RMl
no. 916
1968
COLLECTION,
HANDLING &
SHIPMENT
y
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE/public health service
•"W
COLLECTION, HANDLING, AND SHIPMENT
OF MICROBIOLOGICAL SPECIMENS
JOHN E. FORNEY, PH.D., Editor
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
Public Health Service
HEALTH SERVICES AND MENTAL HEALTH ADMINISTRATION
NATIONAL
COMMUNICABLE DISEASE CENTER
Atlanta, Georgia 30333
7 ^
\ ^
This is a revision of a similar publication
entitled “Collection, Handling, and Shipment
of Diagnostic Specimens” previously issued
under the same publication number.
Public Health Service Publication No. 976
First Printed December 1962
Revised November 1968
For sale by the Superintendent of IJocuinents, U.S. Cioveriunont Printing Ollke
Washington, U.C. 20402 - Price 60 cents
UNITED STATES GOVERNMENT PRINTING OFFICE
WASHINGTON, D. C. : 1968
FOREWORD
A reliable laboratory report as an aid in the diagnosis of disease depends upon the care and thought used in
the collection, handling, and transport of specimens. T oo often physicians and other medical or public health
personnel are only generally familiar with the problems and procedures involved in obtaining and submit-
ting material for laboratory examination.
The objective of this manual is to present procedures which, in the opinion of the staff of the National
Communicable Disease Center — who authored the various sections — have been found to be practical and
productive. Other methods may be equally satisfactory, however, and may, at times, be substituted for
those described. In any case, whenever laboratory examinations are to be carried out in local or State
laboratories, methods acceptable to these laboratories should be used.
It is of utmost importance that the purpose of a procedure for handling a specimen be kept in mind. Thus,
for example, the safest and most expeditious method of transporting specimens to a diagnostic laboratory
may be by automobile rather than by mail or express, thus obviating the need for elaborate packaging. In
contrast, forwarding specimens to a reference library by mail requires procedures which insure viability of
the infectious agent and provide maximum protection to those handling the shipment in transit.
Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the
U. S. Department of Health, Education, and Welfare.
"" 't' ''U A
CONTENTS
Page
Laboratory Services at the National Communicable Disease Center 1
Procedures For Collection, Preparation, And Shipment Of Microbiological
Speciments 2
GENERAL INSTRUCTIONS 2
1. Basic Principles 2
2. Infectious Diagnostic Specimens 2
3. Identification of Specimens 2
4. Packaging of Specimens 2
5. Shipment of Specimens 3
6. Criminal Statute (18 USC 1716 ) Pertaining to Shippers of Diagnostic Materials 3
BACTERIOLOGICAL, MYCOLOGICAL, AND PARASITOLOGICAL SPECIMENS 3
1. Bacterial Specimens 4
2. Mycological Specimens 4
3. Parasitological Specimens 4
VIRAL AND RICKETTSIAL SPECIMENS 4
MISCELLANEOUS SPECIMENS 5
Directions For The Collection Of Specimens For The Laboratory Diagnosis Of
Certain Bacterial, Mycotic, Parasitic, Arthropod-borne, Viral and Rickettsial
Diseases 6
BACTERIAL DISEASES 6
1. Anaerobes (diseases due to) 6
2. Anthrax 7
3. Brucellosis 7
4. Diphtheria 8
5. Enteric Infections 8
a. Typhoid Fever 8
b. Salmonellosis 9
c. Shigellosis 9
d. Enteropathogenic Escherichia coli 9
6. Gonorrhea 10
7. Hemolytic Streptococcus Infections 10
8. Hemophilus Infections of the Pharynx and Conjunctiva 12
9. Leptospirosis 12
10. Meningitis 12
11. Plague 13
12. Syphilis 13
13. Tuberculosis 15
14. Tularemia 16
MYCOTIC DISEASES 16
1. Cutaneous Fungus Infections 16
2. Subcutaneous Fungus Infections 17
3. Systemic Fungus Infections 17
a. Actinomycosis 17
b. Coccidioidomycosis 18
c. Histoplasmosis 18
d. Fluorescent Antibody (FA) Staining 19
PARASITIC DISEASES 19
1. Blood Parasites 19
2. Intestinal Parasites 22
V
CONTENTS— Continued
Page
ARTHROPOD-BORNE DISEASES (SPECIMENS FOR IDENTIFICATION OF ARTHROPODS) 27
1. Ectoparasites 27
2. Flies and Mosquitoes 27
3. Arthropod Specimens Submitted for Virus or Rickettsia Isolation 27
VIRAL AND RICKETTSIAL DISEASES 30
1. Preparation and Shipment of Diagnostic Specimens 30
2. Minimum Data to be Supplied With Specimen 31
3. Laboratory Methods in Diagnosis 32
4. Interpretation of Laboratory Results 32
5. Packing and Shipment of the Specimens for Rabies Diagnosis 33
TABLES
Table
1. Location of Parasites or Their Diagnostic Stage Within the Body 19
2. Immunodiagnostic Tests for Parasitic Infections 23
3. Preserving and Handling Insects and Other Arthropods 27
4. Diagnostic Procedures for Viral and Rickettsial Diseases 34
FIGURES
Figure
1. Properly and Improperly Prepared Blood Films 21
2. Parasitological Examination of Stool Specimens 24
3. Use of Cellulose-Tap>e Slide Preparation for Diagnosis of Pinworm Infections 26
4. Arthropods of Medical Importance 28
5. Methods of Collecting Live Insects 29
APPENDICES
Appendix
I Postal Requirements for Shipping Diseased Tissues and Other
Spiecimens 36
II Selected Transport Media and Reagents 38
III Acceptable Containers for Use in Shipping Specimens 40
IV Reference Diagnostic Services Available at the National Communicable
Disease Center 41
V Request for Viral and Rickettsial Reference Service (P.H.S.
Form 3.332) 43
VI
Laboratory Services at the National Communicable Disease Center
The laboratories of the National Communicable Disease Center (NCDC) serve as a national reference or
consultative facility and accept specimens from only the State public health laboratories and Public Health
Service facilities. Specimens which cannot be examined locally should be sent to the State laboratory where
they may be processed or, if the requested service is not available at the State level, the State laboratory
director may then forward either the original specimen or the pure culture to the NCDC. Certain services
available at NCDC are of value in epidemic situations only, and submission of single cultures from isolated
cases would serve no useful purpose. Requests for serotyping of Group A streptococci and phage typing of
Salmonella, Shigella, or staphylococci from such cases serve to illustrate the point. Acceptance of diagnostic
specimens from private physicians or institutions and local health departments is not authorized.
The volume of specimens submitted for virological examination has increased to the point that it is impos-
sible for NCDC to examine routine specimens and, at the same time, provide the support needed for de-
velopment of virological facilities within the State laboratories and assist the States in the solution of their
epidemiologic problems. Because assistance to the States in the above matters is of major importance and
a basic responsibility of NCDC, the resources of NCDC virology laboratories must be directed to these
ends. Therefore, the Laboratory Program limits its acceptance to specimens having epidemiologic im-
plications.
Since virology is still a rapidly developing science, diagnostic problems may occasionally arise which require
assistance from the NCDC laboratories even though the case has no immediate clear-cut epidemiologic im-
plication. In such instances, the State laboratory director should consult with the Chief of the Virus Reference
Unit at the NCDC.
From the viewpoint of the practicing physician, this policy should work no hardship since most virological
diagnosis is retrospective and of little or no value in the treatment of the individual patient.
1
Procedures for Collection, Preparation, and Shipment of Microbiological Specimens
GENERAL INSTRUCTIONS
1. Basic Principles. In obtaining and submitting
diagnostic specimens, basic principles should be
observed. Principles applicable to specimens
submitted for isolation and identification of the
etiologic agent include:
a. Select appropriate specimens.
b. Obtain specimens as early in the illness as
possible.
c. Identify the specimen properly.
d. Request specific test(s) desired or indicate
etiologic agent suspected.
e. Protect viability of organisms during transit.
Other principles applicable to specimens sub-
mitted for serological examination are:
a. Collect blood and serum samples in sterile
tubes and maintain sterility in handling. Use
leakproof stoppers on all tubes.
b. Collect paired specimens at appropriate in-
tervals because, with a few exceptions, they
are desirable and because, in suspected viral
infections, they are mandatory. Since a rise
in antibody titer is a more reliable diagnostic
sign than the mere presence of antibodies,
the first specimen should be collected as
early after onset as possible and the second,
usually two or three weeks later.
c. Protect whole blood from freezing under
any circumstances.
In general, specimens submitted as smears for
examination should:
a. Be allowed to dry completely.
b. Be packed properly to prevent breakage.
2. Infectious Diagnostic Specimens. Rapid trans-
port of infectious diagnostic specimens is of vital
importance in communicable disease control.
Fortunately, no regulations hamper such move-
ment as long as one follows the rigid Postal
Regulations which pertain to the preparation of
the specimen for shipment.
The NCDC defines infectious diagnostic speci-
mens as:
a. All specimens of human or animal excreta,
secreta, tissue, tissue fluids, or hair which
contain or are suspected of containing the
live causative agent of a human disease or
an animal disease transmissible to man, and
which are shipped or mailed to a diagnostic
or research laboratory for isolation and
identification of the etiological agent.
b. Pure cultures or concentrated isolates or
vectors of etiological agents shipped from
the isolating or collecting laboratory to a
specialty laboratory for identification and
typing, or further research, or both.
c. Pure cultures of known etiological agents
which are used as reference cultures or as
antigens in diagnostic laboratory procedures.
3. Identification of Specimens. Identify individual
specimen tubes or containers by encircling them
with typed or penciled legends on adhesive tape.
Give patient’s name, type of specimen, and date
of collection. This is particularly important with
clear fluids and paired sera. Ink, hall-point pen,
wax, or indelible pencil should not be used be-
cause writing done with them becomes illegible.
Include with the shipment a legible copy of a list
of the specimens giving identifying name or
number, date obtained, and tests desired.
4. Packaging of Specimens. Proper packaging not
only protects the specimen in transit but also
the personnel handling it. Protection is espe-
cially important in the case of breakage.
Never Mail Viable Specimens in Petri Plates.
Never Enclose Dry Ice in
Hermetically Sealed Containers.
A safe packaging procedure which complies
with Public Health Service Regulations and the
requirements of the Universal Postal Union
follows:
a. Enclose the specimen in a bottle or tube of
thick glass sealed with a rubber stopper or
2
paraffin-treated cork. Bottles are preferred
because of their greater shock resistance, but
heavy-walled tubes are acceptable if prop-
erly packed. Closure by fusion is also accept-
able.
b. Place the glass container in an airtight and
watertight tin to absorb any leakage, pack
absorbent cotton, vermiculite, sawdust, or
other suitable absorbent material around the
glass container.
c. Pack the can in a cardboard container with
crumpled newspaper or other shock-resisting
insulating material and wrap for shipment.
d. Individual tubes must be shipped in con-
tainers providing sufficient space for shock-
absorbing material all around the tube. If
several tubes are packed in the same can,
wrap them individually in absorbent material
such as soft paper or cloth to provide ade-
quate cushioning between the tubes.
e. SCREW-CAP TUBES ARE NOT RECOMMENDED
for blood, serum, or other fluid specimens
because leakage frequently occurs, particu-
larly when outside pressure decreases during
air transportation. Screw-cap tubes are ac-
ceptable for agar or similar cultures.
f. Bottles or tubes for specimens should be of
hard glass and of 2 ml. or larger capacity.
g. Screw caps should have a resilient, sealing
gasket or insert and should be secured by
tape.
h. When screw-capped jars are used for ship-
ping larger specimens, the resilient cap lining
should achieve airtight and fluid-tight clos-
ure, and the cork or cap should be secured
in place with a metal collar or adhesive tape.
Gas-forming cultures of yeast are an excep-
tion to this rule.
i. Regular #3 household cans or pressure-
sealed paint cans are useful for shipment of
bottles or multiple specimens. Household
cans should be sealed by roll crimping the
lid with a home-canning device. Pressure-
sealed paint cans have the advantage of not
requiring a crimping device. The large sized
paint cans are practical for large quantity
shipments and may be used as outer con-
tainers, as required by the Convention of the
Universal Postal Union.
5. Shipment of Specimens.
a. Mark shipments with “Perishable,” “Packed
in Dry Ice,” “Refrigerated Biologic Ma-
terial,” “Fragile,” or some other suitable
designation. Standard labels should be used
if available.
b. For long distances, ship all specimens by
air mail or air express. Air freight should not
be used when speed is essential. If possible,
assure that the shipment is given priority
over nonperishable items.
c. Specimens submitted to the laboratory
should be accompanied by an appropriate
report form giving name and age of patient,
source of specimen, disease suspected, brief
statement of clinical symptoms, and, in the
case of cultures, tentative identication.
d. Delay may be avoided by addressing ship-
ments to the laboratory unit involved; for
example, “Virus Reference Unit,” “Enteric
Bacteriology Unit,” “Venereal Disease Re-
search Laboratory,” etc.
e. Shipment of specimens should be timed so
that they will not arrive at the laboratory on
or just before a weekend or holiday; this
will help avoid possible deterioration.
f. In some localities, surface mail, bus or rail-
way express may be faster than air transport;
but, in any case, the most rapid method
should be used.
6. Criminal Statute (18 USC 1716) Pertaining to
Shippers of Diagnostic Materials. This statute
is of interest to all shippers of diagnostic ma-
terials whether the materials are potentially
pathogenic or not. Even if spillage occurs from
non-pathogenic materials but injures or dam-
ages mail, equipment, or personnel, the shipper
may face prosecution even though there is no
question of hazard from an infectious agent.
The value of meticulous packaging, with suffi-
cient absorbent material around the specimen to
prevent fluid leakage, extends well beyond the
major concern of preventing accidental infec-
tion.
BACTERIOLOGICAL, MYCOLOGICAL,
AND PARASITOLOGICAL SPECIMENS
For the purpose of this manual, the significant differ-
ence between primary specimens and cultured bac-
teria and fungi is that primary specimens are usually
shipped intrastate only, whereas most shipments to
NCDC or other out-of-State reference laboratories
consist of pure cultures. The same general rules and
principles covering handling and shipment of pure
3
cultures also apply to specimens obtained from the
patient or any other source.
1. Bacterial Specimens. Agar slant or stab cul-
tures, using freshly prepared media free of
excess moisture, are most practical. Stab cul-
tures are best for anaerobes. If the bacteria have
low shipping tolerance (as does Neisseria, for
example), fresh and relatively heavy growths on
either blood agar or brain heart infusion slant,
moistened with a drop of blood, or a chocolate
agar slant should be forwarded. The tube should
be closed with either a rubber stopper or
paraffin-treated cork secured in place with ad-
hesive tape. A screw-cap tube is also acceptable,
provided the cap is wrapped and sealed with
tape. The tape should be applied in the direction
of the threads to avoid loosening the cap. Viable
cultures should NEVER be shipped in Petri
plates.
2. Mycological Specimens. Yeast cultures which
develop gas are the single exception to the rule
that specimens should be placed in tubes or
bottles sealed with rubber stoppers or paraffin-
treated corks. In this instance, the culture tube
should be plugged with a dry, nonabsorbent,
tight fitting cotton plug, long enough to extend
into the tube about one inch. This plug should
be held in place with adhesive tape in such a
manner that gases will escape, but the plug will
not work loose. Slant cultures on firm agar are
preferred over stab cultures. Other fungal cul-
tures must be shipped only in sealed tubes or
bottles.
3. Parasitological Specimens. Stool specimens to
be examined for intestinal protozoa should be
submited in two parts: one in polyvinyl alcohol
(PVA) fixative solution, the other in formalin.
In suspected cases of filariasis or trypanoso-
miasis, whole blood should be heparinized and
sealed in a tube as for bacterial specimens. It
may be necessary to submit samples of arthropod
ectoparasites in two portions: one for identifica-
tion, the other for viral or rickettsial isolation.
.Mosquitoes and flies should be sent to the
laboratory unmounted, preferably in pill boxes
between layers of cleansing tissue but never
between layers of cotton. If viral or rickettsial
isolation is to be attempted, the arthropod speci-
mens must be collected alive, sealed in ampules,
and stored and shipped on dry ice. Cyanide or
chloroform jars must not be used because viral
and rickettsial agents are inactivated by these
materials.
