHador
ETHIOPIA M MOG» f-S AZBFTF Head Nauona
TuberiuKHrtC ontrol Progj am me Mmoiry erf Htkith
P.O. Boa 1234, Addis Ababa
FEDER Al KF Pl BI.K OF C.F.RMaN*
* . ■ • a.
Sectctan General Dam «c he a Zen talkmnUM ntf Beaampt.jog
'<
.">• ».
Pobpeohusenstraaae 14c 2V0U Hamburg •<
FINLAND
P FaNI Sturciati kienerai
Suomen Tuberkuk»o«n v astusumitytaitsiya r y
Kale*ankatu9 (HUGO Helsiaki 19
FRANC E Dr P Ot Rtl t A
t:o- (.ener-1
< •i(nne Natnrfi^i ant t a Iubercutosc e* les Mautdiot
66 bo U hr 1 a rd Salat Michel. 7Mo* Parrs
GAMBIA Mif
•>..•- M
■
i Hea>- Banjet
t.ERM AN DEMOCRATIC RFPtBLH
*•
Sekieto Gesellscnad tor Bionchopne^ix «.<.« una
' - Blkamsrr 1069 Warea 'Murrtx*
GHANA f he Nar«»nal • Hganszing S<s reiar, Ghtam Society for
r>. >' • .tnb.»
'_ P O Boa 2*»G2. Accra
GREEt E Dr ■> PoLY/:OGOPO< IOS Pnrndeni NaI...n..
As,. . ...
Agatrui l
H2 Mecogwa Street. Acheaa
Gl ATE MALA S.a I Ml NOZ PLAZA Secret a rut Ext u» a
I gaV*..n.ii nn ; . .'abcrcuki> 9A ( alle V ’ G-65.
Zona I. Guatemala ( A
l.lfNEA A* ...n F utx . . . *< M.n s»e> • !r a Same ( aaakF*
HAITI Or M K ' BIN GEt’RGFS Pte-oticnie A.MHx.ia(M>n
Hd'(< :nnm
i Fdber*ulnae Delmaa 31 Na Ml B P Pe?
Purr au Prince
HON DI R AS
HONGKONG M
». • F« ..
-.e.tri.. The Hrng
Kong Tuberculosis.. C bev A Haar D ^ tscs Aivxiiiwn
266 Queen t Road Eaaf. Hong Kong
Hl Nt, aRY
K . h ' ' ' L.ea . Sct e-.a-. Hung* an
Medical Assoc wuon for lubcrcuiuats «nd Puimcrwi, Doeaam.
Plheno I I RudaprM 1029
Illi AND Dt f B1ONDA1 «.MI Mwuititf.t
r Tn
Deparunen. o| Lung Diseases and (uDenutaxA
Baronaltg 4’ iut R»,kja*'k
INDIA Mr P n RaMAN v<xreUtfy (General
The Fubercukrsu Association of India
I Red ( ross Ruad New Befall 1IWOI
INDONESIA Dr AS GL NARDI he .'
l/>. . < The
Indonesian Tube'-ui> ,
• , . ...
JI Arco Raya 8 II.
Keniang Jakarta Selslao
IRAN Hr M * ABB ASM TKt« ANI x < ,u. ■ 4. «, . nt
Mi. m
Hejhh F ehet an
IRAQ V-it» I.eneral An- bcrcul. *.t and (. heal Dtgea->e*
Sik-cg
i.( 2. ay ton Hat A; Kindi, harkh P <» Sac 6191.
Mansour Baghdad
IRE I AND i» I i I AM V Due.
Peamoanl t best Hmpttai NewctuUte C • Dublin
ISRAEL Dt M Mt NOES P.r.deni An ’
.
*
r .. .
Ruppinnineet 14a. P C» Bee W24. Fel * • • •
ITALY Pi< 1 M I I i ' HE SI Scirt^i'i <>en<r«l F <• u- i. < n •
liubana donico va Titbcrcukrv e te Muhuisc ffotmunar* Vk.imJi,
V.m Eato 2 4 .M1M2 Roma
IVORY IOASI M«
TVNON M x Nl.Ot A p ?» i. •
( <mtnc AnlJtubc>cuieus de -a Cdtdd Howe
8 P V J.’l \ bid] an
JAPAN M
KONNO t
'
Th. j.-jp *n
x •
htkkafcu ' obo Uai
VI : Mitakkbo I < Kocnr (bi-alaKu lafc.-tdl
JORDAN Mr . I . RIF A 1 .k)
' a Sj- . ..
