WO2007068951A1

WO2007068951A1 - An improved method of preparing a biological specimen for examination

Info

Publication number: WO2007068951A1
Application number: PCT/GB2006/004716
Authority: WO
Inventors: Richard Banfield Hicks
Original assignee: Diasys Europe Ltd
Current assignee: Diasys Europe Ltd
Priority date: 2005-12-16
Filing date: 2006-12-15
Publication date: 2007-06-21
Anticipated expiration: 2008-06-16
Also published as: GB0525591D0; GB2433219A

Abstract

A measured quantity of sheep's faeces is placed in a mixing vessel (10) together with a saturated salt solution. The faeces are broken up and the mixing vessel is agitated to produced a substantially homogeneous solution. The mixing vessel (10) is stoppered by a closure (13) through which a tubular filter (14) extends into the solution so that a proportion of the solution enters the filter. When the assembly is now inverted only the filtrate within the filter is decanted. The solution external to the filter remains in the mixing vessel by the creation of sub-atmospheric pressure above it.

Description

"AN IMPROVED METHOD OF PREPARING A BIOLOGICAL SPECIMEN FOR EXAMINATION"

This invention relates to an improved method of preparing a biological specimen for examination and particularly, but not exclusively, to a method of preparing a faecal specimen for an examination to determine the proportion of parasitic eggs in the faeces, particularly but not exclusively the faeces of domesticated animals such as sheep.

Gastrointestinal parasites impair the growth of grazing ruminants, reducing food intake and the efficiency of feed utilisation. Sub-clinical disease can lower the quality of the carcass and reduce milk production.

During the 1950s and early 1960s much research was undertaken to reduce epidemics of gastrointestinal parasites in grazing ruminants, sheep being the primary focus. This involved the creation of 'safe' pasture, land that alternates between the growing of arable crops and the grazing of cattle. The system 'broke' the life cycle of the faecal parasite by removing the parasite host but put severe constraints on the use of the land.

The advent of highly effective broad-spectrum drugs in the early 1960s radically changed the approach to parasitic control. Frequent treatments provided an effective and relatively cheap solution.

By the 1980s it became evident that certain parasites were developing resistance to these drugs and that a policy of parasitic control was the only long-term solution. Veterinarians worldwide are now encouraging farmers to adopt parasitic control measures to maintain the viability of the sheep industry.

The principle of control is to maintain acceptable levels of parasitic infection in animals rather than adopting a policy of total elimination. Drug use is reduced with little or no detrimental effect to the animal or its value. Control is achieved by measurement i.e. the counting of faecal worm eggs in a known weight of faeces. McMaster egg counting techniques are used worldwide and were developed extensively by the Central Veterinary Laboratory in the UK. They are the techniques of choice for faecal worm egg counting in Veterinary Parasitology.

The technique makes use of the fact that eggs float and separate from faecal matter in saturated solutions of various salts (NaCl, MgSO4, and ZnSO4). Helminth eggs from a 45ml faecal suspension (3g of faeces and 42ml of saturated salt solution) are counted in a McMaster counting chamber. The volume of the counting chamber grid (0.15ml which equates to 1/10Og of faeces) in each of the two chambers of the slide allows eggs per gram of faeces to be determined (one egg counted = 100 eggs per gram of faeces).

Three grams of faeces and 42ml of water are traditionally used to make the faecal suspension rather than Ig and 14ml. This is to minimise sampling error arising from the fact that eggs are not evenly distributed through the faeces.

The existing protocol has the following steps: -

I) Weigh 3 g sheep faeces. 2) Break up with spatula.

3) Tip into wide mouth jar containing 45 glass beads. (Wash jar and beads between each test)

4) Add 42ml of water. Secure lid.

5) Shake vigorously for 30s. The agitated glass beads break down the sample. 6) Pour contents through a 150 micron stainless woven mesh into a collection bowl. (Wash mesh and bowl between each test)

7) Aliquot 2 x 15ml volumes from bowl into 2 x 15ml tubes.

8) Centrifuge tubes to compact faecal matter.

9) Pour off contents. 10) Replace with saturated salt solution.

I 1) Vortex / shake tubes to re suspend faecal matter. 12) Pipette suspension from each tube. Fill each chamber of the McMaster slide from a different tube.

To minimise the variation in results the method described requires manual dexterity and training and an understanding of the physical principles involved. Careful cleaning of the equipment is required to prevent cross contamination between samples.

The standard McMaster procedure relies on the practised skill of the lab technician to pour the faecal suspension from a large receptacle into a relatively small tube. This procedure requires a funnel; it is an unpleasant task and exposes the technician to a potentially dangerous aerosol.