NOTES: Special precaution in regard to safe
packaging is required when shipping
the etiological agents of especially
dangerous diseases such as plague,
cholera, anthrax, tularemia, and coc-
cidioidomycosis. Label tubes or bot-
tles containing such organisms with
precautionary labels and enclose
them in double metal containers.
In some instances, reference cultures
of bacteria are maintained either in
the sand desiccated or lyophilized
state. Such cultures are shipped in the
same manner as agar cultures.
VIRAL AND RICKETTSIAL
SPECIMENS
If isolation of a virus is to be attempted, the source
of the specimen should be carefully selected, and the
specimen should be obtained during the early, acute,
febrile phase of illness.
Depending on the circumstances, the material for
virus isolation may be either nasal or throat washings,
sputum, feces, cerebrospinal fluid, scrapings, aspira-
tions from lesions, or tissues from autopsies. Blood,
spinal fluid, and tissue should be handled aseptically.
Isolations of rickettsia may be obtained from whole
blood. Unless soecimens for virus isolation can be
delivered to a laboratory within three hours, it is
mandatory that they be frozen and kept frozen during
shipment. An exception is a specimen suspected of
containing respiratory syncytial virus. If freezing is
impossible, tissue specimens may be placed in
buffered glycerin for transport; but the results may
be equivocal. This procedure is impossible with body
fluids.
Possible delays in transit necessitate the use of
sufficient dry ice in the shipping container to insure
freezing for 48 hours in excess of the normal transit
time.
Since changes in atmospheric pressure during air
shipment may force stoppers from tubes, resulting in
loss of the specimen, only tight fitting corks or soft
rubber stoppers should be used; and, in any case, the
cork should be anchored with adhesive tape. When a
highly infectious disease such as psittacosis or small-
pox is suspected, several layers of gauze soaked in
4% formalin should be wrapped around the tube or
bottle containing the specimen before it is placed in
a metal, leakproof shipping case. Substitution of
10% cresol solution for the formalin is permissible.
Specimens for serologic tests indicative of viral in-
4
fection must be taken aseptieally, and 10 to 15 ml.
of blood should be drawn. Paired specimens are es-
sential to diagnosis as a rise in specific antibody titer
in the course of illness and convalescence is the only
definitive serologic evidence of current infection. The
first specimen should be taken during the acute phase
of illness and the second, two to four weeks later,
although the optimum times for the collection of
specimens vary with the disease. After the blood has
clotted and the serum has separated, remove the
serum promptly and aseptieally, and immediately re-
frigerate or freeze it. Do not add a preservative, as
this would destroy the serum’s usefulness.
MISCELLANEOUS SPECIMENS
1. If bacterial or fungal serology only is desired,
draw 10 ml. of blood. This amount wiU yield
sufficient serum because of the smaller number
of antigens used in the tests.
2. Sera intended for only bacterial tests or fungal
serology may be preserved by adding merthiolate
to make a final concentration of 1 : 10,000 (0.01
ml. of 1% merthiolate to each ml. of serum).
3. When an infection has been treated with an
effective antibiotic, antibody development may
be suppressed or delayed, and, in such cases, a
third blood specimen taken late in convalescence
may be helpful.
4. A presumptive diagnosis on the basis of a high
serologic titer in a single convalescent blood
specimen may be possible. This procedure is
most often useful in confirming an epidemic
situation where paired specimens are also avail-
able from a significant number of individuals.
This is also true in cases of rare diseases such
as glanders, typhus (not Brill’s disease), or
yellow fever, in which previous exposure is only
a remote possibility.
5
Directions for the Collection of Specimens for the Laboratory Diagnosis of
Certain Bacterial, Mycotic, Parasitic, Arthropod-Bome, Viral and
Rickettsial Diseases
In any epidemic situation involving the collection of
specimens for laboratory examination, the epidemi-
ologist should consult the director of the laboratory
where the work is to be done to make sure that: (a)
the tests to be requested are available, (b) the cul-
ture media and reagents required will be ready when
needed, and (c) the collection and submission of
specimens is scheduled at a rate that will not overtax
the facilities.
BACTERIAL DISEASES
1. Anaerobes (diseases due to). The major areas
of public health interest in which anaerobic
bacteria are implicated are: 1 ) wound infection,
2) food poisoning outbreaks due to Clostridium
botulinum, 3 ) food poisoning due to Clostridium
perfringens, 4) tetanus, and 5) infections with
non-sporeforming anaerobic bacteria. Specimens
may be as diverse as food, blood, tissue, cere-
brospinal fluid, or materials from wounds, ab-
scesses, and serous cavities. Culture and toxin
testing of food samples should be attempted in
a local laboratory only if the personnel are ex-
perienced in anaerobic techniques. Otherwise,
the specimen should be forwarded to the near-
est competent reference laboratory. Certainly,
however, a smear of the original food specimen
should be made for Gram staining since study
of this smear will give the laboratorian a census
of the relative numbers and different kinds of
bacteria present. Such information may be im-
portant in concluding whether a presumptively
significant organism was recovered during
culture.
a. Food specimens for isolation of Cl. botu-
linum and/ or toxin
Specimens should be collected and main-
tained at the same temperature as in the
natural state for transport to the laboratory
or shipment to a reference laboratory. Lab-
oratory testing consists of detection and
typing of Cl. botulinum toxin in the food as
well as isolation and identification of the
causative organism.
b. Food specimens for enumeration of Cl.
perfringens
Quantitation of Cl. perfringens from food
should be performed in a local laboratory as
soon as possible after the food is collected.
If the material must be shipped, it should
be refrigerated at 4°C. and maintained at
this temperature during transport. At 4°C.,
some organisms will die, thus causing lower
counts. This fact should be kept in mind in
interpreting counts. Freezing of specimens
drastically lowers counts, and shipment with-
out refrigeration permits multiplication of
the organisms, thus producing unusually
high counts.
c. Specimens from human sources
Clinical material must be cultured immedi-
ately after collection since some anaerobes
die very rapidly upon exposure to oxygen
in a nonprotective environment. Where
possible, aspirated fluids arc preferable to
swabs. Never allow material on swabs to
dry out. If a specimen cannot be cultured
immediately, it is helpful to place the ma-
terial in a medium containing a reducing
6
agent, such as fresh thioglycollate broth, at
room temperature for a period not exceed-
ing two hours.
A Gram stained smear should be prepared
on all specimens, and each type of organism
seen in smears should be isolated in pure
culture at the local laboratory. Pure culture
isolates can be shipped to a reference labora-
tory for final identification. Thioglycollate
medium, enriched with 10% normal rabbit
serum, is preferable for isolation of the non-
sporeforming anaerobes, and chopped meat
medium (Appendix II) is preferable for the
sporeforming anaerobes. It is important to
plate specimen material directly on fresh
blood agar plates since some fastidious an-
aerobes may be overgrown by other organ-
isms in broth medium. Broth cultures are
incubated 24 hours, preferably in an
anaerobe jar; Gram stains are prepared, and
a second set of blood agar plates is inocu-
lated. Blood agar plates should be incubated
in an anaerobe jar for a minimum of 48
hours. Isolated colonies should then be
picked to chopped meat or thioglycollate
medium for isolation of pure cultures.
For shipment of pure cultures to a reference
laboratory, actively growing cultures in
screw-cap tubes of chopped meat medium,
thioglycollate medium, or semi-solid thio-
glycollate medium with 0.5% added agar
can be used. Prior to shipment, a one-half
inch column of sterile 5% agar should be
layered over the culture to act as a seal, and
the cap should be sealed with waterproof
tap>e.
2. Anthrax. Anthrax is primarily an infectious
disease of animals; however, man may be in-
fected through contact with infected animals or
animal products. The majority of cases are cu-
taneous infections, but pulmonary and intestinal
infections may be seen. “Industrial anthrax”
occurs in workers exposed to carpet wools,
goat hair, or skins originating in areas where
the disease is prevalent among animals. Labora-
tory diagnosis is made by smear examination,
culture of the organism, animal inoculation, or
fluorescent antibody technic.
Specimens for laboratory diagnosis
a. Smears may be made from sputum, blood,
other fluids, or tissues, and then air dried
and heat fixed.
b. Blood, vesicle fluid, and scrapings from the
base of the lesion, regional lymph nodes, or
other organs, taken in as clean a manner as
feasible, may be cultured on plain or blood
agar or inoculated into animals.
c. Tissue impression smears, sputum, other
clinical material and cultures may be stained
with fluorescent antibody for specific identi-
fication of the organism.
d. Aqueous extracts of soil samples, heat-
treated to destroy vegetative cells, may be
cultured or used for animal inoculation.
All types of examinations should be made
in the nearest competent laboratory because
delay in the shipment of fluids or tissues may
result in contamination of the specimen and
death of the organism.
3. Brucellosis. This disease is found primarily in
goats, cattle, and swine. Man contracts the
disease either by direct contact with diseased
animals or through consumption of infected
milk and milk products. Although the three
organisms. Brucella melitensis, Brucella abortus,
and Brucella suis, most often infect goats, cows,
and pigs, they are not host specific, and all in-
fect man as well. The disease occurs in acute,
subacute, and chronic forms; and all three types
are produced by any of the species of Brucella.
Infection occurs most frequently by direct in-
vasion of the organism through the intact in-
testinal mucosa; but stockyard workers, farmers,
and veterinarians are often infected through the
skin by direct contact with living or dead tissues
of infected animals. Laboratory diagnosis is
made by agglutination tests, animal inoculation,
or recovery of the organism in cultures.
a. Specimens for isolation of the agents
Draw 10 to 15 ml. of blood directly into a
“B-D” blood culture medium bottle and
send it to a local laboratory for incubation.
Due to the intermittent appearance of or-
ganisms in the blood stream, it may be
desirable to take additional specimens at in-
tervals of several hours or days, depending
on the condition of the patient.
b. Specimens for serologic tests
Since an agglutination titer of about 1:100
or higher is believed indicative of brucellosis,
successive blood specimens taken three to
six weeks after onset of illness are con-
sidered sufficient for diagnostic purposes.
7
4. Diphtheria. Specimens for diphtheria should be
collected from both the nose and throat. Two
sterile swabs should be used for each person
cultured, one swab to obtain a specimen from
the throat lesions or tonsillar crypts and the
other to collect materials from the nasopharynx.
Swabs should be kept separate during transport
to the laboratory.
Specimens for isolation of the agent
To obtain nasal and throat specimens, the swab
should be introduced into the nares at a right
angle to the plane of the face, inserted com-
pletely into the nasopharynx, and rotated over
the pharyngeal surface near or in the tonsillar
fossa. Because a nasal membrane is a particu-
larly good source of organisms, the swab should
be placed at the margin of the membrane, if
present, and the specimen taken without exces-
sive bleeding.
Preferably, the initial isolation of organisms
suspected of being Corynebacterium diphtheriae
should be carried out in a competent laboratory
near the source of the specimens. If it will re-
quire more than two hours to get the specimens
to the laboratory, they should be inoculated to
Loeffler’s slants and incubated overnight before
transporting them. The nasopharyngeal and
throat swabs should be rotated gently over the
surface of separate slants of the medium. Care
should be taken not to break the surface of the
medium. The swab should then be placed in a
separate tube to be sent to the laboratory with
the slant.
On arrival in the laboratory the inital swab is
inoculated to a Loeffler’s slant, a tellurite agar
plate, and a blood agar plate. If the specimen
is received on Loeffler’s medium, a smear is
made for microscopic examination, and a rep-
resentative sample of the growth is inoculated
to tellurite and blood agar plates. The use of
blood agar is necessary to demonstrate the pres-
ence of Group A streptococci, or strains of C.
diphtheriae which may be inhibited on the tel-
lurite medium.
In carrier surveys, both swabs may be streaked
gently and repeatedly over the surface of a single
slant. Be sure to rotate the swabs between the
thumb and forefinger as the swabs arc drawn
back and forth over the medium. Streaking of
tellurite plates with the initial swabs taken in
carrier surveys is of little advantage. On occa-
sion, inhibitory lots of Loeffler’s medium have
been encountered due to the presence of anti-
biotics in the serum from which the medium
was prepared. Precaution should be taken,
therefore, to make sure that all lots of the
medium to be used will support growth of C.
diphtheriae. Diagnosis from smears made
directly from the patient should never be at-
tempted since many other organisms morpho-
logically resembling C. diphtheriae occur in the
normal or diseased throat.
5. Enteric Infections. In suspected cases of ty-
phoid fever and salmonellosis, specimens sub-
mitted for culture include blood, feces, and
urine. Rectal swabs may be obtained by using
an ordinary cotton-tipped applicator. Blood
serum for agglutination tests is not recom-
mended because such tests provide little or no
significant information. Blood cultures and ser-
ologic tests are not done in shigellosis. The mul-
tiplicity of fluids suggested as transport media
for specimens to be cultured indicates that none
is ideal nor markedly superior to others, and,
therefore, isolation of the agent should be at-
tempted in the nearest competent laboratory.
a. Typhoid fever
Specimens to be collected for isolation of
Salmonella typhi
( 1 ) Blood taken early in the course of ill-
ness is the specimen of choice, but the
probability of recovery of the organism
decreases rapidly after 7 to 10 days of
illness. Ten to 15 ml. of blood
should be drawn directly into a “B-D”
blood culture medium bottle (the vol-
ume of broth should be at least 10
times the volume of blood) and should
be examined for growth after 3, 5, 7,
and 1 4 days’ incubation. If the blood is
not drawn directly into the culture
medium, an anticoagulant should be
added.
(2) Stool examination is accomplished by
adding approximately two grams, or a
portion the size of a small marble, of
formed stool to one ounce of Sachs
30% glycerol in buffered physiological-
saline in a two-ounce, screw-cap bottle
or “alkaselzer” bottle. Shake the bottle
vigorously to emulsify the specimen
before shipment. If the stool is liquid,
add 2 ml. of the specimen to the pre-
servative in the bottle. Be sure to in-
clude in the specimen any bits of
mucosa or mucus present in the stool.
8
Before shipment of the specimen, make
sure there is no leakage from the bottle.
Amies’ modification of Stuart’s trans-
port medium may be used.
(3) A “midstream” specimen of urine is
collected as cleanly as possible, after
cleansing the genitalia with soap and
water and drying the area. Place the
specimen in a tightly closed container
for immediate delivery to the labora-
tory. In typhoid fever, stool cultures
are more often positive than urine cul-
tures.
(4) Specimens of food suspected of con-
taining Salmonella typhi should be
placed in ice cream cartons or other
suitable containers and immediately
refrigerated. During transport to the
laboratory, refrigeration must be main-
tained.
b. Salmonellosis
Food poisoning may be caused by many
members of the Salmonella group. Prelimi-
nary epidemiological investigation should
reduce to a minimum the number of sus-
spected foods likely to be responsible for any
given outbreak, and indiscriminate collec-
tion of samples is unwarranted. Such in-
quiries will indicate the food consumed in
common by those made ill, and, although
such evidence is not infallible, it is at least
presumptive.
Specimens to be collected for isolation of
the agent
( 1 ) If food from sealed containers is sus-
pect, unopened containers of the same
production lot should be submitted for
testing. Also, representative samples of
the suspected food should be trans-
ferred to a sterile sample bottle or ice
cream carton and refrigerated during
transit to the laboratory.
(2) Blood specimens for culture from pa-
tients in food poisoning are of limited
value.
Blood cultures may be of value in the
severe enteric, typhoidal, and septi-
cemic types of salmonellosis. In such
cases 10 to 15 ml. of blood may be
drawn into a tube containing an anti-
coagulant or into a bottle of blood
culture medium, as for typhoid fever.
(3) Fecal specimens, if obtained early dur-
ing the acute stage of the disease, are
the specimen of choice and should be
collected as for typhoid fever. If the
specimens are to be mailed to the lab-
oratory, they should be handled in the
same manner as fecal specimens to be
examined for 5. typhi.
(4) Serologic studies on the sera of patients
are not indicated since the results may
be equivocal and impossible to inter-
pret.
c. Shigellosis
Numerous members of the dysentery group
of organisms are responsible for enteric dis-
ease. Their isolation is not difficult, and
identification is accomplished by biochem-
ical and serologic studies.
Specimens to be collected for isolation of
the agent
( 1 ) Blood specimens should not be sub-
mitted for cultural examination in
suspected cases of shigellosis.
