© VILE LABORATORY DIAGNOSIS A SELF-INSTRUCTIONAL COURSE INTRODUCTION U.S. Department of Health, Education, and Welfare .i cytoplasm Locate the (ectoplasm) 1. nucleus . nuclear membrane . cell wall . vacuole . cytoplasm a. endoplasm b. ectoplasm cytoplasm (endoplasm) Ov WN FUNCTION characteristics=——————> FEEDING 1. FEEDING: note how the il cl pseudopodia engulf the food 2. MOVEMENT: note how the cell is pulled forward by the extended pseudopodia 3. REPRODUCTION: note MOVEMENT how the cell multiplies by binary fission Remember: all cells may be characterized by: STRUCTURE & FUNCTION REPRODUCTION If you already knew the information contained on this page, you may skip to page 7—otherwise continue your study of the above material on the next page. The drawing below represents the STRUCTURE of the cell. In this case it is an AMEBA, a unicellular animal. Using the following information, you draw arrows to and label the cell’s STRUCTURES: NUCLEUS—the most prominent structure in the cell NUCLEAR MEMBRANE—the membrane surrounding the nucleus CELL WALL— the membrane surrounding the cell VACUOLE—-cavity inside the cell CYTOPLASM (endoplasm, ectoplasm)—material inside the cell The drawings below represent FUNCTIONS of the cell (ameba). Using the following information, you write in the FUNCTION represented by each drawing: FEEDING—by surrounding food with pseudopodia MOVEMENT—by thrusting of pseudopodia REPRODUCTION—by binary fission 785-020°—64——2 3 Now you draw an ameba and label it according to STRUCTURE (nucleus, nuclear membrane, cell wall, vacuole, endoplasm, ectoplasm) : Draw three amebae and label them according to FUNCTION (feeding, movement, reproduction): Turn back to page 1—check and correct your work. Without looking back, represent both STRUCTURE and FUNCTION of an ameba with labeled drawings: 4 Check and correct your work. If you skipped pages 3-5 and made any critical errors on this page, GO BACK to page 3 and follow instructions. Unit 2 DIAGRAM . INGESTED LIFE CYCLE This section will teach you the LIFE CYCLE of Entamoeba histolytica. Read carefully but do not try to memorize the following: E. histolytica is the only ameba that is definitely known to produce disease in man. This pathogenic organism exists in two forms: the protected CYST and the active TROPHOZOITE. Amebae (usually cysts, occasionally trophozoites) are INGESTED by man, ordinarily in contaminated water or food. Trophozoites ingested will be killed by stomach acids, since they have no protective covering. CYSTS are carried to the lower ileum, where the amebae EXCYST. The resultant trophozoites reach the lumen of the colon, MULTIPLY, and may INVADE and destroy the tissue of the colon wall (by secreting an enzyme) or INVADE and MULTIPLY in other organs (lungs, liver, brain, etc.) by way of the circulatory systems. Trophozoites may MULTIPLY in the lumen of the colon, the colon wall, and other organs they invade. Trophozoites in the lumen may either ENCYST or remain trophozoites and in either form PASS in feces; trophozoites are most common in dysenteric stools. Most trophozoites disintegrate soon after passage; cysts may be ingested and the life cycle continued. On the opposite page is a labeled LIFE CYCLE CHART of E. histolytica. Look at the diagram below; it shows the steps of the LIFE CYCLE CHART in simplified form. With your pencil, circle the steps on the CHART that correspond to those on the diagram; do this now: INVADE MULTIPLY EXCYST v MULTIPLY PASE INVADE - ENCYST MULTIPLY PASS Before leaving this page you should be able to recall these key words: INGESTED EXCYST—MULTIPLY—INVADE ENCYST—PASS Lire INFECTIVE STAGE oy INVADE od CYCLE > MULTIPLY (9 ) (abscesses in liver, lungs, etc) CHART vO mature cysts INGESTED in lower ileum EXCYST ; matures | via circulation EXTERNAL ENVIRONMENT | INTERNAL ENVIRONMENT (MAN) in lumen of colon "N MULTIPLY | may return may INVADE DIAGNOSTIC STAGES colon wall and PASS \ [in feces) MULTIPLY mature (i: g some trophozoites ENCYST immature \i PASS and disintegrate 10 A partially labeled Life Cycle Chart of E. histolytica is shown on the opposite page. The SHADED area of the chart shows in part how amebae move within MAN and how they may produce disease (by invasion). NOW YOU DO THIS: 1. To complete the Life Cycle Chart, show that the amebae leave man and enter the EXTERNAL ENVIRONMENT. Show this by using the underlined letters to complete the appropriate labels in the chart. Show (complete the labels) that while in the colon some of the trophozoites ENCYST. Show that some of the amebae may remain trophozoites. Show the three DIAGNOSTIC STAGES (in feces) of the ameba. . Show that while in the DIAGNOSTIC STAGE (in feces) trophozoites PASS and disintegrate. . Show that while in the DIAGNOSTIC STAGE (in feces) the cysts PASS and that the immature cyst later matures. Now, check your labeling by the chart on the preceding page. Correct any errors. RNAL ENVIRONMENT STAGES D Pe mature immature P and dis fees sneer] some trophozoites EN some re trophozoites 11 12 A convenient way to represent the life and actions of E. histolytica is by a LIFE CYCLE CHART like the partially completed one on the opposite page. NOW YOU DO THIS: 1. To begin the Life Cycle Chart show the ameba in the INFECTIVE STAGE (top of chart) being INGESTED. 2. Show that the cysts are in the INTERNAL ENVIRONMENT (MAN). 3. Show that the cysts EXCYST in the lower ileum. 4. Show that trophozoites MULTIPLY in the lumen of the colon; that they may INVADE the colon wall and MULTIPLY ; and that they may INVADE and MULTIPLY via the circulation causing abscesses in the liver, lungs, etc. 5. Now, show the subsequent steps in the Life Cycle by completing the labels. Check your work by the chart on page 9. Correct any errors. LIFE INF__ STAGE may IN and CYCLE MUL (ab in liver, lungs, etc) CHART mature cysts NG 1 ! in lo il EX in | | | I | 1 via cir I EXT ENVY ccc } IH EN (MAN) | in lu——of c | ! MU | may return 1 | 1 1 | may IN DI S c wall and cin some trophozoites EN immature AC — PASS and dis 13 14 NOTE: Did you remember to check and correct your work on the previous page? If not, do it now. —_——— Demonstrate your understanding of the Life Cycle of E. histolytica by labeling the chart on the opposite page. Start at the top of the chart. Use the key words as a guide: INGESTED EXCYST—MULTIPLY—INVADE ENCYST—PASS Check and correct your work. LIFE may | and CYCLE M CHART (ab——— in liver, lungs, etc) mature ¢ IN in | il EX I E and some inl may IN colon wall and M 15 ==") Cae Sa lee - hi - + iE] = DERE ) ? - a ey ih tne igi - ga Garis aa wa. =. eh EE m4 Bi rire = "= 28 pegmpAr p gE. . ; = ied = =f Mem fae Tet = re Lar Ek Be mn ral cL = = _ a2 - B - - . = h ~— a : EE pe oF et fh =n Ss fry. oy. ily = Picture the Life Cycle Chart in your mind and demonstrate your understanding by outlining the steps (in narrative form) of the Life Cycle of Entamoeba histolytica. Check and correct your work. 17 18 You have now completed PART I of the course, ‘Amebiasis: Labo- ratory Diagnosis.” If PART II is available, you should take it now. It will teach you to identify intestinal amebae. REFERENCES You may refer to the charts and books listed below for further information: Brooke, M. M., and Melvin, D. M. 1960. Life cycle charts: common intestinal protozoa of man. Department of Health, Education, and Welfare, Public Health Service, Com- municable Disease Center, Atlanta. Belding, D. L. 1958. Basic clinical parasitology. Appleton-Century-Crofts, New York. Faust, E. C., Beaver, P. C,, and Jung, R. C. 1962. Animal agents and vectors of human disease. 2d ed. Lea & Febiger, Philadelphia. Oo RESULTS OF FIELD TRYOUTS—PART | The table summarizes data on the use of this part of Ame- biasis: Laboratory Diagnosis by a wide range of trainees under “field” conditions. Field tryouts were held at the following places: Emory Medical School, Atlanta, Ga. first-year medical students. Grady Memorial Hospital, Atlanta, Ga. Participants: 27 medical technology trainees and their instructor. Tulane University and Charity Hospital, New Orleans, La. Participants: 13 parasitology graduate students, 7 medical technology trainees, and 6 advanced scientific personnel. Jackson Memorial Hospital, Miami, Fla. Participants: 29 medical technology trainees, 12 medical tech- nologists, and 3 advanced scientific personnel. Murrell Dobbins Technical School, Philadelphia, Pa. Participants: 26 medical technology trainees, 15 laboratory directors, 7 medical technologists, and 2 physicians. State Junior College of Broward County, Fort Lauder- dale, Fla. Participants: 87 undergraduate students. The field tryouts were conducted in a formal classroom setting. Representatives from the Laboratory Branch and the Instructive Communications Unit gave general instruc- tions on the use of the materials and observed the tryouts. Each trainee was given a package containing two envelopes; Envelope #1 contained a pre-lesson test, Part I: ‘Life Cycle of Entamoeba histolytica,” and Part II: ‘‘Identifica- tion of Intestinal Amebae’’; Envelope #2 contained Part III: “Laborabory Procedures,” a post-lesson test, and a questionnaire (to obtain information about relevant back- ground of trainees and to elicit opinions of training ma- terials). Items in Envelope #1 were used (in the order shown above) during a 4-hour session on Day 1; items in Envelope #2 were used during a 4-hour session on Day 2. Participants: 36 All materials were turned in at the end of each session. This was the procedure followed except in Philadelphia and Fort Lauderdale, where the two 4-hour sessions were on the morning and afternoon of the same day. Of the 271 trainees participating, 242 completed Parts I and II. Reasons for failing to finish ranged from lack of interest to lack of time. Student participation was es- sentially voluntary, but too often the trainees had not been told exactly what to expect. Also, using these ma- terials in a formal classroom setting under any time restric- tions is contrary to recommended use (see Specifications on the inside front cover and in the Introduction to the Course). Each participant can be classified as belonging to one of seven groups according to the trainee’s current position, past experience, and training as they relate to the subject matter covered in the lesson. Three groups belong to the primary trainee population: Group A: 15 scientific personnel with bachelor’s degree and at least one course in parasitology. Group B: 14 scientific personnel with bachelor’s degree and no formal training in parasitology. Group C: 36 first-year medical students with no parasitology training except in general college biology. Three groups belong to the secondary trainee population: Group D: 14 advanced scientific personnel, usually with graduate degrees and much experience. Group E: 37 medical technology trainees with high school education, several with 1 year of college. Each had at least 4 weeks of formal parasitology training. Group F: 46 medical technology trainees with high school education, several with 1 year of college. None had any previous parasitology training. One group was outside specified trainee population: Group @G: 80 junior college students who had had only college biology. Data from 6 field tryouts of Part I: *‘Life Cycle of Entamoeba histolytica” Completion time Pre-test scores™ Post-test scores™ Group No. median range median range median | range ’ Percent | Percent | Percent | Percent Ad 15 20 lth. cored alan I 1 42-100 100 71-100 Beam 14 33 MA. veges 27 min.—1 hr. 25 min...........- 57 0-100 100 57-100 Cate 36 33 ili eran 22 min.~1 br. 33 min........... 29 0-71 100 71-100 Dunn 14 20 Min... tt vsnsans 12min~-35 min. ..c.ccaveuua-a 71 57-100 100 57-100 Bas 37 Smilin... Le a 1I3min—~1he. 15min... ...... 57 28-100 100 57-100 Boor 46 d0mih. onsen annn 24 min~58 min... .crvvevena- 29 0-71 100 29-100 ge 80 44 HN. ik me mm 10 min Lh. cme m——— 29 0-85 100 43-100 *The pre- and post-lesson tests were the same; credit on the post-test was given only for answers exactly as they were taught by the lesson; on the pre-test, credit was given for answers generally synonymous or compatible with those taught by the lesson. Public Henlth: Service. Publication No. 1127 Paved AMEBIASIS EIDE EE NASER a self- instructional lesson (31) INTESTINAL AMEBAE U.S. Begartment of Health, Education, and Welfare Public Health Service The information on this page is primarily for administrators and instructors SPECIFICATIONS Training Objectives After taking this lesson, the student, using a microscope and properly prepared slides, will be able to— 1. recognize classic morphological characteristics of intestinal amebae, and 2. differentiate and identify by stage, genus, and species classic examples of the seven intestinal amebae: Entamoeba histolytica, Entamoeba hart- manni, Entamoeba coli, Entamoeba poleckz, Endolimaz nana, Iodamoeba butschlii, and Dientamoeba fragilis. Primary Trainee Population 1. Laboratory-technician trainees with biology training at the college level. 2. Medical students. 3. Parasitology students. Secondary Trainee Population Anyone interested who has— 1. knowledge of basic biology, 2. skill in basic microscopy, and 3. ability to read at the college level. Individualization Provided Depending upon the experience of the individual— 1. unless contraindicated, the student should take PART I, “Life Cycle of Entamoeba histolytica,” before taking PART II of the course, and 2. the student should be allowed to proceed through these lessons at his own pace; he may skip certain pages when so instructed in the text. 3. Although this lesson was designed for intensive study by a “beginning” student, it can be an effective review for more advanced students. For review only, study the ‘“demonstration’’ pages and skip ‘“practice’’ pages (these pages will be obvious to you). 4. The lesson can be used to best advantage during times most convenient for the student. Approximate Learning Time Experience has shown that this lesson requires from 1% to 2% hours actual study time (no time limit implied). Restrictions and Limitations 1. The lesson does not attempt to present the clinical picture of amebiasis or the significance of the seven intestinal amebae. 2. When possible, students using this lesson should be apprised of the special characteristics of this instructive method and admonished to follow in- structions precise—to respond and to use the Answer Booklet as directed. 3. For maximum effectiveness, the lesson should be followed up as soon as possible with actual laboratory experience. Results of Field Tryouts—see inside back cover. Additional information about this lesson can be found in the Introduction to the Course. IDENTIFICATION OF INTESTINAL AMEBAE Part Il of the three-part course “Amebiasis: Laboratory Diagnosis” AN INSTRUCTIVE COMMUNICATION U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Communicable Disease Center Atlanta, Georgia 30333 A Joint Effort of LABORATORY BRANCH and CURRICULUM AUTHORITY: M. M. Brooke, Se. D. Chief, Laboratory Consultation and Development Section SUBJECT MATTER EXPERT: Russell K. Carver, Parasitologist Parasitology Training Unit TECHNICAL REVIEWER: Dorothy Mae Melvin, Ph. D. Chief, Parasitology Training Unit TRAINING BRANCH INSTRUCTIVE COMMUNICATIONS UNIT: Robert L. Reynolds, Chief J. H. Harless, Analyst-Writer Andrea D. Lawrence, Junior Analyst-Writer Frances H. Porcher, Editor Theresa McDaniel, Editorial Assistant PRODUCTION ASSISTANCE: Fran Chesser K. Jane Paull John P. Gust, Jr. CONTRACT ADVISOR: Thomas F. Gilbert, Ph. D. Public Health Service Publication No. 1187, Part II UNITED STATES GOVERNMENT PRINTING OFFICE, WASHINGTON: 1964 For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. 