WO2006000813A2

WO2006000813A2 - Testing kit

Info

Publication number: WO2006000813A2
Application number: PCT/GB2005/002525
Authority: WO
Inventors: Mark Justin Millington Rawe; Graham Hunter; Stephen John Lawrence
Original assignee: Hunter Scientific Limited
Priority date: 2004-06-26
Filing date: 2005-06-27
Publication date: 2006-01-05
Also published as: GB0414376D0; WO2006000813A3; GB2412748A; GB2412748B

Abstract

A testing kit for determining sperm count in a semen sample suitable for home use, and a method of determining sperm count using the aforesaid kit, the kit comprising, a microscope, a sample support for receiving the sample, a sample support locating means, a graticule being visible through a lens of the microscope, a coffer slip for placing over the sample on the sample support, illuminating means, and a spacer for maintaining a pre-determined distance between the sample support and the cover slip; wherein the sample support locating means is adapted to locate the sample support in a pre-determined position with respect to the microscope such that when the kit is in use, Both the sample and graticule are located within an optical path of the microscope.

Description

Testing Kit

BACKGROUND

a. Field of the Invention

The present invention relates to a male fertility testing kit for determining sperm count and which is suitable for home use by an untrained user. The invention also relates to a method of using such a kit.

b. Related Art

There is an increasing awareness amongst the male population that, in general, sperm counts are falling for various reasons. Around 1 in 6 couples of child- bearing age have problems in conceiving and around 50% of these problems are sperm related. Males often find it more difficult to seek medical advice relating to fertility problems despite the fact that it is much easier to test a male for fertility problems than a female. It can be embarrassing for a male to be asked to produce a semen sample in a clinic for testing which may be one reason for their reluctance to seek professional advice.

There is currently a colorimetric home sperm testing kit available for assessing male fertility. However, this kit only allows a user to determine if his sperm count is above or below 20 million per ml by a colour change. Publications produced by the World Health Organisation (WHO) state that a male is sub fertile if his sperm count is less than 20 million per ml. This kit will not enable a user to determine how far above or below this threshold his sperm count is. In addition, this kit does not enable a user to determine if the sperm are healthy. Using this test, the sperm analysed could be dead, immotile or morphologically irregular. These are all factors which are significant in determining a male's fertility.

It is an object of the invention to provide a male fertility testing kit that addresses some of the above issues.

SUMMARY OF THE INVENTION

According to the present invention there is provided a testing kit suitable for home use for determining sperm count in a semen sample, the kit comprising: a) a microscope, b) a sample support for receiving a sample, c) a sample support locating means, d) a cover slip which, in use, is arranged to substantially cover a portion of a sample on the sample support , e) a graticule on one of the sample support and cover slip, f) illuminating means, and g) a spacer for maintaining a pre-determined distance between the sample support and the cover slip such that, in use, the cover slip, spacer and sample support co-operate to provide a predetermined depth of sample between the cover slip and sample support; wherein, in use, the sample support locating means is arranged within the microscope such that when a sample support having a cover slip arranged substantially covering a sample is arranged in contact with the sample support locating means, the sample and graticule are arranged such that they overlay one another within an optical path of the microscope so that they can be viewed together by a user, the illuminating means being arranged to illuminate the sample and graticule.

According to another aspect of the invention there is provided a method for determining sperm count in a semen sample comprising the steps of: a) providing a testing kit comprising: a microscope, a sample support for receiving a sample, a cover slip, a sample support locating means, a graticule being located on one of the sample support and cover slip, illuminating means, and a spacer; b) arranging a sample of semen between the sample support and the cover slip using the spacer to maintain a pre-determined distance between the sample support and the cover slip such that there is a pre-determined depth of sample between the cover slip and sample support; c) arranging the sample support in the sample support locating means such that the sample and graticule overlay one another within an optical path of the microscope; d) illuminating the sample using the illuminating means; e) using the microscope to view the sample and the graticule within the optical path and make observations relating thereto; and f) using the observations to determine an estimated sperm count.

The sample is placed on the sample support, covered by the cover slip and then the sample support is arranged in contact with the sample support locating means. In use, the sample support locating means is located in a first position in which the sample support is located in a pre-determined position with respect to the microscope and there is no need for a user to adjust the position of the sample support in order to be able to see the sample and/or graticule as this position ensures that both the sample and graticule are located within the optical path of the microscope and are therefore visible. This makes the testing kit easier to use for an untrained user and the likelihood of errors is reduced.