(2) Fecal specimens should be submitted
for attempted isolation of the infecting
organism at any stage of illness, but
specimens will yield more successful
isolations if they are obtained in the
acute phase of the disease. Examina-
tion of a series of fecal specimens is
important because the appearance of
the organism in the stool may be in-
termittent. The specimen should be
handled as one for typhoid fever.
(3) Rectal swab specimens are sometimes
the most convenient specimens from
which to attempt recovery of dysentery
bacilli. This is especially true when ex-
amining inmates of institutions, hos-
pitalized patients, or infants and chil-
dren. Preferably, culture plates should
be inoculated immediately after the
specimen is taken, but the plates must
be hand-carried to the laboratory for
incubation; otherwise, the specimens
should be transported to the laboratory
as promptly as possible by placing the
swab in Amies’ modification of Stuart’s
transport medium.
d. Enteropathogenic Escherichia coli
A variety of agents may cause the diarrheal
diseases of the newborn and infants. Thus,
epidemics and sporadic cases of “summer”
diarrhea, infantile enteritis, or diarrhea of
9
the newborn may be caused by Salmonella,
Shigella, or viruses. Many otherwise unex-
plained epidemics of infantile diarrheal dis-
ease in which certain serotypes of Escher-
ichia coli were recovered have, however,
occurred. For these particular serotypes,
the term enteropathogenic E. coli, or EEC,
is commonly used. In order to control the
spread of infantile diarrhea, the causative
organism must be accurately and rapidly
identified.
Earlier attempts at differentiation of the
enteropathogenic coli strains were unsuc-
cessful because only biochemical methods
were employed and, as is now known, differ-
ent E. coli serotypes produce identical bio-
chemical reactions. These biochemically
similar organisms fall naturally into certain
groups, and serologic methods must be used
to determine the serotypes within each bio-
chemical group.
The determination of E. coli serotypes is
dependent on the determination of the “O,”
“B,” and “H” antigens of the bacterium.
The E. coli responsible for the disease can
be distinguished from the E. coli constitut-
ing the normal intestinal flora only by use
of specific antisera.
Specimens to be collected for isolation of
the agent
Fecal material should be collected either
as a stool or on a rectal swab before anti-
biotic therapy is begun. The stool specimen
or rectal swab should be placed in a screw-
cap vial containing 30% buffered glycerin-
saline solution, or (if there will be more
than a two-hour delay before it can be
planted in the laboratory) on Amies’ mod-
ification of Stuart’s transport medium.
Specimens for enteropathogenic E. coli di-
agnosis by FA technic.
Specimens preserved with any fluids con-
taining glycerol are not satisfactory for FA
staining. A separate buffered swab (as sup-
plied with Amies’ transport medium) should
be sent to the laboratory in a separate tube
containing no preservative or medium.
6. Gonorrhea. Difficulties in the laboratory diag-
nosis of gonorrhea are increased when the
clinician fails to exercise care in securing suit-
able exudates for examination. This is especially
true in chronic gonorrhea of women and in “test
of cure’’ where only minimal numbers of gon-
ococci may be present in the exudates obtained
from endocervical, Skene’s, and Bartholin’s
glands. The exudate is taken with sterile cotton-
tipped applicator sticks; the cotton tip should
be small enough to enter easily the urethra and
cervical os. Sometimes a platinum loop is found
more suitable for taking the small amount of
exudate from the urethra or cervix.
For culture examination the exudate should be
rolled out on part of the agar surface as soon
as it is obtained. Care must be exercised in
rolling the swab over the surface so as not to
penetrate the relatively soft agar. If agar plates
cannot be supplied to the clinician, the swabs
may be introduced into sterile test tubes con-
taining a little broth.
The specimen should be sent at once to the
laboratory. If the clinician inoculated the plates,
they are further streaked in the laboratory or, if
swabs are sent in carrying tubes, plates are im-
mediately inoculated and streaked. The longer
the time interval between collection and inocula-
tion of the specimen on the culture medium,
the greater the decrease in the number of
positive cultures. If cultures are collected
sporadically over a period of several hours,
they should be placed in a closed candle ex-
tinction jar.
For the primary cultivation of specimens from
the urethra, cervix, vagina, or rectum, the
Thayer-Martin medium selective for gonococci
is recommended. Antibiotic supplements of
polymyxin B-ristocetin or of vancomycin-colis-
tin-nystatin for use in the selective medium may
be obtained from commercial sources.
Subcultures of oxidase-positive colonies of gram-
negative diplococci should be made on chocolate
agar slants in screw-capped tubes and incubated
for 18 to 24 hours in a candle extinction jar.
After this time, if gram-negative diplococci in
pure culture are present, the cap should be
tightened and the tube mailed to the State health
laboratory for identification or transmittal to
the Venereal Disease Research Laboratory.
7. Hemolytic Streptococcus Injections. Specimens
usually submitted are material from the anterior
nasal or nasopharyngeal areas, or from the
throat, or pus, sputum, spinal fluid, discharges,
exudates, urine, blood, and milk. The technic
of swabbing an area to be cultured is as im-
portant in the isolation of streptococci as is the
cultivation of the specimen taken. In strepto-
coccal infections of the upper respiratory tract,
the organisms may be cultured from the nose or
throat specimen. When the specimen is from the
10
throat, care should be taken to sample an area
of inflammation or exudate. If only one speci-
men can be taken, it should be from the throat.
In carrier surveys, nasopharyngeal specimens
are preferable. Unless a suitable transport
method is used, swabs should be planted within
not more than four hours after the specimen is
taken. All other specimens should be refrigerated
from the time taken until they are cultured,
a. Specimens for isolation of the agent
Initial isolation of streptococci should, if
possible, be carried out in a competent
laboratory close to the source of the
specimen.
(1) Anterior Nasal, Nasopharyngeal, and
Throat Specimens:
Material taken from the upper respira-
tory passages is the most common type
of specimen cultured for hemolytic
streptococci. Individual sterile swabs
are used and anterior nasal specimens
are obtained by introducing the swab
into the nares for about an inch. To
obtain a nasopharyngeal specimen, the
tip of the nose should be elevated, and
the swab, moistened with broth or
saline, should be introduced along the
floor of the nasal cavity, under the
middle turbinate, to the pharyngeal
wall. Be sure to touch any exudate
present. With the tongue depressed,
pass a dry swab over the tonsils and
pharynx, being sure not to touch the
tongue. If inoculation of an isolation
medium must be delayed beyond four
hours, use one of the following trans-
port methods ;
(a) The swab may be inserted into a
sterile screw-cap tube containing
indicator silica gel.
(b) A blood agar slant in a screw-cap
tube may be streaked with the
swab which is then left in place
during transport.
(c) Several filter paper transport kits
are available from commercial
sources. The swab should be rolled
and scrubbed onto the piece of
filter paper and discarded. The
filter paper should be allowed to
air dry for 3 to 4 minutes; it
should then be refolded into its
carrier paper, and returned to the
envelope.
(2) Pus, Sputum, Spinal Fluid, Discharges,
Exudates, Urine:
A Gram stain of the specimen at the
local or hospital laboratory will in-
dicate roughly the number of organisms
present, and if there are only a few, the
fluid may be centrifuged and the sedi-
ment cultured. Streak the blood agar
plates so as to obtain well-isolated
colonies and inoculate a tube of en-
riched infusion broth.
(3) Blood
After decontaminating the skin with
4% iodine followed by wiping with
70% alcohol, use a sterile syringe to
draw about 10 ml. of blood and add it
to a flask containing 2 ml. of 3%
sterile sodium citrate or 1.0 mg. of
heparin. After mixing, transfer the
specimen aseptically to 100 ml. of
blood culture broth. A Loeffler slant or
a blood agar plate may also be inocu-
lated. If a plate is used, limit the
inoculum to a small area of the medium
and take the plate to the laboratory
where streaking for isolation can be
done properly with a wire loop. Do not
mail Petri dishes. The addition of an-
tagonists to media for blood cultures
from patients undergoing chemotherapy
and the addition of penicillinase in
cultures from patients receiving peni-
cillin are usually unnecessary.
(4) Milk
Refrigerate the specimen for as much
of the time as possible before it is
cultured. If refrigeration is impossible,
mix the specimen with Vs volume of
glycerol. Culture suitable dilutions in
blood agar pour plates in duplicate,
mixing the sample and melted medium
in the plate so as to get isolated
colonies. Incubate one plate aerobically
and one under anaerobic conditions,
b. Streptococcus grouping by fluorescent anti-
body (FA) technic
Smears made directly from the patient are
unsatisfactory for examination by FA tech-
nic. For this examination, specimens may be
transported in the same manner as for cul-
ture. The swab should be placed in Todd-
Hewitt broth for enrichment purposes for
two to three hours at 37 °C, before the FA
smears are made.
11
c. Specimens for streptococcus tjping
Ver\’ few laboratories are in a position to
type streptococci. If isolates are to be sent
to the NCDC for typing, they should be sub-
mitted only after consultation with the Strep-
tococcus Unit of the Laboratory Program
and then, as pure cultures on blood agar
slants.
8. Haemophilus Injections of the Pharynx or
Conjunctiva
Specimens for the isolation of the agent
Blood, sputum, throat, or nasopharyngeal swabs,
and purulent exudates are collected in the
customary manner, using aseptic technic where
feasible. Since use of swabs made of wooden
sticks or wire inhibit the growth of these organ-
isms, small swabs made of cotton attached to
some inert, synthetic material such as nylon or
Teflon, which is not inhibitory, should be used.
Teflon tubing has proved to be satisfactory for
this purpose. A small, sterile, dry swab may be
rotated gently over the conjunctiva or pharyn-
geal mucosa to obtain some of the exudate if
any is present. The swab should be immediately
placed in a tube of semisolid agar for transport
to the laboratory where it is incubated for about
4 hours. Plates of transparent agar and blood
are then streaked for isolation of the organism.
Blood should be inoculated into a flask of broth
with a large surface area. Sputum and throat
specimens are cultured on one of the transparent
agars as well as on blood agar. The swab should
not be left in the tube of semisolid agar during
incubation.
9. Leptospirosis. This disease should be considered
in all cases of febrile illness of unknown origin
and it may suggest aseptic meningitis, or non-
paralytic ]X)liomyelitis. Rats, dogs, cattle, swine,
and many wild animals are common animal
reservoirs. Infection is transmitted to man by
urine containing the agent and occurs when
leptospires enter the body through the mucous
membranes of the mouth, nose, throat, eyes,
lungs, or abraded skin.
a. .Specimen.s for the isolation of the agent
Blood, urine, and cerebrospinal fluid may be
collected for culture and/or animal inocula-
tion with a view to recovery of the organism.
Blood taken during the febrile stage of
illness is the most reliable for culture, but
spinal fluid collected within the first 10 days
of illness also may be cultured. Inocu-
late the freshly-drawn specimen directly
into several tubes of a suitable semisolid
medium such as Fletcher’s. Multiple tubes of
media should be used and small inocula, 1 to
3 drops of blood to 5 ml. of medium,
planted. As Fletcher’s medium remains
stable for 3 to 4 months, the laboratory may
supply it to the physician. It can be inocu-
lated at the bedside, and then shipped by
mail. Voided urine if diluted, may be
cultured directly into semisolid medium. Five
10-fold dilutions in buffered saline or a suit-
able broth are first prepared. Since quantita-
tive dilutions are not necessary, a single
2.0 ml. syringe with a 20-gauge needle may
be used to prepare these dilutions in the
syringe. Draw up to 0.1 ml. of urine and 0.9
ml. of diluent for the first dilution; expel all
but 0.1 ml. of this dilution, planting one
drop into 5 ml. of medium. For the second
dilution, again draw 0.9 ml. of diluent into
the syringe. Plant one drop of the mixture
into 5 ml. of medium again expel all but
0.1 ml. Repeat with the third, fourth, and
fifth dilution. Otherwise, inoculate into such
test animals as weanling hamsters or guinea
pigs.
Any pure cultures isolated should be shipped
in semisolid media to a reference laboratory
for confirmation by serologic tests,
b. Specimens for serologic tests
Microscopic or macroscopic agglutination
tests are the most common serologic pro-
cedures used for the diagnosis of leptospi-
rosis. To detect a rise in titer, a specimen
of blood should be taken at two different
times: one during the first week or acute
phase of illness, the other ten days or two
weeks later. Maximum titers are usually
reached by the third or fourth week. While
serodiagnostic tests are of value in con-
firming past or current leptospiral infection,
paradoxical reactions may occur, and de-
termination of the infecting serotype can be
made only by isolation and serologic identi-
fication of the leptospires.
10. Meningitis. Any organism capable of invading
human tissues can cause infection of the men-
inges. As used here, the term “meningitis” refers
to an inflammation of the meninges, brain, or
spinal cord associated with increased cell counts
in the spinal fluid. Characteristic of spinal fluid
in bacterial meningitis is a predominance of
polymorphonuclear cells and a decrease in
dextrose content.
12
Representatives of the following species or
groups of bacteria are most often involved in
meningitis: 1) Neisseria meningitidis, 2)
Haemophilus influenzae, 3) Diplococcus pneu-
moniae, 4) Streptococcus pyogenes, 5) Staphyl-
ococcus aureus, 6) Proteus species, 7) Pseudo-
monas species, 8) Escherichia coli, 9) Myco-
bacterium tuberculosis, 10) Listeria monocyto-
genes, 11) Mimeae, and 12) Flavobacterium
meningosepticum. Meningitis may also be caused
by viruses, fungi, spirochetes, or protozoa.
Specimens for the isolation of the infectious
agent
Spinal fluid, blood, nasopharyngeal swabs,
petechial scrapings, and less frequently ventric-
ular, cisternal, or subdural fluid, are usually
submitted for laboratory examination. The time
in the course of the infection at which the speci-
men is taken, the temperature at which it is held,
and the amount of the inoculum used for
cultures are all important. Spinal fluid should be
taken as soon as meningeal symptoms appear;
blood for cultures, as soon as infection is sus-
pected; petechial scrapings for culture, in either
doubtful or postmortem cases. Nasopharyngeal
swabs are cultured for carrier detection. All
specimens for isolation of the meningococcus
should be transmitted to the laboratory with a
minimum of delay. If nasopharyngeal swabs are
not planted on plating media immediately, they
may be transported in a tube containing 0.5 ml.
heart infusion broth. Blood for culture may be
drawn directly into a vacuum bottle containing
appropriate medium and taken to the laboratory
for incubation.
1 1 . Plague. From time to time epidemics of plague
have swept over large areas of the world,
temporarily paralyzing all forms of human
activity. Plague is primarily a disease of rats and
wild rodents. It is transmitted from animal to
animal by the bites of infected fleas. Man serves
only as an accidental host. The pneumonic type
of the disease can, however, be spread from man
to man by droplet infection without the inter-
vention of an insect vector,
a. Specimens to be collected for isolation of the
agent
Bubo fluid, portions of bubo, spleen, bone
marrow, sputum, blood, or ectoparasites may
be submitted for cultures in cystine broth or
on blood agar slants or plates. Do not ship
plates. Original specimens should be shipped
in double containers with screw tops. The
disease may be identified in decayed or
mummified carcasses by precipitin tests or
the fluorescent antibody technic,
b. Diagnosis of plague by FA technic
This procedure appears to be reliable and
rapid yielding results in one hour, in the
examination of bubo exudate, blood from
human cases, tissue impression smears, or
cultured organisms. However, if clinical
material is injected into laboratory animals,
about two days must be allowed for the
development of organisms within the animal
before the FA test may be made or isolation
by culture begun.
Plague bacteriophage may be used to differ-
entiate between plague and pseudotubercu-
losis organisms.
12. Syphilis.
a. Guide for submitting blood specimens for
the Treponema Pallidum Immobilization
(TPI) test
( 1 ) Criteria for requesting the TPI test
This test should be requested only on
specimens from patients who are diag-
nostic problem cases: (a) with reac-
tive nontreponemal tests and no history
or clinical evidence of syphilis; or (b)
with nonreactive nontreponemal tests
and suggestive evidence of syphilitic
infection.
(IF THE PATIENT HAS RECEIVED
ANY INJECTED ANTIBIOTICS WITHIN
ONE MONTH OR ORAL ANTIBIOTICS
WITHIN ONE WEEK OF THE DRAW-
ING OF THE BLOOD SPECIMEN, AN
INVALID OR INCONCLUSIVE FIND-
ING IN THE TPI TEST MAY RESULT.)
(2) Collection of the blood specimen
(a) Collect at least 5 ml. of blood with
a sterile syringe and needle.
