WS/T 634-2018Detection of intestinal protozoa - Iodine staining smear method (English PDF)

5.1 Reagents

Iodine solution (see Appendix A for preparation methods).

5.2 Materials

5.2.1 Fecal sampling box (clean, wide mouth, covered).

5.2.2 Slides.

5.2.3 Cover slips (18 mm x 18 mm).

5.2.4 Bamboo sticks.

5.2.5 scorpion (medical dentate, 12.5 cm, elbow).

5.2.6 Measuring cylinder (100 mL).

5.2.7 Volumetric flask (100 mL).

5.2.8 Brown reagent bottle (100 mL, covered, matte).

6.1 Sample preparation

Place 5 g to 10 g of fresh feces in the stool sample box, identify the number on the stool sample box, patient name, date of collection, sample the test should be carried out within 24 h. If it is not detected in time, it should be stored at 4 ° C for less than 72 h.

6.2 Sample testing

Near the center of the slide, drop 1 drop of the prepared iodine solution. Pick up the mung bean-sized stool with a bamboo stick, place it in iodine and mix well, and apply it to the diameter.

A uniform film of about 1 cm, the thickness of which is slightly identifiable through the faecal film. Covered with a cover slip, placed on a microscope Check below (see Appendix B).

6.3 Observation under the microscope

Use the objective lens 40× to observe and find the intestinal protozoan cysts or oocysts in order from left to right and top to bottom (see Appendix B and Appendix C).

6.4 Result determination

All the smears were observed to be positive for the cyst or oocyst, and no cyst or oocyst was found to be negative. Encapsulation or oocyst size, shape, Number of nuclei, shape and distribution of perinuclear staining plasmids, shape and location of nucleoli, presence or absence of fibril and fibril arrangement, glycogen block and chromosome the morphological characteristics determine the species (see Appendix D and Appendix E).

Appendix A

(informative appendix) Preparation of iodine solution A.1 formula Potassium iodide 4.0 g, chemically pure.

Iodine 2.0 g, chemically pure.

Distilled water 100 mL.

A.2 Preparation method Dissolve 4.0 g of potassium iodide in 30 mL of distilled water, then add 2.0 g of iodine. Dissolve and dilute to 100 mL with distilled water. Mix well.

Store in a brown reagent bottle with a stopper.

Appendix B

(informative appendix) Intestinal protozoan detection B.1 stool examination Near the center of the slide, drop 1 drop of prepared iodine solution, pick up the fresh mung bean size stool with bamboo stick, put it in iodine solution and mix well.

The manure film with a diameter of about 1 cm is relatively uniform. The thickness of the fecal membrane is slightly identifiable through the faecal film. Covered with coverslips, Check under the microscope. First use the objective lens 10 × to find the field of view, and then use the objective lens 40 × to observe, if necessary, use the objective lens 100 × to observe the capsule or the structural features of the oocysts were measured using a micrometer to measure the size of the cyst or oocyst. Find intestinal parasites in order from left to right and top to bottom or oocysts. All the smears were observed to be positive for the cyst or oocyst, and no protozoan cyst or oocyst was found to be negative. Encapsulation or oocyst Size, shape, number of nuclei, shape and distribution of perinuclear staining plasmids, shape and location of nucleoli, presence or absence of fibril and fibril arrangement, glycogen Morphological characteristics such as blocks and pseudochromosomes are used to determine the species.

In order to avoid the missed inspection, the feces should be collected the next day for more than 3 consecutive times. For some chronic patients, the fecal examination should last for 1 week to 3 weeks, multiple times.

Check to make sure there are no missed patients. The cysts or oocysts of common intestinal protozoa are more common in patients with chronic diarrhea and in forming feces, while trophozoites are more common.

For loose stools or feces with pus and blood, trophozoites are usually treated with direct saline smear.

The container for the feces should be clean and prevent contamination and drying, the feces should not be mixed with urine, etc., the number on the stool sample box, the patient Name, date of collection, and testing within 24 hours.