Preferably, the graticule is provided on the sample support such that in use the sample will be adjacent the graticule. This helps to ensure that whenever the sample is in focus, the graticule will also be in focus as the sample and the graticule are in close proximity on the sample support. Specifically, it is preferred that the graticule is laser etched onto a surface of the sample support. - A -

The cover slip is preferably pre-fixed to the sample support with the spacer between the sample support and cover slip as this reduces the likelihood of user error when applying the cover slip over the sample. If the cover slip is pre-fixed, it is preferred that the spacer is substantially circular and substantially encloses the graticule. The spacer in such an arrangement preferably includes at least one filling aperture and a vent aperture allowing a sample to be drawn through the filling aperture across the graticule by capillary action, with the air initially between the cover slip and sample support being expelled through the vent aperture. The sample may be introduced using a pipette at the filling aperture. A fixed cover slip arrangement may be easier for a user than placing the sample on the sample support and then placing a cover slip on top, as cover slips may be very delicate and can be quite difficult to manipulate. It may also prove difficult to ensure that the user arranges the cover slip using the spacer to determine correctly the distance between cover slip and sample support.

The spacer is preferably between 4 μm and 20 μm in depth. Sperm are approximately 4 μm in diameter and the spacer therefore should be greater than 4 μm in order to allow them to swim about so that it is possible to see which ones are motile. By having the spacer greater than 4 μm, the sperm are not likely to be squashed if the cover slip is applied to the sample support after the sample is in place, and, if the cover slip is pre-fixed to the sample support the spacer will be sufficient to allow the sperm to be drawn into the space between the cover slip and sample support. It is preferred that the spacer is less than 20 μm in depth so that the chance of having sperm swim over the top of one another is minimised. It is much easier to count the sperm if they are in a substantially single layer. Alternatively, and more preferably, the spacer may be between 8 and 12 μm inclusive in depth. Most preferably, the spacer will be 10 μm in depth.

If the cover slip is not pre-fixed to the sample support, it is preferred that the spacer is located on the sample support; however it may alternatively be located on the cover slip. It is preferred that both the graticule and the spacer are located on the sample support as this means that the cover slip can be a standard, clear cover slip which may be cheaper to produce.

The spacer may be a printed layer and preferably it is a screen-printed layer. However, it may also be printed by any other form of printing such as ink jet printing or hot foil printing. The spacer may alternatively consist of a layer integrally moulded on the surface of the slide or be formed by any other suitable process. A screen-printed layer is preferred as this provides an accurately sized spacer which is cheap to produce.

The spacer is preferably substantially circular or annular in shape.

It is preferred that the illuminating means is a light emitting diode (LED). LEDs have several advantages over standard filament bulbs such as a longer life span, they are more compact and have a lower power consumption. In addition, they are more efficient and therefore give off less heat than a filament bulb. This may be particularly important in this application as it is important not to heat the sample too much as this may kill the sperm. However, the illuminating means may also be any other form of visible light source.

The LED may be a green LED as this has been shown to give the best light for viewing the sperm as the human eye is more receptive to green light. However, it could also be any other type or colour of LED which allows a user to view the sample. A white LED has also been found to be particularly suitable for this apparatus. It is possible that the illumination could be a white (or other colour) light and that the lenses may be coloured, or include a colour filter such that the light visible to a user is of a different colour to that emitted by the illuminating means.

Preferably, the sample support locating means is a recess in a base of the microscope, the recess being substantially complementary in shape to the periphery of the sample support. A peripheral edge of the recess may additionally include grip portions to expose a least a portion of a side of the sample support so that the sample support may be more easily removed from the recess acting as the sample support locating means by a user.

The sample support may be made of a plastics material such as polycarbonate so that it is safer and less hazardous than a sample support made out of glass. This renders the sample support more appropriate for home use. In addition, it may be cheaper to manufacture. However, the sample support may be made out of glass or any other suitable material.

The sample support may be a disc i.e. circular in shape. A disc has fewer sharp edges than a rectangular sample support which again makes it safer for home use. Additionally, the sample support (whatever shape it may be) may be of a greater thickness than a standard laboratory glass slide thereby making it easier to pick up from a flat surface.