20402—Price 65 cents “Amebiasis: Laboratory Diagnosis” CONTENTS Introduction to the Course PART I Life Cycle of Entamoeba histolytica PART II Identification of Intestinal Amebae PAGE Preface v Pre-Lesson Assessment vi How To Use This Lesson vii Introduction 1 Unit 1 Laboratory Report 3 Unit 2 Differentiation of Characteristics of Amebae 7 Unit 3 Identification of Entamoeba and Its Species 27 Unit 4 Identification of Other Genera and Species 38 Unit 5 Review of Ameba Identification 44 Unit 6 Similar Organisms and Artifacts 49 Unit 7 Review of Identification of Amebae and Artifacts 51 Unit 8 Related Microscopy 53 Summary 57 PART III Laboratory Procedures [=] i} i} El EK - - v [i PREFACE This lesson is PART II of the three-part course, “Amebiasis: Laboratory Diagnosis.” Although suitable for independent use, it can be used most effectively as part of the total course. The lesson teaches the differentiation and identification of the seven intestinal amebae (and four common artifacts). While no written materials, pictures, or drawings could possibly present all the variations of amebae or serve as a substitute for personal instruction in the laboratory, this lesson is designed to shorten and make more efficient use of classroom time in preparation for laboratory training. This lesson is student-oriented and specifically designed to produce post-lesson behavior at a level consistent with the objectives stated in the SPECIFICA- TIONS. Over and above the materials that are taught by the lesson to fulfill those objectives, certain additional information pertinent to the general field has been included on the advice of the Curriculum Authority. However, this lesson does not presume to be all-inclusive. To assure yourself that the lesson is appropriate to your needs, read the SPECI- FICATIONS (inside front cover) and look over the questions of the PRE- LESSON ASSESSMENT on the next page. PRE-LESSON ASSESSMENT Before you go any further, see if you can answer the following questions to determine whether you need to take PART II: 1. What are the names (genus and species) of the amebae inhabiting the digestive tract of man)? 2. What is the usual number of nuclei in the mature cyst and in the troph- ozoite in each of the intestinal amebae? 3. What are the diagnostic characteristics of E. histolytica (cyst and troph- ozoite) ? 4. What are the diagnostic characteristics of the other intestinal amebae? 5. How can histolytica and hartmannz be differentiated? 6. Can you reconstruct (draw) the classic picture of the intestinal amebae? 7. Can you reconstruct (draw) at least three artifacts that resemble amebae found in fecal specimens? 8. How should findings of a microscopic examination for amebae be reported to the requesting institution or physician? 9. What microscopic objectives should be used for the types of slide prep- arations? 10. What illumination and searching technique is best for identification of amebae? If you cannot fully answer the above questions, you should take this lesson. If you can answer them, you are ready to go on to PART III, “Laboratory Procedures.” HOW TO USE THIS LESSON This is a self-instructional lesson; its purpose is to teach not to test. You will need only this booklet, a pencil, and your own time (from 1% to 2% hours) to complete the lesson. Do not hesitate to write your answers or make notes in the booklet. REMEMBER This lesson has been prepared so that the amount of reading necessary is minimal and yet most meaningful. Therefore, it is very im- portant that you follow these instructions: —Read EVERYTHING carefully -—Proceed through a page or para- graph ONE STEP AT A TIME —Do not skip anything unless you are specifically told to do so —Do exactly WHAT you are told to do WHEN you are told to do it FOLLOW THE INSTRUCTIONS vii Have you removed the ANSWER BOOKLET from the front of this book? If not, do it now. Use it throughout the lesson—when instructed—to check the answers you have given. INTRODUCTION Microscopic identification of Entamoeba histolytica in fecal specimens is the only reliable technique for confirming the diagnosis of amebiasis in a patient or in the carriers of the pathogenic species. LE. histolytica is the only species with pathological significance; however, you will learn to identify the others—ZFEntamocba hartmanni, Entamoeba coli, En- tamoeba polecki, Endolimaxz nana, Iodamoeba bitschlii, and Dientamoeba fra- gilis—so that you can differentiate between them and the disease-producing species. A report of their presence—by name—indicates to the requesting physician that his patient has ingested material contaminated with feces. Correct identification of the seven species of amebae infecting the colon of man depends as much on proper preparation of slides and systematic microscope technique as it does on knowing and observing the morphology of the species. Given a slide to examine, you must first ADJUST THE MICROSCOPE correctly. This entails setting it to get optimal illumination, varying the focal levels to bring out morphological detail, and using the ocular calibrations correctly. Next, a systematic search of the slide enables you to FIND THE ORGANISM. Examination then enables you to IDENTIFY THE STAGE (whether tropho- zoite or cyst); then, IDENTIFY THE GENUS (Entamoeba, Endolimaz, Toda- moeba, or Dientamoeba) by the presence or absence of peripheral chromatin and/or chromatoid bodies; then, IDENTIFY THE SPECIES (histolytica, hartmanni, coli, polecki, nana, bitschlii, or fragilis). The last thing you do is FILL OUT LAB REPORT (name the organism found, or state “no amebae found” or “unidentified amebae found”). LEARN the six general steps: ADJUST MICROSCOPE FIND ORGANISM IDENTIFY STAGE IDENTIFY GENUS IDENTIFY SPECIES FILL OUT LAB REPORT S525 50 BD 735-022°—64——2 The SIX GENERAL STEPS for accurate identification of amebae are listed below but not in the correct order. With your pencil, NUMBER the steps to show the order in which they should be done: FILL OUT LAB REPORT IDENTIFY SPECIES IDENTIFY STAGE — ADJUST MICROSCOPE FIND ORGANISM IDENTIFY GENUS Check and correct your work. USE THE ANSWER BOOKLET. Unit 1 LABORATORY REPORT When you have completed the microscopic examination of all the slides, written the EXAMINATION NOTES, and noted the FINDINGS, such as ! EXAMINATION NOTES “no amebae found” OR Initial P t Cult . . . Case Wet Mount a Stain S1ide Wet i ii ot. ‘“anidentified amebae found [give stage] ”’ He wan san han | see acolo OR ta | on ane — | pln | ste the genus, species, and stage of so | Aa avn A | wn te amebae found, for example: Jy 3 . » “2 ve ee | one es Entamoeba coli cysts found % \ then, the last step is to FILL OUT THE LAB REPORT and send it to the requesting physician or institution. Thelab report below has been filled out toshow that Entamoeba coli cysts were found: — Z. owe 3/2/e3 Requested by. HL OMle Laboratory Report : Frm STOOL EXAMINATION for PARASITES This is an example of EXAMINATION NOTES kept throughout your microscopic examination: EXAMINATION NOTES Case ++ / 3/ 4/63 Initial Wet Mount no Concentrate Wet Mount [Fea — Or ebme Culture Stain Slide Wet Mount Perm. St. [r—— fe #2 ¥v/e3 os pe £ § Fra te ee £ Faspala readed ) Ge funet_ _— Sr po / The last thing you do is FILL OUT LAB cases below: Case #1 Nome PAnaune . (A. Date _8/¢ es Requested by. a OD Sle Laboratory Report : STOOL EXAMINATION for PARASITES \ REPORT; do this for both Case #2 Name Zelpide. C. ete 3/4t/ie3 R soy ils. O. Rhis Laboratory Report : STOOL EXAMINATION for PARASITES Check and correct answers. Did you use your Answer Booklet to check and correct your answers on page 5? If not, do it now. Complete the laboratory diagnosis for case #1: EXAMINATION NOTES Permanent Culture Stain Slide Wet Mount | Perm. St. Fe Jo armcbae | acta amelne (rt Concentrate Case vere Praca K vere J2/43 Requested bya. OD Law Laboratory Report : STOOL EXAMINATION for PARASITES Check and correct answers. Unit 2 DIFFERENTIATION OF CHARACTERISTICS OF AMEBAE You have learned to FILL OUT LAB REPORT from examination notes. Now you will learn what to look at to IDENTIFY the STAGE, GENUS, and SPECIES of an ameba. STAGE (trophozoite or cyst) is determined by the overall shape of the organism and the presence of certain bodies in its cytoplasm. GENUS is determined by the presence or absence of PERIPHERAL CHRO- MATIN in the nucleus and/or CHROMATOID BODIES in the cytoplasm. SPECIES is determined by the characteristics of the KARYOSOMAL CHROMATIN, peripheral chromatin, chromatoid bodies, and characteristics of the cytoplasm. TROPHOZOITE CYST RBC karyosomal chromatin peripheral chromatin chromatoid body I< The two drawings below show you PERIPHERAL CHROMATIN in the nuclei of two organisms. The nuclear structure is basically the same in both cysts and trophozoites. CO OO The PERIPHERAL CHROMATIN The PERIPHERAL CHROMATIN in these 4 nuclei is in these 5 nuclei is FINE & EVENLY dispersed. COARSE & IRREGULAR. In each blank below, WRITE THE LETTERS of the drawings that fit the description: C. O O FINE & EVENLY dispersed peripheral chromatin ———COARSE & IRREGULAR peripheral chromatin Check and correct answers. 735-022°—64——3 i Look at the nuclei of these organisms. The chromatin in the center of the nuclei is KARYOSOMAL CHROMATIN (KC); that on the outer edge is PERIPH- ERAL CHROMATIN (PC). All nuclei have KC, not all of them have PC. Pp The KARYOSOMAL CHROMATIN (KC) in these nuclei is SMALL & CENTRIC. The PERIPHERAL CHROMATIN (PC) is (CHECK ONE): — fine & even coarse & irregular — missing The KC (karyosomal chromatin) is LARGE & ECCENTRIC. The PC (peripheral chromatin) is: — fine & even coarse & irregular — missing The KC is LARGE & BLOT-LIKE. The PCis: fine & even coarse & irregular missing The KC is LARGE with ACHROMATIC GRANULES. The PCis: fine & even coarse & irregular — missing The KC is BROKEN & CLUMPED. The PC is: —_ fine & even coarse & irregular missing Check and correct your answers. 11 Some CYSTS may have CHROMATOID BODIES. A. This CYST has ROD-SHAPED CHROMATOID BODIES with ROUNDED ENDS. \ The KC is: —_ large & eccentric \ ____ large & blot-like small & centric broken & clumped large with achromatic granules CHROMATOID BODIES with SPLINTERED ENDS. The KC is: large & eccentric — large & blot-like small & centric broken & clumped — large with achromatic granules CHROMATOID BODIES with ANGULAR ENDS. The KC is: large & eccentric — large & blot-like small & centric broken & clumped — large with achromatic granules Other CYSTS have no CHROMATOID BODIES. D. (De The KC is: The KC is: large & eccentric — large & blot-like small & centric broken & clumped — large with achromatic granules _____ large & eccentric — large & blot-like small & centric broken & clumped _ large with achromatic granules No TROPHOZOITE has CHROMATOID BODIES. F. ® The KC of this trophozoite is: ___ large & eccentric — large & blot-like small & centric broken & clumped large with achromatic granules Check and correct your answers. 13 YOU describe the organism by checking the correct description: A. G55 Nl 14 . PERIPHERAL CHROMATIN is: . KARYOSOMAL CHROMATIN is: . CHROMATOID BODIES have: . PC is: . KC is: . CHROMATOID BODIES have: . PC is: . KCis: 3. CHROMATOID BODIES have: — missing — fine & even coarse & irregular —— large & eccentric — large & blot-like small & centric broken & clumped — large with granules splintered ends — rounded ends angular ends (missing) — missing fine & even coarse & irregular — large & eccentric large & blot-like small & centric broken & clumped — large with granules splintered ends — rounded ends angular ends (missing) — missing — fine & even coarse & irregular — large & eccentric — large & blot-like small & centric broken & clumped — largewithgranules splintered ends — rounded ends angular ends (missing) . PC is: . KC is: . CHROMATOID BODIES have: PCs: . KC is: . CHROMATOID BODIES have: . PCis: . KC is: . CHROMATOID BODIES have: Check and correct answers. — missing —fiie & even coarse & irregular — large & eccentric — large & blot-like small & centric broken & clumped — large with granules splintered ends — rounded ends angular ends (missing) — missing — fine & even coarse & irregular — large & eccentric — large & blot-like small & centric broken & clumped — large with granules splintered ends — rounded ends angular ends — (missing) — missing — fine & even coarse & irregular — large & eccentric — large & blot-like small & centric broken & clumped — large with granules splintered ends — rounded ends angular ends (missing) 15 In the various slides you will examine, the nuclear structure of many organisms may not be visible or characteristic; you will have to consider other factors in making identifications. Look at the drawings of the organisms below and read the description of their CYTOPLASM or its CONTENTS: CYST with large INCLUSION MASS near the nucleus CYST with large GLYCOGEN VACUOLE TROPHOZOITE with INGESTED RBC (red blood cells) DIFFERENTIATION of ENDO- and ECTOPLASM PROGRESSIVE MOTILITY exhibited TROPHOZOITE with INGESTED BACTERIA (“dirty” cytoplasm) 735-022°—64— 4 17 Some characteristics have already been indicated for distinguishing CYSTS from TROPHOZOITES. Generally the overall shape and the contents of the cytoplasm are used: CYSTS NOTE: Only cysts will contain —chromatoid bodies —aglycogen vacuole —inclusion mass Most cysts will have —smooth and rounded walls —more than one nucleus TROPHOZOITES NOTE: Only trophozoites will —contain bacteria and food particles ——contain RBC —show motility Most trophozoites will be —more irregular —uninuclear The nature of the CYTOPLASM and its CONTENTS as identifying character- istics is stressed below. CHECK the correct statement to identify organism characteristics and STAGE: A. It has: The STAGE is: It has: The STAGE is: It has: It has: The STAGE is: The STAGE is: cyst trophozoite ingested RBC large inclusion mass large glycogen vacuole cyst trophozoite ingested RBC large inclusion mass — large glycogen vacuole ingested bacteria cyst trophozoite ingested RBC ingested bacteria progressive motility no differentiation of endo- and ectoplasm differentiation of endo- and ectoplasm cyst ——trophozoite ingested RBC ingested bacteria progressive motility — differentiation no differentiation of endo- and ectoplasm Check and correct answers. 19 20 GO TO THE NEXT PAGE Some of the characteristics of the organisms shown on the opposite page are described below. YOU write the letter or letters of the drawing(s) in the blank beside the phrase thatdescribesitscharacteristics (be sure to consider the STAGE): 1. Trophozoite with small centric KC and evenly distributed PC 2. Trophozoite with large eccentric KC and coarse PC — 3. Cyst with large eccentric KC and coarse PC — 4. Cyst with small centric KC and evenly distributed PC — 5. Cyst with blot-like KC — 6. Trophozoite with blot-like KC — 7. Cyst with chromatoid bodies with rounded ends 8. Cyst with chromatoid bodies with splintered ends 9. Cyst with chromatoid bodies with angular or pointed ends — 10. Cyst with an inclusion mass — 11. Cyst with a large glycogen vacuole — 12. Trophozoite with ingested RBC — 13. Trophozoite with ingested bacteria (‘“dirty’’ cytoplasm) 14. Trophozoite with 2 nuclei — 15. Trophozoite with large KC with granules around KC 16. Cyst with large KC with granules near KC — 17. Trophozoite exhibiting progressive motility Check and correct answers. 