WO2001051099A1

WO2001051099A1 - Coverslip holder

Info

Publication number: WO2001051099A1
Application number: PCT/IB2001/000137
Authority: WO
Inventors: Bianca Hellias; Hans Floesser; Carlos Dotti
Original assignee: European Molecular Biology Laboratory
Priority date: 2000-01-14
Filing date: 2001-01-12
Publication date: 2001-07-19
Also published as: AU2001230434A1

Abstract

A holder (1) for holding a planar article, such as coverslips or slides during sterilisation prior to their use in preparing cell cultures or for adhering or attaching cells thereto, for example, in neurobiology. The holder (1) comprises a substantially planar frame portion (10) having article receiving positions (16) and associated article retaining means (26). The article receiving positions each comprise a cavity (18) defined by an annular recess within the frame portion (10), the cavity's central portion being a void (20), the article for sterilisation fitting within the cavity (18), and the retaining means (26) being adapted to retain the article within the cavity (18). The article retaining positions (16) can be provided on both sides of the frame portion (10), the retaining positions (16) on each sides (13, 14) forming a coplanar array of article retaining positions (16). A kit comprising a plurality of holders (1) is also provided. The kit may include a stand portion (34) onto which the holders (1) may be stacked together.

Description

COVERSLJP HOLDER

The present invention relates to a holder for holding a planar article during sterilisation. In particular the holder is for holding coverslips or slides during their sterilisation prior to their use in preparing cell cultures or for adhering or attaching cells thereto, for example, in neurobiology.

In order to adhere a cell to a slide or a slide coverslip, various preparation steps must be undertaken. In particular, it is of the utmost importance that the slide or coverslip is sterile to avoid bacterial contamination of cell culture. The sterilisation process usually requires an initial wash and sterilisation step.

For example, a conventional procedure for the preparation of coverslips for the attachment of primary culture cells involves an initial step of inserting coverslips into porcelain staining racks, rinsing with water and then soaking in nitric acid for around 16-24 hours. The coverslips are then washed extensively with distilled water before being dried in a hot oven. The coverslips are then covered with aluminium foil and sterilised by dry heat

For use, sterile forceps are used to distribute the coverslips into tissue culture dishes in a laminar flow hood. This involves a handling step. For primary cell cultures such as neuronal cell cultures, bone-derived cells, myoblasts or endothelial cells, and also for obtaining a stronger attachment of "simpler" cells which are subjected to harsh experimental treatments, for example, membrane permeabilization for nuclear import- export assays, some form of matrix is required for successful attachment of cells to occur. Coverslips are thus often coated with a compound such as poly-lysine. This procedure requires further handling steps: firstly paraffin drops must be applied to each coverslip, for example, to prevent direct contact between neurons and glial cells during co-culture. Each coverslip is then covered with 3-4 drops of the poly-lysine solution in borate buffer overnight, before being aspirated, and the coverslips rinsed with sterile water

One problem associated with conventional methods of preparing coverslips is that there are a number of stages during the preparation in which the coverslips must be handled. Not only does this make the process of sterilisation more laborious, but the chances of the coverslips becoming infected by microbes increases significantly. Coverslips can be purchased pre-sterilised; coverslips of this type are provided within blister-package strips by Eppendorf-Netheler-Hinz GmbH under the trademark CELLocate®. However, these coverslips are sterilised by conventional means using an autoclave or UV light, and therefore lack the highly clean and hydrophobic surface that is required for the even adhesion to glass of matrix components applied in aqueous solution. An appropriate surface is only guaranteed by exposing the coverslips to strong acid or base (for example, nitric acid) and then drying and sterilising the coverslips using a high temperature exposure. Accordingly, the fact that these coverslips are provided sterilised is in fact meaningless for users that work with primary cell cultures, since the coverslips must still be cleaned, coated and re-sterilised as discussed above.

