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Chapter 27 : Macroscopic and Microscopic Examination of Fecal Specimens

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Abstract:

This chapter discusses about macroscopic examination and microscopic examination of fecal specimens. The microscopic examination of the stool specimen, normally called the ova and parasite examination, consists of three separate techniques: the direct wet smear, the concentration (sedimentation and flotation), and the permanent stained smear. For laboratories using iron hematoxylin stains in combination with sodium acetate-acetic acid-formalin (SAF)-fixed material and modified acid-fast stains for , , and , this modification represents an improved approach to current staining methods. Specialized stains include Modified Kinyoun’s acid-fast stain (cold method), modified Ziehl-Neelsen acid-fast stain (hot method), carbol fuchsin negative stain for , rapid safranin method for , rapid safranin method for , using a microwave oven. The low cost of the reagents, the simple staining protocol, and the rapid microscopic examination make the method using auramine O stain suitable for screening unconcentrated fecal specimens. The detection of spp. and the microsporidia from stool specimens has depended on two separate stains. However, a method is now available that stains both organisms, an important improvement since dual infections have been demonstrated in AIDS patients This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods, and considerably reduces the time necessary for microscopic examination.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27

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Figures

Image of Figure 27.1
Figure 27.1

Method of scanning direct wet film preparation with a 10× objective. Note that the entire coverslip preparation should be examined before indicating the examination is negative. (Illustration by Nobuko Kitamura.)

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.2
Figure 27.2

Direct wet smear with saline. (Top row) trophozoite (left), cyst (right); (second row) sp. (probably ) (left), central body form (right); (third row) trophozoite (left), cyst (right); (fourth row) immature oocyst (left), cyst (right); (bottom row) cyst (left), cyst (right).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.3
Figure 27.3

Direct wet smear with saline and iodine. (Top) cyst with iodine; (middle) cyst with saline (left), cyst with iodine added (right); (bottom) cysts with iodine. Note that more detail can be seen once the iodine is added to the wet mount. Also, when iodine is used, the glycogen vacuole stains dark (brownish gold to brown) in the cysts and is clearly visible.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.4
Figure 27.4

Commercially prepared D’Antoni’s iodine; most commercial suppliers can provide this iodine solution.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.5
Figure 27.5

Fecal concentration procedures: various layers seen in tubes after centrifugation. (A) Formalin-ether (or ethyl acetate). The sediment should be well mixed, and a drop of sediment should be examined using the 10× low-power objective and the 40× high dry power objective. (B) Zinc sulfate (the surface film should be within 2 to 3 mm of the tube rim). Material from both the surface film and the sediment must be examined before the specimen is indicated as negative. Heavy or operculated helminth eggs do not float. (Illustration by Nobuko Kitamura.)

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.6
Figure 27.6

Sedimentation concentration. (Left) Unfertilized egg. (Right) egg.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.7
Figure 27.7

Flotation concentration. (Upper) egg (left), egg (right). Note that both of these eggs in the top row are operculated and do not float in the zinc sulfate flotation concentration method; the opercula pop open, and the eggs fill with fluid and sink to the bottom of the tube. (Lower) Hookworm egg (left), egg (right). These eggs concentrate using the flotation method and can be seen in the surface film. However, remember that both the surface film and the sediment must be examined by this method before reporting the final ova and parasite examination results.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.8
Figure 27.8

Method used to remove the surface film in the zinc sulfate flotation concentration procedure. (A) A wire loop is gently placed on (not under) the surface film. (B) The loop is then placed on a glass slide. (Illustration by Nobuko Kitamura.)

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.9
Figure 27.9

(Upper) FPC JUMBO large concentration tubes and connector system (Evergreen Scientific). (Lower) Small concentration tubes and FPC HYBRID connector system (Evergreen Scientific).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.10
Figure 27.10

Stool collection vial and funnel used in fecal concentration (Hardy Diagnostics).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.11
Figure 27.11

(Top) PARA-SED concentration system with both small and large tubes (Medical Chemical Corp.). (Middle) SED-CONNECT system with collection vials and various reagents (Medical Chemical Corp.). (Bottom) Filter attachment system (Medical Chemical Corp.). Note the screen, which is a substitute for the gauze in the traditional gauze/filter concentration method. See also MICRO-SED.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.12
Figure 27.12

(Upper left) MACRO-CON concentration system (Meridian Bioscience). (Upper right) SPINCON concentration system (Meridian Bioscience). (Lower) Funnel used in fecal concentration (Meridian Bioscience).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.13
Figure 27.13

Countertop workstation that automates the microscopic analysis of fecal concentrates (DiaSys Corp.).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.14
Figure 27.14

Dual-flow-cell Optical Slide Assembly (DiaSys Corp.) that fits into the stage clips of any standard upright microscope.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.15
Figure 27.15