It is preferred that the microscope is a dark-field microscope because unstained sperm show up brighter under dark field illumination than under bright-field illumination. It is preferred not to stain the sperm because this will kill them and make it hard to tell if the sperm in view are healthy; in particular, it may not be possible to tell if the sperm are motile. It should be understood that the term "dark field microscope" is intended to mean any form of microscopy in which only light reflected by the sample and/or graticule enters the objective lens and is intended to include so called pseudo dark field microscopes which do not include complex optics to avoid direct lighting of the subject, but simply have the illuminating means being arranged to substantially avoid direct light from the illuminating means entering the objective lens.

When a pseudo dark field microscope is to be used, it is preferred that the illuminating means is in a fixed and pre-determined orientation with respect to the graticule on the sample support. If the sample support is not placed in the correct orientation for such microscopy, then the graticule and/or sample may not be wholly visible. The graticule should preferably be positioned so that the gridlines are at a 45° angle to the light coming from the illuminating means. Such issues of orientation can be substantially reduced by using a plurality of light sources arranged around the graticule so that at the gridlines of the graticule will be illuminated irrespective of the orientation of the graticule. Preferably there are at least 3 light sources spaced around the sample location.

It should also be noted that, if the sample support is a disc, or any other shape that could be inserted into a sample support locating means in a plurality of orientations, and the microscope is a dark field microscope, the sample support may additionally have means for ensuring that it is placed in the correct orientation with respect the illuminating means. This may be any form of projection or cut-out which interacts with a complementary cut-out or projection on the sample support locating means which together ensure that the sample support is placed in the correct orientation. Alternatively, there may be an arrow or other visible indication printed on it which corresponds to another printed indication on the sample support locating means which indicates the correct orientation of the sample support.

Optionally, the position of the illuminating means may be adjusted so that the microscope alternatively functions as a bright-field microscope, which some people may prefer.

It is preferred that the microscope comprises three main body parts, those being a housing, a base and a neck. Fabricating three main parts can be relatively simple and can provide a robust product without the need for tooling for a plurality of parts.

It is preferred that the base includes the illuminating means and an associated power supply, and houses the sample support locating means. It is preferred that the base also includes a switch for activating and de-activating the illuminating means so that the power supply can be preserved. It is intended that this device will be portable, so the power supply preferably comprises an energy storage means within the base to avoid the need to connect to an external power supply such as mains electricity. The energy storage means may be a battery, capacitor or other suitable means and may be rechargeable.

The neck preferably comprises all of the optical lenses required for the microscope. Preferably the neck contains three lenses which together provide the user with a substantially fixed magnification of between 20Ox and 30Ox and preferably comprises a fixed objective lens.

The housing preferably engages with the base and neck such that, when in use, the neck is held such that a sample on a sample support in the sample support locating means is within the optical path of the microscope. It is preferred that the housing engages with the base by resting on a top surface thereof, preferably being guided by a shoulder or projection so that the housing is correctly aligned with the base. The housing preferably cooperates with the base and neck to substantially enclose a volume such that, in use, the sample is substantially not exposed to ambient illumination. In such an embodiment, the housing acts in a dual role of correctly arranging the neck relative to the base and also substantially preventing ambient light illuminating the sample.

Preferably the housing and neck cooperate to provide focussing of the microscope. Focussing may be achieved in several ways, but is preferably achieved by engaging the neck with the housing using a screw thread on the neck such that the distance between the neck and base can be adjusted by rotating the neck relative to the housing to adjust the screw thread, thereby adjusting the distance between the objective lens and the sample.

To use the three piece microscope, a user would preferably be provided with the neck coupled to the housing in a predetermined position and a separate base. The user would locate a sample support and sample in the appropriate position in the sample support locating means and engage the housing with the base. It is preferred that the predetermined position of the housing and neck is such that the sample and graticule are substantially in focus when the housing is engaged with the base. The user can adjust the focus of the microscope by rotating the neck relative to the housing such that the distance between the neck and base is altered, thereby adjusting the focus of the microscope.