AfHiluftc uIi.s.h Xvh.- ■
■ '.•lim, PO But *G< Amaase
KENYA i'> I A 'Ll iK.
. »•
(he Pi
I o»
P O Bot 2U'*Sl Nairobi
kt WAIT Dr M AMRLTAWAB I>mw
AdmirHSira/uW. Ministry >j( PuBIk Health.
LEBANON Dr TOl Hi AL AW AR Secretaire General
Assooatxm Libanatw de Lum centre la rubercutose. Centre
Hespiialir-r dr Bfcanne*
LIBYAN ARAB JAMAHIRIYA fhe Acting Secretary
Central Committee of TB and Respiratory Diseases Secretariat
for Health
Dr A W KHALI! P O Box 2470. Benghazi
LlXFMBl RG Dr J GOEDER i President Ltgue
Luxembowgeowc-de Prevention et d'Action Medito-Sociales.
Centre Victor Hugo. 7 Av Victor Hugo,
Lux erabourr-Ltmpertsberg
M ADAGAM AR Chef Disiston f ubcrcufase. C omite Malagasy
ebittre la Tuberculosc. c/o I nstitut d‘Hygiene Seriate, Avenue
de la Rriinfan, Tananarive
MALAWI? Tuberculosis Department Ministry of Health,
P.O. Box 30577, Lilongwe 3
MALAYSIA: Mr NG KON CHEE. Secretary General.
Malaysian .Associauon for the Prevention of Tuberculosis
P.O. Box 684, Kuala Lumpur
MALL Dr A K SANG ARE. Secretaire General.
Comity AnuiubcrcuicuxduMali. BP 700. Bamako
MEXICO Sr E. ORELLANA MORA Director Ejccunvo
Comile Naonnal de Lucha contra M Tuberculosis
bandies 4‘h 2* piso. Apartado Postal No 5-471, Mexico 6 D.F.
MOROCCO: Prof M BOl’ZEKRL Prudent I .gue Mz^rne
cun tee b Tubcrculose 4, Boulevard Treble** B P 462, Rabat
MOZA.MBIQUE:ChefcdeStcijlo Se
a CooperacSo International. Minweno
C.P. 264. Maputo
NEPAL Mr DfcVENDRA B PRADHAN Secretary
Nepal Tuberculosis Association. P.O. Bo* 1494
Kahmati. Kathmandu
(fetor. Kaj
NETHERLANDS; D> MA _
Ncdtrbndse Centrals Vcreniging fat
Tufrercutosc. Pastbus 146. 2501 CC Den Haag
NEW ZEALAND: Dr J McLEOD. PreMiirgiiret Hospital. Christchurch
-NIL AR AG LA Jefe del Depwtamenm
Ministwb tfe Salud; Managua
NIGER: M ifl Ministrc de la Same. MinteUnj
dc.teSanr<i. Niamey
NIGERIA:Dr J A IDOW
Nrgdris Anutubereufasis >
P M B. 12611. Lagos
NORW AV; Secretary Gen
Tolkehclsen, Postbvks 712
O.M AN: Ministry of Heuli I
PAKISTANI Dr SAFE
Pgkfiioii N-aiionai Tuf»«u
Sjakamkrpura Peshawar City
PANAMA: Dr. f M(
Apartado
Organiiaeid ~
Panama 1
PVRAOLVVS, A FADLALA I'rcdentc.
Paragdaya Anutuberculbsa Avenlda Colin 282 (Ah
de Correo 625. Asunddn
PERI Or < MENOOZA EOWING
. Peruana de Tisiologfa. NeumbJogfa y Enfcrm
Domingo Casanova 116, Mnce. Lima
PHILIPPINES: Dr A G
Th.ePhilipptne TuberculostiSocteiy Ifw . Qucwn m.
Eulogvo Rodrigue* Ave (Espana Ext- >. Quezon Cit;
P.O Box 281. Manila
POLAND: Prof P KRaKOWKa. President, Soaetd Polonaise
du Phnsiopneumalogjc uL Pluck a 26. 01 118 Warszawa
PORTUGAL Dr DAS NEVES ALMEIDA. Serv^ de LuU
AntifabcLixiniw-SLAT Avenida 24de Juitw, Lisboa 2
REPUBLIC OF KOREA M. DAE RON KIM, Secretary
Gcmfcrai, The Korean National Tubdrculnsts AkjtoctaiKm-.