(b) Transfer the blood to a sterile test
tube and stopper with a paraffin-
coated cork. Uncoated corks or
rubber stoppers are unsatisfactory.
NOTES:
Unless a vacuum tube with a nontoxic
rubber stopper is used, the rubber
stopper should be replaced immedi-
ately after collection of the specimen.
Vacutainer tubes with nontoxic rubber
stoppers are available from Becton-
Dickinson (Catalog #4719, descrip-
tion #3200 NT.)
13
BACTERIAL CONTAMINATION REND-
ERS SPECIMENS UNSATISFACTORY FOR
TESTING. SERA SHOULD NOT CONTAIN
PRESERVATIVE OR ANTICOAGULANT.
(c) Secure the stopper in the tube with
adhesive tape.
(d) Place the blood specimen in a
mailing container with the com-
pleted clinical history form and
mail it immediately to the State
public health laboratory.
(e) A clinical history form on the
patient should be submitted to the
State laboratory with the speci-
men. This should include infor-
mation on evidence or history of
syphilis, other treponematoses or
venereal diseases in the patient or
family, previous therapy, a record
of previously performed serologic
tests for syphilis on serum or
spinal fluid specimens, and evi-
dence of diseases or conditions
other than syphilis.
(3) Preparation of the sterile serum sample
(a) The State public health laboratory
will aseptically separate and trans-
fer the serum to a sterile tube
stoppered with a paraffin-coated
cork.
(b) The sterile serum sample will then
be forwarded immediately to;
The National Communicable
Disease Center
Venereal Disease Research
Laboratory
Atlanta, Georgia 30333
(4) Reporting of results
Test results will be reported to the
State public health laboratory for for-
warding to the submitting physician.
f5) Public Health Service facilities
These facilities have special instructions
and forms for submitting specimens
directly to the Venereal Disease Re-
search Laboratory.
b. Darkfleld examinations for Treponema pal-
lidum
Darkfield examination is an examination of
exudate from suspected syphilitic lesions
with a compound microscope equipped with
a darkfield condenser. The darkfield exami-
nation should be performed on any rash or
lesion suspected of being syphilitic, and,
ideally, it should be performed immediately
after the specimen is collected. If this exami-
nation cannot be performed locally, contact
the State department of public health labora-
tory and/or the venereal disease control
section for information on the availability
of this service.
c. Fluorescent antibody darkfield (FADF)
examination for T. pallidum
( 1 ) Clean the suspected syphilitic lesion
with a gauze sponge wet with tap water
or saline. Dry the area and abrade it
with a dry sponge. It may be necessary
to squeeze the base of the lesion to pro-
mote the appearance of serum. For dry
lesions, apply a large drop of saline and
emulsify the surface material with a
toothpick or bacteriological loop.
(2) Collect the specimen in a dry capillary
tube (optimum dimensions — 75 x
1.5 mm.).
(3) With a 70%-alcohlol sponge, wipe off
the top of the capillary tube which con-
tacted the lesion and seal both ends of
the tube with clay, paraffin, Critocaps,
or a small flame.
(4) For mailing specimens to a central
laboratory, place the capillary tube in
a cardboard slide-mailing container,
seal with cellophane or masking tape,
OR, place the capillary tube in a test
tube, stopper, place in a mailing con-
tainer, and pack to protect against
breakage.
FREEZING SPECIMENS BEFORE
MAILING TO A CENTRAL LAB-
ORATORY IS NOT NECESSARY.
(5) When specimens are received in the
laboratory for examination, the capil-
lary tube (in the mailing container),
plus the identification slip, is placed in
a freezer (— 20°C. to — 40°C.) until
the specimen is completely frozen. If
the specimen is to be examined imme-
diately, the capillary is removed from
the mailing container for more rapid
freezing. After freezing, thaw the speci-
men at room temperature and examine
it according to the FADF procedure.
d. Blood specimens for the nontreponemal
14
tests, the one-fitth volume Kolmer with
Reiter protein antigen (KRP) and Fluores-
cent Treponemal Antibody (FTA) tests.
(1) ColJection tubes should be clean, dry,
and sterile to prevent contamination
and hemolysis of the specimen. Vacu-
um tubes or tubes with paraffin-coated
corks may be used.
(2) At least 5 to 8 ml. of blood should be
drawn, placed in the tube aseptically,
and allowed to clot at room tempera-
ture. Store the specimen in the refrig-
erator until the specimen is sent to the
laboratory. Specimens should not be
placed in the mail over long weekends
or holidays when delivery may be de-
layed.
NOTE: Hemolysis may be caused by wet
or dirty syringes, needles, or tubes;
chemicals; freezing; or extreme
heat.
( 3 ) If serum is submitted to the laboratory,
submit information as to whether or
not it has been heated (giving time and
temperature) and if preservatives have
been added.
e. Spinal fluid specimens for nontreponemal
tests and total protein determinations
(1) Collection tubes should be clean and
sterile. (Merthiolated tubes may be
prepared for the collection of the spec-
imens: prepare a 1% aqueous solution
of Merthiolate; place 0.1 ml. in a clean,
sterile tube; and dry in a desiccator
over CaCL. This compound curtails
bacterial growth without interfering
with the nontreponemal tests for syphi-
lis and does not affect the results ob-
tained with the turbidimetric methods
for determining total proteins in spinal
fluids.) Stopper with paraffin-coated
corks.
(2) Collect 2 to 8 ml. of spinal fluid asepti-
cally.
(3) If the specimen is centrifuged before
sending it to the laboratory, note the
original condition or appearance of the
specimen on the request slip.
NOTE: Specimens grossly contaminated
with blood or bacteria are unsatis-
factory for testing.
13. Tuberculosis. Since Mycobacterium tuberculosis
may invade any organ of the body, such varied
specimens as sputum, gastric washings, pus,
urine, or spinal fluid may be sent to the labora-
tory for examination. Sputum is the specimen
most frequently submitted. Patients must be
taught the difference between saliva and sputum
and told that an early morning specimen is
generally most productive. They should also be
cautioned against contaminating the exterior of
the container, as such contamination creates a
hazard for all who handle the specimen.
Pooling of sputum for several days is not desir-
able because it may be toxic to tubercle bacilli
present and the degree of contamination may
be increased. Specimens must be sent through
the mails in sterile screw-capped containers
having resilient rubber liners in the caps. Pack-
aging must be in double mailing containers. The
need for examination of repeated specimens
should be stressed, and the results of a single
negative specimen should never be accepted as
conclusive evidence of the absence of disease.
Specimens submitted in unsterile containers,
pill boxes, fruit jars, ointment jars, or in paper
envelopes or on pieces of gauze are utterly use-
less and will not be examined by the laboratory.
Pathogenic fungi capable of causing pulmonary
mycosis are sometimes encountered during cul-
ture work for tuberculosis. While certain sap-
rophytic fungi also survive the processing of
specimens, none of the fungi isolated should be
casually discarded as harmless contaminants;
instead, they should be referred to a competent
mycology laboratory for identification.
Specimens to be collected for isolation of the
agent
a. Sputum, the thick yellowish-green exudate
from the lungs, and not saliva, should be
collected in whatever type of sterile con-
tainer is furnished by the laboratory, and
in no other. A pinch (about 50 mg.) of
sodium carbonate added to the container
will help to suppress the multiplication of
contaminants. Be sure the sputum is de-
posited within the container without soiling
the exterior. Make sure the closure is leak-
proof and forward the specimen to the
laboratory in the double mailing container.
b. Gastric washings, collected in the morning
on a fasting stomach, should be transported
to the laboratory as promptly as feasible.
The more prompt the examination the
greater the chance of recovering the tubercle
bacillus. If the specimen is to be sent through
the mails, some attempt should be made to
15
neutralize the acidity with buffering tablets
or sodium carbonate.
c. Urine may be contaminated with acid-fast
saprophytes present on the external genitals,
so a catheterized specimen is preferable. If
catheterization is impractical, a midstream
sample, collected in a sterile container, after
careful washing of the external genitals,
may be acceptable.
d. Other materials such as samples of spinal,
pleural, or synovial fluid, and pus, may be
collected for diagnostic examination. Asepn
tic collection in sterile tubes and the addi-
tion, where indicated, of an anticoagulant
such as ammonium oxalate, are essential to
maintain the specimen in a fluid state.
14. Tularemia. The laboratory diagnosis of tula-
remia may be accomplished by either a) the
agglutination test, or b) cultural examination.
The extreme infectivity of Pasteurella tularensis,
however, makes routine culture inadvisable,
and agglutination tests are the method of choice
in the diagnostic laboratory.
a. Specimens for isolation of the agent
Culture of P. tularensis from any one of the
natural hosts such as ground squirrels, wild
rabbits, wild mice, quail, or other animals
should not be attempted except in a ref-
erence laboratory. If such an animal is
shipped, the body should be wrapped in
cloth soaked in cresol, placed in a container
with dry ice, and forwarded to the labora-
tory immediately. Identification of the or-
ganism in smears made from the blood or
tissues of the host is not possible; but in
human cases, cultures may be made from
any of the organs or from the sputum, blood,
or exudates, and the organism may be
identified by specific agglutinating antisera.
Sp>ecific FA conjugate may also be used.
b. Specimens for serologic tests
Blood should be drawn aseptically, allowed
to clot, and the serum removed for agglu-
tination tests. Care should be taken to assure
that the blood is not hemolyzed due to ex-
posure to excessive heat or cold in transit
to the laboratory. To demonstrate a rise in
antibody titer, preferably at least two blood
specimens should be obtained — the first
during the acute stage of the disease, and the
second about three weeks later. Precipitin
tests arc also possible; but these, together
with FA tests, probably will be available
only in a reference laboratory.
MYCOTIC DISEASES
Fungus diseases are conveniently classified as cuta-
neous, subcutaneous, and systemic infections. The
etiologic agent is identified by study of its mor-
phology as seen in clinical material and by its mor-
phological and physiological characteristics as ob-
served in pure culture. Serological methods, for
example, agar gel precipitin tests, agglutination tests,
and FA techniques, are also of value in the identifi-
cation of certain pathogenic fungi.
Specimens taken for laboratory examination vary
with the disease and the tissues involved. Specimens
include sputum, gastric washings, spinal fluid, blood
smears, citrated blood, blood serum, bone marrow,
pus, pleural fluid, urine, biopsy or surgical sp>ecimens,
scrapings from edges of lesions, skin scrapings, hair,
and nail clippings. If competent local laboratory as-
sistance is available, it should be used for the proper
collection of clinical materials. Since many clinical
materials such as sputum, lesion exudates, and tis-
sues are readily overgrown by contaminating bacteria
and saprophytic fungi, it is usually wise to make
cultures on selective and nonselective media and to
ship these, rather than the clinical materials, to the
reference laboratory. The use of penicillin and strep-
tomycin or chloramphenicol as antibacterial inhib-
itors in the specimen may be helpful if cultures can-
not be made before shipment.
It must be borne in mind, however, that certain of the
pathogenic fungi, particularly Nocardia asteroides
and Actinomyces israelii, are sensitive to antibacterial
antibiotics. Furthermore, others, particularly Histo-
plasma capsulatum, die out rather quickly in clinical
material. Refrigeration of the specimen in transit will
help to overcome this difficulty. In general, refrigera-
tion of specimens in transit serves not only to
maintain viability of fungus pathogens but also to
decrease growth of contaminants.
1 . Cutaneous fungus infections. Careful choice of
specimens for laboratory study is important. The
Wood’s lamp is useful in the collection of speci-
mens in tinea capitis infections since hairs in-
fected by most members of the genus Micro-
sporum frequently exhibit fluorescence under a
Wood’s lamp. However, in tinea capitis due to
Trichophyton species, infected hairs usually do
not fluoresce.
a. .Specimen collection for laboratory examina-
tion
Fluorc.scent hairs, or nonlluoresccnt hairs
which are broken off and appear dis-
eased, should be plucked with a sterile
forcep, or, if diseased hair stubs arc not
16
apparent, the edges of a scalp lesion should
be scraped with a sterile scalpel. Skin lesions
should first be cleansed with 70% alcohol
to reduce bacterial and saprophytic fungi.
Scrapings should be made from the outer
edges of skin lesions. In infections of the
nails, the friable material beneath the edge
of the nails should be scraped out, or por-
tions of abnormal appearing nail should be
scrap>ed or clipped off, and saved for exami-
nation and culture.
Enclose hair specimens, skin scrapings, or
nail clippings or scrapings in clean paper
envelopes and label them with the patient’s
name or specimen number. Enclose these
envelopes in larger heavy paper envelopes
for mailing to the laboratory. Do not put
specimens in cotton plugged tubes, as the
specimen may become trapped among the
cotton fibers and lost. Do not put specimens
into closed containers such as rubber
stoppered tubes as this keeps the specimen
moist and allows overgrowth of bacteria and
saprophytic fungi.
2. Subcutaneous fungus infections. Procedures for
isolating the etiologic agents of chromoblastomy-
cosis, mycetomas, and sporotrichosis are
obvious. Granules, pus, or serosanguinous
fluid from lesions or draining sinuses should be
inoculated on several tubes of Sabouraud dex-
trose agar with and without chloramphenicol and
cycloheximide.
3. Systemic fungus infections. Systemic mycoses
are not contagious and most are chronic and
evolve slowly. Since time is usually not of vital
importance, it may be profitable to consult with
a mycology laboratory to determine what type of
specimen to collect in a given case, and how it
should be handled. Examining unstained prepa-
rations of clinical material as well as stained
smears is of practical value in determining the
appropriate types of media to inoculate and the
correct incubation temperatures. It is usually
wise to inoculate both simple media, such as
Sabouraud dextrose agar, and enriched media,
such as brain heart infusion agar. Each medium
should be made with and without antibiotics.
Infections with Histoplasma capsulatum and
Coccidioides immitis present special problems
to the laboratory worker. In the case of histo-
plasmosis it is often difficult to isolate the
causative fungus because the organisms are often
few in number and tend to die out in the clinical
materials. In the case of coccidioidomycosis the
fungus agent is relatively easy to isolate. It is
highly infectious, however, and many laboratory
workers hesitate to attempt its isolation. Sug-
gestions on methods for the isolation and
handling of H. capsulatum and C. immitis are
given below,
a. Actinomycosis
If isolates of suspected anaerobic to micro-
aerophilic Actinomyces species are to be sent
to a reference laboratory for identification,
the following methods are recommended.
( 1 ) Liquid media
(a) Inoculate culture into freshly
boiled TST enriched* thioglycol-
late broth. Incubate at 37° C. for
3 to 4 days. If growth is apparent
at that time, seal by pouring over
the surface of the medium about
3 ml. of a sterile melted mixture of
paraffin and vaseline (50% paraf-
fin & 50% vaseline).** Seal the
top of the tube with a sterile
rubber stopper, or use a screw-cap
tube sealed with masking tape.
(b) If Actinomyces Maintenance
Broth*** is used, inoculate the
tube of broth and incubate under
pyrogallol-carbonate seal for 3 to
4 days. If growth is apparent,
transfer the liquid with the culture
to a clean sterile tube, and seal
with vaseline-paraffin mixture as
descibed above.
THE IMPORTANT POINTS
TO REMEMBER ARE:
(1) Actinomyces cultures should
not be more than 3 to 4 days
old before shipment. Send
them by airmail if possible.
(2) Liquid or semisolid cultures,
should be sealed with vase-
line-paraffin mixture before
shipment.
* Thioglycollate with TST
Thioglycollate broth
(with dextrose and indicator) 29.5 gms.
Trypticase Soy broth 1.5 gm.
Tryptose broth 1.25 gm.
Distilled water 1000.00 ml.
** A stock vaseline-paraffin mixture may be prepared by
melting together equal amounts of each. After mixing
well, it may be distributed in 3 ml. amounts in plugged
tubes for sterilization. These may be stored for use as
needed.
*** This medium is recommended for fastidious strains,
which do not grow well in thioglycollate broth. It is
available in desiccated form from BBL, Division of Bio
Quest, P. O. Box 175, Cockeysville, Md., 21030.
17
b. Coccidioidomycosis
Direct microscopic examination of wet, un-
stained clinical material should be made, but,
regardless of the direct findings, cultures
and/or animal inoculations are indicated.
