CONCENTRATION
`
`SPECIMEN
`
`METHODS
`
`SELECTIONOF
`
`PROCEDURESFOR
`
`PRES
`
`SPECIMENS
`OF
`
`MICROSCOPY
`
`MOUNTS
`
`WILS
`
`GOVU
`
`HE 20.7008 : In 8/2/982
`
`782
`
`UNIVERSITY OF
`
`MINNESOTA LIBRARY
`
`MAY 13 1985
`
`DEPOSITORY PUBN .
`
`U.S.-G. P. O. - D - 295
`GOVERNMENT PUBLICATIONS DIVISION
`
`CDC
`
`LAB MANUAL
`
`LABORATORY
`
`PROCEDURES
`
`FOR
`
`THE DIAGNOSIS
`
`OF
`
`INTESTINAL PARASITES
`
`U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
`
`PUBLIC HEALTH SERVICE
`
`Centers for Disease Control
`
`Geneoscopy Exhibit 1026, Page 1
`
`

`

`Geneoscopy Exhibit 1026, Page 2
`
`Geneoscopy Exhibit 1026, Page 2
`
`

`

`LABORATORY
`
`PROCEDURES
`
`FOR
`
`THE DIAGNOSIS
`
`OF INTESTINAL PARASITES
`
`Dorothy M. Melvin , Ph.D.
`
`and
`
`Marion M. Brooke , Sc.D.
`
`THIRD EDITION , 1982
`
`Reprinted January 1985
`
`U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
`
`PUBLIC HEALTH SERVICE
`
`CENTERS FOR DISEASE CONTROL
`
`LABORATORY IMPROVEMENT PROGRAM OFFICE
`
`LABORATORY TRAINING AND CONSULTATION DIVISION
`
`ATLANTA , GEORGIA 30333
`
`HHS Publication No. ( CDC ) 85-8282
`
`Geneoscopy Exhibit 1026, Page 3
`
`

`

`Use of trade names is for identification only and does not constitute endorse-
`
`ment by the Public Health Service or by the U.S. Department of Health and
`
`Human Services
`
`For sale by the Superintendent of Documents , U.S. Government Printing Office
`
`Washington , D.C. 20402
`
`ii
`
`Geneoscopy Exhibit 1026, Page 4
`
`

`

`PREFACE
`
`This manual is based on material developed for courses in the
`
`"Laboratory Diagnosis of Parasitic Diseases , " which have been pre-
`
`sented by the Centers for Disease Control (CDC ) since 1945. The
`
`authors have been associated with these courses since their begin-
`
`ning and have evaluated a great deal of material in preparing labor-
`
`atory manuals and brochures for student use . This manual contains
`
`information on practical procedures for performing parasitologic
`
`examinations in public health and medical laboratories . It includes
`
`information on collecting and processing specimens , methods of
`
`examination , and establishing reliable routine laboratory regimens
`
`for different types of laboratories . Information on collecting and
`
`shipping specimens for serologic testing is also included , although
`
`immunodiagnostic procedures are not discussed . The diagnostic
`
`stages of intestinal helminths and protozoa have been described by
`
`the authors in another publication : Morphology of the Diagnostic
`
`Stages of Intestinal Parasites of Man ( Brooke and Melvin , 1978 ) ,
`
`and are not included here .
`
`Like previous editions this third edition of the manual is divided
`
`into two sections : ( 1 ) a text section in which the available diagnostic
`
`procedures and methods are described and evaluated , and ( 2 ) a tech-
`
`niques section which gives step-by-step directions for diagnostic pro-
`
`cedures, formulas for solutions , and methods for preparing and
`
`examining specimens . The techniques section also contains informa-
`
`tion on microscopy , the calibration of ocular micrometers , quality
`
`control , and safety precautions .
`
`Some of the material in this manual has been taken from Ame-
`
`biasis : Methods in Laboratory Diagnosis by Marion M. Brooke (out
`
`of print) and the self-instructional lesson prepared by CDC , Ame-
`
`biasis : Laboratory Diagnosis : Part III: Laboratory Procedures ( see
`
`Reference Materials in QUALITY CONTROL for source) .
`
`The authors acknowledge the contributions of numerous asso-
`
`ciates over the years and thank the various people who have assisted
`
`in the preparation of the manual , particularly Miss Edwina B. Davis ,
`
`Chief, and Mr. Philip Thompson , Publication Activities , Center for
`
`Infectious Diseases , who edited the material , Dr. John Krickel ,
`
`Director of the Laboratory Training and Consultation Division , who
`
`helped in developing the format of the original manual , Mrs. China
`
`Christian who typed the manuscript , and Mr. Ed Biel , artist , Graphics
`
`iii
`
`Geneoscopy Exhibit 1026, Page 5
`
`

`

`Section , Publication Management , who prepared most of the illus-
`
`trations .
`
`Dorothy M. Melvin , Ph.D.
`
`Marion M. Brooke , Sc.D.
`
`iv
`
`Geneoscopy Exhibit 1026, Page 6
`
`