21 22 GO TO THE NEXT PAGE WRITE the letter of the drawing (on the opposite page) beside the appropriate description given below (remember, the description may apply to more than one drawing): 1. Trophozoite with small centric KC and evenly distributed PC 2. Trophozoite with large eccentric KC and coarse PC 3. Cyst with large eccentric KC and coarse PC 4. Cyst with small centric KC and evenly distributed PC — 5. Cyst with blot-like KC ___ 6. Trophozoite with blot-like KC 7. Cyst with chromatoid body with rounded ends 8. Cyst with chromatoid bodies with splintered ends _ 9. Cyst with chromatoid bodies with angular or pointed ends — 10. Cyst with an inclusion mass 11. Cyst with a large glycogen vacuole _______ 12. Trophozoite with ingested RBC 13. Trophozoite with 2 nuclei 14. Trophozoite with granules around large KC ___ 15. Trophozoite exhibiting progressive motility 16. Cyst with granules near large KC Check and correct answers. 23 24 h Hid ARIS GO TO THE NEXT PAGE The pictures shown on the opposite page are photographs of actual slide preparations. Write the letter (or letters) of the photograph beside the de- scription of its characteristics below: 1. Trophozoite with small centric KC and evenly distributed PC 2. Trophozoite with large eccentric KC and coarse PC — 3. Cyst with large eccentric KC and coarse PC 4. Cyst with small centric KC and evenly distributed PC 5. Trophozoite with blot-like KC — 6. Cyst with chromatoid bodies with rounded ends 7. Cyst with chromatoid bodies with splintered ends 8. Cyst with an inclusion mass 9. Cyst with a large glycogen vacuole — 10. Trophozoite with ingested RBC 11. Trophozoite with 2 nuclei - 12. Trophozoite with large KC with granules 13. Trophozoite exhibiting progressive motility Check and correct answers, 25 Unit 3 IDENTIFICATION OF ENTAMOEBA AND ITS SPECIES Look at the outline below; it shows the IDENTIFICATIONS you will make: 1. STAGE: cyst OR trophozoite 2. GENUS: Entamoeba OR other J \ 3. SPECIES: histolytica Endolimax nana hartmanni Todamoeba biitschlii coli Dientamoeba fragilis poleckt You have learned what characteristics (PC, KC, chromatoid bodies, etc.) to look for in identifying the organisms. You will now learn how to differentiate the organisms by GENUS and SPECIES using the characteristics you have learned. When you know the STAGE of an organism, you can immediately narrow the GENUS possibilities by simply noting the presence of peripheral chromatin and/or chromatoid bodies. Organism is GENUS Entamoeba —> has PC and/or chromatoid bodies Endolimazx Organism is GENUS Jodamoeba > no PC or chromatoid bodies a Dientamoeb YOU note whether the organisms below have PC and/or chromatoid bodies, then write “Entamoeba” or “other” under them: Check and correct your answers. 27 » = Below are drawings representing both STAGES. Remember that the presence or absence of PC and/or chromatoid bodies determines GENUS. You indicate which organisms belong to GENUS Entamoeba by writing “E” in the blank: Check and correct answers. 29 30 You indicate blank: which of the organisms are Entamoeba by writing “E” in the Check and correct answers. Entamoeba histolytica and Entamoeba hartmanni cannot be differentiated except by SIZE and the fact that hartmanni does not ingest RBC. Differential size of other organisms is important in their identification, but it is only with histolytica and hartmanni that size is so often decisive. When you have found an ORGANISM, identified the STAGE as cyst or as trophozoite, identified the GENUS as ENTAMOEBA, identified the SPECIES as histolytica or hartmannt, you then measure the organism with the OCULAR MICROMETER in the microscope to differentiate the SPECIES (histolytica or hartmanns). IF the organism is MORE THAN 11 microns in diameter, it is probably histolytica. IF the organism is LESS THAN 9 microns in diameter, it is probably hartmanna. On the basis of the above general rules, identify the species (fill in the blank) of each organism pictured below: 7 MICRONS 12 MICRONS 8 MICRONS 13 MICRONS d(C) fool ! IMPORTANT 11 and 9 microns are given as general dividing lines between the two species, but there is much overlapping in size. If an organism found is within the 9-11 range, at least 25 more organisms should be found and carefully measured. They may then be identified as either histolytica or hartmanni or as a combina- tion of the two species (see table): Specimens A B C No. of organisms measured 25 25 25 11 microns or more (diam.) 19 0 11 9 microns or less (diam.) 0 19 i2 Identification histolytica | hartmanni| histolytica & hartmanni 31 The genus ENTAMOEBA includes the species: histolytica, hartmanni, colt, and polecki. The table below describes characteristics of the organisms. Eh. refers to both histolytica and hartmanni since they have only one major difference and that is SIZE. the characteristics of E.h. are SYMMETRICAL (e.g., centric KC, even PC, rounded chromatoid bodies, round RBC). NOTE THAT (coli and polecki have the same nuclear structure, but can be differentiated in the CYST stage by chromatoid bodies and number of nuclei; polecki also has the distinctive inclusion mass. E.h. E.c. E.p. | KC small large centric eccentric fine coarse PC even irregular IN CysTe| 18 1 CHROMATOID . rod-shaped irregular angular BODIES 1N rounded ends splintered ends pointed ends 7 “OTHER” RBC oy inclusion mass BODIES (in trophozoite) a (in cyst) 7 PR dirty cytoplasm CYTOPLASM (contains bacteria, food particles, etc. £3 MOTILITY progressive *NOTE: Knowing only the number of NUCLEI in a CYST does not IDENTI- FY unless the number is 5-8, in which case it will be Z.c. (see table). Very rarely does histolytica have more than 4 nuclei. Number of NUCLEI in TROPHOZOITE has limited diagnostic value; all trophozoites have one nucleus except D. fragilis, which may have more than one. Using the table on the opposite page WRITE E.h., E.c., E.p., or E.c. or E.p., to indicate the SPECIES of the drawings below: Check and correct answers. 33 34 IDENTIFY the SPECIES of the organisms on the opposite page. Refer to the table on page 32 only after you have done as much as possible without help. Recall that these are the characteristics you look at to IDENTIFY E.h., E.c., and K.p.: —NUCLEAR STRUCTURE (PC & KC) —NUMBER of NUCLEI in CYST (14, 1-8, or 1) —CHROMATOID BODIES (rounded, splintered, or pointed) —OTHER BODIES (RBC & inclusion mass) —MOTILITY (progressive) —CYTOPLASM (dirty, ete.) E. histolytica (or hartmannt) has “symmetrical” characteristics. REMEMBER THAT coli and polecki TROPHOZOITES appear the same. Check and correct answers. 35 hu Use the appropriate abbreviations to identify the organisms pictured below: Check and correct answers. 37 Unit 4 38 IDENTIFICATION OF OTHER GENERA AND SPECIES You have learned to IDENTIFY all of the SPECIES of the GENUS Entamoeba found in the intestinal tract of man. The table below summarizes the char- acteristics that will enable you to differentiate the ‘other’ three amebae; each is of a different GENUS: —FEndolimax nana —Iodamoeba biitschlii —Dientamoeba fragilis the three amebae designated as “other” have no PC or chromatoid bodies. fragilis has only the trophozoite stage. NOTICE THAT It is the ONLY trophozoite that may have two nuclei. To help you remem- ber, think: ; : broken into two nuclei “fragile” (fragilis) <_{ oken KC E.n. Lb. D.f. KC large and | large with achromatic broken and blot-like granules beside KC clumped 1 I le | a CYTOPLASM * Age 2 Te oe renee (only suggestive) NUCLEI IN kkk TROPHOZOITE** *NOTE: This suggestive characteristic indicates only that the organism is POSSIBLY 1.5. **NOTE: None of the CYSTS of the three “other” amebae can be differen- tiated by number of nuclei alone (however, butschliz usually has one). *#*NOTE: The binucleate state of fragilis trophozoite is a result of incomplete division. This state will not always be seen. Remember, no cyst of this species is known to exist. Use the table on the opposite page to IDENTIFY the organisms pictured below. WRITE in the appropriate blanks E.n., 1.b., POSS. 1.b., or Df. (3 ecient I. Check and correct answers. 39 40 NAME the genus and species (E.n., I.b., POSS. Lb., or Df.) of the organisms pictured on the opposite page. Do not refer to the table until you have com- pleted as many as you can without help. broken into two nuclei” broken KC” “fragile REMEMBER { © look at KC, cytoplasm, and number of nuclei in the trophozoite stage. some characteristics indicate only a POSS. Check and correct answers. 41 42 With abbreviations, identify the genus and species of organisms on this page: A. Check and correct answers. The lesson so far has made no effort to teach you to pronounce and spell the names of the seven amebae. nunciation guides. Now do this: Given below are the amebae names and pro- 1. look at the first word in Column I and pronounce it ALOUD several times; 2. cover Column I and look at the first word in Column II, pronounce it ALOUD several times; 3. cover Columns I and IT and write (spell) in Column III the word you have just learned to pronounce; 4. erase any incorrect spelling and repeat the steps until you have pro- nounced and spelled the word correctly. I IT IIT SAY ALOUD SAY ALOUD COVER AND SPELL enta ME bah . . . . . . . . Entamoeba . . . . Go through all three steps (I, II, and III) for each word before you go to the next word. his toh LIT ikah . . . . . histolytica hart MAHN nee . . . . . hartmanni KOlee. . . . ...... wh . .. =. po LECKY. . . . . . . . polecki . . . . . en doh LYE max . . . . . . Endolimax . . . . NAYmal . . ox + 2 + » DAB « = + « + » eye OH dah mee bah . . . . Jodamoeba . . . . BOOTCH lee eye . . . . . biitschlii . . . . dye EN tah mee bah . . . . Dientamoeba . . . FRAJillis . . . . .. .. fragilis . . . . . 43 Unit 5 REVIEW OF AMEBA IDENTIFICATION Below and on the opposite page are all the amebae to be found in the digestive tract of man; some are typical, some exhibit variations. all have cyst and trophozoite stages except D. fragilis REMEMBER ( Entamoeba has PC and/or chromatoid bodies E. histolytica has “symmetrical” characteristics YOU write the genus and species (no abbreviations) below each organism: 44 Check and correct answers. 45 46 Identify, with full name, the genus and species of each ameba: Check and correct answers. 47 " a i aE Unit 6 SIMILAR ORGANISMS AND ARTIFACTS Some of the most difficult problems in the correct identification of amebae in stool specimens are caused by the presence of free-living amebae, other proto- zoa, and the many confusing elements whose structures are similar to those of amebae. Some of these “artifacts’” are pictured below. Look closely and note how their characteristics differ from the classic amebae you have learned: polymorphonuclear leukocyte E. histolytica cyst artifact artifact ARTIFACT RESEMBLES but CYTOPLASM and NUCLEUS is less dense is coarser, more elongated epithelial cell trophozoite of E. coli and polecki is more granular, not as vacuolated ; has no ingested particles is coarser, larger plant cell (Blastocystis hominis) cysts (many species) has larger central vacuole or mass has several granules around edge of cell macrophage (stained) when unstained, E. histolytica trophozoite is coarser with less distinctive pseudopodia is larger with chromatin particles 49 50 Examine the pictures of the ARTIFACTS shown below. Place the identifying letter beside the appropriate description: — A plant cell (Blastocystis hominis) —An epithelial cell (resembling Z. coli trophozoite) — A macrophage —— A polymorphonuclear leukocyte with nuclei resembling those of I. histolytica Check and correct answers. Unit 7 REVIEW OF IDENTIFICATION OF AMEBAE AND ARTIFACTS The photographs below include examples of all the amebae you have learned to identify; there are also some artifacts and atypical amebae included. IDENTIFY by filling in the blanks (write out genus and species of amebae): D. E. F G. H. 1. J. RK. L. CHECK and CORRECT your answers. If you made no ) 4 errors, skip to p. 53. Otherwise continue on the next page. 51 52 IDENTIFY these amebae (full name of genus and species) and artifacts: Check and correct answers. Unit 8 RELATED MICROSCOPY Review the SIX GENERAL STEPS: ADJUST MICROSCOPE FIND ORGANISM IDENTIFY STAGE IDENTIFY GENUS for organism IDENTIFY SPECIES FILL OUT LAB REPORT So be You have now learned how to IDENTIFY the organism and FILL OUT LAB REPORT. However, many times you cannot make a diagnosis on the basis of one organism. You must often FIND enough organisms to obtain a composite picture of characteristics in order to make a diagnosis. for example: In the initial wet mount you may find an organism that shows progressive motility, suggesting that it is possibly FE. histolytica, but you must FIND other organisms that demonstrate diag- nostic nuclear structures and/or cytoplasmic characteristics of that species to make a definite diagnosis. Several different types of slide preparations are sometimes necessary, since certain preparations reveal some structures and characteristics better than others. 53 ADJUSTING MICROSCOPE There remains for consideration in this lesson the first general step, ADJUST MICROSCOPE, which includes the following: —calculating the OCULAR CALIBRATIONS for each objective —maintaining optimal ILLUMINATION —selecting appropriate OBJECTIVES for each slide OCULAR MICROMETER CALIBRATION CHART Low Power High Power Oil Imm. Co . 16 mm. 4 _mm, L.8 mm. This is a typical units microns units microns units microns OCULAR MICROMETER CALIBRATION CHART: 1 14.0 1 3.21 1 1.35 2 28.0 2 6.42 2 2.70 3 442.0 3 9.63 3 4.05 4 6860 49 157 21 49 66.15 50 700.0 50 /60.50 50 67.50 Size as a differentiating characteristic has not been stressed in this lesson except for E. histolytica and E. hartmanni; however, as your experience in- creases you will become aware that, despite considerable overlap, relative sizes and ranges of sizes are valuable indices in delimiting all the various species. Before the microscope can be used for the examination and any measuring done, the values of the units of the OCULAR MICROMETER in the lens of the microscope are calculated for each OBJECTIVE and recorded on a chart similar to the one shown above. While examining each slide, optimal (reduced) illumination must be maintained to make the morphological details of amebae stand out. Too much light will obliterate the details. According to the characteristics of each slide, you must select the correct OBJECTIVE: If STAINED SLIDES: Examine under OIL IMMERSION. To help you remember, think: “OIL STAINS” If UNSTAINED SLIDES: Scan under LOW POWER; change to HIGH POWER when an organism is found. (Use OIL IMMERSION for greater magnification if necessary.) 