During the testing process, the staff should be safe to prevent the feces from polluting the environment. The utensils should be cleaned and thoroughly disinfected after each use.

Waste should be disposed of in accordance with medical waste disposal requirements.

Figure B.1 Smear preparation and viewing sequence diagram

B.2 Marsh's flagellate the larvae of the larvae of P. striata are pear-shaped or lemon-shaped, with a length of 6.5 µm to 10.0 µm. The wall is thin and has a nucleus. The organelle includes cells.

Cytostomal fibrils, moving substrates and axonemes. For M. larvae, fresh feces should be collected for iodine staining.

Color, observed under the objective lens 40× and the objective lens 100× to improve the chance of finding the capsule.

B.3 Isospora

The oocysts of the genus Bemis are small, the color is shallow, and the worms are easily lysed in vitro, which is easy to miss. Preserved with 2.5% potassium dichromate Fecal specimens can kill all bacteria, help detect oocysts, and preserve specimens for a long time.

B.4 Cryptosporidium

Cryptosporidium oocysts are round or elliptical and vary in size from 4 µm to 6 µm depending on the species. The wall of the capsule is smooth and colorless.

Contains 4 sporozoites. The sporozoites are crescent-shaped, 1.5 µm × 0.8 µm in size, and one in the nucleus, located in the posterior part of the worm. Oocyst contains residual body 1 , consisting of a pile of particles and a hollow bubble. Cryptosporidium oocysts have a high rate of missed detection in iodine smears, commonly used for acid-fast staining Detection.

In patients with acute cryptosporidium, the diarrhea feces contain more oocysts, which can be usually iodine staining smear. Such as checking with patients or livestock when the exposed population and the patients after the treatment are reviewed, the concentration method can be used to increase the detection rate. When it is difficult to judge, it is necessary to use the auramine-phenol staining and change.

Identification by special staining such as good acid resistance staining.

B.5 colon pouch ciliates the colonic ciliate cysts are round or oval, with a diameter of 40 µm to 60 µm and are pale yellow or light green. The wall of the capsule is thick and transparent.

The nuclei can be seen after staining. The newly formed capsule clearly shows the movement of the trophozoite in the capsule. The discharge of colonic pouch ciliate cysts is often intermittent.

Therefore, it is necessary to check repeatedly.

Appendix C

(informative appendix) Common intestinal protozoan cyst or oocyst morphology pattern

Figure C.1 Pattern of common intestinal protozoan cysts or oocysts

Note. This figure only shows the morphology of common intestinal protozoan cysts or oocysts.

Appendix D

(informative appendix) Common intestinal protozoa and their capsular or oocyst morphological characteristics D.1 Amoeba the amoeba in the parasitic human intestine mainly includes E. histolytica, E. histolytica, P. eleganide, Amoeba, and Amoeba.

B. sinensis, amiola, amoeba, diaspame, amoeba, etc. Encapsulation of amoeba in the tissue and other amoeba in the intestine the difference is shown in Table D.1. E. histolytica and dispana amoeba are indistinguishable in morphology. In.1997, the WHO Special Committee recommended microscopy.

Microscopically, the cysts containing quadruplexes should be identified as E. histolytica or Despana amoeba, and isozymes, ELISA and PCR can be further used.

Analyze and other methods for identification.

Table D.1 Identification characteristics of human amoeba cysts (iodine staining)

Insect species

Morphological characteristics Diameter/µm shape nuclear cytoplasm Amoeba 10~35 1~8 round, occasionally 8 or more, nuclear Membrane thickness, most of the nucleoli are biased Brown immature cysts, visible brown glycogen foam;

The tip is thin and irregular, fragmented, and has a strong refractive index and is white.