Another problem with these coverslips is their great expense. This means that most research laboratories tend to protect their budget by performing the laborious task of sterilising and coating coverslips manually. Furthermore, even the CELLocate® coverslips cannot be reused without re-sterilisation.

It would thus be desirable to provide a means to simplify the preparation of sterile coverslips for use in cell culture procedures.

According to the present invention there is provided a holder adapted to retain one or more substantially planar articles during sterilisation thereof, comprising a substantially planar frame portion having one or more article receiving positions and associated article retaining means.

Preferably, the article retaining means are retractable. For example, the article retaining means may be at least one set of rotatable clips having a substantially D shaped profile, the clips being arranged around the, or each, article receiving position such that they can retain the. or each, article in one orientation, and release the, or each, article at a second orientation.

The article retaining means may be in the form of one or more rods extending across at least a portion of the, or each, article receiving position. The rod or rods may be retained on the frame portion by insertion through rings or by clips thereon.

Where there is a plurality of article receiving positions, a network of rods, for example in a grid or a grill formation, may be provided as the retaining means. The rod(s), or the grid or ~>

grill, sandwich the substantially planar article between the retaining means and the substantially planar frame portion.

The article retaining means may comprise a second planar frame portion. Preferably the second planar frame portion has one or more vents corresponding to the one or more article receiving position. In this embodiment, the two frame portions may conveniently be rectangular or square. Frame clips or pegs may be provided to hold the two frame portions together, thereby sandwiching the article or articles between the two frame portions. The sandwiched articles are preferably provided in recesses to be free to move within the recess.

Chamfered corners may be provided on each of the frame portions to allow correct alignment of the two frame portions.

Preferably, the, or each, article receiving position is adapted to receive a coverslip.

In an alternative construction, the, or each, article receiving position is adapted to receive a microscope slide.

Preferably, an aperture extends through the frame at the, or each, article receiving position. The aperture allows full sterilisation of both sides of the article.

Preferably, the, or each, article receiving position comprises a cavity defined by an annular recess within the frame portion, the cavity^'s central portion being a void defining the aperture, the article for sterilisation fitting within the cavity, and the retaining means being adapted to retain the article within the cavity. Since the mouth of the cavity is recessed, there is formed a shoulder on which the edges of the article can rest during use.

Preferably, the article, when retained at the article receiving position, is retained loosely therein so that upon immersion in a fluid, all surfaces of the article are brought into contact with the fluid.

Preferably, one or more article receiving positions is provided on both sides of the frame portion. This allows, for example, a slide or slide coverslip to be inserted on each side of the frame portion. Preferably, the retaining means comprises at least one spring-mounted clip that is biased to a position for retaining an article in its article receiving position.

Preferably, the holder is constructed from a chemical and heat resistant material such as polytetrafluoroethane (PTFE), Perfloro-Alkoxyalan (PFA) or PFA/PTA. Chemical and heat resistant metals could also be used. This is necessary to allow the holder to be subjected to both strong acid and strong alkali and to temperatures in excess of 225°C.

Preferably, a plurality of article retaining positions are provided on one side of the frame portion, the plurality of article retaining positions forming an array. Each article retaining position in the array may be coplanar with each other article retaining position in the array.

The retaining means may comprise a plurality of clips and pegs positioned in alternating fashion around the, or each, article retaining position. This allows two or more holders to be stacked upon each other without corresponding clips on each adjacent holder interfering with each other. Further, if the pegs are kept as short as the clips, the two adjacent holders will stack more compactly.

The present invention also provides a kit for holding planar articles for sterilisation thereof, comprising a plurality of these holders. The holders are adapted, in use, to be stacked together.

Preferably, the kit further comprises a stand portion adapted to support the holders when in a stacked arrangement. The stand portion may comprise one or more fingers and each holder may comprise one or more corresponding stacking apertures, so that in the stacked arrangement, the finger (or fingers) extends through the stacking apertures of the holders. Generally, the holders are stacked together vertically onto the stand portion.