The sample support locating means and sample support preferably cooperate to prevent a sample support being arranged upside down in the sample support locating means. This could be achieved in a number of ways, for example using visual indications or by the sample support and sample support locating means having complementary shapes such that arranging the sample support in the sample support locating means is not readily possible by a user. This may be achieved by a chamfer around the edge of the sample support such that a surface of the sample support has a larger cross sectional area than an opposing surface and the sample support locating means being provided with a complementary angled inner edge. Such an arrangement, possibly in conjunction with, or instead of, visual indicators, could help to reduce the chances that a user will place the sample support into the sample support locating means upside down.

It is preferred that the sample support locating means is secured in a fixed position with respect to the base. However, it is envisaged that the sample support locating means could comprise a drawer or other movable member that has a first position in which, in use, a sample support arranged in the sample support locating means is positioned in the optical path of the microscope and a second position in which arranging of the sample support in the sample support locating means is facilitated.

Other aspects and benefits of the invention will appear in the following specification, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described, by way of example, and with reference to the following drawings in which:

Figure 1 is a cross-sectional view of a microscope;

Figure 2a is a plan view of a sample support;

Figure 2b is a side view of the sample support of Figure 2a;

Figure 3a is a plan view of a different sample support;

Figure 3b is a side view of the sample support of Figure 3a;

Figure 4 is a close up view of a graticule and spacer on a sample support; and

Figure 5 shows a cross section through a microscope having three main sections.

DETAILED DESCRIPTION

Figure 1 shows a schematic side view of a microscope 10 used in a testing kit suitable for home use for determining sperm count in a semen sample. The microscope 10 has a viewing tube 12, a platform 18 and a base 34. A body 11 of the microscope 10 may be made of plastic which may be injection moulded, aluminium or any other lightweight material. In addition, the microscope 10 may have rubber feet (not shown) to provide extra grip to a surface on which it is placed so that it may not easily be knocked during use. An eyepiece 13 is located at the top of the tube 12 and an objective lens 16 is located at the bottom of the tube 12. The eyepiece 13 contains an ocular lens 14. The lenses may be made of either glass or plastic. The focus of the ocular lens 14 may be adjustable by twisting the eyepiece 13. If this is the case, the eyepiece 13 may be located on a screw thread (not shown) inside the tube 12 so that when the eyepiece 13 is turned, the position of the ocular lens 14 is finely adjusted. Overall, it is envisaged that the microscope will have approximately 30Ox magnification.

The platform 18 is located below the viewing tube 12 and carries a sample support locating means 25 in which a sample support or slide 20 may be placed. The sample support locating means 25 ensures that, in use, the sample support 20 is located in a pre-determined position with respect to the microscope such that the sample is located within an optical path of the microscope. A semen sample 22 can then be placed upon the sample support 20 and covered with a cover slip 24. In this embodiment, the sample support is a plastic circular disc and the sample support locating means 25 is a layer on top of the platform 18 having a circular aperture into which the disc 20 fits. In an alternative embodiment, the sample support locating means may consist of a number of locating portions which project from the platform 18 and which determine the position of the sample support. For example, if the sample support is rectangular in shape, the sample support locating means may consist of a number of right angular-shaped locating portions. The sample support locating means may be any means which serve to position the sample support in the correct position within the optical path of the microscope so that both the sample and graticule can be viewed by a user.

A spacer 26 maintains a pre-determined distance between the sample support 20 and the cover slip 24 thereby ensuring that a known depth of sample 22 is achieved. In this embodiment, the spacer 26 is an annular screen-printed layer located on the sample support 20. The screen printed layer 26 is 10 μm in depth. The spacer 26 further acts as a seal ensuring that the sample 22 stays within its confines, so that a known volume of sample is achieved. The screen printed layer additionally functions to indicate to the user the position in which the sample should be placed. The microscope further has illuminating means which, in this embodiment, is a light emitting diode (LED) 28 located substantially below and to the side of the platform 18. There is an aperture 30 in the platform 18 which allows light from the LED 28 to pass up and through the sample support 20 located on the platform 18. In this embodiment, the sample support 20 is translucent. The microscope 10 will also have an on-off switch (not shown) for switching on the LED 28. The translucent sample support 20 allows light from the LED to pass up and through it. Due to the position of the LED no light will enter the objective lens 16 directly but will instead pass directly through the sample support and past the lens. The only light that will enter the objective lens is that whose course is altered when it meets the sample 22 and grid lines forming the graticule 36. This light will be reflected and/or refracted by the sample and/or grid lines. The image formed will therefore have a dark background and the sample and graticule/grid lines will appear white.