Work flow diagram for fecal specimens. The total examination includes the direct mount, concentrate, and permanent stained smear (fresh specimen) or the concentrate and permanent stained smear (preserved specimens).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.16
Figure 27.16

Trichrome staining. Option 1, for use with smears prepared from fixatives containing mercuric chloride. The iodine is used to remove the mercuric chloride, and the subsequent two alcohol rinse steps remove the iodine. Thus, prior to staining, both the mercuric chloride and iodine have been removed from the smear. Options 2 and 3, for use with smears prepared from fixatives containing no mercuric chloride (the user can select option 2 or 3; there is minimal to no difference).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.17
Figure 27.17

Guess the identity of these intestinal protozoa! See p. 830 for the answers.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.18
Figure 27.18

Intestinal protozoa in the genus (stained with trichrome stain). Rows, from top to bottom, show trophozoites (note ingested RBCs), / trophozoites (the morphology does not allow differentiation between pathogenic and nonpathogenic ), / cysts, trophozoites (note uneven nuclear chromatin), and cysts (note the rough ends of chromatoidal bars and nuclear characteristics).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.19
Figure 27.19

Intestinal protozoa (stained with iron hematoxylin stain). (Upper) trophozoites (note the fragmented nuclei). (Lower left) trophozoite (note the large karyosome). (Lower right) cyst (note the large glycogen vacuole and “basket” nucleus).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.20
Figure 27.20

Iron hematoxylin stain incorporating the carbol fuchsin step. Note the modified acid-fast oocysts and the cyst.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.21
Figure 27.21

Work flow diagram for fecal specimens (coccidia). The most important part of the procedure would be the concentrate ( sp.) and the permanent stained smear, using one of the modified acid-fast techniques ( and spp.).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.22
Figure 27.22

Quality control slides for performing modified acid-fast stains ( and spp.) (Medical Chemical Corp.).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.23
Figure 27.23

stained with modified acid-fast stain. Note the range of color intensity; some oocysts resemble “wrinkled cellophane.”

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.24
Figure 27.24

(8 to 10 μm), (4 to 6 μm), and artifact (~2 μm) stained with modified acid-fast stain. Note that the oocyst did not stain (modified acid-fast variable) and has the “wrinkled-cellophane” appearance, while both the oocyst and the artifact did stain modified acid-fast positive. These artifacts are frequently seen; it is very important to measure objects seen in the modified acid-fast smears to confirm that they are actual parasites and not artifacts.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.25
Figure 27.25

(Upper) Immature oocyst stained with modified acid-fast stain; (lower) immature oocyst (left) (note that the entire oocyst retains the stain) and mature oocyst containing two sporocysts (right).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.26
Figure 27.26

(Top) Multiple microscopes with different attachments, including fluorescence (bottom right) (courtesy of Olympus America Inc.). (Middle) stained with auramine O fluorescent stain (courtesy of Thomas Hänscheid). (Bottom) spp. stained with auramine O fluorescent stain (left, 40× objective; right, 100× oil immersion objective) (courtesy of Thomas Hänscheid).

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.27
Figure 27.27

(Upper) Microsporidian spores in a stool specimen concentrate sediment stained with a modified trichrome stain. The spores range from about 1.5 to 2.0 μm in diameter. Note the horizontal lines, indicating the presence of a polar tubule. (Lower) Microsporidian spores from a nasopharyngeal aspirate stained with a modified trichrome stain. Note the presence of the polar tubule within several of the spores.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.28
Figure 27.28

Microsporidian spores in a urine sediment, stained with a modified trichrome stain. Note that some of the spores are intracellular. A urine specimen tends to be “cleaner” than stool; therefore the spores may be easier to see and identify in urine or any specimen that contains less artifact material than stool.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Image of Figure 27.29
Figure 27.29

spp. and microsporidian spores in an acid-fast trichrome stain. Note the size differential.

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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References

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Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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REVIEW Direct Smear

REVIEW Direct Smear

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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REVIEW Concentration

REVIEW Concentration

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Quantitation of Parasites, Cells, Yeasts, and Artifacts

Quantitation of Parasites, Cells, Yeasts, and Artifacts

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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Quantitation of Parasites, Cells, Yeasts, and Artifacts

Quantitation of Parasites, Cells, Yeasts, and Artifacts

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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REVIEW Permanent Staimed Smears

REVIEW Permanent Staimed Smears

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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REVIEW Modified Acid–Fast Smears

REVIEW Modified Acid–Fast Smears

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27
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REVIEW Modified Trichrome–Stained Smears

REVIEW Modified Trichrome–Stained Smears

Citation: Garcia L. 2007. Macroscopic and Microscopic Examination of Fecal Specimens, p 782-830. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch27

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