The present invention also provides a method of sterilising a slide or slide coverslip using the holder of the present invention.

Use of the holders of the present invention to handle slides or coverslips gives a reduced risk of contamination of the culture by human handling. For example, using the porcelain racks of the prior art, slide coverslips are handled when inserting in the racks for the initial acid wash, when transferring the coverslips for poly-lysine treatment and when transferring coverslips to cell culture dishes for cell plating. In the holders of the present invention, handling is required only once, when the coverslips are still dirty, and involves the step of inserting them into the holder for the acid wash. All subsequent steps can be performed without ever touching the coverslip. Furthermore, cells can be directly plated with the coverslips still in the holders. This greatly reduces the risk of contamination, since the holder is handled, rather than the coverslips.

A further problem with prior art methods is that in porcelain racks, it is very common for coverslips to stick to each other during the washing step, and prior to drying. This causes the surface of many of the coverslips to dry imperfectly. Therefore, the poly-lysine does not spread evenly, which consequently leads to an inhomogeneous surface for cell attachment. This does not occur in the holders of the present invention, as each coverslip is individually retained in a "fixed'^" position.

Porcelain racks are expensive to manufacture since they require an initial moulding step, followed by a firing step. In contrast, the holders of the present invention can be fabricated out of plastics materials, for example, PTFE or PFA-TFA. They can therefore, for example, be injection-moulded in large numbers, meaning that the holders can be manufactured inexpensively.

Further, in view of the ease with which different moulds can be designed for injection moulding of plastics, the holders can easily be adapted for retaining, for example, coverslips of different sizes, and even the holders can be made of different sizes to fit into. for example, 3cm, 6cm, 10 cm or custom larger petri dishes.

Conventional porcelain racks each hold 12 coverslips (if put in zig-zag up to 24, although this can greatly increase the number of coverslips that stick to each other, therefore highly increasing the chances of bad substratum spreading). Each holder of the present invention may conveniently be arranged, in accordance with the first embodiment, to retain 8 coverslips, and in accordance with the third embodiment, 96 coverslips, although other shapes and sizes of holders could be designed for production of other numbers of sterilised coverslips. For example, industrial use may require substantially larger holders to be made. Because the holders can be stacked, for example, ten holders on one stand portion, a greater number of coverslips can be processed at the same time. The holders of the present invention, when fabricated from plastics, can be made highly resistant to acids and heat and importantly, do not crack or break after falling. In such an eventuality_, the coverslip articles will also be retained within the holder and will not fall out if the holder is dropped.

Under routine culture conditions on coverslips. even' time a petri dish is taken out of the incubator_, for example, to check cell growth, contamination and viability^, the resulting movement of fluid generally causes sliding of the coverslips. This will often result in adjacent coverslips becoming partially or totally overlapped, thereby causing the cells in the _"sandwiched" coverslip to die. In contrast, using the holders of the present invention. this cannot happen. At any angle of orientation, the coverslips are retained in position.

Slides and coverslips for use in the present invention need not be glass. Plastic coverslips. for example, can also be processed.

The present invention will now be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1 is a plan view of a bottom side of a holder in accordance with a first embodiment of the present invention;

Figure 2 is a partial section through a frame portion of a holder in accordance with a first embodiment of the present invention;

Figure 3 is a partial section showing grooves adjacent a cavity in the frame portion of Figure 2;

Figure 4 is a plan view of a top side of the holder of Figure 1 ;

Figure 5 is a cross section through line 5-5 of Figure 4:

Figure 6 is a perspective view of a holder in accordance with a first embodiment of the present invention;

Figure 7 is a front elevational view of a rotatable clip for snap-fitting into the frame portion of Figure 5;

Figure 8 is a plan view of the clip of Figure 7: Figures 9, 10 and 11 are perspective, plan and front elevational (with partial section) views, respectively, of a screw driver for adjusting the rotational orientation of clips on the frame portion;

Figure 12 shows a perspective view of a prior art coverslip handler:

Figure 13 shows a perspective view of a kit comprising a plurality of holders stacked onto a stand portion:

Figure 14 shows a third embodiment of a holder in accordance with the present invention:

Figure 15 shows the holder of Figure 14 part assembled:

Figure 16 shows the embodiment of Figure 14 fully assembled;

Figures 17 and 18 show in detail the holder and the retaining means, respectively, of the holder of Figure 14; and

Figures 19 and 20 are cross sectional views through the holder of Figure 14.