`

`.xiii
`
`XV
`
`1
`
`1 1 1
`
`5
`
`5 6
`
`6 7
`
`7 9
`
`•
`
`·
`
`·
`
`.
`
`•
`
`10
`
`10
`
`15
`
`16
`
`16
`
`•
`
`16
`
`17
`
`18
`
`..18
`
`19
`
`19
`
`20
`
`CONTENTS
`
`ILLUSTRATIONS
`
`INTRODUCTION
`
`SPECIMEN COLLECTION
`
`FECAL SPECIMENS
`
`Collection ...
`
`Normally Passed Specimens
`
`Multiple Specimens
`
`Purged Specimens .
`
`Enema Collection
`
`Transportation and Preservation .
`
`Unpreserved Specimens
`
`Preserved Specimens .
`
`Helminths ..
`
`Culture Medium for Shipping
`
`Packing Specimens for Mailing •
`
`Storage and Handling of Fecal Specimens
`
`OTHER SPECIMENS
`
`Sputum
`
`Urine
`
`Anal Swabs
`
`Vaginal and Urethral Swabs
`
`Duodenal Drainage Material
`
`Sigmoidoscopic Material .
`
`Aspirated Material
`
`Biopsied Material
`
`POSTAL SERVICE MAILING REGULATIONS
`
`•
`
`21
`
`22212
`
`27
`
`7
`
`28
`
`30
`
`30
`
`30
`
`31
`
`32
`
`33
`
`METHODS OF EXAMINATION
`
`FECAL SPECIMENS
`
`Macroscopic
`
`Microscopic
`
`Wet Mounts
`
`Saline ·
`
`Temporary Stains
`
`Concentration
`
`Flotation
`
`Sedimentation
`
`V
`
`Geneoscopy Exhibit 1026, Page 7
`
`

`

`Technical Errors
`
`Permanent Stains
`
`Protozoa
`
`Helminths
`
`Cultivation
`
`·
`
`Protozoa
`
`Helminths . ·
`
`Other Procedures
`
`Cellophane Thick Smear
`
`• ·
`
`Egg Hatching Technique .
`
`.
`
`Filtration Procedure
`
`Estimation of Worm Burden .
`
`OTHER SPECIMENS .
`
`Sputum and Urine
`
`Anal Swabs .
`
`Vaginal and Urethral Material
`
`•
`
`Duodenal Drainage Material
`
`Sigmoidoscopic Material .
`
`Aspirated Material
`
`Biopsied Material
`
`SELECTION OF PROCEDURES FOR LABORATORY
`
`REGIMENS
`
`PROCEDURES FOR REFERENCE DIAGNOSTIC OR
`
`PUBLIC HEALTH LABORATORIES
`
`Fecal Specimens .
`
`•
`
`Other Specimens ...
`
`PROCEDURES FOR HOSPITAL AND CLINIC
`
`LABORATORIES ..
`
`Feces and Sigmoidoscopic Material
`
`Normally Passed Specimens
`
`Purged Specimens
`
`Sigmoidoscopic Material
`
`Combination of Specimens
`
`Other Types of Specimens
`
`Aspirated Material
`
`•
`
`Anal Swabs ..
`
`Urine , Vaginal Swabs , Duodenal Drainage Material ,
`
`and Sputum
`
`·
`
`•
`
`•
`
`·
`
`•
`
`35
`
`35
`
`35
`
`38
`
`39
`
`39
`
`40
`
`42
`
`42
`
`42
`
`42
`
`42
`
`44
`
`44
`
`44
`
`44
`
`45
`
`46
`
`47
`
`47
`
`49
`
`49•
`
`49
`
`50
`
`50
`
`52
`
`52
`
`54
`
`54•
`
`·
`
`54
`
`55
`
`55
`
`55
`
`55
`
`PROCEDURES FOR POSTTREATMENT SPECIMENS
`
`•
`
`56
`
`REPORTING RESULTS •
`
`57•
`
`vi
`
`Geneoscopy Exhibit 1026, Page 8
`
`

`

`TECHNIQUES
`
`PRESERVATION OF SPECIMENS
`
`·
`
`FORMALIN
`
`Preparation .
`
`Technique for Using
`
`•
`
`PVA-FIXATIVE ..
`
`Preparation
`
`Technique for Using
`
`• •
`
`MICROSCOPY
`
`EQUIPMENT
`
`ILLUMINATION AND ALIGNMENT
`
`•
`
`CARE OF THE MICROSCOPE
`
`CALIBRATION OF OCULAR MICROMETER
`
`DIRECT WET MOUNTS
`
`PREPARATION OF MOUNTS
`
`·
`
`Unpreserved Feces
`
`Sealing Preparation
`
`Formalin Preserved Feces
`
`•
`
`Concentrated Specimens
`
`Other Types of Material
`
`EXAMINATION
`
`SOLUTIONS
`
`Iodine Solutions
`
`Dobell and O'Connor's Iodine •
`
`Lugol's Iodine
`
`Merthiolate- Iodine -Formaldehyde ( MIF ) Solution
`
`Quensel's Stain Solution
`
`...
`
`Buffered Methylene Blue Solution
`
`CONCENTRATION PROCEDURES
`
`CALCULATION OF CENTRIFUGAL SPEED
`
`FLOTATION PROCEDURES
`
`Modified Zinc Sulfate Centrifugal Flotation Technique
`
`Preparation of Solution •
`
`Technique
`
`Fresh Fecal Specimens
`
`·
`
`Formalin -Preserved Fecal Specimens
`
`vii
`
`59
`
`59
`
`59
`
`60
`
`61
`
`62
`
`64
`
`67
`
`67
`
`68
`
`69
`
`71
`
`75
`
`75
`
`. 75
`
`. 76
`
`76
`
`78
`
`79
`
`80
`
`82
`
`82
`
`82
`
`83
`
`84
`
`86
`
`88
`
`91
`
`91
`
`93
`
`93
`
`93
`
`94
`
`94
`
`96
`
`Geneoscopy Exhibit 1026, Page 9
`
`

