to be 22 and in a diarrheic stool, 12. This yields a ratio of 1:2:4 (formed:mushy:diarrheic). It is best to convert all counts to a "formed stool" basis by multiplying the count obtained from mushy stools by 2 and from liquid stools by 4.

INTERPRETATION OF RESULTS

Egg counts are applicable chiefly to infections with nematodes such as Ascaris, hookworm, and Trichuris, where the eggs are passed more or less continuously. Quantitative egg outputs have been used primarily in estimating hookworm burdens, particularly N. americanus burdens, and the procedure was originally developed for use in determining densities of infections with this worm in population groups. The interpretation of results, therefore, usually relates to hookworm, but it could be applied to other infections in which the egg output per gram of feces is known.

The number of N. americanus worms necessary to produce clinical symptoms varies from person to person and depends on several factors. As stated, a female Necator will shed an average of 44 eggs per gram of formed feces. Thus, a technique which can determine the number of eggs in a gram of feces in a given sample enables one to calculate the number of female worms present. Since males and females are usually found in almost equal numbers, doubling the original calculation yields the approximate total worm infection. Various workers consider that when there is a burden of 100 or more hookworms, clinical symptoms are likely to occur. This intensity level is represented by 2,600 or more eggs per ml of feces and is considered a moderate infection. In their hookworm surveys, Keller et al. (1934, 1940) used the following classification:

For routine purposes, it is usually sufficient to indicate the clinical classification based on eggs per ml of feces. The number of worms present need not be calculated.

STANDARD SMEAR EGG COUNTING TECHNIQUE

The direct smear technique for egg counting involves using a light meter or photo-electric foot-candle meter in preparing a fecal smear of a known standard density.

APPARATUS (fig. 28)

1. Light meter. The meter used should have the window on the same side as the scale.

2. A lamp, adjustable in height, suspended directly over the window of the meter. Either a 75- or 100-watt bulb may be used. 3. Block about 15-mm thick, 2 to 3 inches long, and with a central hole 16 mm in diameter. The inner surface of the hole may be covered with black ink to reduce light reflection and interference. The block is placed over the meter window to reduce the opening and to provide a uniform light area.

The apparatus can be standardized or calibrated by using barium sulfate suspensions as standards.

Figure 28.

Lamp and Light Meter Apparatus for Standard Smear Egg Count

Lamp

Ring Stand with Clamp

Wooden Block

Light Meter

PREPARATION OF SOLUTIONS

Stock Solutions

Solutions of 2N sodium sulfate (Na2 SO4) and 1 N barium chloride (BaCl2) are prepared by analytical methods.

In a 1-liter volumetric flask, dissolve the Na2 SO4 in a part of the water, then add water to the 1-liter level.

2. BaCl2 · H2O

Distilled water to make

Prepare as for Na2 SO4 solution.

122.16 gm

1000.0 ml

3. Prepare salt-glycerin mixtures by mixing (separately) 2 parts of each solution with 1 part of pure glycerin. The salt-glycerin mixtures are stored separately and mixed in varying proportions to give the desired concentration standard.

Standards

The barium chloride solution is added to the sodium sulfate solution slowly, drop by drop, with constant stirring. The proportions used for various standards are as follows:

CALIBRATION OF LIGHT METER

1. Adjust the light to a predetermined reading on the scale (Beaver used 20 foot-candles as a setting).

2. Place a clean 3x1-inch slide over the opening of the block covering the light meter window.

3. Using a pipette which delivers 0.05 ml per drop (a regular medicine dropper is usually satisfactory), place a drop of the thoroughly mixed barium sulfate suspension in the desired concentration on the slide over the block opening. The 1/1,000 (1 mgm) or 1/500 (2 mgm) standards are preferred for routine purposes.

4. With an applicator stick, carefully spread the suspension to exactly fit the opening. If the standard extends beyond this area, the slide should be discarded and a new one prepared. 5. Read the scale. Subsequent fecal preparations should be of sufficient density to cause the same light reduction.

6. With some meter scales, it may be easier to adjust the light so that a known, easily read scale reading is the end point. The main considerations are to determine the reduction in light and intensity with the desired standard concentration and to prepare fecal mounts of the same density.

7. After adjusting the light and calibrating the apparatus, the position should not be changed. Periodic checkings are necessary, however, to be sure that current fluctuations or jarring have not affected the adjustments. In some cases, a voltage regulator is desirable.

PREPARATION OF THE FECAL SMEAR

A 1-mgm or 2-mgm fecal suspension is usually easier to examine than thicker mounts, and the light meter should be calibrated with the corresponding standard (1/1,000 or 1/500).

1. Using a pipette which delivers a 0.05-ml drop (medicine dropper), place a drop of distilled water on a clean slide directly over the block opening.

2. With an applicator stick, emulsify feces in the water, spreading the film to fit the block opening exactly, until the reading on the scale is the same as that of the corresponding standard. If the preparation extends beyond this area or too much feces is added, it should be discarded and a new slide prepared.

3. Cover with a 22-mm square coverslip and, beginning with one corner, systematically count the number of eggs in the preparation. Do not seal the mount, since the paraffin may obscure some eggs.

4. Use of a 5X ocular will decrease the examination time. Be careful not to overlap fields and recount eggs already counted. 5. To obtain eggs-per-gram count, multiply the number of eggs counted by 1,000 if a 1-mgm preparation is used or by 500 if a 2-mgm preparation is used. No correction factors for soft or loose stools, as required by the Stoll count, are necessary. If no eggs are found in the 2-mgm preparation, report “less than 500 eggs per gram." A 3-mgm (or heavier) preparation is too thick to count the eggs accurately.

6. If several counts are to be made, the meter should be checked with the standard at intervals to make sure the readings have not altered. This is especially important if the flow of electric current fluctuates. A voltage regulator may be used if the current fluctuation is pronounced.

USE OF NONSTANDARDIZED SMEAR FOR EGG COUNTS

If no light meter is available, a rough estimate of the worm burden can be made from ordinary direct wet mounts prepared for routine examination. Experienced technologists usually prepare smears containing 1.5- to 2-mgm of feces. For practical purposes, the number of eggs per gram can be estimated by using 750 for a multiplication factor in step 5. Some laboratories report simply "eggs-perpreparation" without attempting to convert the count to eggs-pergram. This is satisfactory so long as the person receiving the information can interpret the results. For example, 1 or 2 eggs per smear may represent a very light infection while 15 or 20 may represent a heavy infection.

INTERPRETATION OF RESULTS

Egg counts are interpreted in the same manner as with the Stoll dilution count. In both cases, the counts are estimates and provide an estimate of the existing worm burden rather than a specific count.

•KATO THICK-SMEAR TECHNIQUE FOR QUANTITATIVE DETERMINATIONS

The Kato thick-smear technique is a simple quantitative method based on direct examination of feces (Martin and Beaver, 1968). The sample size is about 25 times greater than that used in the