54 FINDING THE ORGANISM Under the second general step FIND ORGANISM are included the following: —SYSTEMATIC SEARCHING of the slide —Varying FINE ADJUSTMENT —Varying FOCAL LEVELS to see all details —Varying intensity of illumination SYSTEMATIC SEARCHING with overlapping fields is essential for seeing all organisms on a slide. for example: up and down searching ) with overlapping fields During the systematic searching of the Saline Wet Mount the FINE ADJUST- MENT should be turned back and forth. This turning will make the walls of some of the organisms appear to glitter, thus helping you find them. When an organism is found, the FOCAL LEVEL should be VARIED, so that all the structures of the three-dimensional amebae can be seen. Because reduced illumination is not optimal for all structures, varying the illumination is often necessary. Check (+) all the important points to remember in ADJUSTING the MICROSCOPE and FINDING the ORGANISM: Calculate the ocular calibrations for each objective Reduced illumination is generally best for seeing the charac- teristics of amebae Vary illumination if necessary Stained slide: use oil immersion Unstained slide: use low power, then high power, maybe oil immersion Searching is done systematically Focal levels varied to see all structures Fine adjustment turned to see refractility Check and correct answers. 55 When you are examining slides, there are many things you must remember. CHECK (v/) each correct statement below: —— 1. The ocular calibrations must be calculated for the oil immersion objective only. 2. The ocular calibrations must be calculated for all objectives. 3. Reduced illumination is generally better for observing character- istics of amebae. 4. Bright illumination makes structures clearer. 5. Stained slides are examined under: oil immersion. — low then high power (maybe oil immersion). high then low power (maybe oil immersion). 6. Unstained slides are examined under: oil immersion. low then high power (maybe oil immersion). high then low power (maybe oil immersion). 7. The best slide searching technique is random with fixed focal depths. 8. The best slide searching technique is systematic with varied focal depths. 9. Some organism walls are highly refractile in saline wet mounts. Check and correct answers. 56 SUMMARY Using drawings and photographs you can now identify the classic forms of the seven species of ameba infecting the digestive tract of man. You can also recog- nize some of the variations in morphology encountered. With the knowledge you have gained and with a minimum of laboratory experience, you will be better fitted to identify the species. Remember: 1. Proper use of the microscope is vital—there must be optimal il- lumination, appropriate objectives for the slide, and a systematic searching procedure. 2. The structures you can see clearly will depend upon the type of slide you are examining; therefore, it is important to include several preparations in your lab regimen. 3. Too often you cannot positively identify a given organism; the charac- teristics seen are either vague or only suggestive. For any given slide, if no single organism presents a diagnostic picture, it will be necessary to make a COM POS- ITE PICTURE from several organisms. Even the composite picture may be only suggestive and not diagnostic. 4. Make notes on an appropriate form throughout each examination and record suggestive and diagnostic findings of all species observed. 5. If the organism has prominent peripheral chromatin and/or chromatoid bodies, it is usually one of the species of the genus Entamoeba. (a) FE. histolytica is the only ameba that will typically ingest RBC. It will not usually ingest bacteria. The characteristics are symmetrical. (b) E. hartmanni is smaller than histolytica and does not ingest RBC. (¢) E. coli, as a mature cyst, has more than four nuclei. (d) FE. polecki cannot be differentiated from coli as a trophozoite, but its cyst is characterized by the large inclusion mass and one nucleus. 6. Endolimax nana is characterized by the large karyosomal chromatin. 7. Todamoeba biitschlii cyst has the large glycogen vacuole. 8. Dientamoeba fragilis is known only in the trophozoite stage and usually has two nuclei. 9. A rough estimate of size can be obtained by comparing any organism with an RBC, but for precise measurements the ocular micrometer is used. 10. There are many artifacts present in stool specimens; look out especially for plant cells, polymorphonuclear leukocytes, and macrophages. 11. The Entamoeba gingivalis trophozoite may be confused with the trophozoite of FE. histolytica. However, the former inhabits only the mouth area and will not be found in stool specimens. 57 58 You have now completed PART II of the course, “Amebiasis: Laboratory Diagnosis.” If you plan to take PART III, which will teach you the various laboratory procedures that are essential to the diagnosis of amebiasis, you should wait a day (but no more than two days) before doing so. REFERENCES You may refer to the articles and books listed below for further information: Articles Brooke, M. M., G. Otto, F. Brady, E. C. Faust, T. T. Mackie, and H. Most. 1953. An analysis of a memorandum on the diagnosis of amebiasis. Am. J. Trop. Med. Hyg. 7: 593-612. Burrows, R. B., and G. E. Klink. 1955. Endamoeba poleck: infections in man. Am. J. Hyg. 62: 156-167. Freedman, L., and R. Elsdon-Dew. 1959. Size as a criterion in the human intestinal amebae. Am. J. Trop. Med. Hyg. 8: 327-330. Gleason, N. N., M. Goldman, and R. K. Carver. 1963. Size and nuclear morphology of Entamoeba histolytica and Entamoeba hartmanni trophozoites in cultures and in man. Am. J. Hyg. 77: 1-14. Books Craig, C. F. 1944. The etiology, diagnosis, and treatment of amebiasis. Williams and Wilkins Co., Baltimore. 1948. The laboratory diagnosis of protozoan diseases. 2d ed. Lea & Febiger, Philadelphia. Hoare, C. A. 1950. Handbook of medical protozoology. Williams and Wilkins Co., Baltimore. Markell, E. K., and M. Voge. 1958. Diagnostic medical parasitology. W. B. Saunders Co., Philadelphia. Spencer, F. M., and L. S. Monroe. 1961. The color atlas of intestinal parasites. Charles C. Thomas Co., Springfield, Ill. U.S. GOVERNMENT PRINTING OFFICE: 1964 = ox Tw RESULTS OF FIELD TRYOUTS—PART II The table summarizes data on the use of this part of Amebiasis: Laboratory Diagnosis by a wide range of train- ees under ‘‘field”’ conditions. Field tryouts were held at the following places: Emory Medical School, Atlanta, Ga. Participants: 36 first-year medical students. Grady Memorial Hospital, Atlanta, Ga. Partici- pants: 27 medical technology trainees and their instructor. Tulane University and Charity Hospital, New Orleans, La. Participants: 13 parasitology graduate stu- dents, 7 medical technology trainees, and 6 ad- vanced scientific personnel. Jackson Memorial Hospital, Miami, Fla. Partici- pants: 29 medical technology trainees, 12 medical technologists, and 3 advanced scientific personnel. Murrell Dobbins Technical School, Philadelphia, Pa. Participants: 26 medical technology trainees, 15 laboratory directors, 7 medical technologists, and 2 physicians. State Junior College of Broward County, Fort Lauder- dale, Fla. Participants: 87 undergraduate students. The field tryouts were conducted in a formal classroom setting. - Representatives from the Laboratory Branch and the Instructive Communications Unit gave general in- structions on the use of the materials and observed the tryouts. Each trainee was given a package containing two envelopes; Envelope #1 contained a pre-lesson test, Part I: “Life Cycle of Entamoeba histolytica,” and Part II: “Identification of Intestinal Amebae’”’; Envelope #2 con- tained Part III: “Laboratory Procedures,” a post-lesson test, and a questionnaire (to obtain information about relevant background of trainees and to elicit opinions of training materials). Items in Envelope #1 were used (in the order shown above) during a 4-hour session on Day 1; items in Envelope #2 were used during a 4-hour session on Day 2. All materials were turned in at the end of each session. This was the procedure followed except in Phila- delphia and Fort Lauderdale, where the two 4-hour sessions were on the morning and afternoon of the same day. Of the 271 trainees participating, 242 completed Parts I and II. Reasons for failing to finish ranged from lack of interest to lack of time. Student participation was essentially voluntary, but too often the trainees had not been told exactly what to expect. Also, using these ma- terials in a formal classroom setting under any time re- strictions is contrary to recommended use (see Specifications on the inside front cover and in the Introduction to the Course). Each participant can be classified as belonging to one of seven groups according to the trainee’s current position, past experience, and training as they relate to the subject matter covered in the lesson. Three groups belong to the primary trainee population: Group A: 15 scientific personnel with bachelor’s degree and at least 1 course in parasitology. Group B: 14 scientific personnel with bachelor’s degree and no formal training in parasitology. . Group C: 36 first-year medical students with no parasi- tology training except in general college biology. Three groups belong to the secondary trainee population: Group D: 14 avanced scientific personnel, usually with graduate degrees and much experience. Group E: 37 medical technology trainees with high school education, several with 1 year of college. Each had at least 4 weeks of formal parasitology training. Group F: 46 medical technology trainees with high school education, several with 1 year of college. None had any previous parasitology training. One group was outside specified trainee population: Group @G: 80 junior college students who had had only college biology. Data from 6 field tryouts of Part lI: ‘Identification of Intestinal Amebae" Completion time Pre-test scores* Post-test scores ™ Group No. median range median range median range Percent | Percent | Percent | Percent Bhi sm iin 15 lhr. 46min. tL . ... 45 min.-2 hr. 40 min_________ 58 18-89 90 75-98 1 = 14 1 hr. 30 min... 1 hr. 20 min.-2 hr. 20 min____ 30 0-85 92 39-98 Coden 36 1hr. 47 min________ Ihrs3hr. Smin... 1. =. 2 0-14 87 42-100 Dion 14 hte erent 34 min.—1 hr. 56 min_________ 76 44-97 98 67-100 Ee 37 Thr. 36 min... ....... 55 min~3 hr. 10 min.......... 48 0-73 94 60-100 War 46 2 Worse ge 1 hr. 24 min.-3 hr. 45 min____ 4 0-65 91 40-100 Brena 80 2hr.5min. Lc. 45 min~3 hr________________ 0 0-19 85 30-100 *The pre- and post-lesson tests were the same; credit on the post-test was given only for answers exactly as they were drug I the lesson; on the pre-test, credit was given for answers generally synonymous or compatible with those taught y the lesson. Public Health Service RR NER EE ya Te UE Ss D. ANSWER BOOKLET for IDENTIFICATION OF INTESTINAL AMEBAE Part Il of the three-part course “Amebiasis: Laboratory Diagnosis” Take this booklet out before starting the lesson. Examine it carefully, then put it where you can reach it easily to check your work as you take the lesson. EE A: ANSWERS FOR PAGE 2 6 FILL OUT LAB 1 ADJUST MICROSCOPE REPORT 5 IDENTIFY SPECIES 2 FIND ORGANISM 3 IDENTIFY STAGE 4 IDENTIFY GENUS PAGE 5 Case #1 Case #2 Laboratory Report : E. hid. Aophy PAGE 6 Laboratory Report : PAGE 9 A & C FINE & EVENLY DISPERSED B&D COARSE & IRREGULAR PAGE 11 fine & even coarse & irregular missing . missing missing HUORE PAGES 12 AND 13 A. small & centric D. large with achromatic granules B. large & eccentric E. large & blot-like C. large & eccentric F. broken & clumped 735-022°-—64 PAGES 14 AND 15 A. = Bmw coarse & irregular large & eccentric splintered ends fine & even small & centric rounded ends C. 1. missing 2. large & blot-like 3. (missing) D. 1. missing 2. large with granules 3 . (missing) WN = WN . Missing broken & clumped . (missing) . coarse & irregular large & eccentric angular ends = > 0 = [SY Oo A. cyst large inclusion mass B. cyst large glycogen vacuole C. trophozoite ingested RBC progressive motility differentiation of endo- and ectoplasm . trophozoite ingested bacteria no differentiation of endo- and ectoplasm = > a t= No pd A and Ql=lgi=l OU ih Ww ND == gz Lp = “iQ = 2 (Note: particles in J are not bacteria) J 12. E,F, H, and T 13. l~ QZ [= on ae! > D = DN oo > E 2 B and C 3. Fai Gs B Lo B F7 H Cs A *Trophozoites sometimes appear to be almost round. A. Entamoeba — = — p—_ » oS © === C. Entamoeba NN = O p— w J 13. D 14. A 15. H 16. PAGE 29 G. __ K. H. “ER A. “E” E. “E” Lr B. “E” F. “E» J. e G. “E” K. “E” D. “E” H,___ EE T= PAGE 33 A Fe F. Ec. J. Ke.or Ep B. Eh. G. Eh. K. E.h. C. Ep H Ep. L. Ec.or Ep D. Ep. I. Ee. M. Eh. = > a = Ig Jk > SQ : S Q = FIZ og by Sl SI on | SS G. POSS. Lb. H. 13. I Dy. PAGE 42 So SS = HORE =I sy SN PAGES 44 AND 45 A. Dientamoeba fragilis B. Endolimax nana C. Entamoeba coli or Entamoeba poleckr D. Entamoeba histolytica or E. hartmanni E. Endolimaz nana F. Entamoeba coli G. Iodamoeba biitschlii H. Entamoeba polecki I. Entamoeba histolytica J. Todamoeba biitschlii PAGES 46 AND 47 HoQ WE hartmanna Entamoeba histolytica Entamoeba coli or Entamoeba polecki Entamoeba colr . Entamoeba cols Entamoeba histolytica or F. G. Hn LL J. Endolimax nana K. Entamoeba histolytica or Entamoeba histolytica or hartmanni hartmanni L. Entamoeba poleckr Todamoeba biitschlii M. Dientamoeba fragilis Dientamoeba fragilis N. Todamoeba biitschliv Todamoeba biitschlii O. Entamoeba coli P. Endolimaz nana = > Pp! = ot S D A plant cell C An epithelial cell A A macrophage B A polymorphonuclear leukocyte = > : = ot pd ovo mE Dientamoeba fragilis Entamoeba coli or Entamoeba polecki Entamoeba coli . Entamoeba histolytica plant cell Todamoeba biitschlit Entamoeba histolytica or hartmanni Entamoeba coli Todamoeba biitschlin Endolimax nana . Entamoeba histolytica or hartmanni Entamoeba coli or Entamoeba polecky EC He PAGE 52 A. Entamoeba coli E. ITodamoeba biitschliz 1. Entamoeba polecki B. Entamoeba histolytica F. Entamoeba coli or J. Entamoeba C. Entamoeba histolytica Entamoeba poleckr histolytica or or hartmanni G. Iodamoeba biitschlii hartmanni D. Dientamoeba fragilis H. plant cell K. epithelial cell L. Entamoeba coli or Entamoeba poleckt PAGE 55 You should have checked all points. PAGE 56 v2, 5. _V oil immersion v8. v.53 6. _v low then high v9. power (maybe oil immersion) U.S. GOVERNMENT PRINTING OFFICE: 1964 . A ih Lad “ id AMEBIASIS LABORATORY DIAGNOSIS, part lil a self- instructional lesson LLL CYL LITHIA U.S. Department of Health, Education, and Weifare Public Health Service The information on this page is primarily for administrators and instructors. SPECIFICATIONS Training Objectives After taking this lesson, and in an appropriate laboratory setting, the student will be able to— 1. prepare four basic slides used in the diagnosis of amebiasis: direct (initial) wet mount, concentrate wet mount, culture wet mount, and permanent stain slide; . label fecal specimen; 3. specify (list) which of the four procedures to include in a regimen for central diagnostic laboratories and for clinic and hospital laboratories; and 4. specify (list) the bases for rejecting improperly processed specimens. no Primary Trainee Population 1. Laboratory-technician trainees with biology training at the college level. 2. Medical students. 3. Parasitology students. Secondary Trainee Population Anyone interested who has— 1. knowledge of basic biology, 2. skill in basic microscopy, and 3. ability to read at the college level. Individualization Provided 1. The student should be allowed to proceed through the lesson at his own pace and may skip certain pages when so instructed in the text. 2. The parts of the course, “Amebiasis: Laboratory Diagnosis,” should be taken in order. Whether a trainee takes Parts I and II before taking this part will depend upon the experience and training of each individual; all parts should be taken unless contraindicated. 3. Although this lesson was designed for intensive study by a ‘“beginning’’ student, it can be an effective review for more advanced students.” For review only, study the ‘“‘demonstration’ pages and skip ‘practice’ pages (these pages will be obvious to you). 