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Chapter 3 : Macroscopic and Microscopic Examination of Fecal Specimens

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Abstract:

  • Macroscopic Examination
  • Microscopic Examination (Ova and Parasite Examination)
    • Direct wet smear
    • Concentration (sedimentation and flotation)
    • Permanent stained smear
  • Specialized Stains for Coccidia (, [], and Species) and the Microsporidia
    • Modified Kinyoun's acid-fast stain (cold method)
    • Modified Ziehl-Neelsen acid-fast stain (hot method)
    • Carbol fuchsin negative stain for (from W. L. Current)
    • Rapid safranin method for
    • Rapid safranin method for , using a microwave oven
    • Auramine O stain for coccidia (from Thomas Hänscheid)
    • Modified trichrome stain for the microsporidia (Weber—green)
    • Modified trichrome stain for the microsporidia (Ryan—blue)
    • Modified trichrome stain for the microsporidia (Kokoskin—hot method)
    • Acid-fast trichrome stain for and the microsporidia

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Figures

Image of Figure 3.1
Figure 3.1

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.) Note: All methods contained in the figures can be found in . doi:10.1128/9781555819002.ch3.f1

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.2
Figure 3.2

Direct wet smear with saline. (Top row) () trophozoite (left), cyst (right); (second row) sp. (probably ) (left), spp. central body form (right); (third row) trophozoite (left), cyst (right); (fourth row) immature oocyst (left), cyst (right); (bottom row) cyst (left), cyst (right). doi:10.1128/9781555819002.ch3.f2

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.3
Figure 3.3

Direct wet smear with saline and iodine. (Top) cyst with saline (left), cyst with iodine (note chromatoidal bars with sharp ends) (right); (next row) egg in saline (left), egg with iodine added (right); (next row) cyst with saline (left), cyst with iodine (right); (bottom row) spp. in saline (left), spp. in iodine (right). 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. doi:10.1128/9781555819002.ch3.f3

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.4
Figure 3.4

Commercially prepared D'Antoni's iodine; most commercial suppliers can provide this iodine solution. Do NOT USE Gram's iodine for the parasitology procedures. doi:10.1128/9781555819002.ch3.f4

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.5
Figure 3.5

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. The amount of sediment should not be excessive in either the sedimentation or flotation procedure. Heavy or operculated helminth eggs do not float. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch3.f5

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.6
Figure 3.6

Sedimentation concentration. (Left) Unfertilized egg. (Right) egg. doi:10.1128/9781555819002.ch3.f6

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.7
Figure 3.7

Flotation concentration. (Upper) egg (left), egg (right). Note that both of these eggs in the top row are operculated and WILL 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. doi:10.1128/9781555819002.ch3.f7

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.8
Figure 3.8

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.) doi:10.1128/9781555819002.ch3.f8

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.9
Figure 3.9

(Upper) FPC JUMBO large concentration tubes and connector system (Evergreen Scientific). (Lower) Small concentration tubes and FPC HYBRID connector system (Evergreen Scientific). doi:10.1128/9781555819002.ch3.f9

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.10
Figure 3.10

Stool collection vial and funnel used in fecal concentration (Hardy Diagnostics). doi:10.1128/9781555819002.ch3.f10

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.11
Figure 3.11

(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. doi:10.1128/9781555819002.ch3.f11

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.12
Figure 3.12

(Upper left) MACRO-CON concentration system (Meridian Bioscience). (Upper right) SPINCON concentration system (Meridian Bioscience). (Lower) Funnel used in fecal concentration (Meridian Bioscience). doi:10.1128/9781555819002.ch3.f12

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.13
Figure 3.13

Countertop workstation that automates the microscopic analysis of fecal concentrates (Apacor Ltd. USA, Brooklyn, NY). doi:10.1128/9781555819002.ch3.f13

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.14
Figure 3.14

Dual-flow-cell Optical Slide Assembly (Apacor Ltd USA, Brooklyn, NY) that fits into the stage clips of any standard upright microscope. doi:10.1128/9781555819002.ch3.f14

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.15
Figure 3.15

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). doi:10.1128/9781555819002.ch3.f15

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Algorithm 3.1
Algorithm 3.1

Method for the preparation of TOTAL-FIX-preserved fecal specimens (Universal Fixative) for the concentration, permanent stained smear, fecal immunoassays, and special stains for the coccidia and microsporidia. (a) This fixative contains NO mercury, NO formalin, and NO polyvinyl alcohol (PVA); use one-third stool and two-thirds fixative and MIX WELL. Allow 30 min prior to processing. (b) The EIA and rapid cartridge detect antigen; sample the clear fluid at the top of the vial containing the stool/fixative mixture. If the vial is shaken, make sure it stands on the counter for 5 to 10 min to allow the particulate material to settle out; work with the clear fluid only. (c) TOTAL-FIX has been tested with a number of molecular methods; however, there is no confirmation that the specimen will work with every possible available method. (d) Rinse fluids (water, saline, formalin) will PREVENT permanent stained smears from staining correctly; rinse fluids can ONLY be used for the final concentration step (after original sediment has been removed for permanent stained slide preparations—STEP 2). (e) Since the direct fluorescent antibody immunoassay (DFA) detects the actual cysts (rare trophozoites—much less intense fluorescence) and oocysts, this test sensitivity is enhanced if centrifuged sediment is used for testing; the same advantage is seen using modified acid-fast stains for (use sediment). Remember, do not use RINSED sediment, but sediment from the original centrifugation. (f) Examine entire 22-by-22-mm coverslip at low power (10× objective); examine one-third to one-half coverslip at high dry power (40× objective).