Safety precautions: Petri plate cultures
should never be made because they may lead
to laboratory infection. Cultures should be in
large test tubes which give broad agar sur-
faces for inoculation. Specimens held for any
length of time, or shipped to the laboratory,
should be in tubes containing an antibiotic
to retard growth of contaminating organisms.
If the laboratory worker does not have the
proper experience or equipment for handling
the highly infectious cultures of C. immitis,
the animal inoculation or “indirect method”
for demonstration of the fungus should be
used (see below).
( 1 ) Specimen collection for laboratory
examination
(a) Merthiolated serum from serial
blood specimens should be ex-
amined by the complement fix-
ation test. Add merthiolate to
retard growth of contaminants.
(b) Sputum, pus, pleural fluid, and
gastric washings treated with the
antibacterial antibiotics, chlor-
amphenicol or a combination of
p>enicillin and streptomycin, may
be inoculated directly into mice or
cultured on a selective medium
such as that containing cyclohexi-
mide and chloramphenicol. Pre-
liminary identification of cultures
by microscopic examination must
be confirmed by animaj inocula-
tion. Unless safety hoods are
available, however, it is advisable
to use the animal inoculation or
“indirect method” for the demon-
stration of C. immitis. By this
method, the handling of the highly
infectious mycelial phase of this
fungus is avoided.
(2) Animal inoculation of culture.s sus-
pected of being C. immitis and the “in-
direct method” for demonstration of
C. immitis from the clinical materials.
Mice may be inoculated intrapcritone-
ally and guinea pigs intratesticularly
with culture suspensions, or directly
with clinical materials which have been
treated with antibacterial antibiotics.
The male guinea pig is the animal of
choice in determining whether a
mycelial culture is or is not C. immitis,
or whether the clinical material con-
tains elements of this fungus. Intra-
testicular injection of these animals with
C. immitis gives rise to an orchitis and
examination of fluid withdrawn from
the testes will reveal the tissue form of
C. immitis. If guinea pigs are not avail-
able, inject mice intraperitoneally and
examine lesions or lymphatic exudates
for the fungus.
c. Histoplasmosis
If possible, culture media should be inocu-
lated immediately following collection of
clinical material. If the specimens are to be
shipped or held for any length of time, they
should be refrigerated because these organ-
isms die out rapidly when exposed to higher
temperatures. An antibiotic such as chlor-
amphenicol should be added to specimens
that are to be shipped.* Recovery of H.
capsulatum by direct culture from clinical
materials is not always difficult, but the
chances of recovery are greatly increased if
mice are inoculated and sacrificed for culture
at the end of two weeks.
( 1 ) Specimen collection for laboratory ex-
amination
(a) Blood smear examination is un-
profitable except in acute, dissem-
inated cases.
(b) Citrated blood is of value for
culture and animal inoculation in
acute, disseminated cases.
(c) Merthiolated** serum from serial
blood specimens, taken at 3- to 4-
week intervals, should be tested
by complement fixation and agar
gel precipitin tests.
(d) Sputum specimens*** should be
collected in sterile bottles to which
chloramphenicol has been added.
A minimum of six specimens
should be collected from each
patient before histoplasmosis is
ruled out.
* See appendix II, page xx
*• See appendix II, page xx
*** Sputum brought up from bronchial areas by having the
patient use correct postural drainage procedures.
18
(e) When sputum cannot be obtained,
gastric washings may be sub-
mitted. They should be collected
in a sterile bottle containing
chloramphenicol.
(f) Spinal fluid is taken only if there
is cerebral meningeal involvement.
It is collected in a tube or bottle
containing antibiotic.
(2) Animal inoculation
Intraperitoneal inoculation of mice
with clinical materials greatly increases
the probability of demonstrating the
presence of H. capsulatum. Sacrifice the
animals after 2 weeks and culture the
liver and spleen on cycloheximide agar
at room temperature and on blood agar
without antibiotics at 37° C.
d. Fluorescent Antibody (FA) Staining in
Mycology
In certain reference laboratories, FA tech-
nics are regularly used in conjunction with
conventional methods of identification.
Currently these procedures have been most
successfully applied in medical mycology in
the identification and detection of Sporo-
trichum schenckii, Blastomyces dermatitidis,
Cryptococcus neoformans, H. capsulatum,
and C. immitis.
Specimens for examination by the fluorescent
antibody technique are obtained from such
varied sources as blood, spinal fluid, sputum,
lesion exudates, sternal marrow, scrapings
from edges of ulcers and abscesses, and
aspirated fluid from abscesses and tissue. If
the laboratory is close by, such specimens
can be submitted directly to the laboratory.
If these materials have to be shipped to a
distant laboratory, smears should be pre-
pared for shipment. Sputum should be
enzymatically digested (by parasentin or
trypsin) and smears made from the centri-
fuged sediment. Such smears are made
directly within etched circular (1 cm. di.)
areas of glass slides and are then allowed to
air dry. These preparations are fixed by heat.
Other materials such as exudates or tissues
do not require digestion. These smears are
made directly within the etched areas of
glass slides, are allowed to air dry and are
then fixed by heat. Spinal fluid or gastric
washings should be centrifuged and smears
made from the sediment.
PARASITIC DISEASES
The diseases caused by animal parasites may be
conveniently considered as blood, intestinal, or tissue
infections; and laboratory diagnosis may be made by
direct examination of the specimen or indirectly by
serologic tests. The specimens submitted for labora-
tory examination may be smears of bone marrow,
liver, spleen, or whole blood, aspirates from lesions,
vaginal or urethral secretions, bile, sputum, duodenal
drainage, anal swabs, feces, or portions of the
parasite itself.
1 . Blood Parasites. Proper collection and handling
of specimens to be examined for blood parasites
are important since inadequate or poor samples
may lead to erroneous conclusions. Not all the
organisms usually grouped as blood parasites are
diagnosed from blood. In certain instances,
spinal or peritoneal fluid, aspirates and biopsies
of organs and tissues are also used. The speci-
men to be obtained depends on the location of
the parasite or its diagnostic stage in the body.
Table 1 shows the location of parasites or their
diagnostic stage within the body.
TABLE 1
Location of Parasites or their
Diagnostic Stage within the Body
PERIPHERAL BLOOD OTHER
Within
RBC
In
Plasma
Within
Leuco-
cytes
to
Bone
Marrow
Lymph
Nodes
Spinal
Fluid
Plasmodia spp. (4)
+
+
Trypanosoma gambi-
ense and T.
rhodesiense
-h
-1-
+
Trypanosoma cruzi
-1-
-t-
Leishmania donovani
-1-
-f
-h
Leishmania tropica
and L. brasiliensis
+
Filaria-microfilariae
(5 spp.)
+
-f-*
+*'
Onchocerca volvulus
-1-
Toxoplasma gondii
-f
+
+
*Loa loa microfilariae may be found in calabar swellings
**Wuchereria bancrofti and Brugia malayi microfilariae
a. Blood specimens
Two typ>es of specimens are collected for
recognition of those parasites whose diag-
nostic stages are found in peripheral blood,
(1) dried blood films for staining and (2)
19
whole blood samples. This applies in ma-
laria, trypanosomiasis, and filariasis (with
the exception of onchocerciasis).
( 1 ) Blood films
Citrated, oxalated, or clotted blood
should not be used to make blood films
except in emergencies. In thin films the
blood is spread over a relatively large
area in a thin layer; in thick films, it is
concentrated in a small area. For rou-
tine diagnosis, the thick film is prefer-
able since it permits the examination
of a large amount of blood. However,
parasite morphology is more distinct
and typical in a thin film. For this rea-
son, a thin and thick film combination
on the same slide is recommended.
Thin films are prepared by pricking the
ear or finger after the area has been
cleansed with gauze, not cotton, soaked
in 70% alcohol, and allowed to dry
completely. A single drop of blood is
deposited near one end of a slide and
spread as though making a preparation
for a differential count. Thick films are
made by either touching the under sur-
face of the slide to a fresh large drop of
blood on the finger, without touching
the skin, and rotating the slide to form
a film about the size of a dime. Alter-
natively, several drops of blood may be
deposited close together near one end
of the slide and puddled with the corner
of a slide, applicator stick, or toothpick.
The films are allowed to air dry in a
horizontal position protected from dust
and insects. Thin films dry rapidly but
thick films require 8 to 12 hours. Prop-
erly and improperly prepared films are
shown in Figure 1.
(2) Whole blood
If concentration methods are to be used,
blood is collected by venipuncture, and
sodium citrate or heparin is added to
prevent clotting. Aseptic technic is es-
sential in the collection of the sample.
Do not use preservatives since they may
kill the organism and make cultivation
or animal inoculation impossible.
(3) Time of collection
Time of specimen collection in malaria
is important but less so than in filaria
infections. Parasites are most numerous
in malaria about midway between
chills, but their morphology is some-
what less characteristic. One specimen
taken at this time and a second 5 to 6
hours later is ideal.
Because of the nocturnal periodicity in
certain filaria infections, notably by
Wuchereria bancrofti and Brugia ma-
layi, the specimen should be taken be-
tween 10 p.m. and 2 a.m. Infections
with W. bancrofti or Wuchereria paci-
fica in the southwest Pacific area exhibit
no such periodicity. In Loa loa there
is a diurnal periodicity, and specimens
should be collected between 10 a.m.
and 2 p.m. Blood collection should also
be correlated with the stage of the in-
fection. For example. Trypanosoma
cruzi is found in the blood only during
the acute, febrile stage of Chagas’
disease. The same is true for infections
with Trypanosoma gambiense and Try-
panosoma rhodesiense.
b. Body fluid, aspirates, and biopsies
Certain blood parasites are diagnosed by the
examination of body fluids and tissues rather
than by direct examination of blood. Thus,
biopsies of liver, spleen, bone marrow, and
lymph glands must be studied to demon-
strate the parasite. This is true in leishma-
niasis, filariasis (onchocerciasis), and some-
times in malaria and trypanosomiasis. In
some cases of onchocerciasis, tissue biopsies
may reveal microfilariae when examination
of skin from the nodule is negative. Localiza-
tion of the nodules on the upper or lower
part of the body influences distribution of the
microfilariae and dictates selection of the
biopsy site which may be the shoulder, calf,
thigh, or another part of the leg.
Aspirates of lymph glands or ulcerative skin
areas are collected for diagnosis of leish-
maniasis, trypanosomiasis, or filariasis. Cere-
brospinal, hydrocele, pericardial, pleural,
and peritoneal fluids may be used for the
diagnosis of trypanosomiasis, filariasis, or
toxoplasmosis.
Handling of tissues or fluids depends on the
examination to be made. If cultures or ani-
mals arc to be inoculated, asepsis should be
practiced, and the media or animals should
be inoculated as soon as possible after the
specimen is collected. If inoculation is de-
layed, the specimen should be stored in the
refrigerator.
20
PROPERLY MADE SLIDES
I
21
Too thin Improperly placed Damaged by flies
Tissue for sectioning should be placed in a
fixative as soon as it is removed from the
body.
c. Shipment of specimens
Dry blood or tissue smears may be placed in
slide boxes with tissue between and over the
slides to prevent breakage. Slides may be
wrapped in bundles, slide over slide, in toilet
tissue with one or two layers of tissue be-
tween adjacent slides. The slides may be
packed in a mailing container or box with
enough protective material to prevent break-
age, but these containers should be packed
in a box with additional packing material.
d. Storage of specimens
Prompt examination of blood specimens is
desirable because blood loses its affinity for
stain after 3 to 4 days. Unstained slides may
be refrigerated a week, but, if staining is to
be delayed, thin films should be fixed
with methyl alcohol and thick films dehemo-
globinized in buffered water before storage.
Subsequent staining requires special technic.
Prompt refrigeration of whole blood is nec-
essary if it is not to be examined immedi-
ately. Microfilariae remain alive in blood a
week or more under refrigeration. Tissue or
fluid smears, like thin blood films, may be
stored in the refriegrator, after fixation with
methyl alcohol, before being stained.
Stained blood films, tissue impression
smears, or fluid smears may be preserved by
covering the film with a coverslio or a coat-
ing of clear Diaphane or other neutral
mounting medium.
e. Immunodiagnostic tests in parasitic diseases
The immunodiagnostic tests employed in
parasite diagnosis are, in general, modifica-
tions of procedures in common use, that is,
complement fixation, precipitin, hemag-
glutination, flocculation, or fluorescent anti-
body technics.
Various types of immunodiagnostic tests
and the present status of their applicability
in a variety of parasitic diseases are shown
in Table 2. Specimens collected for serologic
diagnosis of parasitic infections are taken as
for other types of serologic tests. The serum
should never be inactivated. If examination
is to be delayed, 0.01 ml. of 1% aqueous
solution of borated merthiolate per ml. of
scrum may be added f Appendix II). If the
serum is to be used also for virus serology,
it must not be merthiolated.
2. Intestinal Parasites. Diseases caused by intesti-
nal parasites are diagnosed mainly by the exami-
nation of fecal specimens, but urine or sputum
may be used for diseases caused by certain
species. Properly collected and preserved speci-
mens are of utmost importance since old, or
poorly preserved materials are of little value in
establishing a diagnosis and may lead to errone-
ous conclusions.
a. Fecal specimens
The stool should be collected in a clean con-
tainer or on clean paper and transferred to
a suitable container such as a half-pint waxed
carton with an over-lapping lid. Urine must
be excluded since it will destroy trophozoites
if they are present. Feces deposited on soil
are not satisfactory due to the possible pres-
ence of free-living larvae and other con-
taminants which may confuse the diagnosis.
The specimen should be taken to the labora-
tory at once; or if the examination will be
delayed, a small portion, in addition to the
carton specimen, should be placed in PVA
fixative (polyvinyl alcohol) (Appendix II)
to preserve trophozoites. Administration of
barium, magnesia, or oil prior to collection
will render a specimen unsatisfactory for ex-
amination.
b. Mailed specimens
Specimens sent through the mail must be in
containers which meet postal regulations for
infectious materials.
One-ounce, screw-cap vials placed in a metal
case with a screw cap and enclosed in a
cardboard container with a metal screw cap
are satisfactory. The vials should be sealed
with adhesive tape around the top to prevent
leakage. They should then be packed in suit-
able absorbent materials to prevent break-
age from shock transmission and to absorb
entirely any leakage that would result if
breakage should occur. Mailed specimens
require use of a preservative, and a two-vial
method of collection and shipping is ad-
vocated. One vial contains 5% or 10%
formalin, the other PVA fixative. Thus the
laboratory has a formalinized specimen that
can be examined for cysts and helminth eggs
and a PVA-fixed specimen that can be ex-
amined for trophozoites, and to a lesser de-
gree, for cysts. The method of handling a
stool specimen for parasitological examina-
tion is shown in Figure 2.
22
Table 2
IMMUNODIAGNOSTIC TESTS FOR PARASITIC INFECTIONS
TESTS
PARASITIC
DISEASES N.
Intraderma 1
Complement
Fixation
Precipitin
Agglutination
Flocculation
Hemagglutination
Latex
Agglutination
Fluorescent
Antibody
Methylene Blue
Dye Test
Ascariasis
@
@
®
®
T richinosis
•
•
•
•
®
®
Toxocariasis
@
©
Cysticercosis
•
•
Echinococcosis
•
•
®
•
•
Schistosomiasis
•
•
@
®
®
Clonorchiasis
®
©
Paragonimiasis
®
®
Filariasis
®
®
Chagas' disease
@
•
©
©
©
Leishmaniasis
•
®
Toxoplasmosis
•
•
©
®
®
•
Amebiasis
®
©
O) Under experimental investigation
*)u sed for diagnosis but requires further evaluation for routine use
^ Generally accepted useful routine diagnostic test
23
PARASITOLOGICAL EXAMINATION OF STOOL SPECIMENS
24
DO NOT COVER THE STOOL WITH CRESOL
OR OTHER DISINFECTANT.
DO NOT CONTAMINATE THE SPECIMEN WITH
URINE, DIRTY WATER, OR EARTH.
DO NOT FILL THE EMPTY VIAL MORE
THAN HALF FULL.
LABEL EACH VIAL WITH THE PATIENT’S
NAME AND ADDRESS.
c. Collection of multiple specimens with and
without catharsis
Because of the intermittent passing of para-
sites from the host, examination of multiple
specimens is desirable. Ascaris, hookworm,
and Trichuris eggs appear almost daily in
feces. Cysts of Entamoeba histolytica and
eggs of certain of the helminths such as
Schistosoma species and Diphy Robot hr ium
latum are passed intermittently. These ir-
regularities emphasize the need for collec-
tion of at least three specimens spread over
10 to 14 days.