4. The lesson can be used to best advantage during times most convenient for the student. Approximate Learning Time Experience has shown that this lesson requires from 13 to 21% hours actual study time (no time limit implied). Restrictions and Limitations 1. The lesson does not attempt to teach all possible procedures, but only a minimum number that have been found to be effective. 2. The lesson does not teach the detailed steps of concentration, cultivation, and trichrome staining procedures. 3. When possible, students using this lesson should be apprised of the special characteristics of this instructive method and admonished to follow in- structions precisely—to respond and to use the Answer Booklet as directed. 4. For maximum effectiveness, the lesson should be followed up as soon as possible with actual laboratory experience. Results of Field Tryouts—see inside back cover Additional information about this lesson can be found in the Introduction to the Course. LABORATORY PROCEDURES Part lll of the three-part course “Amebiasis: Laboratory Diagnosis’ AN INSTRUCTIVE COMMUNICATION U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Communicable Disease Center Atlanta, Georgia 30333 WLS A Joint Effort of LABORATORY BRANCH and CURRICULUM AUTHORITY: M. M. Brooke, Sc. D. Chief, Laboratory Consultation and Development Section SUBJECT MATTER EXPERT: Russell K. Carver, Parasitologist Parasitology Training Unit TECHNICAL REVIEWER: Dorothy Mae Melvin, Ph. D. Chief, Parasitology Training Unit TRAINING BRANCH INSTRUCTIVE COMMUNICATIONS UNIT: Robert L. Reynolds, Chief J. H. Harless, Analyst-Writer Andrea D. Lawrence, Junior Analysi- Writer Frances H. Porcher, Editor PRODUCTION ASSISTANCE: Fran Chesser K. Jane Paull CONTRACT ADVISOR: Thomas F. Gilbert, Ph. D. Public Health Service Publication No. 1187, Part III UNITED STATES GOVERNMENT PRINTING OFFICE, WASHINGTON: 1964 For sale by the Superintendent of Documents, U.S. Government Printing Office ‘Washington, D.C. 20402—Price 60 cents PART 1 PART 11 PART III Unit 1 Unit 2 Unit 3 Unit 4 Unit 5 Unit 6 “Amebiasis: Laboratory Diagnosis" CONTENTS Introduction to the Course Life Cycle of Entamoeba histolytica Identification of Intestinal Amebae Laboratory Procedures Preface Pre-Lesson Assessment How to Use This Lesson Introduction Collection and Processing of Specimens Labeling Specimens Preparation of Slides A. Preparation of Wet Mount Slides B. Preparation of Permanent Stain Slides Rationale for Cookbook Procedures A. Cookbook: Trichrome Staining Technique B. Cookbook: Formalin-Ether Concentration Technique C. Cookbook: Cultivation of Intestinal Protozoa When to Cultivate Selection of Procedures for Laboratory Regimens Summary iii PAGE vi 14 20 22 29 35 36 42 48 55 57 63 ps] i 2 Fd or 54 ELL Es a a _ Tk or Co i =. = R= oo a Ny Fa PREFACE This lesson is PART III of the three-part course, ‘Amebiasis: Laboratory Diagnosis.” Although suitable for independent use, it can be used most effectively as part of the total course. The lesson teaches the proper method of collecting and processing fecal speci- mens, the method of preparing certain basic slides, and the rationales for other basic procedures used in the microscopic identification of amebae. Two possible regimens made up of combinations of the various procedures are also taught. This lesson is student-oriented and specifically designed to produce post-lesson behavior at a level consistent with the objectives stated in the SPECIFICA- TIONS. Over and above the materials that are presented by the lesson to fulfill the objectives, additional information pertinent to the general field has been included on the advice of the Curriculum Authority. However, this lesson does not presume to be all-inclusive. To assure yourself that this lesson is appropriate to your needs, read the SPECIFICATIONS (inside front cover) and look over the questions of the PRE-LESSON ASSESSMENT on the next page. PRE-LESSON ASSESSMENT Before you go any further, see if you can answer the following questions to determine whether you need to take PART III: 1. What factors determine the order in which a large number of fecal specimens are examined? 2. What information should be recorded on every container when fecal specimens are collected and when they are received in the laboratory? 3. What information should be recorded on each prepared slide? 4. What procedures increase the chances of identifying intestinal amebae? 5. What techniques should be included in a laboratory regimen for the diagnosis of amebiasis? 6. What reagents are used on wet mounts and what purpose does each serve? 7. What differences are there in the preparation of permanent stain slides from fresh and from preserved specimens? 8. Can you outline a minimum laboratory regimen that will efficiently serve to recover and demonstrate intestinal amebae? 9. What are the rationales for performing each step of the formalin-ether concentration technique, cultivation procedures, and the trichrome staining technique? 10. Under what circumstances should a culture be done as an aid in identifying intestinal amebae? 11. What is the proper method of collecting, preserving, and shipping fecal specimens? If you cannot answer the above questions fully, you should take PART III. If you can answer the questions, you will not need to take this lesson. HOW TO USE THIS LESSON This is a self-instructional lesson; its purpose is to teach not to test. You will need only this booklet, a pencil, and your own time (from 1% to 2% hours) to complete the lesson. Do not hesitate to write your answers or make notes in the booklet. REMEMBER This lesson has been prepared so that the amount of reading necessary is minimal and yet most meaningful. Therefore, it is very important that you follow these instructions: —Read EVERYTHING carefully —Proceed through a page or para- graph ONE STEP AT A TIME —Do not skip anything unless you are specifically told to do so —Do exactly WHAT you are told to do WHEN you are told to do it FOLLOW THE INSTRUCTIONS Have you removed the Answer Booklet from the front of this book? If not, do it now. Use it throughout the lesson— when instructed—to check the answers you have given. INTRODUCTION When you examine fecal specimen slides for amebae, the ease with which you are able to find and then identify an organism is largely determined by the effectiveness of the LABORATORY PROCEDURES. The first thing you must do after you receive the properly collected and shipped specimens in the laboratory is LABEL the SPECIMENS. The specimen from each patient constitutes a case: it may be one fresh sample, two preserved samples (in formalin and in polyvinyl alcohol-fixative), or various combinations of these. To put cases in order you must determine and label consistency of fresh specimens of the same age, arrange cases with fresh specimens in order of consistency and place cases with preserved specimens after them, number in the above arranged order (this is the order in which you will examine the specimens), and label each sample with the date. The next and most involved step in the regimen is to PREPARE SLIDES, In the course of the examination of each specimen, you will prepare a series of slides (two to six), depending on how the specimen is received and the success of the diagnostic techniques used. You will label and apply specimen (fresh or preserved) to all the slides. You will use reagents (saline and iodine, iodine, or Quensel’s solution) on some; to others you will apply a permanent stain. You will cover (mount or seal) all slides. RECOVERY PROCEDURES make up the next general step in the regimen. You may RECOVER amebae from a specimen by concentration and cultivation. A concentrate is always prepared; a culture may be prepared. The final step is always to NOTE FINDINGS for each slide. This is done by entering on the Examination Notes, at the appropriate time, the case number, date, and findings for the slide. On the opposite page is a schematic representing the four general steps; study it carefully but do not try to memorize: Before turning the page, be sure you can recall at least the four general steps of the LABORATORY PROCEDURES: —LABEL SPECIMENS —PREPARE SLIDES —RECOVERY PROCEDURES —NOTE FINDINGS SCHEMATIC OF LABORATORY REGIMEN , . watery (w) determine consistency loose (I) of fresh samples of soft (s) the same age; label formed (f) with fresh samples in order of consistency arrange corer LABEL number with preserved samples last in random order SPECIMENS date eee al | containers case number label slide TT date saline and iodine iodine only .* use reagents . Quensel’s solution PREPARE SLIDES apply specimen (F) polyvinyl alcohol - fixative (PVA) fresh _ formalin preserved ** stain (permanent) mount seal RECOVERY << specimen PROCEDURES culture specimen NOTE case number FINDINGS = or findings 735-023°—64——2 LABEL SPECIMENS Shown on right is a case with two samples, which have been labeled (both caps and containers) : ‘case number consistency date CASE Y Fresh sample PVA sample The other case has one fresh sample; YOU label it (cover and container): 1. the case number is #2 > 2. the consistency is soft, :—————> 3. the date is 3/4/63 ——————> Fresh sample PREPARE SLIDES The slide on the right was prepared from the fresh sample of case #1 on 3/4/63; slides have been labeled with number & date the number and date. Reagents have been put on the slide, and SPecimen has been applied. It has been sealed § : SPecimen Y YOU label the bottom slide: 1. with #1 and 3/4/63 -——————> 2. show SPecimen applied ————> 3. show the slide dried and stained : > 4. show the slide mounted J :————> Nom mount here) RECOVERY PROCEDURES Place a check mark (v/) in the blank to show that the specimen was concentrated: — SN CONCENTRATION Place a check mark in the blank to show that a culture was needed in this case: CULTIVATION NOTE FINDINGS & b Findings are generally entered in the Examination Notes as you examine each slide of a case. The Examination Notes below show findings for Case #1 already written in. YOU write in the following findings for Case #2 on 3/4/63: Initial Wet Mount: Feoesidly Z bictlyhiea Fropabe., Concentrate Wet Mount: £ he Zolofrca eyed a Permanent Stain Slide: E£. biatrly five. Bogda aud eid Frum Culture Wet Mount: ar] Aeeded_ Culture Permanent Stain Slide: wry cede EXAMINATION NOTES Initial Concentrate Permanent Culture Case Wet Mount Wet Mount Stain Slide Wet Mount Perm. St. ~~ Check and Correct Your Work. Did you use the Answer Booklet to check and correct your work on pages 4 and 5? If not, do it now. Unit 1 COLLECTION AND PROCESSING OF SPECIMENS To ensure the best possible chances for identifying intestinal amebae, the fecal specimen must be COLLECTED and PROCESSED properly. The COLLECTOR of the specimen is responsible for seeing that it is properly collected and correctly preserved; or if the fresh specimen is to be examined, he must see that it gets to the laboratory as quickly as possible. He should label the container with the name and age of patient and the date and time of passage. The LABORATORY TECHNICIAN must assume responsibility for the ex- amination for parasites; therefore, he must see that the specimens he receives are adequate in quantity and in satisfactory condition for microscopic examination. IMPORTANT BOTH the collector and laboratory technician must at all times take care to do two things: 1. protect from disintegration the fragile trophozoites that may be present in the specimen 2. keep the specimen free from elements that would make it unsuitable for examination When the COLLECTOR receives a request to collect a fecal specimen for in- testinal parasite examination, he should go through certain steps to assure that the specimen will be suitable for use in the laboratory. The chart below sum- marizes the correct actions of the COLLECTOR and the reason for performing each step: STEP REASON Make sure that the specimen is free from such purgatives as mag- nesia and oil, or chemicals such as barium and bismuth. These elements in the stool render the specimen unsatisfactory for examina- tion—the preparation will be unclear. Collect in half-pint carton, free from urine, water, and dirt. Urine and water destroy trophozoites; dirt may contaminate the specimen with free-living organisms. Label carton with identifying in- formation and TIME OF PAS- SAGE. Trophozoites may deteriorate if there is a long delay before laboratory examination. If specimen is to be mailed, pre- serve in PVA and in formalin using the 2-vial method.* OR Carry specimen while still body- warm to the laboratory. OR If examination may be delayed, preserve part of specimen in PVA. This permits recovery of all stages of the organism. Organisms are more easily identified in fresh specimen; trophozoites are more motile in warm specimen. PVA preserves trophozoites so that specimen is suitable for staining for months. *Study the following schematic of the 2-vial method for mailing specimen: 1 PART SPECIMEN to w= 3 PARTS PRESERVATIVE Feces in dry§ 1 PART SPECIMEN to corfon 3 PARTS PRESERVATIVE Formalin Pack & mail with filled out form attached to inner tube The following chart summarizes the actions and responsibilities of the LABORATORY TECHNICIAN after receiving the fecal specimen: STEP REASON Examine the specimen grossly for abnormal-appearing areas (with mucus, blood, ete.), macroscopic organisms, and consistency. Abnormal areas are more likely to contain amebae. The more liquid the stool, the greater the probability of infection. Evaluate the specimen for exami- nation suitability.* If found un- suitable, request proper specimen from the sender. Improperly mixed or preserved speci- men does not allow organisms to be seen adequately. Presence of undesirable medicinal ele- ments makes the preparation on the slide unclear. Urine or water destroys trophozoites; dirt contaminates the specimen. Prepare slides from fresh specimen while still body-warm; or if delayed, refrigerate. Refrigeration decelerates bacterial growth and thus minimizes the dis- integration of trophozoites. *Specimen is unsuitable for examination if: —not enough preservative used (specimen will be thick and gummy) —improperly mixed (small, hard fecal material in preservative) —undesirable medicinal elements (oil, magnesia, barium, bismuth, ete.) in stool —non-stool elements in specimen (urine, water, disinfectants, or dirt) Before leaving this page be sure you can answer correctly (to yourself) the following questions concerning the actions of the collector and the technician when collecting and processing specimens for examination: x: » 5. Why should specimen be free from undesirable medicinal elements? From urine, water, dirt? Why is the 2-vial method used? What is the correct proportion of specimen to preservative in the 2-vial method? What does the COLLECTOR do if the specimen is to be carried to the lab, but will be delayed? What does the TECHNICIAN do if his examination of fresh specimen will be delayed? 6. What does the technician look for in the gross examination of fresh specimen? 7. On what conditions will the tech- nician evaluate the fresh specimen as unsuitable and what will he do then? 8. Why does the technicain try to examine a fresh specimen while still body-warm? 9. What happens to the trophozoites if the specimen is too old or not preserved? = 1 et seal ry . ’ So - “iy . . 