E. histolytica

10~20

1~4 circles, occasionally 8, visible core Renhe nuclear membrane, small nucleoli, centered, Thin nuclear membrane Brown immature cysts, visible brown glycogen, dark in the center, the edges are lightly colored and blurred; sometimes uncolored bars are visible Quasi-chromosome, obtuse at both ends Polecene amoeba 10~15 1 round, faintly visible, slightly larger nucleolus, Multiple deviation About half of the inclusion bodies have 1~2 inclusion bodies; the number of chromosomes is large and the shape Diverse and irregular Amoeba 5~10 1 to 4 ovals, visible nucleoli and nuclear membrane, Nucleolus accounts for a large proportion of nuclear membrane Slightly thick Brown immature cysts, brown glycogen vesicles;

Thin rod-shaped, both ends are like rice grains, the number is 4~6 at most.

Not colored, showing a refractive blank B. iodine amoeba 5~20 Irregular, 1 oval, occasionally 2, nuclear vague 1 dark brown, large, round, clear-edged glycogen Small medullary amoeba 5~10 elliptical or round 1~4, brown saccharide vesicles visible in nucleolar clear immature cysts D.2 Ciliate the cystic ciliate capsule of the colon is round or oval, with a diameter of 40 µm~60 µm. The wall of the capsule is thick and transparent, and the nucleus can be seen after staining. new the formed capsule can clearly see the trophozoite in the capsule, and the cilia on the surface are clearly visible, and then gradually disappear. Very few cysts are formed in the human body, so less see.

D.3 Flagellate

The flagellates in the parasitic human intestines are mainly Giardia lamblia Stile (also known as duodenum Giardia).

Larval flagellate, Trichomonas vaginalis, Trichomonas vaginalis and Trichomonas sinensis. The difference between the capsules of Giardia lamblia and other flagellates in the intestine See Table D.2.

Table D.2 Differences between Giardia lamblia and other flagellate cysts in the intestine

Insect species

Morphological characteristics Shape wall size/µm nuclear cell spiral groove axis flagella Blue Jad caterpillar Oval cyst wall thicker, cytoplasmic Contraction Significant gap (8~12) ×(7~10) 2 or 4 - - 2 cytoplasm can be seen in the middle body, Flagella, axial structure Mai's lip flag Pear shaped or Lemon shape Thinner, but the front end is quite thick, Dyeing visible front end has Papillary transparent bubble 6.5~10 1 have (dyeing See fiber branch Supported cell mouth) Yes - the cell mouth contains short flagella and both sides of the fiber the worm is a thick layer of double-layered (4~9) × (3~4), often located in the package.

Central sac

Have (stained

Show)

- - Self-nuclear edge visible inside the capsule Rigid cyst Two filaments stretched out Human intestinal trichomoniasis or Round Double layer (6~8) × (3~4) 1~4, most 2 or four, symmetric Distributed at both ends - - - The wall is clear, the flagella and Filaments are usually not visible Chinese trichomoniasis pear-shaped double layer 6 × 3 1 Show) - - The middle body is visible in the cytoplasm, Flagella, axial structure D.4 Blastocystis hominis the genus Bacillus is a round or oval shape with a size of 2 µm to.200 µm, with an average of 4 µm to 15 µm. The worm has obvious walls and the cytoplasm of the protozoa.

It is dyed yellow or light brown, the nucleus is dyed dark brown, there are 3~7 particles in the periphery, the size and shape of the particles change, and the nucleolus is visible.

Chromosomes, glycogen blocks, etc. There are five types of Blastocystis hominis.

D.4.1 vacuole type (also known as central bubble type) the worm body is round or oval, with a diameter of 2 µm~200 µm, with an average of 4 µm~15 µm. There is a large transparent vacuole in the center, and the surrounding cytoplasm A crescent-shaped thin band is formed, in which there are 2 to 4 nucleus in the nucleus, and a blocky heterochromatin is accumulated in the nuclear membrane.

D.4.2 Amoeba type (also known as deformation type) the size of the worm is 2.6 µm ~ 7.8 µm, the shape is variable, and the pseudopod protrusion is visible. The cytoplasm contains many small granular substances and no cell membrane.

D.4.3 Particle type

The worm is slightly larger than the vacuole type, and the cytoplasm is filled with granular substances.