`

`555880
`
`97
`
`97
`
`100
`
`101
`
`101
`
`102
`
`102
`
`102
`
`103
`
`104
`
`105
`
`108
`
`108
`
`108
`
`108
`
`111
`
`111
`
`111
`
`111
`
`113
`
`115
`
`116
`
`116
`
`116
`
`116
`
`116
`
`117
`
`117
`
`118
`
`118
`
`118
`
`119
`
`119
`
`Concentrated Brine Flotation Technique
`
`Preparation of Solution
`
`•
`
`Technique
`
`SEDIMENTATION PROCEDURES
`
`Gravity Sedimentation Technique
`
`Centrifugal Sedimentation Technique
`
`Feces
`
`Urine
`
`Acid-Ether Sedimentation Technique
`
`Preparation of Solution
`
`•
`
`Technique
`
`Formalin-Ether Sedimentation Technique
`
`Unpreserved Specimens
`
`Formalin-Preserved Specimens
`
`Formalin-Ethyl Acetate Sedimentation Technique
`
`•
`
`Merthiolate- Iodine -Formaldehyde Concentration
`
`Technique ( MIFC )
`
`Gravity Sedimentation
`
`MIFC Centrifugal Method
`
`STAINING PROCEDURES
`
`FECES ...
`
`Preparation of Smears
`
`Unpreserved Specimens
`
`PVA-Fixed Specimens
`
`·
`
`Examination ..
`
`Quality Control
`
`...
`
`Preparation of Solutions
`
`Schaudinn's Fixative .
`
`·
`
`Iodine Alcohol
`
`•
`
`Mordant
`
`.
`
`Stains .
`
`Stock Hematoxylin Stain
`
`•
`
`Hematoxylin Staining Solution
`
`Trichrome Stain
`
`Chlorazol Black E Stain
`
`Destaining Solutions . .
`
`Ferric Alum Solution .
`
`Phosphotungstic Acid Solution
`
`Saturated Aqueous Picric Acid Solution
`
`·
`
`Acid Alcohol
`
`Carbol-Xylene
`
`•
`
`119
`
`119
`
`120
`
`120
`
`viii
`
`Geneoscopy Exhibit 1026, Page 10
`
`

`

`Lithium Carbonate , Saturated Aqueous Solution
`
`...
`
`120
`
`Techniques
`
`Heidenhain's Iron-Hematoxylin Stain •
`
`Unpreserved Specimens ·
`
`PVA-Fixed Material
`
`Stain Reactions
`
`Iron -Hematoxylin-Phosphotungstic Acid Stain
`
`•
`
`Technique
`
`Stain Reactions
`
`Trichrome Stain
`
`Unpreserved Specimens
`
`•
`
`PVA-Fixed Material
`
`Stain Reactions
`
`Chlorazol Black E Stain
`
`Fecal Smears
`
`Protozoa in Tissue
`
`Stain Reactions
`
`OTHER MATERIALS
`
`ADULT HELMINTHS
`
`•
`
`•
`
`Collection of Specimens
`
`From Feces
`
`From Tissue
`
`From Animal Hosts
`
`Preparation of Solutions
`
`Fixatives .
`
`Stains
`
`Staining Technique .
`
`Preparing Worms
`
`•
`
`Fixing Worms
`
`Staining Procedure
`
`Clearing Helminths Without Staining
`
`Preparation of Solutions
`
`Technique
`
`Dehydration-Clearing Helminths for Permanent Mounts
`
`CULTIVATION PROCEDURES
`
`•
`
`INTESTINAL PROTOZOA
`
`Media .
`
`Modified Boeck and Drbohlav's Medium
`
`Egg Yolk -Infusion Medium
`
`·
`
`Liver Infusion Agar Medium
`
`ix
`
`120
`
`120
`
`121
`
`122
`
`123
`
`124
`
`124
`
`125
`
`125
`
`127
`
`128
`
`128
`
`129
`
`129
`
`130
`
`131
`
`131
`
`132
`
`132
`
`132
`
`132
`
`132
`
`133
`
`133
`
`134
`
`135
`
`135
`
`137
`
`138
`
`139
`
`139
`
`140
`
`141
`
`.143
`
`143
`
`143
`
`143
`
`146
`
`148
`
`•
`
`•
`
`Geneoscopy Exhibit 1026, Page 11
`
`

