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. If fatty acid crystals have been seen before heating and globules appear after heating, report as “fatty acids increased.”
Procedure for the Qualitative Test for Fecal Fat (Sudan Black IV)
1. Place a small aliquot of stool suspension on a clean glass slide.
2. Mix 2 drops of 95% ethanol with the suspension on the slide.
3. Add 2 drops of Sudan Black IV Stain to the suspension on the slide, and mix well.
4. Cover the suspension with a coverslip, and examine microscopically for the presence of large orange or red droplets.
Results and Patient Reports for the Qualitative Test for Fecal Fat (Sudan Black IV)
1. Fatty acids are present as lightly staining flakes or needle-like crystals that do not stain.
2. Soaps appear as nonstaining formless flakes, coarse crystals, or rounded masses.
3. Neutral fats appear as large orange or red droplets. If 60 or more stained droplets (neutral fats) are seen per 40× (high-power) field, it is a presumptive finding that the patient has steatorrhea.
Procedure Limitations for the Qualitative Test for Fecal Fat
1. The formation of large needle-like crystals as the preparation cools after heating does not necessarily mean that the original globules were fatty acid. Sudan III forms very short needle-like crystals in bunches as it dries.
2. Very few, if any, neutral fat globules are seen in a normal stool specimen. The presence of large amounts of neutral fat should raise suspicion that the patient has ingested mineral oil or castor oil, thus causing a false-positive result.
3. Do not count the fat that is present in vegetable cells.
Quantitation of Reducing Substances (Clinitest)
Clinitest is a reagent tablet test based on the classic Benedict’s copper sulfate reduction reaction, combining ingredients with an integral heat-generating system. Clinitest provides clinically useful information about carbohydrate metabolism by determining the amount of reducing substance in urine or stool. Reducing substances convert the cupric sulfate (CuSO4) to cuprous (Cu2O) oxide and cause a change in solution color ranging from blue through green to orange (Fig. 4.11). Unpreserved stool is required; the specimen should be placed in the refrigerator if there is a delay in testing. Specimens collected more than 48 h earlier or specimens that are dried out should be discarded, as fresh specimens should be collected. Although this is a relatively old method, there are references in the literature (34).
Figure 4.11 Color range for stool Clinitest reactions: blue (negative) to orange (positive). doi:10.1128/9781555819002.ch4.f11
Clinitest Tablets
Clinitest reagent tablets (store tablets at room temperature in a plastic bag)
Comparative color chart that comes with the tablets
Deionized water
Chek-Stix positive control
Quality Control for Quantitation of Reducing Substances (Clinitest)
1.
