RMl

no. 916

1968

COLLECTION, HANDLING & SHIPMENT

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U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE/public health service

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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

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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.

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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

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CONTENTS— Continued

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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.

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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

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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

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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-

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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.

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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

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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.

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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.

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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

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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

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throat, care should be taken