2. Rinse the proglottid with tap water, and blot it dry on paper towels.
3. To prepare for the injection, hold the proglottid between your thumb and first finger or on the surface of the counter (harder to do).
5. Very carefully, penetrate either the central uterine stem at the end where the proglottid has become unattached from the chain or penetrate the side uterine pore with the end of the needle.
6. GENTLY inject some India ink; it should penetrate the uterine branches very quickly. IF THE INJECTION DOES NOT IMMEDIATELY OCCUR, DO NOT FORCE THE INJECTION.
7. You can try the other entry location, again GENTLY injecting the ink.
8. After the injection, rinse the gravid proglottid with tap water, blot dry on paper towels, and place the proglottid between two microscope slides separated at the edges by thin pieces of cardboard.
9. Fasten the preparation by placing rubber bands at each end of the slides so that the segments become somewhat flattened.
10. Observe the number of uterine branches on ONE SIDE ONLY where they come off the main uterine stem; generally Taenia saginata has 12 or more branches, while Taenia solium has approximately 8 or fewer branches.
11. After the identification has been made, the proglottid can be left between the two slides (place a rubber band around the slides), dehydrated through several changes of ethyl alcohol (50, 70, 90, and 100%), cleared in two changes of xylene, and mounted with Permount for a permanent record.
12. After the xylene step, the proglottid will be stuck to one of the two slides; do not try to remove it (it is very brittle and will crack) but merely add the Permount to the proglottid and then add the coverglass.
Results and Patient Report from the India Ink Injection Procedure
Tapeworm proglottids may be recovered (either singly or several attached together).
1. Report as:
Example: Taenia saginata gravid proglottid present
Procedure Notes for the India Ink Injection Procedure
1. Remember that T. solium eggs are infective (cysticercosis). The identification to the species level may not be possible until after the ink injection is completed.
2. Wear mask, laboratory coat, and gloves when performing this procedure.
3. Fresh, unpreserved proglottids are recommended; preserved proglottids can be used, but they are harder to inject.
4. Make sure the proglottid is blotted dry prior to the injection; otherwise, it may be difficult to hold onto the proglottid—it can slip through your fingers.
5. DO NOT FORCE THE INK INJECTION. If the ink does not easily flow into the branches, it may be because the proglottid is not gravid, but only mature. Also, the needle may not be in the right place.
6. IF THE INK INJECTION IS FORCED, it is more likely you may create an aerosol, thus increasing the danger for exposure to viable T. solium eggs.
7. Remember that after the xylene step, the proglottid will be stuck to one of the two slides; do not try to remove it (it is very brittle and will crack) but merely add the Permount to the proglottid and then add the coverglass.
Procedure Limitations for the India Ink Injection Procedure
1. If the proglottid is fresh, it may have begun to disintegrate, thus making the ink injection more difficult.
2. If the proglottid is mature AND NOT YET GRAVID, the branches may not inject properly.
3. If the ink injection is not successful, one can try some of the mounting methods found in chapter 9.
Qualitative Test for Fecal Fat
The microscopic examination of stool with the addition of Sudan III is a very simple, quick, and widely used technique to screen the specimen for fat. Fresh, unpreserved fecal material is required. If there is a time delay prior to testing, the specimen should be refrigerated. Specimens collected more than 48 h earlier or specimens that are dried out should be discarded, and fresh specimens should be collected. Although this technique is quite old and the original paper may be difficult to obtain, it was published in 1961 (32).
Sudan Black IV Stain, also known as scarlet red, was introduced by Michaelis in 1901 as a fat stain. It is a dimethyl derivative of Sudan III, which makes it a deeper and more intense stain, but it has similar physical properties and is fat soluble. This stain has been widely used as a screening method because it is easy to use and correlates well with quantitative methods.
Sudan Black IV Stain is used as a qualitative method to detect the presence of fecal fat. Normally the stool does not contain more than 20 g of fat daily. In patients with steatorrhea, fat malabsorption occurs, and greater quantities of fat are detected in the stool. This procedure, when performed carefully and consistently, is a simple method of detecting this condition in the patient (33). Other dyes such as Sudan II, Sudan III, Oil Red O, and Sudan Black B can be used as well.
Sudan Stain
Quality Control for the Qualitative Test for Fecal Fat
1. Follow routine procedures for optimal collection and handling of fresh fecal specimens for parasitology.
2. Run a QC sample with each batch of patient tests as in procedure below.
3. As a positive control, mayonnaise is used; red-stained fat globules should be observed microscopically.
4. As a negative control, water is used; no fat globules are observed.
5. Record all QC results. If the QC results are unacceptable, the test must be repeated and documented on the corrective action sheet.
Procedure for the Qualitative Test for Fecal Fat (Sudan III)
1. Mix a small amount of stool with an equal amount of 95% ethanol in a test tube. Mix well.
2. Add 2 drops of Sudan III stain to the stool-ethanol mixture. Mix well, and let stand for a few minutes.
3. Using a pipette, remove a drop from the test tube and place it on a slide. Cover with a coverslip.
4. Using the microscope, examine the slide for globules of fat stained red (neutral fat) and needle-like crystals (fatty acid).
5. Add several drops of glacial acetic acid to the test tube. Remove a drop from the test tube and place it on a slide. Cover with a coverslip.
A. Gently heat the slide over a flame. (Do not boil.)
B. Observe again on the microscope for globules of fat stained red (Fig. 4.10).
Figure 4.10 Fat droplets seen in the fecal fat test. The color can range from red to orange (pale to more intense), and the droplets can vary in size. doi:10.1128/9781555819002.ch4.f10
Results and Patient Reports for the Qualitative Test for Fecal Fat (Sudan III)
Red-stained fat globules or fatty acid crystals are seen microscopically. Report the specimen as “fat not increased” or “fat increased” depending on the number of globules seen.
1. If fewer than 100 globules/high-power field (hpf) are seen before or after heating, report as “fat not increased.”
2. If more than 100 globules/hpf of fat are seen before or after heating, report as “fat increased.”
3.