WS/T 631-2018Detection of Schistosoma japonicum miracidium - Egg hatching method after nylon mesh bag concentration (English PDF)

4.1 Light incubator

Light intensity. 1 Lx ~ 5000 Lx

4.2 Microscope

Optical microscope. 40 × ~ 400 ×

4.3 Magnifier

Magnification. 10×

5.1 Hatching water

Use clean water with no protozoa, residual chlorine below 0.3 mg/L and pH 6.8-7.2, see Appendix A.

5.2 Nylon bag

57 µm/25.4 mm (260 mesh) nylon bag, see Appendix B.

5.3 Sieve

The pore size is 250 µm/25.4 mm (60 mesh).

5.4 Flask

250 mL or 500 mL triangular or long neck flasks.

5.5 Other

Tongue depressor or bamboo stick, clip, capillary straw, slide, 2% iodine, etc.

6.1 Sample preparation

Collect the fresh feces of the subject, weigh not less than 30 g, and store it in a closed container with a wide-mouth cover and anti-leakage cleansing.

This information (name, number, date of inspection) is marked outside the container. Samples collected should be submitted for inspection in time. If they are not detected in time, they should be Store under refrigeration at 4 ° C for no more than 24 h.

6.2 Sample testing

6.2.1 Acceptance registration

Check the weight, quality and label of the feces, check the number after registration, and register.

6.2.2 Water rinse

6.2.2.1 Lay the sieve on the nylon bag and clip the lower mouth of the nylon bag with a clip.

6.2.2.2 Take 30 g of the stool of the subject and place it in a sieve. Rinse with water, tongue depressor or bamboo stick to adjust the slurry, and filter the feces into the nylon bag.

6.2.2.3 Remove the sieve, continue to rinse the dung in the bag with water, and gently shake the nylon bag with a tongue depressor or bamboo stick to accelerate the filtration until it is filtered out.

The liquid becomes clear.

6.2.2.4 Move the lower mouth of the nylon bag into the mouth of the flask, remove the clip, rinse the filter residue in the bag with the incubation water into the flask, and continue to incubate.

Use water to fill 1 cm from the mouth of the bottle.

6.2.3 Butter hatching

Place the flask containing the filter residue in a light incubator (20 ° C ~ 30 ° C) or incubate at the same room temperature, at 3 600 Lx ~ 3 800 Lx light Under the intensity, hatching for 4 h to 12 h (range 0.5 h to 24 h), need to observe 2 to 3 times, see Appendix C.

For each batch of experiments, schistosomiasis-positive feces should be set as a control.

6.2.4 observation

Remove the flask, the bottle mouth is facing the light source, the backing is on a black background, and the plankton is observed in the range of 0 to 4 cm below the water surface.

The observation time is not less than 3 min. If necessary, use a magnifying glass to observe. After the hair is found, it is sucked out with a capillary pipette and evenly divided onto the glass slide.

Observe the swimming state, then add 1 drop of iodine to fix and observe the morphology under the microscope (40 × ~ 100 ×). Buttercup should be associated with protozoa in the water for identification, see Appendix D.

In order to improve the detection rate, such as negative hatching, the sediment can be examined for eggs.

6.3 Result determination

It was found that the sputum of Schistosoma japonicum was positive.

Appendix A

(informative appendix) Hatching water, utensils and waste disposal A.1 hatching water treatment A.1.1 Tap water treatment Put tap water in an open container for 24 h to allow it to naturally dechlorinate or add thiosulfur in tap water.

Sodium (4 mg/L to 8 mg/L) was used after standing for 30 min.

A.1.2 Well water treatment If the water quality is turbid, it can be left to clarify or add alum to clarify the water quality (30 mg/L ~ 40 mg/L). If it contains Protozoa in water, can be added to 65% effective chlorine bleach tablets (3.4 mg/L) or 30% effective chlorine bleach (7 mg/L) for insecticide, Then it was allowed to stand for 24 h for natural dechlorination or desulfation of sodium thiosulfate.

A.1.3 Determination of residual chlorine Take 100 mL of treated water in a Nessler colorimetric tube, add o-toluidine 5 mL and mix for 2 min to 3 min, and then use the residual chlorine standard tube.