The principal object of the present invention is to provide a simplified procedure offering numerous benefits over the techniques currently employed.

In accordance with one aspect of the invention there is provided a method of preparing a biological specimen for examination, comprising mixing a biological sample with liquid in a mixing vessel, agitating the mixing vessel, closing the mixing vessel with a closure which is penetrated by a hollow filter extending into the mixing vessel, the filter having a closed end remote from the closure and opening through the closure, so that at least a portion of the length of the filter is submerged in the solution in the mixing vessel, permitting a proportion of the solution to enter the filter and inverting the mixing vessel to decant said proportion from the mixing vessel without permitting the escape of solution external to the filter within the mixing vessel.

Preferably a collection vessel is fitted to the open end of the filter to provide a closed system and after decanting filtrate into the collection vessel the latter is removed from the filter and mixing vessel and filtrate is pipetted from the collection vessel to the chambers of a McMaster slide. Preferably particles of a material harder than the biological sample are included in the mixing vessel to assist in breaking up the sample when the mixing vessel is agitated.

The particles may be glass beads or more preferably the particles are polymer granules with a density greater than 1.5.

Preferably the tubular filter is of the kind described and illustrated in the Specification of U.S. Patent No. 6,296,763.

Preferably the liquid comprises a saturated salt solution. It may also comprise a surfactant or detergent.

In accordance with another aspect of the present invention there is provided apparatus for use in carrying out the method of any one of the six immediately preceding paragraphs, the apparatus comprising an open-topped mixing vessel and a closure for the mixing vessel which is penetrated by a hollow filter, the filter extending from the side of the closure presented inwardly of the mixing vessel, in use, having a closed end remote from the closure and opening through the closure.

The apparatus may further comprise a collection vessel releaseably connected to the closure to close the open end of the filter.

The filter is preferably of the kind described and illustrated in the Specification of U.S. Patent 6,296,763.

A preferred embodiment of the invention will now be described by way of non-limitative example with reference to the accompanying drawings, in which:

Figure 1 illustrates a mixing vessel in sectional elevation; Figure 2 illustrates the mixing vessel of Figure 1 after it has been stoppered by a closure which includes a tubular filter and a detachable collection tube, and Figure 3 illustrates the assembly of Figure 2 following its inversion.

Referring to the drawings, a quantity of sheep's faeces 11 measured by weight, for example three grams, is broken up with a spatula and introduced into a mixing vessel 10 together with a quantity of a saturated salt solution, for example 42 mis, a surfactant or detergent, a silicone additive and a quantity of polymer granules 12 with a density greater than 1.5. The purpose of the silicone additive is to prevent the surfactant from foaming and the formation of small bubbles in the saturated salt solution. Glass beads may be used in place of the polymer granules.

The mixing vessel 10 is then closed with an imperforate lid 17 which makes a screw- threaded or bayonet-type connection with the open end of the vessel 10. The vessel 10 is then agitated so that the granules 12 break up and disperse the faeces particles to provide a substantially homogeneous solution.

The lid 17 is then removed and replaced by a closure 13 which is penetrated centrally by a tubular filter 14 of smaller cross section than that of the interior of the mixing vessel 10. A collection vessel 16 is screw threaded to the open end of the filter 14 where this penetrates the closure 13.

The filter may be made from a woven synthetic filter fabric such as nylon or polyester supported within an integral polymer cage. Alternatively, and preferably, it is of the kind described and illustrated in U.S. Patent No. 6,296,763. In this case the filter apertures will be rectangular in form. The size of the apertures of the filter is critical. If the apertures are too small eggs will be retained by the filter and faecal debris will rapidly occlude the filter. If the apertures are too large faecal debris will be transferred to the collection tube. Eggs from this sample are likely to be obscured when viewed in a McMaster slide. The optimum area of the apertures in a filter of the kind described and illustrated in U.S. Patent No. 6,296,763 is believed to be within the range from 0.02 mm sq. to 0.16 mm sq. It will be observed from Figure 2 that with the mixing vessel 10 upright the lower part of the filter 14 is submerged in the solution. In a short time equilibrium will have been established and a proportion of the solution including parasitic eggs will lie within the filter 14.

The assembly is now inverted as shown in Figure 3 so that the proportion of the solution within the filter 14 is decanted into the collection vessel 16. Because of the sub- atmospheric pressure thus created in what is now the top region 18 of the mixing vessel the solution external to the filter 14 does not flow through the filter and into the collection vessel.