Referring now to Figures 1, 4 and 6 there is shown a holder 10 in accordance with the present invention.

The holder 1 comprises a frame portion 10 formed from a plastics material. The frame portion 10 is substantially in the form of a circular disc having a top side 12 and a bottom side 14. Figure 1 shows the arrangement of the features on the bottom side 14 and Figure 4 shows the arrangement of the features on the top side 12.

Figure 5 shows a cross-section through the frame portion 10 with all additional features removes for clarity.

The embodiment shown in Figures 1 , 4 and 6 has provided thereon, on each side thereof, four article receiving positions 16. As best shown in the cross section of Figure 5, and the perspective of Figure 6, each article receiving position 16 comprises a cavity 18. The cavity 18 defines an annular recess within the frame portion 10 having shoulders for supporting a planar article therein, such as a glass or plastics coverslip (not shown). A through-hole, or void, extends through the centremost portion of the cavity 18, as signified by reference number 20. This defines an aperture so that during sterilisation, or cleaning, the sterilising or cleaning fluid can circulate fully around the article retained within the cavity 18. Usually the cavity 18 will be deeper than the article to be retained therein, and the diameter of the cavity 18 will also be greater than the diameter of the article. This further allows the article to be loose within the cavity 18 to assist in the even passage of the sterilising, cleaning or other fluid around the article.

The article to be sterilised in the preferred and shown embodiment is a circular disc-shaped coverslip for a slide. However, of course, for non-circular articles, the cavity need not be annular.

Extending tangentially from the perimeter of each cavity 18 there is provided at least one groove 22. As shown in Figure 3, which is a section through any one of the grooves 22, the groove extends from the outer surface of both the top side 12 and the bottom side 14 of the frame portion 10. However, they do not extend all the way through the frame portion 10, but leave a flange 24 across the central diameter of the frame portion 10. The grooves 22 allow, for example, tweezers to be used to place or grasp a coverslip in the cavity 18 using the edges of the coverslip.

In the embodiment shown, around each cavity 18 there is provided two clips 26. Each clip 26 is a rotatable substantially D-shaped member 28 mounted on top of a post 30. A plurality of holes 31 (see Figure 5) are provided about each article receiving position in the frame portion 10 into which the post 30 may be snap-fitted.

Two clips 26 are provided for each cavity 18. The two clips 26 are positioned on diametrically opposed sides of the cavity 18. Corresponding pegs 32 are also positioned at diametrically opposed sides of each cavity, however at a rotated position 90° from the clips 26.

On the top side 12, as shown in Figure 4, the clips 26 are positioned on left and right hand sides of each cavity 18, whereas on the bottom side 14, as shown in Figure 1, the clips 26 are positioned at fore and aft sides of each cavity 18. This non-symmetric arrangement is optional. However, it allows, on stacking two adjacent holders on top of each other, the clips on a top side 12 of a first holder 1 to rest against pegs 32 on a bottom side 14 of a second, i.e. adjacent, holder 1. A result of this arrangement is that interference between adjacent clips 26 on stacked holders 1 does not occur, thereby removing the danger of clips 26 being inadvertently turned during the sterilisation or cleaning process (which involves the circulation of fluid around the clips. Further, by making the pegs 32 shorter than the clips 26. stacked holders 1 will stack more closely together.