Colorimetric determination. The residual chlorine concentration in the water that kills the protozoa in the water should be 0.7 mg/L to 1.0 mg/L, which is consistent with the residual chlorine concentration in the hatchery water.

At 0.3 mg/L.

A.1.4 pH adjustment

The hatching water needs to be tested by pH value. If the water is acidic, it can be adjusted with 10% sodium hydroxide solution; if the water quality is alkaline, it can be used.

Adjusted with 10% hydrochloric acid solution. Adjust to the optimum pH range.

A.2 Incubation treatment Wash the utensils after each use, soak them in hot water at 70 °C ~ 80 °C for 2 min ~ 3 min.

A.3 Waste treatment

Wastes such as containers for storing fecal samples, rinse water after washing, and dregs are treated according to WS 233.

Appendix B

(informative appendix) Nylon bag and sieve making Nylon enamel bag. It has a conical arc shape with an upper diameter of 80 mm, a lower mouth diameter of 15 mm and a depth of.200 mm. The pupil is 57 µm/25.4 mm (260 mesh). The schematic is shown in Figure B.1.

Sieve. Cylindrical with an outer diameter of 90 mm and an inner diameter of 75 mm. The mesh opening is 250 µm/25.4 mm (60 mesh). Schematic diagram B.1.

Figure B.1 Schematic diagram of nylon bag and sieve

Appendix C

(informative appendix) Buttercup incubation time it usually takes 2 to 3 times to observe the hatching of the buttercups, and the observation time varies with the temperature. When the temperature is high, the buttercups hatch quickly, and when the temperature is low, the buttercups are low.

Incubation is slow. 25 °C is the appropriate temperature for the hatchling to hatch.

When the temperature exceeds 30 °C, the first observation can be carried out after 0.5 h to 1 h, and the negative one can observe the second time after 4 h, and observe after 8 h.

The third time; when the temperature is between 26 °C and 29 °C, it can be observed at 4 h after hatching, and the negative ones are observed again for 8 h and 12 h;

When the temperature is between 20 °C and 25 °C, the first time can be observed after 8 hours, the second time after 12 hours, and the third time after 24 hours. Temperature is between 15 ° C and 19 ° C when the time is 12 hours, the first time can be observed, and after 24 hours, the second time can be observed. When the room temperature is above 25 °C, it can be incubated with natural conditions, no Need to warm up. See Table C.1.

Table C.1 Time for observing edulis at different temperatures

Temperature (°C)

Observing the donkey time (h) 1st 2nd 3rd 30~40 0.5~1.0 4 8 26~29 4 8 12 20~25 8 12 24 15~19 12 24 -Appendix D (informative appendix) Identification of protozoa between buttercup and water when observing the edulis, the flask should be directed toward the light source and lined with a black background. Identification under the microscope if necessary. Buttercup the identification points of protozoa in water are shown in Table D.1.

Table D.1 Identification of protozoa in buttercups and water

Identification points, edulis, protozoa the shape of the needle tip is large, the size is the same, slightly long and different in size, flat or round the color is transparent and shiny, with a discolored gray or grayish white, opaque, no refraction the swimming speed swims quickly, keeps going back and forth, moving slowly at a constant speed, stopping when swimming, and uneven speed the direction of the swimming is straight, the horizontal direction, the straight direction, and the straight forward are mostly curved, and there is no certain direction.

Swimming mode

Straight line, fold back after hitting the wall, fold back and straight Uniform forward Intermittent, wave, spiral, springboard and shake Pendulum the range of swimming is wide in the range of 0 to 4 cm below the water surface, and there are above, below and below the water.

references

[1] Wen Liyong. Handbook of schistosomiasis monitoring. Beijing. People's Medical Publishing House, 2014 [2] Wu Guanling. Human Parasitology. 4th Edition. Beijing. People's Medical Publishing House, 2013 [3] Zhou Xiaonong. The basic prevention and control series of human parasitic diseases. Beijing. People's Medical Publishing House, 2011 [4] Department of Disease Control, Ministry of Health. Handbook of Prevention and Control of Schistosomiasis. 3rd Edition. Shanghai. Shanghai Science and Technology Press, 2000 [5] WS 261-2006 Diagnostic Criteria for Schistosomiasis. Ministry of Health of the People's Republic of China.