`

`Charcoal Medium
`
`Commercial Media
`
`Inoculation and Examination Techniques
`
`•
`
`Shipping Medium
`
`...
`
`EXAMINATION AND CULTIVATION OF ASPIRATES
`
`FROM LIVER ABSCESSES ...
`
`CULTIVATION OF TRICHOMONAS VAGINALIS
`
`·
`
`Preparation of Media
`
`Feinberg-Whittington Medium
`
`Modified Diamond's Medium
`
`Technique
`
`HELMINTH LARVAE ·
`
`• •
`
`Charcoal or Sand Culture
`
`Preparation ...
`
`Recovery of Larvae from Cultures
`
`Test -Tube Culture
`
`·
`
`Examination ..
`
`MISCELLANEOUS PROCEDURES
`
`TECHNIQUES FOR DIAGNOSING ENTEROBIASIS
`
`•
`
`Cellulose -Tape Slide Technique
`
`• •
`
`Preparation of Slides
`
`Collection Technique
`
`Examination Technique
`
`Vaseline-Paraffin Swab Technique
`
`Preparation of Swabs
`
`Collection Technique
`
`Examination Technique
`
`KATO THICK - SMEAR TECHNIQUE FOR FECES
`
`•
`
`ESTIMATING WORM BURDENS
`
`...
`
`Dilution Egg Counting Technique
`
`Apparatus ..
`
`Preparation of Solution
`
`Procedure .
`
`Interpretation of Results
`
`Standard Smear Egg Counting Technique
`
`·
`
`Apparatus .
`
`Preparation of Solutions .
`
`Calibration of Light Meter
`
`Preparation of Fecal Smear
`
`Use of Non- Standardized Smear .
`
`Interpretation of Results
`
`149
`
`151
`
`151
`
`152
`
`154
`
`155
`
`155
`
`155
`
`156
`
`156
`
`157
`
`157
`
`157
`
`158
`
`158
`
`161
`
`163
`
`163
`
`163
`
`163
`
`163
`
`165
`
`165
`
`165
`
`165
`
`165
`
`166
`
`167
`
`168
`
`168
`
`169
`
`169
`
`171
`
`172
`
`172
`
`•
`
`173
`
`•
`
`·
`
`173
`
`174
`
`175
`
`175
`
`X
`
`Geneoscopy Exhibit 1026, Page 12
`
`

`

`Kato Thick -Smear Technique for Quantitative
`
`Determinations ..
`
`Quantitative Method for Schistosoma haematobium
`
`Eggs in Urine
`
`Coefficient of Infection for Enterobius Infections
`
`175
`
`177
`
`178
`
`EGG HATCHING FOR SCHISTOSOME MIRACIDIA
`
`.. 179
`
`PROCEDURES FOR MATURING COCCIDIA OOCYSTS
`
`• •
`
`181
`
`EXAMINATION OF RECTAL BIOPSY TISSUE .
`
`Schistosomiasis
`
`•
`
`Amebiasis
`
`TECHNIQUES FOR TRICHINOSIS
`
`·
`
`Compression Technique .
`
`Digestion Technique .
`
`RECOVERY OF HELMINTHS FROM FECES
`
`182
`
`183
`
`183
`
`•
`
`184
`
`185
`
`185
`
`187
`
`RECOVERY OF LARVAE AND EGGS FROM SOIL
`
`.. 187
`
`COLLECTING AND SHIPPING SPECIMENS FOR
`
`SEROLOGIC TESTS FOR PARASITES
`
`QUALITY CONTROL
`
`TRAINING OF PERSONNEL
`
`•
`
`EQUIPMENT ....
`
`TECHNICAL PROCEDURES .
`
`CHECKS
`
`REFERENCE MATERIALS
`
`PARASITE REFERENCE COLLECTION ..
`
`188
`
`191
`
`191
`
`·
`
`192
`
`193
`
`195
`
`196
`
`198
`
`EQUIPMENT AND MATERIALS FOR LABORATORY
`
`DIAGNOSTIC SERVICES IN PARASITOLOGY
`
`199
`
`SAFETY PRECAUTIONS
`
`. 203
`
`PERSONAL HYGIENE AND HEALTH PRACTICES
`
`.. 203
`
`DISPOSAL OF SPECIMENS AND HANDLING OF
`
`SPECIFIC ORGANISMS
`
`...
`
`HANDLING OF DANGEROUS CHEMICALS
`
`·
`
`STORAGE AND DISPOSAL OF FLAMMABLE
`
`SOLVENTS
`
`EQUIPMENT SAFETY
`
`RESPONSIBILITY FOR LABORATORY SAFETY
`
`PUBLICATIONS ON SAFETY
`
`.. 204
`
`.. 206
`
`.. 208
`
`•
`
`•
`
`210
`
`212
`
`212
`
`xi
`
`Geneoscopy Exhibit 1026, Page 13
`
`

`

`REFERENCES
`
`INDEX .
`
`217
`
`. 229
`
`xii
`
`Geneoscopy Exhibit 1026, Page 14
`
`

