Example: Strongyloides stercoralis larvae detected by agar plate culture.
Procedure Notes for Agar Plate Culture for Strongyloides stercoralis
1. If the larvae are too difficult to observe under the microscope and morphologic details are difficult to see, the larvae can be formalin killed within the plate and examined in the formalin-concentrated sediment.
2. Infective larvae may be found any time after the first or second day or even on the first day in a heavy infection. Since infective larvae may be present on the agar, caution must be exercised in handling the plates once the cellulose tape is removed. Wear gloves when handling the cultures.
3. It is important to maintain the plates upright at room temperature. Do not incubate or refrigerate them at any time; this also applies to the fresh stool specimen.
4. Fresh stool is required for this procedure; preserved fecal specimens or specimens obtained after a barium meal are not suitable.
Procedure Limitations for Agar Plate Culture for Strongyloides stercoralis
1. The agar plate culture technique is successful if any larvae present are viable. If the fresh stool specimen is too old, larvae may not survive and a negative result will be reported.
2. Specimens that have been refrigerated or preserved are not suitable for culture. Larvae of certain species are susceptible to cold environments.
Estimation of Worm Burdens and Kato-Katz Thick Film
The only human parasites for which it is reasonably possible to correlate egg production with adult worm burdens are Ascaris lumbricoides, Trichuris trichiura, and the hookworms (Necator americanus and Ancylostoma duodenale). The specific instances in which information on approximate worm burdens is useful are when one is determining the intensity of infection, deciding on possible chemotherapy, and evaluating the efficacy of the drugs administered. With current therapy, the need for monitoring therapy through egg counts is no longer as relevant. However, several methods that can be used if necessary are discussed below. Remember that egg counts are estimates; you will obtain count variations regardless of how carefully you follow the procedure. If two or more fecal specimens are being compared, it is best to have the same individual perform the technique on both samples and to do multiple counts.
Direct-Smear Method of Beaver
The direct-smear method of Beaver is the easiest to use and is reasonably accurate when performed by an experienced technologist. In the original method, Beaver (26) used a calibrated photoelectric cell to prepare a direct smear of exactly 2 mg. For routine purposes, this is impractical, and the procedure has subsequently been modified (27) such that a direct smear of 2 mg (enough fresh fecal material to form a low cone on the end of a wooden applicator stick) of stool is prepared. Egg counts on the direct smear are reported as eggs per smear, and the appropriate calculations can be made to determine the number of eggs per gram of stool.
Dilution Egg Count
The Stoll count (28) is probably the most widely used dilution egg-counting procedure for the purpose of estimating worm burdens. However, because of cost containment and clinical relevance (therapy is often initiated with no egg count data), most laboratories do not offer this procedure.
Stool displacement flasks for use in this procedure are available commercially. These flasks have a long neck with etched lines at 56 and 60 ml to facilitate proper filling with sodium hydroxide and fecal material. If commercial Stoll flasks are unavailable, any flask that can hold the sodium hydroxide solution, a weighed amount of 4 g of stool, and a few small glass beads can be used for a container.
1. In a calibrated Stoll flask, add 0.1 N sodium hydroxide to the 56-ml mark.
2. Add fresh fecal material to the flask so that the level of fluid rises to the 60-ml mark. This amount of feces is equivalent to 4 g of feces.
3. Add a few glass beads, and shake vigorously to make a uniform suspension. If the specimen is hard, the mixture may be placed in a refrigerator overnight before shaking to aid in mixing.
4. With a calibrated pipette, quickly remove 0.15 ml of suspension and transfer it to a slide.
5. Do not use a coverslip; place the slide on a mechanical stage, and count all of the eggs.
6. Multiply the egg count by 100 to obtain the number of eggs per gram of stool.
7. The estimate (eggs per gram) obtained will vary according to the consistency of the stool. The following correction factors should be used to convert the estimate to a formed-stool basis:
Egg counts on liquid specimens are generally unreliable; the most accurate counts are obtained with use of formed or semiformed specimens.
Modified Stoll Dilution Method
1. Fill a 15-ml centrifuge tube to the 14-ml mark with 0.1 N sodium hydroxide.
2. Add stool to bring the liquid contents up to the 15-ml mark.
3. Mix thoroughly with a wooden applicator stick.
4. If the stool is hard, allow the mixture to stand for several hours.
5. Shake to thoroughly mix, and quickly withdraw exactly 0.15 ml from the middle of the suspension.
6. Transfer the material to a slide, cover (it may be easier to count without a coverslip, since occasionally the eggs flow to the outside of the coverslip), and count the eggs in the entire preparation.
7. Multiply the egg count per preparation by 100 to give an uncorrected count of eggs per milliliter. Corrections may be made as in the Stoll dilution method for original stool consistency.
Kato-Katz Thick Smear
Although this method of counting helminth eggs has been modified many times since it was originally published, the protocol seen below provides an acceptable approach (29). Currently, the Kato-Katz is the recommended method of WHO, with the McMaster method being considered an alternative for the quantification of soil-transmitted helminths eggs in human stool (primarily Ascaris, Trichuris, hookworm, Schistosoma mansoni) (35). Although this thick film method has been adapted to estimate worm burden calculations, it is rarely used in routine clinical laboratories. Also, it is unsuitable for the diagnosis of protozoa or helminth larvae and is difficult to perform on hard and/or liquid stools.
1. Coverslips made of wettable cellophane, medium thickness, cut into 22 × 30 mm rectangles (No. 124 PD, E.I. DuPont de Nemours, Inc., Film Department, Wilmington, DE).
2. Glycerine-malachite green solution: 10 ml pure glycerine and 1 ml 3% aqueous malachite green