1 . oo "3 oo “de = ors ta TT) i I B bor Ca! Co = esa ST oy in a - be - pA a Ny aad A CR ) Describe the actions of the collector and the laboratory technician when collecting and processing fecal specimens: complete the statements in the first column with the word or phrase from the second column (use the LETTER only): 1. preserves trophozoites for staining for several months. 2. ____ in the fecal specimen will destroy trophozoites. may contaminate specimen with free-living organisms. 4. The preservative(s) in the recom- mended 2-vial method is (are) a. collector 5. The examines the fresh specimen grossly for blood, mucus, and macro- b ” A . laboratory technician scopic organisms. 6. in the specimen make the prepa- er BVA ration on the slide unclear for d. PVA and formalin examination. e. Water and urine 7. The evaluates the specimen for suitability for microscopic examina- f. Dirt tion. g. Barium, bismuth, oil, 8. The ___ uses the recommended 2-vial magnesia method for preserving and mailing the specimen. h. formalin 9. The ____ informs the sender when a specimen is unsuitable and why. 10. The ____ refrigerates the specimen if it cannot be examined immediately. 11. The makes certain that the pa- tient has not received undesirable me- dicinal elements and that he keeps the specimen urine-, water-, and dirt-free. Check and Correct Your Work. 723-023°—64——3 1 Write in the word or phrase that best completes each of the following statements about the actions of the collector and the laboratory technician when collecting and processing fecal specimens: 1. 2. 10. . Dirt may contaminate the specimen with . The laboratory technician . Urine or water in the specimen will destroy . The collector uses the . The preservative(s) of the 2-vial method is (are) PVA preserves for staining for several months. The laboratory technician informs the if the specimen is unsuitable for examination. the fresh specimen if it cannot be examined immediately. . The collector makes certain that the patient has not received undesirable medicinal elements and that the specimen is kept free from to preserve and mail a fecal specimen. . The laboratory technician examines the specimen grossly for abnormal areas which are and for consistency, because themore the stool the greater the probability of Undesirable medicinal elements such as make the preparation on slide unclear for examination. Check and correct your work. 13 Unit 2 LABELING SPECIMENS When you have before you those specimens which are suitable for microscopic examination and have examined them grossly for blood flecks, mucous threads, color, and macroscopic parasites, you must LABEL SPECIMENS. Recall: t determine consistency. ow 2 of fresh samples of soft {9 the same age; label formed () with fresh samples in order of consistency arrange cose SEL s ‘number with preserved samples last in random order date ————— all containers 14 The pictures below represent containers with fecal specimens sent to the lab- oratory to be examined for amebae. The specimen samples from one patient should always be kept together; they constitute a CASE. Waxed cartons always contain fresh specimens; vials may contain fresh samples (no label), samples preserved in 10%, formalin (labeled ¥), or samples preserved in poly- vinyl alcohol-fixative (labeled PVA). Therefore, do not assume that all vials contain preserved specimens. LOOK at the carton on the right. The fresh specimen con- tained has been examined to determine its consistency: watery, loose, soft, or formed. Because it is watery it will be examined first and has been designated Case #1. The case number, consistency, and date have been written on both the carton and the cover. The next case includes a fresh sample in a vial. Its consist- ency is loose, and therefore it is designated Case #2, to be examined second (if there had not been a watery specimen it would have been labeled #1). Notice that both samples com- prising the case have been labeled. The other case contains no fresh sample and may therefore be designated Case #3 for order of examination. Cases with no fresh sample (both samples pre- served) are randomly assigned numbers because consistency is not a factor. YOU complete the labeling of both vials in Case #3: FRESH SAMPLE CASE 1 consistency FRESH PRESERVED SAMPLE SAMPLE CASE 2 NOTE: The fresh sample has no F or PVA label. PRESERVED PRESERVED SAMPLE SAMPLE CASE 3 NOTE: Here consistency is Y unknown (no fresh sample). 15 PF aniny FY, =a lT 5 Look at the four cases pictured on the right; these are all the cases that were received in one day. They have been arranged in order of consistency of the fresh specimen. Remember that order of examina- tion is watery, loose, soft, formed. Assume that the date is 3/4/63. Now, YOU complete the labeling for all cases: Y (consistency is watery) REMEMBER: Both samples of a case“are given the same number. (consistency is formed) Check and Correct Your Work. 17 ae - - oo i LB em ks = asl = br - fork wT ERE - 0. EE FR - n Le oo Ta “ 5 m oo - 7: i oo = x a a i a Pictured below are the samples of SEVEN DIFFERENT cases for 3/4/63. Complete the labeling: (formed) (loose) Check and Correct Your Work. 728-023°—64——4 19 Unit 3 20 PREPARATION OF SLIDES In this unit you will learn how to use LABELED SPECIMENS to PREPARE SLIDES to examine for amebae. In most instances amebae can be identified by examining two types of preparations; you will prepare: 1. Initial Wet Mount Slide 2. Concentrate Wet Mount Slide You will also prepare: 1. Quensel’s Wet Mount Slide: IF examination of the initial wet mount shows organisms that are possibly trophozoites but the nuclei are not clear. 2. Permanent Stain Slide: IF (a) the presence of trophozoites is suggested by the consistency (watery, loose, soft) of fresh specimen and the presence of blood and/or mucus, (b) necessary for the identification of organisms unidentified in the wet mounts, and (¢c) a permanent record is desired. 3. Culture Slides (wet mount and permanent stain): IF culture has been prepared as result of earlier indication of pos- sible presence of amebae. Specimens may be used to prepare slides as follows: FRESH SAMPLE FORMALIN SAMPLE PVA SAMPLE Initial Wet Mount Initial Wet Mount Permanent Stain Slide Quensel’s Wet Mount Concentrate Wet Concentrate Wet Mount Mount Permanent Stain Slide Culture Wet Mounts Culture Permanent Stain Slide Three general types of solutions (reagents) are used in the preparation of the temporary WET MOUNTS for the identification of intestinal protozoa: —saline sustains both trophozoites and cysts —Dobell’s iodine makes clearer nuclei in cysts —Quensel’s makes clearer nuclei in trophozoites Using the information on the opposite page, CHECK (y/) the sample(s) which can be used for each of the slides below: 1. Initial Wet Mount Slide: — fresh; _F-preserved; —_PVA-preserved 2. Quensel’s Wet Mount Slide: —fresh; __F-preserved; —_PVA-preserved 3. Concentrate Wet Mount Slide: fresh; _ F-preserved; —PVA-preserved 4. Permanent Stain Slide: — fresh; __F-preserved; —PVA-preserved 5. Culture Wet Mount Slide: — fresh; __F-preserved; —_PVA-preserved 6. Culture Permanent Stain Slide: fresh; _ F-preserved; —_PVA-preserved In most instances, amebae can be identified by examining two slides—CIRCLE their names in the list above. Write in the name of the best reagent in each blank below: makes clearer nuclei in cysts. makes clearer nuclei in trophozoites. sustains both trophozoites and cysts. MAKE SURE YOUR ANSWERS AGREE WITH THE INFORMATION ON THE OPPOSITE PAGE 21 Unit 3A PREPARATION OF WET MOUNT SLIDES There are three basic WET MOUNT slides that may be used in the examination for intestinal amebae: 1. INITIAL WET MOUNT 2. CONCENTRATE WET MOUNT 3. CULTURE WET MOUNT A fresh sample and an F-preserved sample are pictured on the right; use » either one to prepare the INITIAL wet mount. The pictured initial wet mount slides on the right have been prepared as follows: 1. The slides have been labeled with case number and date. 2. a. In preparation of fresh sample: —a drop of normal Saline has been placed on the left of the slide. —a drop of Dobell’s Iodine has been placed on the right end of the slide. —applicator sticks have been used to place FResh SPecimen in saline and mix, and in iodine and mix. (CAUTION: Don’t contaminate saline with iodine; use separate sticks.) b. In preparation of formalin sample: —same as above except that the only reagent used is Dobell’s Todine. 3. Both preparations on each slide have been covered and sealed & . (NOTE: It is advisable to seal the coverslips to the slide with heated paraffin and petroleum jelly, 1:1, employing a cotton swab.) Formalin-preserved sample number & date sealed FResh SPecimen FRESH initial wet mount NOTE: 2" x 3* slides used. number & date lodine Formalin SPecimen FORMALIN initial wet mount Remember that Quensel’s Wet Mount Slide may be necessary in some cases. It is prepared from fresh specimen, basically the same as above, on a 2’ x 3’/ slide using a vital staining solution such as Quensel’s as the only reagent. 22 Pictured on the right is the concentrate of the specimen (fresh or formalin) used to prepare the CONCENTRATE wet mount: Y CONCENTRATE Now YOU show the preparation of a WET CONSE IRATED CONcentrate wet mount by writing MOUNT on the pictured slide: ——————> 1. Case No. and date (#1, 3/8/63):—> 2. Iodine and CONcentrated SPec- imen (on the right) : ———> 3. CONCcentrated SPecimen only (on the left): m—— 5 4. Cover and seal § :=————ou—> (NOTE: a pipette is used to transfer the sediment from the tube to the slide.) Pictured on the right are cultures of the fresh specimen used to prepare CULTURE wet mounts: > A separate slide is prepared for each 24-. culture tube. YOU show the prepa- hour ration of both CULTURE wet CULTURES mounts by writing in the pictured CULTURE slides: > WET MOUNT 1. Case No. and date (#1, 3/8/63) :—> 2. Quensels and CULture SPeci- men (on the right end of the slide): > 3. CULture SPecimen only (on the left end): —mor——— 4. Cover and seal § i: ————————> (CAUTION: Use a separate pipette for each culture tube.) 23 24 REVIEW NOTE: The Initial (Formalin), Concentrate, and Culture Wet Mounts do not require saline since the samples for them are liquid enough. To help you remember where the three reagents go, study the following: IODINE INITIAL WET MOUNT (UBS and (Remember: “a fresh initiate needs everything’) SALINE INITIAL WET MOUNT (FORMALIN) ———————>I0DINE (Remember: “formalin fluid enough, needs less”) only CONCENTRATE WET MOUNT > Iodine (Remember: ‘concentrate on I (one) thing’) CULTURE WET MOUNT: >Quensel’s (Remember: “a Quiet cultured person’’) Now check (v/) below all the points you should remember when preparing wet mounts: — Initial wet mounts can be made from either fresh or formalin samples. _— An initial wet mount (fresh) includes both saline and iodine as reagents. — An initial wet mount (formalin) includes only iodine as a reagent. — A Quensel’s wet mount is prepared if examination of the initial wet mount shows organisms that are possibly trophozoites but the nuclei are not clear. — A concentrate of either the fresh or the formalin specimen is used for the concentrate wet mount. — A concentrate wet mount includes only iodine as a reagent. — Use a pipette to place concentrate sediment on slide. — A culture of the fresh specimen is used for the culture wet mounts. — Culture wet mount includes Quensel’s as the reagent. — Use a different pipette for each tube of culture. — Temporary stains (the reagents iodine and Quensel’s) are placed on the right end of the slide. — All reagents are placed on the slide first, then the specimens added. — Use separate applicator sticks to avoid contamination when mixing various reagents with specimens. — Seal slides with heated paraffin and petroleum jelly (1:1) using a cotton swab. — Use 2’" x 3’ slides for all wet mounts. You should have checked all of the statements above as points to remember. If you did not, review the information on the previous two pages. 25 Pictured below are the specimens needed to complete the three unprepared wet mounts. Use the following information to indicate the preparations: 1. All are Case #2 on 3/8/63. 2. Use SP for placement of SPecimen with FR for FResh, F for Formalin, CON fc for CONcentrate, and CUL for CULture. 3. Use S S for Saline, I for Iodine, and and Q for or Quensel’s. ‘“a fresh initiate needs everything” lc 3 > bd RECALL formalin fluid enough, nands logy “concentrate on I (one) thing “a Quiet cultured person” 4. Use § for covering and sealing. INITIAL WET MOUNT CONCENTRATE WET MOUNT CULTURE WET MOUNT 26 Complete the statements by placing the statement number in the appropriate blank(s); there may be more than one number for a blank, and some blanks should be empty: 1. Separate applicator sticks are used 2. A single pipette is used 3. Different pipettes are used 4. A Quensel’s wet mount is prepared 5. The size of the slide used for wet mounts is 6. Reagents are placed on the slide 7. Temporary stains are placed on the 8. Coverslips are sealed to the slide with s— iil ig, —l — — Ts ——— Xe seer A to avoid contamination when mixing various reagents with specimens. to place drops of specimen sedi- ment on concentrate slide. to place drops of specimen on slide from each culture tube. after examination of every wet mount. if examination of the initial wet mount shows unclear nuclei of possible trophozoites. 17? 23%, 2/2 x 37. before specimen is applied. only after specimen is applied. right end of the slide. left end of the slide. heated paraffin and petroleum jelly (1:1) using a cotton swab. Permount, Balsam, etc. 4 Check and correct your work carefully. If you made no bs errors skip to p. 29; otherwise, continue on the next page. 723-023°—64——F5 27 28 Pictured below are the specimens needed to prepare the slides named. YOU indicate the preparation of slides: INITIAL WET MOUNT CONCENTRATE WET MOUNT CULTURE WET MOUNT Check and Correct Your Work. Unit 3B PREPARATION OF PERMANENT STAIN SLIDES Preparing a PERMANENT STAIN slide is often part of the laboratory regimen. The slide is prepared: (a) if consistency of the fresh specimen and the presence of blood and/or mucus suggest the presence of trophozoites, (b) when necessary for identifying organisms unidentified in the wet mounts, and (c) for permanent record. Pictured on the right are the samples that can be used to prepare the permanent stain slide. When a case includes both and the specimen is old or the age is unknown, use the PVA. The pictured permanent stain slides on the right have been prepared as follows: 1. The slides have been la- beled with case number and date. (NOTE: Etching pencil was used to make labeling permanent.) 2. a. For FResh SPecimen, a single applicator stick was used to apply and spread speci- men to a thin, even layer on the middle third of the slide. b. For PVA sample, the above procedures were followed EXCEPT the specimen was spread EDGE-TO-EDGE in the middle third of slide and allowed to DRY for 24 hours at room temperature. 3. The “COOKBOOK” was then used to perform the actual staining procedures. 