The D-shaped member 28 is diametrically spaced from the cavity such that in one orientation, a closed position as shown at 26A of Figure 4, an article within the cavity 18 is retained over its edge portion by the circular portion of the D-shaped member, and at a second orientation, an open position as shown at 26B in Figure 4, an article within the cavity 18 is free to be removed (or inserted) since the flat portion of the D-shaped member does not overhang the cavity 18. Other suitable clips will be apparent to one skilled in the art, for example spring-biased clips or snap open/snap-close clips.

Referring now to Figure 13, a kit in accordance with the present invention is shown. The kit comprises a plurality of holders 1 as described above. They each have a central stacking aperture 40. The holders 1 can be stacked together on a stand portion 34. The stand portion 34 comprises a base portion 36 in the form of a circular disc having venting apertures provided therein to assist in the sterilisation process, by way of allowing circulation of fluid through the base portion. Extending from the base portion 36 there is a finger 38. The f_inger is in the form of a cylindrical rod of sufficient length for a plurality of holders 1 to be threaded thereon, preferably ten. For this purpose, the finger threads through the central stacking aperture 40 of each holder 1.

The kit of the present invention may also comprise a screwdriver as shown in Figures 9 to 11. The screwdriver allows easy grasping and manipulation of each clip 26 in turn. The screwdriver comprises a handle portion 42, a shaft portion 44 and a head portion 46.

The head portion 46 comprises a recess 48, as shown in Figure 10, and in partial section in Figure 11. The recess 48 is of a substantially D-shaped section to correspond with the shape of the head of the clips 26.

In a second embodiment, not shown, a rod, or a plurality of rods in a grid or grill formation are provided as the article retaining means. The substantially planar frame portion has one or more article receiving positions, and the rod or rods, in use, are positioned such that a substantially planar article at an article receiving position is retained in that article receiving position by the rod or rods. The article is sandwiched between the substantially planar frame portion and the rod. This sandwiching of the article can retain the article in the receiving position either in a fixed position, by a squeeze fit, or loosely. For example, the rods, e.g. of the grid or grills, may be so spaced as to provide spaces therebetween that are smaller than the article. This will prevent the article from falling or slipping out of the article retaining position between the rods.

Preferably the rods are formed of a plastics material.

The rod or rods, in use, will generally extend across the whole plate, therefore spanning a plurality of article receiving positions. However, individual rods for each article receiving position could alternatively be provided.

The rod or rods can be kept in place by sliding the rod or rods through rings provided at each end thereof. The rods may be resiliently displaceable for insertion of the article at the article receiving position.

A large number of substantially planar articles can be held in the article retaining positions by use of only a single rod if the rod extends across those receiving positions. However, with the adjustable clips, each clip has to be adjusted in order to retain an article in the article receiving position.

In a third embodiment, as shown in Figures 14 to 20, the holder comprises a first substantially planar frame portion, or coverslip holder 100, having an array of article receiving positions 16 provided therein. A second substantially planar frame portion, or coverslip stop 102, is also provided. It corresponds in size to the first coverslip holder 100 and forms the article retaining means.

The substantially planar frame portion is substantially square and comprises a near complete 10x10 array of article receiving positions defining 96 article receiving positions. However, in other embodiments, not shown, the planar portion could, for example, be provided with a 5 by 10 array of (50) article retaining positions on each side thereof, with a corresponding article retaining means provided for each side of the coverslip holder 100, thereby providing the means for sterilising up to 100 cover slips at a time.

For conventional laboratory purposes, large holders cannot generally be used for the plating of cells, since culture dishes, or petri dishes, of a suitable size are not generally available. However, of course, culture dishes of an appropriate size could be manufactured. The large holders of this third embodiment are generally for use in the preparation of large quantities of coverslips either for labs who perform lots of cell culture or for selling "ready to coat^" coverslips.

The coverslip stop 102 shown in Figure 18 is a substantially planar, square frame of the same size as the coverslip holder 100.