The microscope 10 further has a power source 32 to provide power to the LED 28. In this embodiment, the power source 32 is a lithium battery located in the base 34 of the microscope 10 below the platform. Alternatively, the power source could be one or more standard batteries such as AA or AAA batteries or the microscope may be powered by the mains power supply.

As seen in Figures 2a and 3a, the sample support 20 has a graticule 36 at its centre. In this embodiment, the sample support 20 is made of plastic and the graticule 36 is laser etched onto a surface of the sample support in an area within the substantially annular-shaped spacer 26 which is 10 μm in depth. Figure 2a shows a rectangular-shaped sample support whereas that of figure 3a is circular/disc-shaped. Figures 2b and 3b show that sample support of figure 3 is thicker than that of figure 2. Figure 4 (not to scale) shows an enlarged view of the graticule 36 inside the spacer 26. The graticule 36 is preferably a square grid which is 2 mm x 2 mm in area and having 20 x 20 squares (400), each square therefore being 0.1 mm x 0.1 mm, i.e. 0.01 mm² in area. However, it should be understood that the graticule may be any other shape, size or configuration which allows a user to count a number of sperm within a defined area. As the depth of the sample will be 10 μm (i.e. 0.01 mm) this means that the volume seen in each square of the grid is 0.0001 mm³ or 1 x 10-7 ml. It is envisaged that the sample support may be approximately 30 mm in diameter.

In Figure 3a it can be seen that the spacer 26 includes a filling aperture 27 and a vent aperture 29. These apertures 27,29 allow a cover slip to be fixed to the sample support 20 before a sample is located on the sample support 20. A user can then place a sample adjacent the filling aperture 27 and allow the sample to be drawn across the graticule area 36 by capillary action. Air between the cover slip and sample support 20 will be expelled through the vent aperture 29 as the sample is drawn between the cover slip and sample support 20. It should be understood that the vent aperture 29 and filling aperture 27 are substantially identical and their functions can be swapped if required.

Once the sample has been placed on the sample support 20 located on the platform 18 of the microscope 10, and then covered by the cover slip 24, a user should switch on the light source, in this case the LED 28, and then view the sample and graticule 36 through the eyepiece 13 of the microscope 10. If the sample and graticule 36 are not exactly in focus, the fine focus may be adjusted by turning the eyepiece 13. Once the sample is correctly focused, the user may then count the number of sperm in one or more of the squares 38 of the graticule 36. In this embodiment, each sperm counted in 10 squares 38 represents 1 million per ml. Therefore, if a user counts 20 or more sperm per 10 squares, they are deemed to be fertile (according to figures published by the WHO). However, if they count more than 10 squares and then take an average of the numbers, this will give a far more accurate estimate of sperm count. It is envisaged that the testing kit will further be provided with a CD-ROM including instructions, information and visual examples for ease of comparison. Other items that may be provided in the kit are non-spermicidal condoms, a collection pot, pipettes and extra sample supports and cover slips. The WHO guidelines suggest that if a first sperm test is poor, the male in question should abstain from sex for 72 hours and then repeat the test to see if the result has improved. It is not advisable to re-use sample supports or cover slips as they may become scratched when cleaned which is why additional sample supports and cover slips may be provided. It is also envisaged that it may be possible to connect the microscope directly to a PC or have a separate or built-in image-capturing system which may be connected to a PC. The microscope and/or image-capturing system could be connected to the PC via various methods such as a USB cable or similar, Bluetooth® or infra-red communication means.

It may be that a user is not required to count the number of sperm present; for example if they do not require a particularly accurate estimation of sperm count. In this instance, they may simply view the sperm in relation to the graticule and then refer to the visual examples provided with the testing kit in order to see approximately which range they fall within. Examples of views of the sperm and graticule when the sperm count is in the range of 0-20, 30-50 and 50-70 million per ml etc. may be provided. A user can then estimate approximately which range their sperm count may fall in by means of comparison only.

Figure 5 shows a microscope having a three section body 50 comprising a base 52, a housing 54 and a neck 56.

The neck 56 comprises an elongate, substantially cylindrical tube 58 in which are mounted three optical lenses, an objective lens 60 and two ocular lenses 62,64. In this case the objective lens is a Comar™ part 04AP04, the ocular lenses 62,64 are Comar™ parts 31 AP29 and 42AP29.