4. The slide was mounted and covered immediately after stain- ing. (NOTE: Coverslips are mounted using Permount, Balsam, or other suitable mounting media.) PVA-preserved Fresh sample somple number & date FRESH permanent stain slide CB mount Cookbook) immediately for ini after staining STAINING spread edge-to-edge PVA permanent stain slide NOTE: 1"’ x 3" slides used. 29 Pictured on the right are specimen samples used in preparing permanent stain RE ——— YOU indicate the steps in preparing a permanent stain slide for Case #1 on 3/4/63: 1. Label slides: 2. a. Indicate the application of FResh SPecimen | <—> |: b. Indicate the application of FRESH PVA SPecimen |[€——>]: 3. Indicate the use of a CB (cook- book) for staining: 4. Indicate immediate mounting and covering J : PYA Check and Correct Your Work. 31 32 In the preparation of permanent stain slides from PVA specimen and/or fresh specimen, match the statements begun on the left with the completions on the right. Fill in the blank(s) with the appropriate number: 1. A cookbook is used 2. Mounting and covering should EL 3. PVA specimen should — 4. Fresh specimen should — I . to begin the preparation of the per- manent stain slides. . for the staining procedures after PVA specimen has been applied to slide and allowed to dry for 24 hours. . for the staining procedure after fresh specimen has been applied to slide. be done a few minutes after other completions. . be done immediately after completion of staining procedures. . be spread in thin, even layer to edge of slide. . be spread in thin, even layer. Check and Correct Your Work. You now know how to prepare the basic slides. As each slide is completed you examine it microscopically for intestinal amebae and NOTE FINDINGS. Recall: case number NOTE date FINDINGS be findings For example: EXAMINATION NOTES Initial Concentrate Permanent Culture Case Wet Mount Wet Mount Stain Slide Wet Mount Perm. St. #| E cole E. cote E cote L940 E hier, E tole 34/63 ind, found od 2 a Taoph, E wy z i Zeoph. found Unit 4 RATIONALE FOR COOKBOOK PROCEDURES There are certain procedures in the LABORATORY REGIMEN for finding and identifying amebae that demand absolute accuracy. Since it is not feasible to have you memorize the detailed steps, “cookbooks” (protocols) are provided in the laboratory and should always be used. While you need not memorize detailed steps, you should know the reasons for them, which this unit teaches. REMEMBER Do not try to learn the detailed steps in the cookbook. Learn the reason for doing the things the cookbook tells you to do. 35 Unit 4A 36 COOKBOOK: TRICHROME STAINING TECHNIQUE The “cookbook” TRICHROME STAINING TECHNIQUE which you will follow in the laboratory regimen for diagnosing amebiasis is shown below. Staining has at least two advantages: (1) it increases the possibility of diag- nosis by making certain characteristics clear, and (2) it provides a permanent record of findings. Staining is part of the “Preparation of Permanent Stain Slide” procedure that you have already learned ; making one or more permanent stain slides is part of the laboratory regimen. The Trichrome Staining Technique consists essentially of dipping or placing slides prepared from either fresh or PVA specimens into various solutions for precise periods of time. Read carefully (don’t memorize) the steps of the staining cookbook and pay special attention to the underlined words: TRICHROME STAINING TECHNIQUE STEPS FRESH SPECIMEN PVA SPECIMEN 1. Schaudinn’s fixative 5 min. at 50° C. (already fixed with or polyvinyl alcohol- 1 hr. at room fixative) temperature 2. Alcohol 709, plus iodine 1 minute 10-20 minutes 3. Alcohol 709, (1) 1 minute 3-5 minutes Alcohol 709, (2) 1 minute 3-5 minutes 4. Trichrome stain 2-8 minutes 6-8 minutes Alcohol 909, acidified 10-20 seconds 10-20 seconds 5. Alcohol 959, rinse twice 5 minutes Carbol-xylene 1 minute 5-10 minutes Xylene 1 minute 10 minutes 6. Mount immediately immediately Given below are the key words that are underlined in the steps of the staining cookbook, along with the appropriate rationales. Now, you do this: 1. Look at each key word in turn. 2. Relate it to its step in the cookbook. 3. Now look back and relate the step and the key word to the rationale. KEY WORDS RATIONALES 1. Schaudinn’s fixative precipitates protein materials 2. Alcohol plus iodine removes mercuric chloride crystals 3. Alcohol removes excess iodine and water 4, Trichrome stains structure Acidified Alcohol removes excess stain 5. Alcohol dehydrates and clears slide Carbol-xylene Xylene 6. Mount immediately protects preparation Repeat the study procedure at least once before continuing. 37 GO TO THE NEXT PAGE TRICHROME STAINING TECHNIQUE STEPS FRESH SPECIMEN PVA SPECIMEN ER Sa—" 1. Schaudinn’s fixative 5 min. at 50° C. (already fixed with or polyvinyl alcohol- 1 hr. at room fixative) temperature 2. Alcohol 709, plus iodine 1 minute 10-20 minutes 3. Alcohol 709, (1) 1 minute 3-5 minutes Alcohol 709%, (2) 1 minute 3-5 minutes 4, Trichrome stain 2-8 minutes 6-8 minutes Alcohol 909, acidified 10-20 seconds 10-20 seconds 5. Alcohol 95%, rinse twice 5 minutes Carbol-xylene 1 minute 5-10 minutes Xylene 1 minute 10 minutes 6. Mount immediately immediately 38 Shown below are the key words of the staining cookbook steps. Opposite them are the rationales, but now they are in random order. Match by filling in the blanks with the appropriate key-word numbers: (Refer to the cookbook on the opposite page if you wish.) KEY WORDS RATIONALES 1. Schaudinn’s fixative a. dehydrates and clears slide 2. Alcohol plus iodine — b. precipitates protein materials 3. Alcohol —— c. removes excess iodine and water 4. Trichrome —— d. protects preparation Acidified alcohol 5. Alcohol e. stains structure Carbol-xylene removes excess stain Xylene 6. Mount immediately f. removes mercuric chloride crystals Check and Correct Your Work. 39 n § v y Ii Beas . =P RIS yr [= SER Ta 3 + B = - . ) b lA. 1 + . . a 5 i aks 2 4 ' Li i : a sd ibs gE 4 in gis Ly 1 * bh . \ 0 = T "aa - i. & . - n * I> f i v 4 E ES wf - i is Aadt oe aed L : : x - } nt: ¥ ¥ « i z » ¥ » AM a . To . La ’ ty Ha 2a ¥ # i i i Et . ¥ gp ry . ih fu * « i a . - on: Ny 5 i w . a I - § 2 - v uy =a ¥ 2 = oz dar enc = ' *r ’ eS il 1% . 2 Ili * ‘ » 3 . ’ . a “ = = 1 5 e 1 E Pe i * = 4 . “f % a w . * ' ) . Ig 1A Lm fen dh | ange 4 = te TE AR Shown below are the rationales (in random order) for the staining cookbook. Match by placing the cookbook step number in the appropriate blank: RATIONALES —— a. removes mercuric chloride —— d. removes excess iodine and crystals water — b. stains structure and removes — e. precipitates protein materi- excess stain als — c. protects preparation — f. dehydrates and clears slide TRICHROME STAINING TECHNIQUE STEPS FRESH SPECIMEN PVA SPECIMEN 1. Schaudinn’s fixative 5 min. at 50° C. (already fixed with or polyvinyl alcohol- 1 hr. at room fixative) temperature 2. Alcohol 709, plus iodine 1 minute 10-20 minutes 3. Alcohol 709, (1) 1 minute 3-5 minutes Alcohol 709, (2) 1 minute 3-5 minutes 4. Trichrome stain 2-8 minutes 6-8 minutes Alcohol 909, acidified ~~ 10-20 seconds 10-20 seconds 5. Alcohol 95%, rinse twice 5 minute- Carbol-xylene 1 minute 5-10 minutes Xylene 1 minute 10 minutes 6. Mount immediately immediately Check and Correct Your Work. 41 Unit 48 COOKBOOK: FORMALIN-ETHER CONCENTRATION 10. 11. 42 TECHNIQUE GO TO THE NEXT PAGE FORMALIN-ETHER CONCENTRATION TECHNIQUE . Break up a portion of the fresh specimen (about size of walnut) in saline or thoroughly stir the formalinized specimen. Add enough saline to the specimen to make at least 10 ml. of suspension. . Strain 10 ml. of the suspension through one layer of narrow-mesh or two layers of wide-mesh wet cheesecloth (or gauze) and a paper funnel into a 15 ml. centrifuge tube. Discard funnel and cloth into disinfectant if using fresh. specimen. Centrifuge for one minute at 700-1000 G’s (approximately 2000 rpm in centrifuge with radius of six inches). Decant supernatant. There should be about one ml. of sediment remain- ing. If you have too much sediment, add water, mix thoroughly, and pour off proportionate amount; for example, if you have three ml. of sediment, add water, mix, and pour off 24 of solution. In the future, adjust the amount of saline (Step 1 above) so that the suspension will yield approxi- mately one ml. of sediment at this point. . Repeat the above washing procedure, centrifuge, and decant, as before. If supernatant is “dirty’”’ and a cleaner sediment is desired, the procedure can be repeated a second time. . Add 10 ml. of 109% formalin to sediment and mix thoroughly. If fresh specimen, let stand five minutes. Add three ml. of ether. Stopper tube, invert, shake vigorously (30 sec.—one minute), remove stopper carefully. Centrifuge for one minute at 375 G’s (approximately 1500 rpm). Four layers should result: ether at top, plug of debris, formalin solution, and sediment. Carefully loosen plug of debris from sides of the tube by ringing with an applicator stick; decant top three layers; wipe inside clean of remaining plug debris with cotton swab. Mix the remaining sediment with the small amount of fluid that drains back from the sides of the tube. If you have less than 0.1 ml. of sediment, add just enough saline to bring this quantity to 0.1-0.2 ml. The “cookbook” FORMALIN-ETHER CONCENTRATION TECHNIQUE which you will follow in the laboratory regimen for diagnosing amebiasis is shown on the opposite page. Concentration is a RECOVERY PROCEDURE used to reduce the amount of material to be examined. The relevant material can then be examined more quickly, and it will include a greater concentration of organisms, allowing you to sample a large quantity of feces easily. Concentration is useful in revealing light infections where cysts are present; there is no satisfactory method for concentrating trophozoites. The Formalin- Ether sedimentation (concentration) technique is effective in concentrating protozoan cysts in unpreserved specimens and can also be used satisfactorily with formalin-preserved specimens. The cysts are normal in appearance and can be identified in the unstained and iodine preparations of the concentrate wet mount. Now, read (don’t memorize) the cookbook steps and pay special attention to the underlined words: 3 Now that you have read the cookbook, study the key words and rationales listed below. Remember the study procedure: (1) look at each key word in turn, (2) relate it to its step in the cookbook, and (3) look back and relate the steps and key words to the rationale. KEY WORDS RATIONALES 1. suspension frees organisms from other matter 2. strain removes coarse particles 3. discard into disinfectant avoids contamination 1. centrifuge sediments organisms and heavier particles 5. decant removes soluble and lighter materials 6. repeat . assures adequate wash 7. add formalin, let stand fixes organisms 8. add ether removes fats and oils 9. shake vigorously . . . allows uniform functioning . remove stopper carefully releases pressure gradually 10. loosen plug . . . assures cleaner decanting . . . wipe inside clean keeps debris from sliding into sediment 11. 0.1-0.2 ml. provides enough sediment for two preparations Repeat the study procedure at least once before continuing. 43 44 10. 11. GO TO THE NEXT PAGE FORMALIN-ETHER CONCENTRATION TECHNIQUE . Break up a portion of the fresh specimen (about size of walnut) in saline or thoroughly stir the formalinized specimen. Add enough saline to the specimen to make at least 10 ml. of suspension. Strain 10 ml. of the suspension through one layer of narrow-mesh or two layers of wide-mesh wet cheesecloth (or gauze) and a paper funnel into a 15 ml. centrifuge tube. . Discard funnel and cloth into disinfectant if using fresh specimen. . Centrifuge for one minute at 700-1000 G's (approximately 2000 rpm in centrifuge with radius of six inches). . Decant supernatant. There should be about one ml. of sediment remain- ing. If you have too much sediment, add water, mix thoroughly, and pour off proportionate amount; for example, if you have three ml. of sediment, add water, mix, and pour off % of solution. In the future, adjust the amount of saline (Step 1 above) so that the suspension will yield approximately one ml. of sediment at this point. . Repeat the above washing procedure, centrifuge, and decant, as before. If supernatant is “dirty” and a cleaner sediment is desired, the procedure can be repeated a second time. . Add 10 ml. of 109% formalin to sediment and mix thoroughly. If fresh specimen, let stand five minutes. . Add three ml. of ether. . Stopper tube, invert, shake vigorously (30 sec.—one minute), remove stopper carefully. Centrifuge for one minute at 375 G’s (approximately 1500 rpm). Four layers should result: ether at top, plug of debris, formalin solution, and sed- iment. Carefully loosen plug of debris from sides of the tube by ringing with an applicator stick; decant top three layers; wipe inside clean of remaining plug debris with cotton swab. Mix the remaining sediment with the small amount of fluid that drains back from the sides of the tube. If you have less than 0.1 ml. of sediment, add just enough saline to bring this quantity to 0.1-0.2 ml. Shown below are the key words of the concentration-cookbook steps. On the right are the rationales, in random order. Match by filling in each blank with the appropriate key-word number: (Refer to the cookbook steps on the opposite page if you wish.) KEY WORDS RATIONALES 1. suspension a. assures adequate wash 2. strain —b. frees organisms from other 3. discard into disinfectant 4. centrifuge 5. decant 6. repeat 7. add formalin, let stand 8. add ether 9. shake vigorously... remove stopper carefully 10. loosen plug . . . wipe inside clean 11. 0.1-0.2 ml. ——g — il matter . removes soluble and lighter materials . assures cleaner decanting... keeps debris from sliding into sediment . avoids contamination . sediments organisms and heavier particles removes coarse particles removes fats and oils fixes organisms . allows uniform functioning. releases pressure gradually . provides enough sediment for two preparations Check and Correct Your Work. 45 10. 11. 46 GO TO THE NEXT PAGE FORMALIN-ETHER CONCENTRATION TECHNIQUE . Break up a portion of the fresh specimen (about size of walnut) in saline or thoroughly stir the formalinized specimen. Add enough saline to the specimen to make at least 10 ml. of suspension. . Strain 10 ml. of the suspension through one layer of narrow-mesh or two layers of wide-mesh wet cheesecloth (or gauze) and a paper funnel into a 15 ml. centrifuge tube. Discard funnel and cloth into disinfectant if using fresh specimen. . Centrifuge for one minute at 700-1000 G’s (approximately 2000 rpm in centrifuge with radius of six inches). . Decant supernatant. There should be about one ml. of sediment remain- ing. If you have too much sediment, add water, mix thoroughly, and pour off proportionate amount; for example, if you have three ml. of sedi- ment, add water, mix, and pour off 24 of solution. In the future, adjust the amount of saline (Step 1 above) so that the suspension will yield approxi- mately one ml. of sediment at this point. . Repeat the above washing procedure, centrifuge, and decant, as before. If supernatant is ““dirty’’ and a cleaner sediment is desired, the procedure can be repeated a second time. Add 10 ml. of 109, formalin to sediment and mix thoroughly. If fresh specimen, let stand five minutes. Add three ml. of ether. Stopper tube, invert, shake vigorously (30 sec.