Normally, passed stool specimens spaced
several days apart are preferable to speci-
mens obtained by catharsis or sigmoidos-
copy, since cysts are more likely to be pres-
ent in passed stool specimens. Purged speci-
mens increase the possibility of finding
organisms. A cathartic of sodium sulphate
or buffered phospho-soda is preferable to
magnesium sulphate since such cathartics af-
fect the morphology of the organism less.
Each bowel movement should be collected
separately, numbered serially, and delivered
promptly to the laboratory. Egg, larvae,
cysts, and trophozoites may be found in such
specimens. If examination of the specimen
is to be delayed, add a portion to PVA
fixative.
d. Collection of specimens by sigmoidoscopy
In amebiasis, if stools are negative, material
may be obtained by sigmoidoscopy immedi-
ately following a normal bowel movement,
or if a cathartic is given, after a lapse of 2 to
3 hours. Collect the specimens with a
serologic pipette rather than a cotton swab
by aspirating material from any visible lesion
and the mucosa. Pathologic areas or the
mucosa wall may also be gently curetted.
Examination of sigmoidoscopic specimens
for diagnosis of amebiasis must be immedi-
ate, but after the direct examination, PVA
fixative may be added and the preparation
dried and stained.
e. Collection of specimens other than feces
Sputum specimens as well as stools should
be collected in suspected cases of paragoni-
miasis. Pulmonary amebiasis and echinococ-
cosis may also be diagnosed by sputum
examination. Urine specimens are used in the
diagnosis of Trichomonas vaginalis and
Schistosoma haematobium. The optimum
urine specimen to be examined for the latter
is one passed at or shortly after noon. Vagi-
nal swabs or scrapings are used in diagnosis
of T. vaginalis.* Anal swabs or cellulose
tape specimens are the usual means of col-
lecting the eggs of Enterobius vermicularis,
as shown in Figure 3. Specimens taken be-
tween 10 p.m. and 12 midnight, or in the
early morning before defecation, are best.
Three consecutive examinations are desir-
able, and a short delay in examination of
either the swab or tape specimen makes no
particular difference, but the specimens
should be refrigerated if examination is de-
layed for more than a day. Biopsied material
for the dagnosis of schistosomiasis may be
collected from the colon, rectum, or bladder
and, for amebiasis, from the rectum or liver.
Biopsy is not recommended for echinococ-
cosis because of the danger of complications
resulting from the leaking of hydatid fluid
into the surrounding tissue. Duodenal drain-
age often reveals organisms when stool speci-
mens are negative for Strongyloides ster-
coralis and Giardia lamblia and should be
collected when diagnosis cannot be estab-
lished by fecal examination.
Biopsy material (human gastrocnemius or
deltoid muscle) may be collected for diag-
nosis of trichinosis. The muscle may be
fixed and sectioned or the fixed tissue may be
submitted for examination and sectioning
and staining. Serologic methods are more
commonly used in the diagnosis of trichinosis
than direct examination of tissue for the
larvae. Animal biopsy or autopsy meat or
other foodstuffs of animal origin suspected
of harboring T. spiralis larvae should be
submitted to the laboratory in plastic bags
and liberally covered with sodium borate
powder. The specimen should not be frozen.
The borate preserved meat may then be
subjected to pepsin-HCl digestion for re-
covery of larvae.
* The swabs may be examined as a wet mount or, prefer-
ably, they may be placed in a suitable culture medium.
25
Figure 3
USE OF CELLULOSE-TAPE SLIDE
FOR DIAGNOSIS OF PINWORM
PREPARATION
INFECTIONS
a CtlKjlo««-tope slide preporotion
b. Hold slide ogoinst tongue depressor one inch
from end ond lift long portion of tope from
slide
e Press gummed turfoces ogomst severol oreos f- Replace tope on slide
of pertonol region
g Smooth tope with cotton or gowie
Note: Specimerts ore best obtotned o
few hours after the person hos retired,
perhaps ot 10 or II P.M., or the first
thing In the morning before a bowel
movement or both
26
Skin snips are the specimens of choice in
infections with Onchocerca volvulus as the
microfilariae do not appear in the blood
stream. Tissue fluid obtained by puncture
and aspiration of a nodule may also show
organisms.
f. Serum
Immunodiagnostic tests for intestinal para-
site infection are considered to be useful
routine diagnostic procedures in only a few
diseases, as shown in Table 2.
ARTHROPOD-BORNE DISEASES
(SPECIMENS FOR IDENTIFICATION
OF ARTHROPODS)
Arthropod ectoparasites play an important role in the
transmission of plague, typhus, tularemia, spotted
fever, relapsing fever, and several other diseases. Any
specimen submitted should be divided, one portion to
be used for identification and the other for isolation
of any virus or rickettsia that may be present. The
sample for identification should be preserved in, pref-
erably, 70% alcohol or in 2% formalin. Samples
for isolation should be collected alive and stored on
dry ice until tested. Table 3 shows the method of pre-
serving and handling insects and other arthropods
according to the kind of examination to be made.
TABLE 3
Preserving and Handling Insects and Other
Arthropods
For Identification
Moist
Dry
In 70% alcohol or 2% formalin
Between layers of tissue
or lens paper. Not cotton
Ticks
Mosquitoes
Mites
Flies
Fleas
True bugs
Lice
Wasps, bees
Fly maggots
Moths
Spiders
Mosquito larvae
Small flies
Bed bugs
Ants
Caterpillars
Butterflies
For Demonstration of Microorganisms or Antibodies
Suspend in bottle of 1% sodium chloride solution.
Ectoparasites usually are wingless arthropods. Ticks
and mites have eight legs in the nymphal and adult
stages. Fleas and lice are true insects with six legs.
Fleas are very active jumping insects, compressed
from side to side, with elongated legs adapted for
jumping. Lice are more sluggish insects, depressed
from top to bottom, with legs adapted for grasping
hairs. Illustrations of various kinds of arthropods are
shown in Figure 4.
1. Ectoparasites. Ticks, mites, fleas, and lice
should be collected from bedding, animal bur-
rows, or birds’ nests with fine forceps, aspirators,
or applicator sticks. They may be simply
combed, brushed, or knocked off an infected
animal.
2. Flies and Mosquitoes. These arthropods may be
collected with insect nets, aspirators, chloroform
tubes, cyanide jars, light, bait, or fly traps.
Barns, outbuildings, the under surfaces of
bridges, or other adult resting places are the best
areas in which to find these insects. Tubes for
picking up live insects, holding cages, storage
boxes, and bottles are illustrated in Figure 5.
All sp)ecimens should be submitted unmounted,
and large numbers of specimens may be mailed
in pill boxes or similar containers. Specimens
may be mailed wrapped between layers of lens
pap>er or cleansing tisue with a layer of absorb-
ent cotton next to the top and bottom of the
box to absorb vibrations and prevent breakage.
Do not ship mosquitoes or flies between layers
of absorbent cotton. If sp>ecimens are mounted
for identification, they should be fastened with
clear fingernail polish or liquid cement to an
insect point or an insect pin. Larger flies may be
pinned through the thorax. Poorly mounted
specimens are worse than those unmounted.
3. Arthropod specimens submitted for virus or
rickettsia isolation. For this purpose it is of
the utmost importance that ticks, mites, fleas,
lice, mosquitoes, or other insects be collected
alive, sealed in ampules or rubber-stoppered test
tubes, and stored on dry ice until tested. If dry
ice is not available, temporary storage in a me-
chanical deep freeze chest is satisfactory. Col-
lection or storage in chloroform tubes or cyanide
jars is not permissible because these materials
inactivate many viral and rickettsial agents.
27
Figure 4
Dermacentor andersoni - Rocky Mountain Wood Tick
Ctenocephalides felis - Cot Flea
Pediculus humanus - Human Head and Body Louse
Pthirus pubis ■ Human Crab Loose
Anopholos quadrimaculatus - Malaria Mosquito
28
Figure 5
Collection of insects olive with on ospirototr using breath
to supply the suction.
Bottle of saline (1% Na Cl) for specimens to be examined
for microorganisms of antibodies. Formalin (10% ~ 3.7%
Tubes for picking up insects with an aspirator. formaldehyde) may also be used for storage of specimens.
Pill box for storage of dead insects. cage during transit.
29
VIRAL AND RICKETTSIAL DISEASES
Diagnosis of viral and rickettsial diseases in the lab-
oratory may be attempted by three general pro-
cedures.
1. Isolation and identification of the inciting
agents;
2. Demonstration of a rise in titer of specific
antibodies during the course of the illness;
3. Examination of the infected tissues for
pathologic alterations.
It is rarely possible or necessary to use all three
procedures in diagnostic work. A decision as to which
procedure should be followed is dictated by the
nature of the infection, the stage of the illness when
the patient is first seen, and the amount of informa-
tion the methods will yield in relation to the time,
effort, and expense involved.
Direct microscopic methods are rarely used and are
of limited usefulness except in rabies diagnosis and
a few other special cases. Aside from immunofluo-
rescence, microscopy is usually confirmed by some
other procedure such as serology or isolation of the
agent.
Serologic tests have far greater usefulness in the
diagnostic laboratory than either of the other ap-
proaches. Serologic methods generally yield informa-
tion more rapidly and less expensively than isolation
and identification of an agent, but they do not give
the same degree of assurance of etiologic involve-
ment. In fatal infections, when there has been in-
sufficient time for specific antibody development,
isolation of the causative agent is the only means of
diagnosis. When the virus exists in many antigenic
types, as in the Coxsackie group, it is essential to
isolate the virus from clinical material. Serologic
tests are possible only for those diseases whose
causative agents have been isolated and from which
satisfactory antigens can be produced. Obviously,
some exception must be made for those diseases in
which a biologically nonspecific phenomenon can be
useful in suggesting a diagnosis, as in the Weil-Felix
reaction for certain rickettsioses, and the heterophile
test for infectious mononucleosis. Serologic methods
alone with contribute little to establishing the etiology
of diseases of unknown causation.
The types of specimens that may be submitted for
laboratory examination in viral and rickettsial dis-
eases are more varied than in any other type of in-
fection. Depending on the disease, the specimens in-
clude; nasal swabs, throat swabs, swabs of oral
lesions, saliva, sputum, nasopharyngeal swabs, nose
and throat washings, stool or rectal swabs, cerebro-
spinal fluid, vesicle fluid, pleural effusion fluid, bubo
aspiration fluid, musele biopsy, crusts, scrapings
from fever sores, postmortem tissue and paired sera.
In interpreting isolation results, it should be remem-
bered that the mere presenee of a virus in exereta
does not neeessarily establish the etiologieal relation-
ship of the agent to the disease. Inapparent and
mixed infeetions, particularly with enteroviruses, are
common and the etiologically unrelated virus may
even give rise to antibody formation which coin-
cides with the timetable of the disease.
1. Preparation and Shipment of Diagnostic Speci-
mens
a. Type of Specimen to be Collected
(1) Virus isolation. Materials for isolation
must be freshly obtained and, if pos-
sible, collected with aseptic precau-
tions. Source of materials depends on
the type of clinical disease (that is,
throat swabs or throat washings in
respiratory infections, etc.). Usual
materials include blood, throat wash-
ings, sputum, feces, effusion fluid, tis-
sue biopsies, autopsy tissue, or lesion
scrapings. No preservative or fixative
should be added. After collection, blood
for virus isolation should be frozen
without further treatment.
(2) Serological tests. Serum should be
obtained from clotted blood collected
and kept under sterile conditions. No
preservative need be added. Separating
the serum from the clot at the source
prevents hemolysis which interferes
with some tests. At least 10 ml. of
blood (5 ml. of serum) and preferably
20 ml. of blood should be collected
for serological tests to permit a multi-
plicity of tests, confirmation, or repeti-
tion of equivocal results when neces-
sary.
b. Time to Collect Specimens
( 1 ) Virus isolation. The time in the course
of the clinical disease that specimens
are collected is of utmost importance.
In general, for isolation techniques
the earlier in the acute stage the speci-
men is taken, the better the chance for
successful isolation. The specimen
should certainly be taken while the
patient is still acutely ill and febrile.
(2) Serological tests. Except in outbreaks
of encephalitis, scrum specimens must
be paired since only a rise in specific
30
antibody titer is positive evidence of
current infection with the responsible
agent. The first sample should be col-
lected as early in the illness as possible
and the second from 2 to 4 weeks after
the beginning of convalescence. Even
in arbovirus infections antibody in the
the first serum is only suggestive of
recent infection and must be followed
up with a second serum.
c. Container for Specimen
(1) Virus isolation. Materials for virus
isolation must be frozen if it will take
longer than a few hours to get them to
the laboratory. This requires dry ice.
Dry ice releases CO2 gas which is
deleterious to most viruses; therefore
these specimens must be completely
sealed off (flame-sealed ampules are
best for fluid specimens) . With samples
placed in bottles closed with tightly
fitting rubber stoppers, the tops should
be sealed with at least three turns of
high quality waterproof adhesive tape.
If facilities for freezing stool specimens
are not available, they can be emulsi-
fied in an equal volume of 1 molar
magnesium chloride and shipped in
an insulated container with cans of
frozen water (picnic cooler type) and
packed in wood shavings.
(2) Serological tests. The container must
protect the specimen from loss by
breakage as well as from contamination
and deterioration. Serum samples in
test tubes with tight rubber stoppers or,
preferably, sleeve stoppers (reinforced
by adhesive tape) are satisfactory.
Cork stoppers are quite unsatisfactory,
and the type of screw-cap vials fre-
quently loosen and leakage occurs.
d. Packing and Shipping of Specimens
(1) Virus isolation. Virus isolation sam-
ples once ampuled should be quickly
frozen (on dry ice) and maintained
in the frozen state (preferably at
— 70°C.) until the time for testing.
For safety purposes and to prevent
breakage, each specimen tube should
be individually wrapped in either paper
towels, facial tissues, or other absorbent
padding material. One or more padded
specimens can be placed in a small
cyhndrical shipping container which is
packed directly against 5 to 10 pounds
of dry ice (the amount of dry ice de-
pending upon the distance to be
shipped) in the center of a box large
enough for 6 inches of insulating ma-
terial (paper, cotton, etc.) to surround
it. Shipment should be by the most
rapid means available (usually air mail,
special delivery). The laboratory
should be notified by wire of the con-
tents, mode of shipment, air express
way-bill number, and time of expected
arrival. Whenever possible, specimens
should be shipped so that they will ar-
rive at the laboratory during the work-
ing day and not on weekends.
Birds submitted for psittacosis studies
should be thoroughly soaked in disin-
fectant, placed in a plastic bag, and
then packed in a shipping box with
dry ice. If rapid delivery is possible,
cans of frozen water may be used.
(2) Serological tests. If adequately pro-
tected against spillage and breakage,
serum samples for serological tests
may be shipped without refrigeration
in ordinary mailing containers. How-
ever, these specimens should also be
sent by the most rapid method.
2. Minimum Data to be Supplied With Specimen
Name of Patient.
Age.
Summary of pertinent history including date of
onset, physical findings, and clinical labora-
tory tests.
Virus group suspected.
Type of material submitted and date of collec-
tion.
Indication of other similar cases in family or
vicinity.
Viral or rickettsial vaccines given to patient and
dates administered.
Exposure to animals or insect vectors.
Antibiotic treatment
All specimens should be forwarded to the State pub-
lic health laboratory, accompanied by Form PHS
3.332 (see Appendix V). These forms are available
at your State laboratory and should be filled in as
completely as possible in order to expedite the proc-
essing of your specimen.
31
3. Laboratory Methods in Diagnosis
a. Direct Methods:
(1) Preparation of specimen for inocula-
tion. Certain types of materials, such
as throat washings, sputum, and feces,
must be freed of contaminating bac-
teria and molds before they are used for
inoculation. Ultra-centrifugation can
separate the contaminants at one speed
and concentrate the virus at another.
Filtration, chemical treatment, and
especially the addition of antibiotics
are also useful in removing contami-
nants. An effort should be made how-
ever, to collect materials as free from
contaminants as possible.
(2) Animal inoculation methods. Various
species of animals are inoculated by
different methods (intracerebral, in-
traperitoneal, intranasal, intravenous,
etc.), depending on the type of human
disease being studied and the type of
material available. Symptoms and
pathological responses are then ob-
served. These methods are used in the
isolation of the agents of rabies, psit-
tacosis, lymphogranuloma venereum,
encephalitis, herpes, rickettsial, and
other diseases.