Figures 17 and 18 show preferred dimensions, in mm. for the holder of the third embodiment of the present invention. 96 16mm-round coverslips can be processed with this embodiment. However, by altering the dimensions thereof, it can be made to handle any number of coverslips, of any required shape or size. Foe this reason, although the dimensions shown in Figures 18 and 19 are preferable, they should in no way be taken to limit scope of the present invention as defined in the claims attached hereto.

A corner of both the coverslip holder 100 and the coverslip stop 102 have a chamfered corner 104 so that the two frame portions 100, 102 can be oriented correctly during assembly, as shown in Figure 16.

The coverslip holder 100 is 3mm thick and the coverslip stop 102 is 1mm thick. Preferably the two frame portions are manufactured from Teflon, PTFE or PFA.

The coverslip holder 100 is provided with five bolt holes 106 into which can be screwed mushroom shaped clips or pegs 108, as shown in Figure 20. The pegs 108 are for insertion into pear-shaped holes or keyholes 110 in the coverslip stop 102. As shown in Figures 15 and 16, after placing the coverslips into the article receiving positions of the coverslip holder 100, the coverslip stop 102 is positioned over the coverslip holder 100 with two chamfered corners 104 similarly oriented, and the pegs 108 aligning with the keyholes 110. The peg 108 goes through the large part of the hole 110 in the coverslip stop 102 and then the coverslip stop 102 is displaced so that the peg 108 latches with the keyhole 110. The pegs and keyholes are designed to fit tightly together, so that the coverslip is not easily displaced from the retaining position.

Once assembled, the coverslips cannot fall out of the article receiving positions since the coverslip stop 102 retains them in their positions. Referring now to Figure 19, in accordance with the previous description of the first embodiment, each article receiving position comprises an aperture 112 and cavity or annular recess 114 into which a coverslip can loosely fit. The coverslip rests on the shoulders 116 of the cavity. A slot 120 for tweezers may also be provided for each cavity 1 14, as shown in Figure 17.

A plurality of holders of the third embodiment can also be stacked one on top of another with the peg 108 protruding upwardly from the coverslip. This separated adjacent holders, thereby allowing sterilizing fluid to pass between adjacent holders.

A stand can be used to hold the plurality of holders. The stand for stacking a plurality of these larger coverslip holders 100 can have a square base and, for example, four rods of a desired height, which height determines the number of holders that the stand can hold.

The use of the second or third embodiment simplifies the use of the holder when processing a very large number of coverslips, since adjusting a multiplicity of individual article retaining means for each coverslip can be timely exercise. In the second and third embodiments there are no screws or clips to be adjusted. It is simply a matter of applying and displacing a coverslip stop 102 or rod relative to the coverslip holder 100 to retain the plurality of coverslips in the article receiving positions.

The holders of the present invention greatly reduce the direct handling of coverslips in the preparation of cell cultures. For example, the holders of the present invention can be used in the sterilisation of coverslips using substantially similar steps to those found in the prior art. However, the coverslips do not require any direct handling after the initial insertion of the coverslips within the cavities 18.

One method of use comprises taking an initially "dirty" holder and inserting the coverslips. This can be initially washed and sterilised in a nitric acid bath. Since in the embodiments shown, while the coverslips are retained in position by the retaining means, the upper surface of each coverslip is freely accessible, there is no need to remove the coverslips from the holder 1 in order to attach cells thereto. Further, since there can be a plurality of coverslips on each side of the holder 1, each holder 1 can actually hold and retain twice the number of coverslips that can have cells attached thereto compared to methods as previously used with an equivalently sized petri dish, and without removing the coverslips from the holder 1, i.e. with one less handling step, at least. By stacking a number of holders 1 on the stand portion 34, the holders 1 all being slightly separated from each other by means of the protruding nature of the pegs 32,108, on each stand portion 34 there can be provided a substantial number of coverslips onto which cells have been plated.

