Faecal Egg Counting

(Modified McMaster Technique)

1.

Aim of Procedure:

Faecal egg count using the modified McMaster technique is currently the main measurement used by this laboratory for selection for parasite resistance.

2.

Materials:
Weighing square 60mL glass bottles trays and tray sheets faecal samples sorted into wooden trays small stainless steel spatula electronic balance (suitable to weigh 2g samples to 1 decimal place) paper towel drench gun and water cool room facility

Slide Preparation electric drill and bit for mixing samples plastic container for rinsing bit and pipette sieved pipette Whitlock McMaster slides or Whitlock Universal tissues perspex slide racks saturated salt solution compound microscope with x4 objective and x10 eyepiece. recording book, recording sheets, pen

3.

Method:

3.1 Weighing of Samples

The samples should be weighed, soaked and cooled as soon as possible after arrival (within 24 hours) to prevent further egg development. Tray sheets are placed in the trays used to hold the glass mixing bottles and are preprinted so that bottles sit on a square containing the number used to identify the sample. Individual bottles are not numbered only the tray is labeled. This position number reduces any bias that may be formed by counting on tag number. Weigh 2g (or 2.5g depending on individual lab) of faeces from each sample into the 60ml square glass bottle and replace each on their respective position. Begin at the bottom left of a tray and work left to right and toward the back. Place empty jars upside down on the position of any missing faecal samples.

 Continually cross-check the position numbers and tray sheet numbers when weighing to avoid errors. If there is insufficient faeces record on the worksheet the exact weight of faeces used. At completion of weighing each tray add 14ml of tap water and refrigerate at 4C, for at least 1/2 hour before counting to soften the faeces and prevent egg hatch. A sheep dosing gun attached to a large bottle is useful for adding water to a large number of samples.

3.2 Preparation of Salt Solution

Weigh 6.4kg of salt into a 20L plastic drench container. Add 15L of water (cold water if it is required immediately). Stir the mixture for 10 minutes with a propeller shaft attached to an electric drill. The water will reach saturation before all of the salt is dissolved. Test the specific gravity using a hydrometer. The specific gravity should be 1.2 15L of saturated salt is enough for approximately 400 faecal samples.

3.3 Adding Saturated Salt Solution

Samples that have been prepared with saturated salt solution should not sit for more than 30 minutes before counting as the salt may destroy the eggs. Ensure the faeces have been well mixed before adding saturated salt solution. Using a special bit on an electric drill (min. 2000rpm), mix the sample thoroughly, until the faeces is broken up and the particles are evenly distributed, (duration depends on the initial consistency but usually about 3-5sec/sample is sufficient). Rinse the bit between samples. Add salt solution up to the bottom of the lip (60mL). Add solution to the samples one row at a time (9 samples). This helps determine where you are up to on the tray. Samples should not be allowed to sit for too long in salt solution before counting, as the eggs may be destroyed.

3.4 Preparation of Slides

Using a sieved pipette stir the sample (in a N-S E-W motion NOT in a circular motion) before allowing the material to flow into the pipette. Softly breathe into the counting chambers of the slide (this wets the surface and allows the mixture to enter easily). Transfer the material into a chamber of a Whitlock McMaster slide or Whitlock Universal slide. Fill the chambers of the slide from right to left and with the 'verandah' of the slide facing away from the operator. Work from the bottom left of the tray across and up to the top right. Prepare the slides in rows - 5 rows to a tray and three slides per row. Place the slides from each row on separate slide racks (beginning at the front of the rack and working back) to enable easy identification of which slides are from which samples. Allow about 1 minute between preparation and counting for the eggs to float to the top of the slide.

3.5 Counting
See figure 1 below to identify different worm egg species Place the slide on a compound microscope (x10 ocular lens and x4 objective lens) stage with the verandah facing away from operator. Use the fine focus knob to bring the air bubbles into sharp focus. Begin counting from the top right of the chamber on the right side of the slide (this appears as the bottom left when viewed through the microscope shown in diagram)

chamber 1

chamber 2

chamber 3

chamber 4

Start

For each sample, count and record the numbers of nematode eggs seen. Record nematodirus eggs, coccidia and Tapeworm separately. Count all the eggs within the double line boundaries (8 lanes for the Whitlock McMaster slides and 5 for the Whitlock Universal slides). Normally only half of a slide is counted if many of the samples have high counts (greater than 50 eggs per half slide). This mainly occurs with H. contortus infections. In the recording book enter the position number of the first sample on each row and the counter and preparer of the slides Enter the egg count of each sample as it is completed. The counts should be transferred from rough recording sheets/books to the official counting sheet at least at the end of each tray, preferably at the end of each row. To avoid operator fatigue when counting large batches of samples, the counters and preparer should rotate duties after every tray. Before any tray of bottles is emptied and washed up, be certain that all of the counts for that tray have been entered on the counting sheets. It is critical that the slides be prepared in the correct order as the samples can only be identified by the number on the tray sheet.

PARASITE OOCYSTS AND EGGS ENCOUNTERED IN FAECAL EGG COUNTS AND THEIR RELATIVE SIZES
150 m

100 m

50 m

Coccidium

Strongyloides

Trichuris

Moniezia

Strongylid

Nematodirus

3.5 Calculations and Recording

The total number of eggs per gram of faeces is calculated using the following equation:
Number of eggs counted (60) total volume (mL) Number of eggs/ g faeces = (0.3) Volume of counted chamber (mL) (2) weight of faeces (g)

(Shortcut for conversion = Number of eggs actually counted x 100) (measurements shown in brackets relevant to CSIRO lab procedures, factors may need to be adjusted for different labs) Where less than 2g or 2.5g have been sampled, substitute actual weight into formula. For Nematodirus use the formula and record count separately. Coccidia record + (low) <10 per x100 field ++ (moderate) 10-30 per x100 field +++ (high) >30 per x100 field Tapeworm record + ++ +++ (low) <10 per x40 field (moderate) 10-30 per x40 field (high) >30 per x40 field

4. Key References
Dunn, A.M. (1969) Veterinary Helminthology. William Heinemann Medical Books Ltd. London. pp. 179, 276-278. Georgi, J.R. (1980) Parasitology for Veterinarians (3rd Ed.), WB Saunders Company, Philadelphia, pp. 179. NSW Agriculture (2000) Modified McMaster Procedure for Worm Egg Counts. Whitlock, H.V. (1948) Some modifications of the McMaster helminth egg counting technique and apparatus. J. Counc. Sci. Ind. Res., 21:177-180.