121-156 Dangsan Dung. Youngdeungpo-Ku Seoul
RUMANIA Prof ( AN AS I ASA ft.. President. Suadfa de
SAtDI ARABIA H J
Assistant Ducctei G
M.rmify <n Health. Riyad
SENEGAL Mi 8EYE. Previdem. Aviation Senegal,
de
Lutte Anlitubervuteuse Service de Fhtisiologie
HdfMUride Fann. Dakar
SINGAPORE Or N( SEN GUPT A. Medical Direcitii
I he Singapore AntHubcrcuiosis Association
267 L an ton men t Road. Singapore 0208
SOMALIA- The Advisor for Tuberculosis o Th- Mini
Health. Mwnslry of Heallh. Mogadiscio
SOI TH AFRICA Mr C H GRE ATHEAD. Exedurtw
Director. South African National Tuberculosis Associsik
Leisk House Cor Bree & Rissfk Streets P.O Bo*
Johannesburg
SPAIN. Dr J ROMERu O1AVARRlETA Sub-D
General. Ccntros Sanuanos y Asisifinaaies de lu a ES.N.
< alle Ventura Rodnguez 7 Madrid 8
SRI Lanka m
pd ionseka t.xecouve 5e<
Ceylon NatronaL Association far the Prevention ril Tuberc
5} Sir Marcus Fernando Road, Colombo 7
SUDAN Dr Ml MAHDI. Prcsidem N.A.PT Mfajstry of
Health. P.O Bo* 2101 Khartoum
SWEDEN: Prof A. HANNGREN Secretary Geoe-al Sweden
Notional Association Against Heart .ind Chase Dweusvs
Kungsgatan 54 S 11135, Stockholm
SW1TZER1 AND- Dr. B FAH. SecretaireC emial
Assochukin SutSMf comre la Tubercuioxe et les Maladies
Pldtnonau-es Fhchrrweg 9. Postfach 2246. 1001 Bern
SYRIAN ARAB REP1 BLK Dr x ARAFFH Prcvuiem
Comiie Synen de Defense contruia Tubeaulnse
B P 744 Damas
THAILAND 0r S
1AROEN. Ex*
Secfeiari, The a
FhuiwntJi
1281 Paholyothkn H
TOG0:DrOTiDJ
National
AnttwibBncoleux de h
B P. 7318
TRINIDAD AND FOBAt.O The Mim> e -I Hcuhh
csTHeaiih and F.n* .nmmem Port <rf Spain
FINISIA ,1 R BE NAMM,\« S
mircGend.i
Ligue Nauonaic vomre la Tubeccuiusc el les Maladies
20811 Y riana
e Griidml
Amow<jk»o Nauonaie
re la F uberculosc
SeHmr- Hatun.
UGANDA !
Minrsiry
HeaitF Bo* 16069. wan
rya, Kampala
ARAB EMI
rvulosis Dcparwnom
2 *3, Abu Dhabi
Ki ATKINSON
i tleart zfld Stroke Aasocration
rth Tavistock Squaw
N Z A NIA T he P'rcsttlen i
i abercufasfe. P 0 Bo* 2449
rm
Dares-Salaam
IRLGLAY Dr M
RE Prcsidente.
AntirubercuhMHt, I
Montevideo
i«n P4U Bzoadwa* New V or
AC. KHOMENKO
Soviet Phthisiologists.
berculosls
Hute of the Health Ministry of the LS-SR,
kva 107564
:Dr P ntiRBE, Prcsfttome, l-cdoi^dfto
nes A<nnubi’«rUlt«f8i. de, Venezuela. Fdificle Santa
Anav Av 2. 95-25 lAnieset MHagm). Apt 74, Maracaibo
VIETNAM Dr ND HUONG Duv.mui liwwtiefa
Tuberculosc•. Avenue Hoang Hoa Tham Hanoi
YEMEN Dr Y ADDS AM Director Nanonut Tuber
C cmiroi Programme, Ministry of Health. Sana'a
YLGOSLAVIa Prim Dr M Mi..............
Pneumo ptiUSiologie Yugoskve. Institutza Piuciw
Boi i lubcrkulozli, 21204 Nori Sd
ZAIRE Dr s MBI ' F HANDf ND A President. LtgUC
Nationalt AnBiubercuteu-a H P IU6. Kinshasa I
ZAMBIA Head of fuborculoMS Department
PO Bo* W2U3. Lusaka
BaBWE- National Secretary, RAPT National
P.O Box 2166 Bulawayo
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