D.4.4 Encapsulation type Round or oval, 3 µm~8 µm in diameter; wall thickness, 1~4 nucleus in cytoplasm, containing multiple vacuoles and glycogen and lipid deposition.

D.4.5 complex split type it is rare, its volume is the largest, and it has a proliferation phenomenon. The process of complex division. first, the nucleus is continuously split into multiple nuclei, between the nuclei and the nuclei.

Only a small amount of cytoplasmic connections, the remaining space is a vacuole structure. When the cell membrane is invaginated, it splits into a number of worms of varying sizes.

D.4.6 Other

In addition to the above five types, there are no vacuole type and multi-cavitation type. no vacuole type lacks central vacuole, small cell, diameter 5 µm, no Membrane. The multi-cavitating worm has a diameter of 5 µm~8 µm, and contains a plurality of vesicles of different contents and sizes, and the surface of the worm has a thick surface.

When cultured in vitro, the vacuolar-free and multi-cavitating worms disappeared and turned into vacuolar and granular worms.

D.5 sporozoan

The sporozoans in the parasitic human intestine mainly include Cryptosporidium, Coprinus comatus, Cyclospora, and human intestinal sarcocystis.

D.5.1 Cryptosporidium oocysts it is round or elliptical and has a size ranging from 4 µm to 6 µm. The wall of the capsule is smooth and colorless, and the capsule contains 4 sporozoites. Sporozoite crescent it has a size of 1.5 µm × 0.8 µm and a nucleus located at the back of the worm. The oocyst contains one residue, consisting of a pile of granules and a vacuole group.

to make.

D.5.2 Isospora capsular oocysts it is round or oblong, and its size is (20~33)µm×(10~19)µm. The immature oocyst contains one large, round cell. mature the oocyst contains two elliptical spores, each containing four half-moon sporozoites and one residue.

D.5.3 Cyclospora oocysts it is a non-refractive, glass-like sphere with a diameter of 8 µm ~ 10 µm. The fresh oocyst contains a pale green mulberry embryo with a diameter of 6 µm ~ 7 µm.

It contains 3 to 9 refractive particles with a diameter of 2 µm ~ 3 µm and a film surrounded by a hollow cluster. Mature oocyst contains 2 Sporangia. The sporangia are oval in shape and have a size of 4 µm × 6 µm. Each sporangium contains 2 sporozoites. Cyclospora oocyst size is an important basis for identification with Cryptosporidium.

D.5.4 Human intestinal sarcocyst oocysts it is spherical or short elliptical, the size is (15~19)µm×(15~20)µm, the wall of the capsule is thin, and there are 2 oval thin-walled transparent spores.

Ascospore, each sporangium contains 4 crescent-shaped sporozoites and residues. Due to the thin and fragile wall, it often ruptures in the intestine and enters the feces.

The sporangia are elliptical, colorless and transparent, and the size is (15~19)µm×(8~10)µm.

Appendix E

(informative appendix) Common impurities in the feces that are similar to the morphology of the cyst

Figure E.1 Common impurities in the feces similar to the morphology of the capsule

references

[1] Yu Senhai, Xu Longzhen. Color map of human parasitology [M]. Beijing. China Science and Technology Press, 1992. 1-15 [2] Zhang Jinshun, Happy Zheng. Clinical Parasitology Test [M]. Beijing. People's Medical Publishing House, 2009. 88-90 [3] Duan Yinong, Wang Zhongquan, Fang Qiang, et al. Modern Parasitology [M]. 2 Edition. Beijing. People's Military Medical Press, 2015. 223-225 [4] Zhou Xiaonong. Parasitic disease detection technology [M]. Beijing. People's Medical Publishing House, 2011. 5-9 [5] Wu Guanling. Human Parasitology [M]. 4 edition. Beijing. People's Medical Publishing House, 2013. 133-155 [6] Zhu Xinping, Su Chuan. Human Parasitology [M].8 Edition. Beijing. People's Medical Publishing House, 2013. 252 [7] Robert LS, Janovy J, Schmid GDFoundations of Parasitology[M].8th ed.Singapore. International Editions, 2009.350-363