The neck 56 is mounted to the housing 54 with a screw thread 66. The screw thread 66 allows the neck to be moved along a central axis of the tube 58 relative to the housing by rotating the neck 56 relative to the housing 54.

The housing 54 is arranged on the base 52 and is located relative to the base by a projection 68 from the base such that the central axis of the tube 58 is in a predetermined orientation with respect to the base 52 such that the optical path of the microscope is arranged such that when a sample support, cover slip and sample arranged in the sample support locating means the graticule and sample will be within the optical path and therefore visible to a user after any required focusing.

The base 52 includes a sample support locating means 70 in which is arranged a sample support 72 which includes a cover slip, spacer and sample (shown in more detail in other figures). The base 52 includes a power supply 74, in this case a battery, a processor 76 and LEDs 78. The LEDs 78 are controlled by the processor 76 and powered by the power supply 74. The base 52 further includes a switch 80 for controlling activation of the LEDs 78.

The microscope is arranged such that when the housing 54 is arranged on the base 52 and the neck 56 is mounted to the housing 54 a sample on the sample support 72 arranged in the sample support locating means 70 is in the optical path of the microscope.

While the present invention has been described with reference to specific embodiments, it should be understood that modifications and variations of the invention may be constructed without departing from the scope of the invention as defined in the following claims.

Claims

1. A testing kit suitable for home use for determining sperm count in a semen sample, the kit comprising: a) a microscope, b) a sample support for receiving a sample, c) a sample support locating means, d) a cover slip which, in use, is arranged to substantially cover a portion of a sample on the sample support , e) a graticule on one of the sample support and cover slip, f) illuminating means, and g) a spacer for maintaining a pre-determined distance between the sample support and the cover slip such that, in use, the cover slip, spacer and sample support co-operate to provide a predetermined depth of sample between the cover slip and sample support; wherein, in use, the sample support locating means is arranged within the microscope such that when a sample support having a cover slip arranged substantially covering a sample is arranged in contact with the sample support locating means, the sample and graticule are arranged such that they overlay one another within an optical path of the microscope so that they can be viewed together by a user, the illuminating means being arranged to illuminate the sample and graticule.

2. A testing kit as claimed in Claim 1 , wherein the graticule is provided on the sample support.

3. A testing kit as claimed in Claim 1 or Claim 2, wherein the graticule is laser etched onto a surface of the sample support.

4. A testing kit as claimed in any preceding claim, wherein the spacer is between 4 μm and 20 μm in depth.

5. A testing kit as claimed in any preceding claim, wherein the spacer is between 8 μm and 12 μm in depth.

6. A testing kit as claimed in any preceding claim, wherein the spacer is located on the sample support.

7. A testing kit as claimed in any preceding claim, wherein the spacer is a screen-printed layer.

8. A testing kit as claimed in any preceding claim, wherein the spacer is substantially annular in shape.

9. A testing kit as claimed in any preceding claim, wherein the cover slip is permanently fixed to the sample support.

10. A testing kit as claimed in any preceding claim, wherein the illuminating means comprises a light emitting diode.

11. A testing kit as claimed in any preceding claim, wherein the sample support locating means comprises a recess in a member, the recess being substantially complementary in shape to the periphery of the sample support.

12. A testing kit as claimed in any preceding claim, wherein the sample support is made of a plastics material.

13. A testing kit as claimed in any preceding claim, wherein the sample support is substantially disc shaped.

14. A testing kit as claimed in any preceding claim, wherein the microscope functions as a pseudo dark-field microscope.

15. A method for determining sperm count in a semen sample comprising the steps of: a) providing a testing kit comprising: a microscope, a sample support for receiving a sample, a cover slip, a sample support locating means, a graticule being located on one of the sample support and cover slip, illuminating means, and a spacer; b) arranging a sample of semen between the sample support and the cover slip using the spacer to maintain a pre-determined distance between the sample support and the cover slip such that there is a pre-determined depth of sample between the cover slip and sample support; c) arranging the sample support in the sample support locating means such that the sample and graticule overlay one another within an optical path of the microscope; d) illuminating the sample using the illuminating means; e) using the microscope to view the sample and the graticule within the optical path and make observations relating thereto; and f) using the observations to determine an estimated sperm count.