`

`ILLUSTRATIONS
`
`FIGURES
`
`1. Collection Containers for Fecal Specimens
`
`2. Use of PVA- Fixative and Formalin for Submitting
`
`Stool Specimens . •
`
`3. Containers for Shipping Fecal Specimens
`
`4. Method for Wrapping Slides for Mailing
`
`•
`
`5. Consistency of Feces and Distribution of
`
`Cysts and Trophozoites
`
`6.
`
`Parasitologic Examination of Stool Specimens
`
`•
`
`7. Use of the Microscope Mirror and Condenser
`
`8. Insertion of the Micrometer Disc into
`
`Microscope Oculars
`
`9. Calibration of the Ocular Micrometer .
`
`10. Preparation of Wet Mounts of Feces .
`
`11.
`
`Sealing Mounts with Vaspar
`
`12. Method of Examining Wet Mount
`
`13. Dobell's Iodine , color
`
`14. Zinc Sulfate Technique , Methods of Obtaining Sample
`
`for Examination .
`
`15. Flotation Vial ...
`
`16 . Sedimentation Flask .
`
`17. Formalin-Ether Sedimentation Technique
`
`•
`
`18. Preparation of Smears for Permanent Stains
`
`19. Smears for Staining, Correct Density
`
`20. Iodine -alcohol , color ·
`
`21. Trichrome Stain Set -up
`
`22. Use of Supports for Whole Mounts of Helminths
`
`•
`
`23. Rice Starch Dispenser
`
`24. Baermann Apparatus
`
`4
`
`12
`
`13
`
`14
`
`28
`
`•
`
`51
`
`70
`
`.. 73
`
`74
`
`· •
`
`77
`
`78
`
`·
`
`81
`
`84
`
`95
`
`98
`
`100
`
`107
`
`•
`
`112
`
`114
`
`117
`
`126
`
`142
`
`145
`
`159
`
`25. Test Tube (Filter Paper) Culture for Nematode Larvae
`
`...
`
`160
`
`26. Cellulose -Tape Anal Swab
`
`27. Stoll Flask and Pipette
`
`·
`
`164
`
`169
`
`xiii
`
`Geneoscopy Exhibit 1026, Page 15
`
`

`

`28. Standard Smear Apparatus
`
`29. Side-Arm Flask for Hatching Miracidia
`
`30. Safety Cabinet for Flammable Solvents
`
`TABLES
`
`1. Diagnostic Stages Recovered by Different Methods
`
`2. Laboratory Routine for Parasitologic Examinations .
`
`172
`
`180
`
`214
`
`41
`
`53
`
`xiv
`
`Geneoscopy Exhibit 1026, Page 16
`
`

`

`INTRODUCTION
`
`Accurate clinical diagnosis of intestinal parasite diseases is difficult
`
`and laboratory confirmation is usually necessary . Demonstrating the
`
`diagnostic stage or stages of the parasite by directly examining
`
`specimens is the most reliable method of establishing a diagnosis
`
`of most parasitic infections , although indirect (serologic ) methods
`
`are available for a few in which the organism is not readily demon-
`
`strated , including trichinosis , echinococcosis , extraintestinal ame-
`
`biasis , and chronic schistosomiasis .
`
`This manual is concerned with direct laboratory procedures used
`
`in recovery and identification of intestinal parasites and related
`
`species (atrial, bladder , and lung parasites ) and with the collection
`
`of satisfactory specimens . Competent laboratory work is dependent
`
`on several factors :
`
`1 ) personnel trained in examining specimens and
`
`accurately identifying organisms , 2 ) adequate laboratory facilities ,
`
`including a good microscope , and 3 ) satisfactory specimens . Any
`
`laboratory procedure used for directly demonstrating parasites is
`
`only as reliable as the microscopist who examines the specimen ;
`
`therefore , all persons attempting to make parasitologic examinations
`
`should have intensive training and supervised experience .
`
`The primary purpose of this manual is to present selected methods
`
`which can be used routinely in laboratories performing parasitologic
`
`examinations and which , when performed properly , are reliable and
`
`thorough .
`
`XV
`
`Geneoscopy Exhibit 1026, Page 17
`
`

`

`FFyMm.*™
`
`Geneoscopy Exhibit 1026, Page 18
`
`Geneoscopy Exhibit 1026, Page 18
`
`

`

`SPECIMEN COLLECTION
`
`AND HANDLING
`
`Geneoscopy Exhibit 1026, Page 19
`
`

`

`
`
`x‘+:-’'t.4.4i:.''
`!!:-i—-ma—__—_——_—~~-
`
`;5
`._—-,
`
`Geneoscopy Exhibit 1026, Page 20
`
`Geneoscopy Exhibit 1026, Page 20
`
`

`

`SPECIMEN COLLECTION
`
`AND HANDLING
`
`The usual diagnostic stages of intestinal parasites are helminth eggs
`
`and larvae and protozoan trophozoites and cysts . Specimens ob-
`
`tained for diagnosis should be collected and handled so that these
`
`stages , when present , will be identifiable when the specimens reach
`
`the laboratory . In fact , reliable diagnoses of parasitic diseases begin
`
`with satisfactory materials , and the importance of properly collected
`
`specimens cannot be overemphasized . Inadequate samples and old
`
`or poorly preserved materials are usually of little or no value in
`
`establishing a diagnosis and may lead to erroneous conclusions .
`
`The most common type of body material submitted for parasi-
`
`tologic examination is feces , but other materials such as sputum ,
`
`urine , aspirates , tissue scrapings , and specimens obtained by biopsy
`
`may be submitted in certain cases .
`
`FECAL SPECIMENS
`
`•COLLECTION
`
`NORMALLY PASSED SPECIMENS
`
`To obtain satisfactory material for examination , adequate instruc-
`
`tions and proper containers for collecting fecal specimens must be
`
`provided . Persons who have little or no knowledge of the proper pro-
`
`cedures for submitting samples to the laboratory often make the
`
`collections ; therefore , complete and explicit directions should be
`
`given to the person responsible for obtaining the specimen . Printed
`
`instructions with line drawings to illustrate certain steps are often
`
`included in the collection kits the laboratory supplies . In hospitals or
`
`clinics , brief but specific directions should be supplied to ward or
`
`nursing stations .
`
`Specimens for diagnosis may be divided into three categories :
`
`1 ) those received directly from hospitalized patients ; 2 ) those sub-
`
`mitted by outpatients ; and 3 ) those shipped or mailed to the labor-
`
`atory . In hospital or clinic laboratories , most patient specimens fall
`
`into the first two categories , although proficiency testing specimens
`
`may be received by mail . Public health and reference laboratories
`
`1
`
`Geneoscopy Exhibit 1026, Page 21
`
`