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.16
Figure 3.16

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). doi:10.1128/9781555819002.ch3.f16

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.17
Figure 3.17

Guess the identity of these intestinal protozoa! See p. 76 for the answers. doi:10.1128/9781555819002.ch3.f17

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.18
Figure 3.18

Intestinal protozoa (stained with Wheatley trichrome stain). Rows, from top to bottom (left and right), show trophozoite and ; trophozoite (note the ingested RBCs) and cyst; () trophozoite and cyst; trophozoite and cyst; and trophozoite and cyst. doi:10.1128/9781555819002.ch3.f18

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.19
Figure 3.19

Intestinal protozoa (preserved with TOTAL-FIX, stained with Wheatley trichrome stain). From top to bottom and left to right. Top row: (), 2 trophozoites, 2 cysts. Second row: (), 3 trophozoites, 1 cyst. Third row: , 3 trophozoites, 1 cyst. Note the nuclear variation in the three trophozoites (very common). Fourth row: , 3 trophozoites and 1 cyst. Fifth row: (3 trophozoites) and (1 trophozoite). doi:10.1128/9781555819002.ch3.f19

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.20
Figure 3.20

Intestinal protozoa stained with iron hematoxylin stain. (Top row) trophozoite (left), () cysts (right); (middle row) cyst (note the large glycogen vacuole) (left), trophozoite (note ingested RBCs) (right); (bottom row) cyst (note more than five nuclei) (left), cyst (right). doi:10.1128/9781555819002.ch3.f20

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.21
Figure 3.21

Iron hematoxylin stain incorporating the carbol fuchsin step. Note the modified acid-fast oocysts (4–6 μm) and the cyst. doi:10.1128/9781555819002.ch3.f21

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.22
Figure 3.22

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 ). doi:10.1128/9781555819002.ch3.f22

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.23
Figure 3.23

Quality control slides for performing modified acid-fast stains ( and ) (Medical Chemical Corp.). doi:10.1128/9781555819002.ch3.f23

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.24
Figure 3.24

oocysts (8–10 μm). (Top) Autofluorescent oocysts; (bottom) oocysts stained with modified acid-fast stain. Note the range of color intensity; some oocysts resemble “wrinkled cellophane.” doi:10.1128/9781555819002.ch3.f24

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.25
Figure 3.25

(Left) (8 to 10 μm), (4 to 6 μm), and artifact (∼2 μm) stained with modified acid-fast stain. Note that one of the oocysts did not stain (modified acid-fast variable) and has the “wrinkled-cellophane” appearance, while the other oocyst displays the typical purple/red color; 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. (Right) stained with a hot safranin stain (higher magnification than the image on the left). (Courtesy of CDC.) doi:10.1128/9781555819002.ch3.f25

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.26
Figure 3.26

(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). doi:10.1128/9781555819002.ch3.f26

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.27
Figure 3.27

spp. oocysts stained with auramine O fluorescent stain (using different filters). doi:10.1128/9781555819002.ch3.f27

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.28
Figure 3.28

(Left) Microsporidian spores in a nasopharyngeal specimen stained with a Ryan modified trichrome stain; (right) microsporidian spores in stool stained with a Weber 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. doi:10.1128/9781555819002.ch3.f28

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.29
Figure 3.29

(Left) Microsporidian spores in a urine sediment, stained with calcofluor white stain; (right) spores stained with an organism-specific fluorescent antibody reagent. 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. doi:10.1128/9781555819002.ch3.f29

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Image of Figure 3.30
Figure 3.30

() oocyst and microsporidian spores in an acid-fast trichrome stain. Note the size differential. doi:10.1128/9781555819002.ch3.f30

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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References

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Tables

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Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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REVIEW Direct Smear

REVIEW Direct Smear

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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REVIEW Concentration

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Untitled

Permanent Stained Smears

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Untitled

Quantitation of parasites, cells, yeasts, and artifacts

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
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Modified Trichrome-Stained Smears

Modified Trichrome-Stained Smears

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
Generic image for table
Untitled

Quantitation of parasites, cells, yeasts, and artifacts

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3
Generic image for table
Untitled

Modified Acid-Fast Smears

Citation: Garcia L. 2016. Macroscopic and Microscopic Examination of Fecal Specimens, p 26-76. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch3

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