The collection vessel 16 is a standard, 15 ml centrifuge tube. The dimensions of the mixing vessel 10 and of the filter 14 together with the measured volumes of liquid and solid matter (3 g of faecal matter + 42 ml of the saline solution) introduced into the mixing vessel 10 ensure that up to 4 ml of the solution is decanted from the filter, each ml containing the same number of eggs. If more than 4 ml of the solution is required shaking the mixing vessel 10 while inverted will cause more solution to pass through the filter 14, but again solution will not flow through the filter as long as the mixing vessel 10 is not shaken.

The use of the screw threaded collection vessel 16 is preferred as this provides a fully closed system, but because of the phenomenon just described the filtered proportion of the solution could be decanted into any other container without permitting the escape of excess solution.

The collection vessel 16 is now removed from the closure 13 and filtrate from it is transferred by pipette to both chambers of a McMaster slide and the number of eggs counted. As an example, if 15 ml of filtrate has been decanted and a volume of 0.15 ml is pipetted the number of eggs counted (X 100) will indicate the number of eggs per gram of the faeces from which the sample was taken. The method of the present invention offers inter alia the following advantages over current procedures:

1) A simplified protocol requiring fewer steps. 2) Centrifugation will not be essential.

3) More consistent results.

4) Faster results.

5) Lower test cost due to reduction in technician time.

6) Reduced necessity for dexterity, skill and training of operatives. 7) A precisely measured quantity of filtrate will be obtained.

8) No cleaning required if the device is single-use.

9) A closed process minimising exposure to potentially dangerous aerosols.

In addition the abrupt inversion of the assembly is preferable to any conventional pouring technique, the latter inevitably permitting a degree of precipitation.

Claims

CLAIMS:

1. A method of preparing a biological specimen for examination, comprising mixing a biological sample with liquid in a mixing vessel, agitating the mixing vessel, closing the mixing vessel with a closure which is penetrated by a hollow filter extending into the mixing vessel, the filter having a closed end remote from the closure and opening through the closure, so that at least a portion of the length of the filter is submerged in the solution in the mixing vessel, permitting a proportion of the solution to enter the filter and inverting the mixing vessel to decant said proportion from the mixing vessel without permitting the escape of solution external to the filter within the mixing vessel.

2. A method as claimed in claim 1, wherein the weight of the biological sample and the volume of liquid are so measured in relation to the dimensions of the mixing vessel and filter that up to 12% of the solution enters the filter to be decanted therefrom.

3. A method as claimed in claim 2, wherein the solution in the mixing vessel is 45 ml and the said proportion which enters the filter is 4 ml.

4. A method as claimed in any one of the preceding claims, wherein a collection vessel is releaseably connected to the open end of the filter to provide a closed system and wherein after decanting said proportion into the collection vessel the latter is removed from the filter and mixing vessel and filtrate is pipetted from the collection vessel to the chambers of a McMaster slide.

5. A method as claimed in any one of the preceding claims, wherein particles of a material harder than the biological specimen are included in the mixing vessel to assist in breaking up the biological specimen when the mixing vessel is agitated.

6. A method as claimed in claim 5, wherein the particles are glass beads.

7. A method as claimed in claim 5, wherein the particles are polymer granules of a density greater than 1.5.

8. A method as claimed in any one of the preceding claims, wherein the tubular filter is of the kind described and illustrated in the Specification of U.S. Patent No. 6,296,763.

9. A method as claimed in any one of the preceding claims, wherein the liquid comprises a saturated salt solution.

10. A method as claimed in any one of the preceding claims, wherein the liquid comprises a surfactant or detergent.

11. A method of preparing a biological specimen for examination substantially as hereinbefore described.

12. Apparatus for use in carrying out the method claimed in any one of the preceding claims, the apparatus comprising an open-topped mixing vessel and a closure for the mixing vessel which is penetrated by a hollow filter, the filter extending from the side of the closure presented inwardly of the mixing vessel, in use, having a closed end remote from the closure and opening through the closure.

13. Apparatus as claimed in claim 12, wherein the dimensions of the mixing vessel and of the filter are so chosen that if 45 ml of a solution is introduced in to the mixing vessel 4 ml of the solution will enter the filter.

14. Apparatus as claimed in claim 12 or claim 13 and further comprising a collection vessel releaseably connected to the closure to close the open end of the filter.

15. Apparatus as claimed in claim 14, wherein the collection vessel is a 15 ml centrifuge tube.

16. Apparatus as claimed in any one of claims 12 to 15, wherein the filter is of the kind described and illustrated in the Specification of U.S. Patent 6,296,763.

17. Apparatus for use in carrying out the method claimed in any one of claims 1-11 substantially as hereinbefore described and as illustrated in the accompanying drawings.