`

`2
`
`SPECIMEN COLLECTION AND HANDLING
`
`most often receive shipped specimens . The criteria for satisfactory
`
`material are essentially the same for all three categories, but the
`
`method of submission , described in the section on Transportation
`
`and Preservation , will vary .
`
`In collecting fecal specimens , several factors should be considered :
`
`type of container , size and age of the specimen , and substances that
`
`might interfere with examinations . The container should be clean ,
`
`dry and sufficiently large to accommodate a sample of adequate size .
`
`It should also be leakproof, with a tight-fitting lid to prevent spills .
`
`A half- pint waxed cardboard carton with an operlapping lid is recom-
`
`mended for routine collection (fig .
`
`1 ) . This type of carton can be
`
`defecated into directly , does not leak , and permits labeling directly
`
`on the box . Plastic cups of similar size with snap-in lids are also satis-
`
`factory . Larger sizes of both types of containers are available .
`
`Feces should be passed directly into the container or on clean
`
`paper and transferred to the container for transporting to the labor-
`
`atory . In hospitals , specimens may also be collected in bedpans if
`
`the feces are not contaminated with urine .
`
`Information identifying the patient and the date and . hour of
`
`passage should be recorded on the carton or should accompany the
`
`specimen . The time of passage is especially important , since tropho-
`
`zoites soon die outside the body and other stages may undergo
`
`changes that would hamper identification . Ideally , unpreserved
`
`specimens should arrive in the laboratory within 1 to 2 hours after
`
`passage . Older specimens , especially if they contain trophozoites ,
`
`may be erroneously reported as negative . A suggested identification
`
`label for specimen containers is as follows :
`
`Name
`
`Age
`
`Date
`
`hour
`
`Male ( )
`
`Female ( )
`
`and
`
`AM
`
`PM
`
`stool passed
`
`To permit adequate macroscopic and microscopic examination ,
`
`the entire fecal passage should be submitted to the laboratory . This
`
`not only provides sufficient material for several techniques , but
`
`Geneoscopy Exhibit 1026, Page 22
`
`

`

`SPECIMEN COLLECTION AND HANDLING
`
`3
`
`allows the technologist to select a particular portion of the stool for
`
`examining . For example , soft or mucoid material is preferred for pre-
`
`paring smears for staining and more formed portions are used for
`
`concentration . Since organisms inhabiting the lower colon may often
`
`be present in the outer layer of the fecal bolus, having the entire
`
`stool will allow the technologist to scrape material from the surface
`
`for examining. Surface material is often useful in diagnosing Schis-
`
`tosoma mansoni and Entamoeba histolytica infections .
`
`If the entire fecal specimen cannot be submitted , a quantity of
`
`about 20 to 30 grams (the size of a medium-size walnut , if formed ,
`
`or 2 to 3 tablespoonsful , if loose or watery ) should be provided to
`
`the laboratory .
`
`Several substances , generally referred to as " interfering sub-
`
`stances," will make the specimen unsatisfactory for parasitologic
`
`examinations . Urine and water will destroy trophozoites , and feces
`
`contaminated with either are not suitable for examination . Speci-
`
`mens collected in bed pans should be checked to be sure they are
`
`free of urine . Occasionally , feces are dipped out of toilet bowls and
`
`thus mixed with water . Not only does water destroy trophozoites ,
`
`but there is a danger of coprozoic free -living organisms contamin-
`
`ating the feces and making diagnosis difficult . In addition , feces
`
`deposited on soil ( as may happen with outpatients) are not satis-
`
`factory , since free -living larvae and other contaminants from the soil
`
`may cause errors in diagnosis .
`
`Certain drugs and compounds will also render the stool specimens
`
`unsatisfactory for examination or for detection of organisms . Among
`
`these are antidiarrheal compounds , antibiotics , antacids , bismuth ,
`
`and barium (Juniper , 1962 ) . Specimens for parasitologic diagnosis
`
`should be obtained before these compounds are used , otherwise
`
`collection must be delayed until the effects have passed . Specimens
`
`should not be collected for 7 to 10 days after barium or bismuth
`
`have been given , since crystals and particles of these compounds in
`
`the feces will interfere with examination and may destroy tropho-
`
`zoites by their abrasive action . Antibiotics often cause a temporary
`
`decrease or absence of organisms in the stools and reliable diagnoses
`
`may not be possible for 2 to 3 weeks or more . In addition , stools
`
`should not be collected from patients receiving gallbladder dye until
`
`about 3 weeks afterwards (McQuay , 1969 ) .
`
`Specimens collected in very cold regions should not be allowed to
`
`freeze , since freezing and thawing may destroy protozoan cysts and
`
`trophozoites (Goldman and Johnson , 1950 ; Chang , 1955 ) .
`
`Geneoscopy Exhibit 1026, Page 23
`
`