—one minute), remove stopper carefully. Centrifuge for one minute at 375 G’s (approximately 1500 rpm). Four layers should result: ether at top, plug of debris, formalin solution, and sediment. Carefully loosen plug of debris from sides of the tube by ringing with an applicator stick; decant top three layers; wipe inside clean of remaining plug debris with cotton swab. Mix the remaining sediment with the small amount of fluid that drains back from the sides of the tube. If you have less than 0.1 ml. of sediment, add just enough saline to bring this quantity to 0.1-0.2 ml. Shown below are the rationales (in random order) for the concentration cook- book. Match by placing the step numbers of the cookbook steps (opposite page) in the appropriate blanks: RATIONALES sss. Th poi Ds sms Ce removes fats and oils avoids contamination allows uniform functioning . . . releases pressure gradually . sediments organisms and heavier particles . assures cleaner decanting . . . keeps debris from sliding into sediment . removes coarse particles . removes soluble and lighter materials . provides enough sediment for two preparations i. assures adequate wash j. frees organisms from other matter . fixes organisms Check and Correct Your Work. 47 Unit 4C 48 COOKBOOK: CULTIVATION OF INTESTINAL PROTOZOA GO TO THE NEXT PAGE CULTIVATION OF INTESTINAL PROTOZOA I. Prepare to inoculate specimen: JT. IIT. IV. VI 1. Take two test tubes of warm (37° C.) medium. 2. Add 30 mg. of sterile rice starch to each tube of medium. 3. Add to one of the test tubes enough penicillin and streptomycin to give a final concentration of 200-250 units of each per ml. of overlay. Inoculate specimen: 1. Examine specimen for consistency and for mucous threads, blood flecks, and other abnormal areas of concentration—use such areas in culture. Liquid or semi-solid specimen Formed specimen 2. Take large-bore pipette and Take two applicator sticks and add a add 0.5 ml. of specimen to portion (size of small pea) of the each test tube. specimen to each tube. 3. Mix the inoculum with the Mix with overlay by rubbing speci- overlay, adding as little air men against side of test tube with as possible in the transfer. stick. 4. Seal tube with paraffin-pet- Seal tube with paraffin-petrolatum. rolatum. Incubate specimen at 37° C. for 24 hours. Prepare and examine culture wet mount. Decide whether to cultivate further: 1. If no amebae are found or if amebae are found and not identified, cultivate further; take two tubes of fresh medium and add rice starch to each. Do not add antibiotics. Transfer half the sediment from each tube to fresh tubes. Incubate all four tubes at 37° C.; examine 48 hours later. 2. Prepare and examine wet mounts of all four cultures. If no amebae are found or if amebae found are not identified, transfer half the sediment from each of the two transfer tubes to fresh media, add rice starch (not antibiotics), and incubate the last four cultures at 37° C.; examine 48 and 72 hours later. 3. When amebae are identified, do not cultivate further. Make a permanent stain slide for a permanent record and to facilitate identification of questionable organisms. Transfer the culture sediments to PVA vials and then prepare permanent stain slides and examine carefully under the oil immersion objective—note findings. The “cookbook” CULTIVATION OF INTESTINAL PROTOZOA which you will follow in the laboratory regimen for diagnosing amebiasis is shown on the opposite page. This technique, like the concentration technique, is a RECOVERY PROCEDURE. Cultivation increases the possibilities of finding an organism by permitting the amebae to multiply. Now read (don’t memorize) the steps in the cookbook procedure; pay special attention to the underlined key ass Now that you have read the cookbook, study the key words and rationales listed below. Follow the study procedure: (1) look at each key word in turn, (2) relate it to its step in the cookbook, and (3) look back and relate the step and key words to the rationale. KEY WORDS 1. 11. III. Iv. VI 1. warm medium 2. rice starch 3. antibiotics 1. abnormal areas 2. add specimen 3. mix 4. Seal incubate wet mount, . cultivate further permanent stain slide RATIONALES provides nutrient material, optimal tem- perature provides particulate food for amebae maintains optimal level of bacterial growth likely to contain amebae start culture provides uniform distribution and releases organism into medium lowers tension for better growth provides time and optimal temperature for growth adequate for initial examination increases possibilities of diagnosis provides permanent record and facilitates identification Repeat the study procedure once before continuing. 49 50 11. III. Iv. V1. GO TO THE NEXT PAGE CULTIVATION OF INTESTINAL PROTOZOA . Prepare to inoculate specimen: 1. Take two test tubes of warm (37° C.) medium. 2. Add 30 mg. of sterile rice starch to each tube of medium. 3. Add to one of the test tubes enough penicillin and streptomycin to give a final concentration of 200-250 units of each per ml. of overlay. Inoculate specimen: 1. Examine specimen for consistency and for mucous threads, blood flecks, and other abnormal areas of concentration—use such areas in culture. Liquid or semi-solid specimen Formed specimen 2. Take large-bore pipette and Take two applicator sticks and add add 0.5 ml. of specimen to a portion (size of small pea) of the each test tube. specimen to each tube. 3. Mix the inoculum with the Mix with overlay by rubbing speci- overlay, adding as little air men against side of test tube with as possible in the transfer. stick. 4. Seal tube with paraffin- Seal tube with paraffin-petrolatum. petrolatum. Incubate specimen at 37° C. for 24 hours. Prepare and examine culture wet mount. Decide whether to cultivate further: 1. If no amebae are found or if amebae are found and not identified, cultivate further; take two tubes of fresh medium and add rice starch to each. Do not add antibiotics. Transfer half the sediment from each tube to fresh tubes. Incubate all four tubes at 37° C. for 48 hours. 2. Prepare and examine wet mounts of all four cultures. If no amebae are found or if amebae found are not identified, transfer half the sediment from each of the two transfer tubes to fresh media, add rice starch (not antibiotics), and incubate all six cultures at 37° C. for 72 hours. Pre- pare and examine wet mounts of all six cultures. 3. When amebae are identified, do not cultivate further. Make a permanent stain slide for a permanent record and to facilitate identification of questionable organisms. Transfer the culture sedi- ments to PVA vials and then prepare permanent stain slides and examine carefully under the oil immersion objective—note findings. Shown below are the key words of the culture-cookbook steps. Opposite them Match by filling in the blanks with the appropriate key-word numbers (use roman and arabic; for example: II-4, IV, ete.): are the rationales in random order. KEY WORDS 11. 3. 4. warm medium . rice starch . antibiotics . abnormal areas . add specimen mix Seal ITI. incubate IV. wet mount V. cultivate further VI. permanent stain slide RATIONALES —a. lowers oxygen tension for better growth —b. provides nutrient material, op- timal temperature ——c. provides time and optimal tem- perature for growth —d. provides particulate food for amebae e. adequate for initial examination —f. maintains optimal level of bac- terial growth —g. increases possibilities of diagnosis —h. likely to contain amebae — i. start culture ——j. provides permanent record and facilitates identification —k. provides uniform distribution and releases organisms into medium Check and Correct Your Work. 51 52 L n. IIT. IV. GO TO THE NEXT PAGE CULTIVATION OF INTESTINAL PROTOZOA Prepare to inoculate specimen: 1. Take two test tubes of warm (37° C.) medium. 2. Add 30 mg. of sterile rice starch to each tube of medium. 3. Add to one of the test tubes enough penicillin and streptomycin to give a final concentration of 200-250 units of each per ml. of overlay. Inoculate specimen: 1. Examine specimen for consistency and for mucous threads, blood flecks, and other abnormal areas of concentration—use such areas in culture. Liquid or semi-solid specimen Formed specimen 2. Take large-bore pipette and Take two applicator sticks and add a add 0.5 ml. of specimen to portion (size of small pea) of the each test tube. specimen to each tube. 3. Mix the inoculum with the Mix with overlay by rubbing speci- overlay, adding as little air men against side of test tube with as possible in the transfer. stick. 4. Seal tube with paraffin-petro- Seal tube with paraffin-petrolatum. latum. Incubate specimen at 37° C. for 24 hours. Prepare and examine culture wet mount. Decide whether to cultivate further: 1. If no amebae are found or if amebae are found and not identified, cultivate further; take two tubes of fresh medium and add rice starch to each. Do not add antibiotics. Transfer half the sediment from each tube to fresh tubes. Incubate all four tubes at 37° C. for 48 hours. 2. Prepare and examine wet mounts of all four cultures. If no amebae are found or if amebae found are not identified, transfer half the sediment from each of the two transfer tubes to fresh media, add rice starch (not antibiotics), and incubate all six cultures at 37° C. for 72 hours. Pre- pare and examine wet mounts of all six cultures. 3. When amebae are identified, do not cultivate further. Make a permanent stain slide for a permanent record and to facilitate identification of questionable organisms. Transfer the culture sediments to PVA vials and then prepare permanent stain slides and examine care- fully under the oil immersion objective—note findings. Shown below are the rationales (in random order) for the culture cookbook. Match by placing the cookbook step numbers (opposite page) in the appro- priate blanks: RATIONALES a. lowers oxygen tension for better growth —b. provides uniform distribution and releases organisms into medium c. provides nutrient material, optimal temperature —d. provides permanent record and facilitates identification e. provides time and optimal temperature for growth —f. start culture —¢g. provides particulate food for amebae —h. likely to contain amebae — i. adequate for initial examination —j. increases possibilities of diagnosis —k. maintains optimal level of bacterial growth Check and Correct Your Work. 53 ) . Ea Fi N oo ate bisa 5 haut Sakina wt Een oo » ny oo ¥ Pel oo Unit 5 WHEN TO CULTIVATE Cultivation can be employed to advantage with fresh specimens if there are adequate facilities, time, and interest. Also, the fresh specimen used should be no more than eight hours old. To increase chances of identifying E. histolytica, PREPARE A CULTURE of the fresh specimen whenever there is a suggestion of the presence of E. histolytica: 1. If amebae (cysts or trophozoites) other than E. histolytica have been found (identified). 2. If unidentified (or possible) amebae have been found. 3. If the stool is dysenteric (watery or loose and abnormal in appearance). NOW you check (v/) the conditions, below, under which you would prepare a culture: —1. No amebae found in dysenteric stool. — 2. Dysenteric stool used and E. histolytica trophozoites found. ——3. Unidentified amebae found. — 4. Cyst possibly E. histolytica found. ——5. E. histolytica cysts found. ——6. E. coli cyst found. ——7. No amebae found in formed specimen. Check and Correct Your Work. 55 56 Fill in the blanks with the appropriate number to indicate your action regarding cultivation after you have examined the initial wet mount. DO THIS: WHEN: 1. Cultivate a. no amebae found in formed specimen. 2. Do not cultivate — b. unidentified ameba found. c. dysenteric stool used and no amebae found. — d. E. stolytica trophozoites found. e. E. histolytica cysts found. — ff. E. colt trophozoites found in dysenteric stool. —— g. a possible E. coli trophozoite found. Check and Correct Your Work. Unit 6 SELECTION OF PROCEDURES FOR LABORATORY REGIMENS The specific slide preparations taught in this lesson were selected because they allow for the recovery of intestinal amebae as well as other parasites. The preparations are: initial wet mount concentrate wet mount permanent stain slide culture slides In general, the more kinds of preparations (slides) included in a regimen, the greater will be the number of infections discovered in a given period of time. However, due to economic and time factors, some preparations are recom- mended to be used routinely and others supplementally. The selection of preparation to be used routinely should depend on these factors: 1. need to recover both cysts and trophozoites 2. whether the specimen is fresh or preserved 3. time and facilities available Before leaving this page you should be able to recall the three selection factors. 57 58 Depending upon the location of the laboratories, either FRESH or PRE- SERVED specimens are received for examination. REMEMBER All preparations can be made from FRESH specimen. All but culture preparations can be made from PRESERVED specimen (PVA and Formalin). FRESH specimen is usually available to CLINIC and HOSPITAL labora- tories. The recommended regimen for these laboratories includes the following procedures: ROUTINE PROCEDURE REASON After you have examined the fresh specimen grossly, you then PREPARE a SMEAR and im- merse it in SCHAUDINN’S FIX- ATIVE. fixes slide for permanent staining if necessary PREPARE AND EXAMINE INITIAL WET MOUNT. to see motility of trophozoites and to provide a stain of organisms PREPARE AND EXAMINE CONCENTRATE WET MOUNT. collects cysts in small area PREPARE PERMANENT STAIN if examination of wet mounts shows positive or ques- tionable evidence of parasites; or if specimen is soft or abnormal in appearance. stains structures better and pro- vides a permanent record soft consistency is more indicative of an infection SUPPLEMENTAL PROCEDURE If time and facilities permit, PREPARE a CULTURE. Per- manently stain at least those cultures that reveal organisms in culture wet mounts. increases chances of recovery and identification The procedures should be repeated with several different specimens for each patient, since organisms are passed intermittently. PRESERVED (PVA and formalin) specimen is usually available to CENTRAL DIAGNOSTIC laboratories. The recommended regimen for the laboratories includes the following procedures: ROUTINE PROCEDURE REASON Use the FORMALIN-preserved specimen to prepare a CONCEN- TRATE WET MOUNT. recovers cysts, eggs, larvae Use PVA-preserved specimen to prepare a PERMANENT STAIN. stains both cysts and trophozoites (PVA fixative includes a plastic which causes specimen to adhere to slide, allowing permanent staining) PROCEDURAL MODIFICATIONS Stain only for those cases where positive or questionable evidence was revealed by the concentrate preparation or where the specimen is soft or abnormal (when known). restricts examination to more rele- vant cases (in a survey where many cases must be examined quickly) Use the chart on this and the opposite page to answer the following questions (to yourself): 1. If you are in a clinic or hospital and have examined a fresh specimen grossly, what are the two slides that you will always prepare? When will you prepare a permanent stain? 2. If time and facilities permit, what supplemental procedures will you carry out? 3. In the central diagnostic lab, what are the two slides prepared? Which preserved specimen of the case is used for each? 4. What are the conditions under which you would modify the regimen by not staining all the cases? Which cases would be stained? Before leaving this page, make sure you can answer correctly all the questions. 59 gras a Hobe be; ) a a gh a = oe hil ‘ | UR rr poo 5 . 3 Hay. dns 3 ares > 2 ed ! pt f g os mes ¥ a! ey - 5 i wg 3. he pfu 44 x $0 5 i A, a i wy