(3) Chick embryo inoculation methods.
Embryonic tissue is more susceptible
to invasion by most viruses than adult
tissue. Chick embryos of varying ages
are inoculated by different routes (yolk
sac, allantoic sac, amniotic sac, etc.)
and are observed for death, for specific
pathological lesions, or as a source of
antigenic material for specific serolog-
ical identification against known pos-
itive immune sera (used in influenza,
smallpox, mumps, etc.).
(4) Tissue culture methods. Tissue culture
represents the use of specific tissues
in culture where extracellular factors
of the media can be controlled. This
method has become the laboratory
procedure of choice in the study of
many viruses (those of poliomyelitis,
influenza, chickenpox, etc.) for isola-
tion or for antibody determination and
measurement.
b. Indirect Methods:
( 1 ) Examination of tissues for pathological
change. Certain viruses cause specific
cellular responses characterized by in-
clusion bodies, such as Negri bodies
in rabies, which are diagnostic of active
infection. Some of the larger viruses —
lymphogranuloma venereum, for ex-
ample can actually be seen in tissue
smears as elementary bodies. Fluores-
cent antibody techniques have recently
been applied to the direct examination
of infected tissues or exudates for the
rapid identification of specific viral
antigen. This technique has been most
widely used in the diagnosis of rabies.
(2) Complement fixation tests. Specific
antigens are available for testing
paired acute and convalescent sera in
many viral and rickettsial diseases. In
rickettsial diseases, the complement
fixation test is more specific than the
Weil-Felix proteus agglutination pro-
cedure.
(3) Red cell agglutination tests. Certain
viruses cause red blood cells to ag-
glutinate. The presence of specific anti-
bodies against that virus will inhibit
the agglutination. This type of test is
frequently used in influenza, mumps,
Newcastle virus disease, certain en-
cephalitis infections, and rubella.
(4) Neutralization tests. In the neutraliza-
tion test, the fact that a given virus has
been inactivated or “neutralized” by
specific antibody present in a serum
specimen is demonstrated by the fail-
ure of a susceptible animal or tissue
culture to become infected when in-
oculated with the serum-virus mixture.
This procedure can be used with paired
acute and convalescent sera in any
virus or rickettsial disease for which
there is a susceptible animal or an
appropriate tissue culture system in
which the etiologic agent will multiply.
4. Interpretation of Laboratory Results
a. Virus Isolation:
Failure to isolate a specific virus from a
specimen does not rule out that agent, or an
unsuspected one, as the possible cause of
the illness in question. When a virus is iso-
lated, it signifies current infection (which
32
may be silent) with that virus. It usually,
but not always, means that the current ap-
parent illness is due to that virus. The
results of virus isolation attempts must be
correlated with the clinical, epidemiologic,
and serologic data.
b. Serologic Tests:
Since it has been found that some individuals
already have antibodies against certain
viruses because of previous contact with
that virus, the finding of antibodies in a
single serum sample taken during or after a
particular illness does not prove the etiology
of that illness. However, a definite (4-fold
or higher) rise in antibody titer from the
acute stage of the disease to convalescence
is usually significant. Although serologic
tests yield indirect evidence of virus activity,
they are more frequently informative than
virus isolation attempts. An effort should be
made in all cases to submit paired serum
specimens in order to make a presumptive
serologic diagnosis or to confirm the signifi-
cance of the virus isolation by demonstrating
a rise in antibody titer against the virus dur-
the course of illness.
Because of the large numbers of antigenic
types, serologic studies on paired serum
specimens usually cannot be performed in
suspected enterovirus infections unless a
virus is isolated from clinical material. When
pericarditis or pleurodynia is present and
adequate clinical information is provided,
the microneutralization test for Coxsackie
B viruses can be performed.
In certain situations, a third serum specimen
(taken late in convalescence, 2 or 3 months
after onset) is extremely valuable. This is
a recognized necessity in lymphocytic chori-
omeningitis and most rickettsial diseases. In
lymphocytic choriomeningitis, complement
fixing antibodies may not occur until late in
the course of the disease, approximately in
the eighth week. In rickettsial diseases and
in psittacosis and lymphogranuloma ven-
ereum, the production of complement fixing
antibodies may be suppressed or delayed in
patients who are treated early with broad
spectrum antibiotics.
5. Packing and Shipment of the Specimens for
Rabies Diagnosis
After decapitation of the animal in the field, the
head should be promptly cooled down and kept
cold. Whenever possible, it should be delivered
by messenger. If no messenger service is avail-
able, the head should be packed for shipment
by the fastest common carrier. It should be put
into a suitable watertight metal container and
tightly sealed. This container, in turn should
be put into a larger watertight metal container.
Cracked ice should be packed between the inner
and outer container. The package should be
clearly labelled and shipped to the laboratory
with utmost dispatch.
NOTE: Although freezing the specimen and
shipping it frozen in dry ice (solid carbon di-
oxide) or in nitrogen flasks will preserve the
virus, quick microscopic examination may be
delayed because of the time necessary for the
head to thaw. Frozen portions of brain and
salivary glands are easier to handle in the lab-
oratory than are frozen entire heads. Immedi-
ately upon thawing, the tissues should be pre-
pared for direct microscopic examination for
Negri bodies, for the fluorescent antibody test,
or for the mouse inoculation test.
33
The following information is desirable when
animal heads are received for examination: the
species and breed of the animal; whether it was
in contact with other animals; whether the
animal died or was killed, and, if the latter, the
means used in destroying it; whether the animal
was confined and observ’ed for an appropriate
time before death, and, if so, for how long;
symptoms of rabies, if any; and history of vacci-
nation against rabies.
34
ENTEROVIRUS-ASSOCIATED DISEASE^
35
See page for instructions on shipping birds.
Conjunctival smears are examined for inclusion bodies.
APPENDIX I
Postal Requirements for Shipping Diseased Tissues
and Other Specimens
(Extracts from Parts 124, 125, and 221, United
States Postal Manual)
PART 124
NONMAILABLE MATTER
124.1 INTRODUCTION
.11 DESCRIPTION. Nonmailable matter includes all
matter which is by law, regulation, or treaty stipula-
tion prohibited from being sent in the mail or which
cannot be forwarded to its destination because of
illegible, incorrect, or insufficient address.
.12 APPLICABILITY. The harmful or objectionable
things identified or described in this part are some
of the matter which may not be sent through the mail,
as a matter of absolute prohibition. See part 125
for matter mailable only under special rules or con-
ditions. Notwithstanding any statement contained
in part 124, which covers only some of the items
prohibited in the mail, the burden rests with the
mailer to assure that he has complied with the law.
In addition to the nonmailable items mentioned in
this part, certain other articles are prohibited in the
mail to military post offices overseas (part 127).
.13 PENALTIES FOR VIOLATION. Severe penalties, by
fine or imprisonment, or both, are provided for per-
sons who knowingly mail or cause to be mailed, any
matter which has been declared nonmailable under
law.
.14 NONCONFOR.VIITY WITH POSTAL REGULATIONS.
Regardless of its nature, matter may not be mailed in
any form if done in violation of postal regulations
for such reasons as failure to pay postage, improper
size or weight, improper permits, improper addresses,
etc.
.15 RESPONSiBii.riY OF MAILER. When mailers
are in doubt as to whether any matter is properly
mailable, they should ask the postmaster. Even
though the Post Office Department has not expressly
declared any matter to be nonmailable, the mailer
of such matter may be held fully liable for violation
of law if he does actually send nonmailable matter
through the mail.
124.2 HARMFUL MATTER
.21 GENERAL PROVISIONS OF LAW
Any articles, compositions, or materials, which
may kill or injure another, or injure the mail or other
property, are nonmailable. This includes but is not
limited to;
a. All kinds of poison or matter containing poison.
b. All poisonous animals, except scorpions (see
125.35), all poisonous insects, all poisonous rep-
tiles, and all kinds of snakes.
c. All disease germs or scabs.
d. All explosives, flammable material, infernal ma-
chines, and mechanical, chemical, or other de-
vices or compositions which may ignite or ex-
plode.
* * *
PART 125
MATTER MAILABLE UNDER SPECIAL
RULES
125.1 LEGAL RESTRICTIONS
.11 HARMFUL MATTER
.111 Certain items barred from the mail, as set
forth in part 124, may be mailed if prepared and
packaged in accordance with this part. These are
items not outwardly or of their own force dangerous
or injurious to life, health, or property.
.112 This part covers generally some of the more
common situations; however, the burden rests with
the mailer to assure that he has complied with the
law and that anything shipped by him has been prop-
erly prepared and packaged. The ordinary test of
adequate preparation and packaging is whether the
36
contents of a parcel are safely preserved under ordi-
nary hazards of mail handling and transportation.
.113 Products, materials, and devices are created
or modified with such frequency that the Post Office
Department is unable to issue general rulings in ad-
vance to govern adequate preparation and packaging.
Any mailer may, however, request the Post Office
Department, in advance, for a specific ruling as to
mailability of his item. The request should be ad-
dressed to the local postmaster, who will forward it
to the Classification and Special Services Division,
Bureau of Operations, Washington, D.C. 20260.
.12 APPLICABILITY OF OTHER LAWS
* * *
.122 Any special conditions or limitations placed
on transportation or movement of certain things shall
govern admissibility to the United States mail, when
imposed under law by the U. S. Department of the
Treasury; U. S. Department of Agriculture; U. S.
Department of Commerce; U. S. Department of
Health, Education, and Welfare; Interstate Com-
merce Commission; or any other Federal department
or agency having legal jurisdiction.
.13 PENALTIES
Severe penalties of fine or imprisonment, or both,
are provided by law, for anyone who knowingly de-
posits for mailing or delivery, or causes to be mailed
or delivered, anything declared nonmailable under
law. Failure to comply with the regulations of the
Postmaster General, as prescribed in this part, as
to matter otherwise nonmailable, constitutes a vio-
lation of law.
125.2 CONDITIONS FOR MAILING
.21 GENERAL NATURE OF PRECAUTIONS REQUIRED
.211 The restrictions against mailing of harmful
matter, from which relief is granted by this part, are
intended to prevent damage or harm to postal and
transportation personnel, to prevent damage or de-
struction of other mail and of property, to avoid ob-
noxious odors, and to prevent the spread of disease
and infection. Special preparation and packaging are
required to protect against such contingencies.
.212 Basic precautions, covered generally in this
section, relate to the inner containers holding the
harmful matter, internal cushioning and protection,
and exterior packaging and marking.
.22 LIQUIDS (nonflammable) and powders
.221 Precautions shall be taken in the case of liq-
uids, pastes, salves, ink powders, pepper, snuff, or
other pulverized materials against damage to mail
and property from leakage and against caustic, irri-
tant, toxic, or soiling effect on mail handling per-
sonnel.
.222 Containers shall meet any applicable Inter-
state Commerce Commission or other Federal speci-
fications. Closures must effectively seal the contents
against leakage. Friction tops must be fastened with
solder, clips, or otherwise so that they will not come
off under impact.
.223 Glass or other breakable containers of liq-
uid must be packaged to withstand handling en-
route. The container shall be cushioned inside the
carton to absorb shock and impact. Where feasible,
absorbent material shall be used, to take up all the
liquid in case of breakage.
H= * *
125.7 IDENTIFICATION AND MARKING
.71 IDENTIFICATION OF CONTENTS. The identity or
nature of contents of anything mailed under any of
the provisions of part 125 shall be stated plainly on
the outside of the parcel, as a condition of mailing.
.72 IDENTIFICATION OF MAILER AND ADDRESSEE. The
full name and address of both the mailer and the ad-
dressee shall be written in ink, rubberstamped, or
pasted on the outside of any package whose mailing
is covered by part 125.
.73 LABELS. Any labels required under Federal law
or under any regulations issued by any Federal agen-
cies pursuant to Federal law shall be pasted to the
outside of the parcel.
* *
PART 221
CONDITIONS APPLICABLE TO ALL
CLASSES
221.1 PREPARING AND ADDRESSING
.11 PREPARING
.111 Senders must prepare articles securely, espe-
cially if they are for distant countries. International
mail is handled more often and subjected to greater
pressure and friction than domestic mail, hence it
must be enclosed in strong envelopes or other wrap-
pings.
* * *
.114 Articles other than letters and letter packages
(AO mail) must be prepared in such a way that their
;ontents are sufficiently protected but so as not to
hinder quick and easy inspection of the contents.
They should be placed under wrapper, on a roller,
or between cardboard; in open bags, boxes, enve-
lopes, or containers or in closed, unsealed bags,
boxes, envelopes, or containers provided with fasten-
ers that can be easily opened and reclosed without
being dangerous; or they may be tied with string or
twine in a manner that will permit them to be easily
untied. Sealing of postal union other articles is not
permitted, even if registered, and they must be pre-
pared in such a way that other articles do not run the
risk of being trapped by them.
^ ^ ^
37
221.3 PROHIBITIONS .\SD RESTRICTIONS
.32 RESTRICTED .ARTICLES
* * *
.325 Perishable Biological Materials. Perishable
biological materials, including those of pathogenic
nature, when sent in the postal union mail are ac-
cepted only as LETTER PACKAGES. The follow-
ing conditions apply:
a. Mailing Restrictions
If a country prohibits perishable biological ma-
terials this is shown under Prohibitions in the
country item in the Directory of International
.Mail. The packages must be packed as pre-
scribed in 221.325c and must bear distinctive
violet labels by which they can be readily recog-
nized and receive careful handling and prompt
delivery.
b. Qualification of Mailers
( 1 ) Only officially recognized laboratories may
send or receive letter packages containing
perishable biological materials. Labora-
tories of the following categories are so
designated:
Laboratories of local. State, and Federal
government agencies.
Laboratories of federally licensed manu-
facturers of biologic substances derived
from bacteria and viruses.
Laboratories affiliated with or operated
by hospitals, universities, research facili-
ties, and other teaching institutions.
Private laboratories licensed, certified,
recognized, or approved by a public au-
thority.
(2) A laboratory desiring to mail letter packages
containing materials of this kind shall make
written application on its letterhead station-
ery to the Classification and Special Serv-
ices Division, Bureau of Operations, Post
Office Department, Washington, D.C.
20260, explaining its qualifications and those
of the prospective addressee to send and re-
ceive such materials, and stating how many
packages are to be mailed. On approval, the
mailer will receive a sufficient number of the
violet labels for the contemplated shipments,
c. Packaging
( 1 ) Perishable biological material not of a path-
ogenic nature must be packed in a nonpo-
rous container surrounded by sufficient ab-
sorbent material to take up all the liquid
and must be placed in an outer protective
container where it should fit tightly to avoid
any shifting.
(2) Perishable biological material of a patho-
genic nature must be packed in a tightly
closed bottle or tube or heavy glass wrapped
in thick absorbent material rolled several
times around the bottle or tube and tied at
the ends, sufficient in quantity to absorb all
the liquid; the wrapped container must be
placed in a strong well-closed metal box
constructed to prevent any contamination
outside of it. This metal box must be
wrapped in cushioning material and placed
in an outer protective box where it should
fit tightly to avoid shifting. The outer con-
tainer must consist of a hollow block of
strong wood, metal, or other equally strong
material with a tight lid so fitted that it can-
not open during transportation.
(3) In addition to the requirements in (1) and
(2), packages must comply with the regu-
lations governing the transmission of such
materials in the domestic mail.
(4) The mailer must place on each package one
of the violet labels mentioned in a and b(2).
>|c :|c s|c
APPENDIX II
Selected Transport Media and Reagents
Some biological specimens will withstand shipment
better if placed in a protective medium or if a pre-
servative is added. Likewise, isolated cultures of
bacteria, fungi, or viruses should be placed in or upon
a medium which will enhance or preserve their via-
bility in transit. It this appendix no attempt is made
to list all suitable transport media. The sender should
give careful consideration to the selection of media
in each specific case. Several “transport” media are
available commercially.
Some selected media recommended by the NCDC
are:
1 . Mcrthiolute solution for preserving blood serum
1.4 gm. sodium borate (borax)
1 .0 mg. merthiolate
100.0 ml. H.O
Dissolve the sodium borate in the water first
and then add the merthiolate. Use 0.1 ml. of
tlic above solution per 10 ml. of serum to give
1:10,000.