`

`4
`
`SPECIMEN
`
`COLLECTION
`
`AND HANDLING
`
`Containers for Collection of Fecal Specimens
`
`Figure 1 .
`
`Name
`
`A
`g
`e
`
`Date.
`
`hour
`
`Male
`Female ()
`
`()
`
`and
`AM
`passed
`stool
`PM
`
`Carton for Unpreserved
`
`Specimens Submitted
`
`Immediately to the Laboratory
`
`Two Vials Containing Formalin
`
`and PVA- Fixative Solutions
`
`for Preserving Specimens
`
`which ARE NOT Sent
`
`Immediately to the Laboratory
`
`F
`
`PVA
`
`Geneoscopy Exhibit 1026, Page 24
`
`

`

`SPECIMEN COLLECTION AND HANDLING
`
`5
`
`MULTIPLE SPECIMENS
`
`Because of the intermittent passage of certain parasites from the
`
`host and limitations of available diagnostic techniques , the possibil-
`
`ity of finding organisms is increased by examining multiple speci-
`
`mens (Andrews , 1934 ; Sawitz and Faust , 1942 ; Wykoff and Ritchie ,
`
`1952 ; Swartzwelder, 1952 ; Alicna and Fadell , 1959 ) . A single ,
`
`normally passed specimen reveals only about one-third to one-half
`
`of the species present . Thus , a physician receiving a report of “ no
`
`parasites found " on a single specimen should realize that this does
`
`not exclude the possibility of the patient being infected . In general ,
`
`nematode species such as Ascaris lumbricoides, hookworm and
`
`Trichuris trichiura shed eggs more or less constantly and may be de-
`
`tected daily in feces . Other parasite species , however , especially the
`
`protozoa, are passed irregularly and possibly for only a few days at
`
`a time (Marsden , 1946 ) . The number of cysts of Entamoeba histo-
`
`lytica passed in the stools fluctuates from day to day , with cyclical
`
`peaks occurring 7 to 10 days apart (Tsuchiya , 1932 ; Lincicome ,
`
`1942 ; Kershaw, 1946 ; Stamm , 1957 ) . Giardia lamblia cysts are also
`
`passed intermittently at intervals ranging from 2 to 3 days to 7 or 8
`
`or more (Rendtorff, 1954 ; Danciger and Lopez , 1975 ) . Egg produc-
`
`tion in certain of the helminth infections , particularly the schis-
`
`tosomes and Diphyllobothrium latum, is also irregular . Proglottids
`
`of Taenia species may be passed at intervals . Thus collecting speci-
`
`mens at 2- to 3 -day intervals seems preferable to obtaining them on
`
`successive days .
`
`Workers do not agree on the number of specimens that should be
`
`examined to rule out infections . If time permits , however , at least 2
`
`or 3 normally passed stool specimens , preferably spaced 2 to 3 days
`
`apart , should be examined before one resorts to catharsis or sigmoi-
`
`doscopy . Normally passed specimens are more likely to contain
`
`cysts which can be identified with greater reliability then tropho-
`
`zoites ( Bijlmer, 1948 ; Brooke et al . , 1953 ; Stamm , 1957 ; and Gold-
`
`man , 1964 ) . Since organisms may be rare in passages following purga-
`
`tion for a week or more , normally passed specimens should be
`
`collected before the purge is given .
`
`PURGED SPECIMENS
`
`In some cases , purged specimens (obtained following administra-
`
`tion of a laxative ) may be desirable or necessary . Several workers
`
`have shown that purgation increases the possibility of finding organ-
`
`Geneoscopy Exhibit 1026, Page 25
`
`

`

`6
`
`SPECIMEN COLLECTION AND HANDLING
`
`isms in a single specimen (Andrews , 1934 ; Alicna and Fadell , 1959 ) .
`
`In cases of protozoan infections , however , trophozoites predominate
`
`in purged specimens , and species identification of trophozoites , as
`
`already stated , is usually less reliable than identification based on
`
`cysts . Saline laxatives such as sodium sulfate or buffered phospho-
`
`soda should be used for purgation . Mineral oil , bismuth , or magnesia
`
`compounds are unsatisfactory . Oil globules interfere with examin-
`
`ation , and crystalline debris from bismuth and magnesia compounds
`
`may obscure the organisms or affect the appearance of trophozoites .
`
`The patient receiving catharsis should be directed to collect all
`
`bowel movements in separate boxes numbered in series and to de-
`
`liver them immediately to the laboratory . Postcathartic specimens
`
`should be examined for eggs , larvae , and cysts as well as for tropho-
`
`zoites. If the examination may be delayed or if it is necessary to ship
`
`the specimen to the laboratory , a portion of the material should be
`
`mixed with polyvinyl alcohol (PVA)-fixative , preferably upon
`
`collection or as soon afterwards as possible ( figs .
`
`1 and 2 ) . ( See the
`
`paragraph on Preserved Specimens for additional information on
`
`PVA-fixatives . ) This practice may be advisable even in clinics and
`
`hospitals where , theoretically , the specimens are taken directly to the
`
`laboratory and examined , but where considerable delay may actually
`
`occur .
`
`ENEMA COLLECTION
`
`Specimens may also be obtained by using a saline solution enema .
`
`Since intestinal parasites seldom locate in the lower colon ( Hegner
`
`et al. , 1938 ) , enemas tend to be less efficient than purges for bringing
`
`down organisms . However , saline enema material can be used for
`
`parasitologic examination in lieu of other types of specimens . It
`
`should be handled as suggested for purge specimens .
`
`•TRANSPORTATION AND PRESERVATION
`
`As stated earlier, the method of collecting and submitting fecal
`
`specimens will vary with the location of the patient in relation to
`
`the laboratory . Either unpreserved or preserved specimens may be
`
`submitted .
`
`Geneoscopy Exhibit 1026, Page 26
`
`

