Lynne Shore Garcia

Diagnostic Medical Parasitology

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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.

      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. 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, Chek-Stix is used; the development of a green, yellow, or orange color with a yellow or red precipitate is considered a positive result.

      4. As a negative control, 0.5 ml of deionized water is used; blue color is considered a negative result.

      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 Quantitation of Reducing Substances (Clinitest)

      1. All testing on clinical specimens should be performed in a biological safety cabinet by personnel wearing gloves and a laboratory coat.

      2. Add 1 volume of stool to 2 volumes of deionized water, and mix thoroughly.

      3. Using a disposable transfer pipette, transfer 15 drops of this suspension into a clean test tube.

      4. Drop one Clinitest tablet reagent into the test tube.

      5. Observe the reaction. Do not shake the tube while the chemical reaction is occurring.

      6. Wait 15 s after the reaction stops, then gently shake contents to mix.

      7. Compare the color of the liquid to the color chart in the package insert of the Clinitest tablet reagent (Fig. 4.11).

      8. Discard supplies in appropriate biohazard containers.

      Results and Patient Reports for Quantitation of Reducing Substances (Clinitest)

      1. Negative: clear to cloudy blue color

      2. Positive: compare the liquid color to the color chart that comes with the tablets, and grade the degree of color development to the color chart (trace, 1+, 2+, 3+, or 4+). These results equate to the grams per deciliter of the reducing substance present per sample. The colors range from blue through green through yellow/orange to orange (negative to 4+).

      3. Positive: report as Trace (0.25 g/dl), 1+ (0.5 g/dl), 2+ (0.75 g/dl), 3+ (1.0 g/dl), or 4+ (equal to or greater than 2 g/dl)

      Example: 1+ (0.5 g/dl)

      4. Negative

      Example: Negative

      Procedure Limitations for Quantitation of Reducing Substances (Clinitest)

      1. Clinitest is not specific for glucose and reacts with any reducing substance in stool, including lactose, fructose, galactose, and pentoses.

      2. Interfering substances may affect the results. These include salicylates, penicillin, large quantities of ascorbic acid, nalidixic acid, and cephalosporins.

      3. Failure to observe the reaction at all times may lead to erroneously low results if reducing substances are present in large amounts. If more than 2% sugar is present, a rapid color change may occur during boiling, causing the color to pass rapidly through bright orange to a dark brown or greenish brown.

      References

      2. Garcia LS (ed). 2010. Clinical Microbiology Procedures Handbook, 3rd ed. ASM Press, Washington, DC.

      3. Isenberg HD (ed). 2004. Clinical Microbiology Procedures Handbook, 2nd ed. ASM Press, Washington, DC.

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