38
2. Sabouraud - cycloheximide - chloramphenicol
agar* for selective isolation of pathogenic
fungi.
Composition: Sabouraud dextrose agar (2%
agar content)
Cycloheximide** 0.5 mg./ml.
Chloramphenicol*** 0.05 mg./
ml.
Preparation: 1 liter
a. Suspend 65 gm. dehydrated Sabouraud
dextrose agar and 5.0 gm. agar in 1,000
ml. distilled water. Heat to boUing.
b. Add chloramphenicol (50 mg. suspended
in 10 ml. of 95% alcohol) to above boil-
ing medium. Remove quickly from heat
and mix.
c. Add cycloheximide solution (500 mg. in
10 ml. of acetone).
d. Mix well and distribute in tubes.
e. Autoclave at 118° C. for 10 minutes — no
longer. Slant and allow to harden.
3. Brain heart infusion — cycloheximide-chloram-
phenicol agar for isolation of fastidious fungi,
for example, Histoplasma capsulatum and
Blastomyces dermatitidis, at 25° C. or room
temperature.
Composition: Brain heart infusion agar (2%
agar content)
Cycloheximide 0.5 mg./ml.
Chloramphenicol 0.05 mg./ml.
Preparation: 1 liter
a. Suspend 37 gm. dehydrated brain infusion
agar and 20.0 gm. agar in 1,000 ml. dis-
tilled water. Heat to boiling.
b. Add chloramphenicol 0.05 mg./ml. to the
boiling medium. Remove from heat quick-
ly and mix.
c. Add cycloheximide 0.5 mg./ml.
d. Mix well and distribute in tubes.
e. Autoclave at 118° C. for 10 minutes — no
longer. Tube and slant.
4. Chloramphenicol solution to add to sputum
bottles for isolation of H. capsulatum
Preparation of stock solution.
Suspend 20 mg. chloramphenicol in 10
ml. 95% alcohol. Add 90 ml. of dis-
tilled water. If necessary, heat gently to
complete solution. This is a stable solu-
tion.
* Two essentially similar media are available in dehy-
drated form: Mycosel agar (BBL, Division of BioQuest);
Mycobiotic agar (Difco).
** Actidione (The Upjohn Co., Kalamazoo, Mich.)
*** Chloromycetin (Parke-Davis Co., Detroit, Mich.)
Use: Add 1.0 ml. of the stock solution to each
sterile sputum bottle. This amount is
ample for inhibition of contaminants in
1 to 10 ml. sputum. If the solution dries
in the bottle before use, its effectiveness
is unimpaired. The concentration de-
sired in sputum is approximately 0.2
mg./ml. of chloramphenicol.
5. Preparation of PVA-fixative for preservation
of stool specimens for parasitologic diagnosis
Add 6 gm. of polyvinyl alcohol* (PVA)
powder to 100 ml. of Schaudinn’s fixative at
room temperature, stirring constantly.
Modified Schaudinn’s Fixative:
Glacial Acetic Acid 5.0 ml.
Glycerol 1.5 ml.
Schaudinn’s Fixative (2 parts
saturated aqueous solution
of mercuric chloride and 1
part 95% ethyl alcohol) 93.5 ml.
Heat to about 75° C. or higher until powder
dissolves and suspension clears.
Cooled to room temperature, the solution
should be clear and free of lumps. Solutions
prepared with some lots of PVA may remain
turbid and may exhibit some precipitate
upon cooling. Unless these conditions are
excessive, they will not interfere with satis-
factory use of the solution. PVA-fixative
remains satisfactory for several months and
can be used either at room temperature or
heated to 50° C.
A quantity of specimen is thoroughly mixed
in a vial containing three or more parts of
PVA-fixative. Films for staining can be pre-
pared immediately or months later by spread-
ing two or three drops of the mixture over the
surface of a microscope slide. The smear
should cover about one-third of the slide sur-
face and it should, to reduce peeling during
staining, extend to the edge of the side of the
slide. It is important not to have the films too
thick and to allow them to dry thoroughly. If
the specimen in the vial jells, it can be lique-
fied by heating in a water bath prior to making
the films.
6. Chopped meat medium.
Ground meat (fat free) 500 gm.
Distilled water 1000 ml.
1 normal NaOH 25 ml.
Use lean beef or horse meat. Remove fat and
connective tissue before grinding. Mix meat,
* Gelvatol — available from Shawinigon Resins Co., Spring-
field, Mass.
39
water and NaOH and bring to boil, stirring.
Cool, refrigerate overnight and skim off any
remaining fat. Filter the mixture through two
layers of gauze and spread the meat particles
out to partially dry.
Add sufficient distilled water to the filtrate to
restore 1 liter original volume and add:
Trypticase or peptone 30 gm.
Yeast extract 5 gm.
Potassium phosphate 5 gm.
Glucose 3 gm.
Adjust pH to 7.8 with 1 N NaOH.
Dispense meat particles in 15 x 125 mm.
screw-cap tubes with a small scoop and add the
enriched filtrate. Use about 1 part meat par-
ticles plus 3 to 4 parts liquid (v/v) per tube.
Add a few iron filings to each tube. Tubes
should be more than half full (about 8 ml.
fluid). Autoclave at 121° C. for 15 minutes.
APPENDIX III
Acceptable Containers for Use in Shipping Specimens
1. Unfrozen specimens
For shipping small numbers of specimens (1 to
3 ) of whole blood or serum, any one of the usual
double-mailing containers with a metal bottom
and metal screw cap is satisfactory, providing
the cap is tight. The tubes should be individually
wrapped and identified, and the identifying list
should be wrapped around the outside of the
inner case before this is inserted in the outer case.
Sufficient absorbent cotton should be included
in the inner case to absorb any liquid resulting
from breakage in transit.
Shipment of large numbers of tubes in a card-
board carton is possible if the tubes are indi-
vidually wrapped and sufficient absorbent buffer
material — cotton, shredded pap>er, excelsior, etc.
— is included between the individual tubes and
next to the walls of the carton to absorb shocks
and leakage. The conventional cardboard car-
tons with partitions designed to create a com-
partment for each tube are most acceptable.
2. Frozen specimens
Small numbers of frozen sera or tissue blocks
may be shipped in metal thermos-type con-
tainers or in cartons with dry ice or bags of Sno-
gel or other reversible refrigerant. If the thermos-
type container is used, it should be pre-chilled
with dry ice for several hours before the speci-
mens are introduced. The specimens should be
individually wrapped and identified and packed
in such a way that they will not become loose as
the dry ice evaporates. Enclose the thermos in
an outer carton containing additional dry ice, if
necessary, and adequate insulating material to
hold the temperature for at least 24 hours longer
than the package is expected to be in transit.
Small numbers of frozen specimens may be trans-
ported short distances by placing them* in insu-
lated paper bags — the so-called “Jiffy Bag” used
for ice cream — with a small amount of dry ice.
These bags should be enclosed within one or two
other “Jiffy Bags” of larger size. Frozen bags
of “Snogel” may be used in place of dry ice.
Larger numbers of tubes should be individually
identified and wrapped and packed in cardboard
cartons with dry ice above and below and
an abundance of insulating material around them.
Wrap the carton with several layers of heavy
brown wrapping paper and tie or seal it securely
with tape. This carton should be enclosed in a
larger one which should be wrapped and sealed
before shipment. The package should be shipped
by the most rapid means available.
3. Refrigerated but not frozen specimens
Animal heads should always be refrigerated but
not frozen during transportation to the labora-
tory. If the head is that of a small animal, it
may be placed in a friction top can of proper
size, the lid firmly pressed into position, and the
can enclosed in a large size lard can or other con-
tainer. Cracked ice is used to fill the outer can
and, if available, a small amount of dry ice may
be included as this will help to hold the tempera-
ture down for a longer time without actually
freezing the specimen.
4. Glass slides
Whenever available, slide boxes should be used
with tissue paper stuffed between the slides.
Otherwise, the slides should be individually
wrapped in tissue and shipped in a screw-capped
mailing container with suitable absorbent ma-
terial to prevent breakage. Under no circum-
stances, should slides he mailed in a letter or man-
ila envelope.
40
APPENDIX IV
Reference Diagnostic Services Available at NCDC
Reference services of the various laboratories of
the NCDC are offered to assist the State public health
laboratories. Specimens should not be submitted
for routine diagnostic tests. Reference specimens
may be submitted only by the State health depart-
ment laboratory. They must conform with postal
regulations, as well as with NCDC instructions for
labelling and transmitting specimens, and for pack-
aging specimens for air mail shipments. Specimens
arriving in damaged condition will be destroyed with-
out examination.
The appropriate receiving laboratory should be
checked on the mailing label to insure prompt de-
livery of specimens. Specimens should be mailed so
as not to arrive in the laboratory on a Saturday, Sun-
day, or holiday. Except as indicated on the list be-
low, specimens sent to NCDC laboratories should be
accompanied by a diagnostic report form available
from the NCDC.
Laboratory support to epidemiological investiga-
tions, survey studies, or special research projects
should be arranged by prior consultation with the
Chief of the Laboratory Program.
REFERENCE SERVICES AVAILABLE
RECEIVING LABORATORY
Bacteriology
Identification, toxigenicity testing, and typing of C. diphtheriae
Slide agglutination and microscopic tests for leptospirosis. Submit form
PHS 3.203E with these specimens.
FebrUe agglutination tests
Serotyping of Listeria
Sera for the diagnosis of infections with S. typhi and other Salmonella
typ>es accepted only under exceptional circumstances. Sera for agglu-
tination tests for diagnosis of bacillary dysentery are not accepted
because they are of no value for this purpose.
Bacterial Serology Unit
Grouping of meningococci. Drug sensitivity testing.
Identification of miscellaneous bacteria other than the Enterobacteria-
ceae, streptococci, C. diphtheriae, and mycobacteria. (Miscellaneous
bacteria cannot be accepted for identification unless they are accom-
panied by a complete history. )
Identification of anaerobic bacteria and their toxins.
Bacteriophage typing of staphylococci. (Only epidemiologicaUy related
cultures will be accepted. )
Focal point for processing of bacteriology intrastate laboratory evalua-
tion specimens.
Bacterial Reference Unit
Serological grouping of beta hemolytic streptococci and typing of Group
A and Group B streptococci.
Biochemical differentiation of other streptococci and of pneumococci.
Serotyping of pneumococci.
Micro-antistreptolysin 0 determinations.
Streptococcus Unit
Identification of Salmonella, Shigella, and Arizona types.
Bacteriophage typing of S. typhi, S. paratyphi B, and 5. typhimurium.
Typing of Escherichia cultures from outbreaks of diarrhea in infants.
Typing of Klebsiella cultures in particular instances.
Identification of other enteric bacteria as warranted by clinical and epi-
demiological considerations.
Enteric Bacteriology Unit
Identification of acid-fast bacilli.
Determination of drug susceptibility of acid-fast bacilli.
Tuberculosis Unit
Serotyping of leptospira cultures isolated from human, animal, or en-
vironmental sources.
Veterinary Public Health
Laboratory Unit
(Epidemiology Program)
41
REFERENCE SERVICES AVAILABLE (continued)
RECEIVING LABORATORY
Mycology
Examination of cultures for pathogenic fungi (Dermatophytes, subcu-
taneous and systemic pathogens).
Examination of stained and unstained histological slides for presence of
pathogenic fungi.
Examination of clinical materials from cutaneous mycotic diseases.
(Hair, skin scrapings, nail clippings, etc.)
Examination of cultures and clinical materials of veterinary origin.
Serologic tests for actinomycosis, blastomycosis, coccidioidomycosis,
crv’ptococcosis, and histoplasmosis.
Mycology Unit
Parasitology
Examination of feces, urine, sputum, and other body fluids and aspirates
for evidence of parasitic infection. (Entamoeba histolytica and other
intestinal protozoa, intestinal helminths, schistosomes, Paragonimus
westermani. Echinococcus granulosus, etc.)
Examination of biopsy or autopsy material for Trichinella spiralis. Echi-
nococcus granulosus, etc.
Examination of histological preparations for presence of parasites.
Identification of isolated worms.
Examination of thick or thin blood films, impression smears, tissue sec-
tions, etc., for blood parasites (malarial parasites, hemoflagellates,
filarial worms, etc.)
Isolation of hemoflagellates by cultivation and animal inoculation from
unpreserved whole blood, spinal fluid, tissue, aspirates, etc.
Serologic tests for toxoplasmosis, trichinosis, echinococcosis, extra-intes-
tinal amebiasis, visceral larva migrans, cysticercosis, filariasis, schisto-
somiasis, Chagas disease, and kala-azar.
By special arrangement: examination of impression smears, spinal fluid,
tissue sections, etc., for Toxoplasma; attempted isolation of organisms
from whole blood, spinal fluid, tissues, etc.; serology on adult eye lesion
cases.
Parasitology Unit
VD Laboratory Procedures
Treponema Pallidum Immobilization (TPl) test on serum.
Identification of cultures of Neisseria gonorrhoeae
Under certain special conditions approved by the Venereal Disease Re-
search Laboratory, cultures of gonococci may be accepted for testing
to determine susceptibility to penicillin.
Venereal Disease Research
Laboratory
(Venereal Disease Program)
Virology and Rickettsiology
Reference studies for the identification of infectious agents in tissue cul-
ture fluids, animal tissues, or original clinical materials (including
acute and convalescent serum specimens for serology) submitted for
confirmation or extension of initial findings and examination of speci-
mens of animal origin for studies involving viral agents transmissable
to man. (Form PHS 3.332 should accompany these specimens.)
Virus Reference Unit
42
APPENDIX V
PHS 3.332 (CDC)
REV 3-64
Patient's Name
Address
City
County
Age Race
Occupation
Dote Onset
Clinical Diagnosis
Physician
Address
DEPARTMENT OF
HEALTH. EDUCATION. AND WELFARE
Public Health Service Communicable Disease Center
Laboratory Branch Virology Section
Atlanta, Georgia 30333
REQUEST FOR VIRAL AND RICKETTSIAL REFERENCE SERVICE
State
Sex
Local File No.
State Approval
Referral
Other Source
LABORATORY EXAMINATION REQUESTED:
Suspected Virus Group
VIRUS: Isolation Identification
SEROLOGY OTHER
REASON FOR REQUEST:
Epidemic Investigation
Surveillance Activities
Epidemiologic Survey
Other
OTHER CLINICAL DATA:
DATES OF PERTINENT IMMUNIZATIONS
Adenovirus
Influenza
Polio - Salk: No. ,
Oral: Type
Smallpox Other
Spotted Fever
Typhus
Yellow Fever
Rabies
Measles
EPIDEMIOLOGICAL DATA
Recent Travel (Location)
Family Contacts
Community Contacts
Animal Contacts
Arthropod Contacts: Mosquitos Ticks Sandflies Other Arthropods
Bite
Exposure only
SIGNS AND SYMPTOMS
Fever: Height
Duration
*
Rash : (Type)
Mucous Membrane lesions
Respiratory:
Rhinitis
Pharyngitis
Pneumonic involvement
Cardiovascular:
Myocarditis
Pericarditis
Gastrointestinal:
Diarrhea
Constination
Abdominal pain
Vomiting
(Continued on Reverse Side)
43
Central Nervous System:
Others :
Headache Delerium Seizures Lethargy
Paralysis: Flaccid Spastic
Muscle VVeakness Bulbar Involvement
Meningismus Nuchal Rigidity
Other
Jaundice Myalgia Pleurodynia
Myositis Conjunctivitis
H emorrhagic Phenomena Hepatomegaly
Associated Illness:
Fatal
TREATMENT
Antibiotics
Other
SPECIMENS SUBMITTED FOR EXAMINATION:
Origin: Human Animal Other
Date of
Collection
Throat Washing
Spinal Fluid
Others
Specimen
Stool
Specimen Local File No, Date of Collection
Serum: Acute
Convalescent
Virus Isolate: Source Passage
Tissue Culture
Mouse
Egg
Other
STORAGE OF SPECIMENS: Duration Method
LOCAL LABORATORY RESULTS:
Clinical diagnostic: WBC Differential
CSF: Cell Count % Lymphocytes
Others
Liver Function Studies
Bacteriological Studies;
Pathological Examinations;
Virus Isolation; Specimen Method
Results
Serology ;
Method used; CF Neut. 111
Test Antigen
- . ,
Acute
Coo valeucent
-
Other Pertinent Information;
44
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