`

`SPECIMEN COLLECTION AND HANDLING
`
`7
`
`UNPRESERVED SPECIMENS
`
`Specimens from hospital or clinic patients are usually unpreserved
`
`and should reach the laboratory within 1 to 2 hours after passage .
`
`Since protozoan trophozoites do not multiply or encyst outside of
`
`the body , they degenerate and die soon after passage . Therefore , it
`
`is essential that unpreserved feces be brought to the laboratory with-
`
`out delay . If the patient is having diarrheic stools , specimens should
`
`be received immediately after passage , within 2 to 1 hour . Because
`
`diarrheic specimens are more apt to contain trophozoites than cysts
`
`there is a greater urgency in getting these to the laboratory .
`
`Outpatients also usually submit unpreserved specimens , but in
`
`many cases , unavoidable delays occur which may result in morpho-
`
`logic alterations in parasites , particularly in protozoa . Ideally , there-
`
`fore , preservatives should be used to keep the organisms in good
`
`condition until the material can be examined . If specimens can be
`
`brought to the laboratory within 12 to 24 hours , the patient should
`
`be provided with a carton and a vial ( 20- to 30 -ml capacity ) of PVA-
`
`fixative (Brooke and Goldman , 1949 ) . He/she should be instructed
`
`to pass the stool into the container and to mix a portion with the
`
`fixative . Both the unpreserved and the PVA- fixed specimens should
`
`be submitted to the laboratory .
`
`More information on the use of PVA- fixative will be found under
`
`Preserved Specimens .
`
`If outpatient specimens will be delayed for longer than 24 hours ,
`
`unpreserved feces may be unsatisfactory for reliable diagnoses . In
`
`these cases , specimens should be preserved as described in the fol-
`
`lowing section .
`
`PRESERVED SPECIMENS
`
`Many diagnostic laboratories receive specimens through the mails.
`
`If these are delayed in reaching the laboratory and are not preserved
`
`properly , any organisms present (particularly protozoa ) may be
`
`unrecognizable or completely disintegrated by the time the specimen
`
`is examined . Adequate and proper preservation is , therefore , es-
`
`sential . In addition , the shipping containers must meet postal require-
`
`ments for mailing laboratory materials . These are described in the
`
`paragraph entitled Packing Specimens for Mailing .
`
`Helminth eggs and larvae and protozoan cysts can be preserved in
`
`5 or 10 percent Formalin in a 1 : 3 proportion of stool and fixative .
`
`The 5 percent solution is preferred because cysts , eggs of Hymeno-
`
`Geneoscopy Exhibit 1026, Page 27
`
`

`

`8
`
`SPECIMEN COLLECTION AND HANDLING
`
`lepis nana and Hymenolepis diminuta , and Stronygloides larvae may
`
`become distorted in 10 percent Formalin . Formalin does not satis-
`
`factorily preserve trophozoites and is not recommended for this
`
`stage .
`
`For preserving trophozoites (and cysts) , PVA-fixative should be
`
`used , in a 1 : 3 proportion of stool and fixative . After preservation in
`
`PVA- fixative , trophozoites present will remain suitable for staining
`
`and identification for several months. In the CDC laboratory , speci-
`
`mens are generally usable for 1 year . Different investigators have
`
`demonstrated the effectiveness of PVA-fixative in preserving the in-
`
`testinal protozoa , particularly the trophozoite stage ( Goldman and
`
`Brooke, 1953 ; Myers et al . , 1959 ; Melvin and Brooke , 1962 ; Markell
`
`and Quinn , 1977) .
`
`The PVA-fixative currently in use is Schaudinn's fixative to which
`
`PVA powder , a synthetic resin , has been added . Since Schaudinn's
`
`solution contains mercury , an environmental pollutant , workers are
`
`investigating the use of nonmercuric solutions in fixing protozoa for
`
`permanent staining. Thus , PVA- fixative solutions which do not con-
`
`tain mercuric compounds may be available in the future as substi-
`
`tutes for the present PVA- Schaudinn's fixative . In this manual , how-
`
`ever, the term "PVA-fixative " refers to PVA- Schaudinn's fixative
`
`unless otherwise indicated .
`
`The formula for preparing PVA- fixative , directions for use , and
`
`commercial sources for both PVA powder and fixative solutions are
`
`described in the section on Preservation of Specimens .
`
`For specimens that must be shipped to the laboratory , a two-vial
`
`method which enables the laboratory to recover all diagnostic stages
`
`of the intestinal parasites ( fig . 2 ) is recommended ( Brooke and
`
`Goldman , 1949 ) . One vial of the kit contains PVA-fixative and the
`
`other contains 5 or 10 percent Formalin . Screw- cap vials of 20- to
`
`30-ml capacity are recommended . In the United States , a number of
`
`State public health laboratories have provided such shipment kits
`
`with good results ( Kelly , 1952 ; Harper et al . ,