Of course, the coverslips may be provided already in a sterile condition. A previously sterilised holder 1 can then be instantly provided with the coverslips, just prior to attaching the cells thereto, so that the holder replaces the petri dish.

The holder is preferably formed from chemical and heat resistant materials so that a wide variety of methods of sterilisation and cell culture processing can be conducted with the holders 1. Preferable materials include plastics such as polytetrafluoroethylene (PTFE) which has a melting point of about 327°C. Another plastics material includes perfluoro- alkoxylakane (PFA or PFA/TFA), which has a melting point of between 300°C and 310°C. These two materials also show extreme resistance to degradation by nitric acid, both in its dilute and its concentrated form.

The present invention has been described above purely by way of example. It should be noted that modifications in detail may be made within the scope of the invention.

Claims

1. A holder adapted to retain one or more substantially planar articles during sterilisation thereof, comprising a substantially planar frame portion having one or more article receiving positions and associated article retaining means.

2. The holder of claim 1 , wherein the article retaining means are retractable.

3. The holder of claim 2, wherein the article retaining means are at least one set of rotatable clips having a substantially D shaped profile, the clips being arranged around the, or each, article receiving position such that they can retain the, or each, article in one orientation, and release the, or each, article at a second orientation.

4. A holder according to any one of the preceding claims, wherein the retaining means comprises at least one spring mounted clip that is biased to a position for retaining an article in its article receiving position.

5. A holder according to any one of the preceding claims, wherein the retaining means comprises a plurality of clips and pegs positioned in alternating fashion around the, or each, article retaining position.

6. A holder according to claim 1 or 2, wherein the article retaining means comprises one or more rods extending across at least a portion of the, or each, article receiving position.

7. A holder according to claim 6, wherein the rod or rods are retained on the frame portion by insertion through rings on the frame portion.

8. A holder according to claim 6 or 7, wherein there is a plurality of article receiving positions and a network of rods for retaining articles in the article receiving positions.

9. A holder according to claim 1, wherein the article retaining means comprises a second planar frame portion.

10. A holder according to claim 9, wherein the second planar frame portion has one or more vents corresponding to the one or more article receiving positions.

11. A holder according to claim 9 or 10, wherein pegs are provided for holding the two frame portions together so as to sandwich one or more articles between the two frame portions.

12. A holder according to any one of claims 9 to 11. wherein a chamfered corner is 5 provided on each of the frame portions

13. The holder of any preceding claim, wherein the, or each, article receiving position is adapted to receive a coverslip.

14. The holder of any one of claims 1 to 12, wherein the, or each, article receiving position is adapted to receive a microscope slide.

10 15. A holder according to any one of the preceding claims, wherein an aperture extends through the frame at the, or each, article receiving position.

16. The holder of any one of the preceding claims, wherein the, or each, article receiving position comprises a cavity defined by an annular recess within the frame portion, the cavity's central portion being a void defining the aperture, the article for

15 sterilisation fitting within the cavity, and the retaining means being adapted to retain the article within the cavity.

17. A holder according to any one of the preceding claims, wherein the article, when retained at the article receiving position, is retained loosely therein.

18. A holder according to any one of the preceding claims, wherein one or more article 0 receiving position is provided on each side of the frame portion.

19. A holder according to any one of the preceding claims, constructed from a chemical and heat resistant material.

20. A holder according to claim 19, wherein said material is selected from the group of plastics comprising polytetrafluoroethylene (PTFE) and perfluoro-alkoxyakane 5 (PFA/PTA).

21. A holder according to any one of the preceding claims, wherein a plurality of article retaining positions are provided on one side of the frame portion, the plurality of article retaining positions forming an array.

22. A holder according to claim 21 , wherein each article retaining position in the array is coplanar with each other article retaining position in the array.

23. A holder substantially as hereinbefore described with reference to any of Figures 1 to 8.

24. A holder substantially as hereinbefore described with reference to any of Figures 14 to 20.

25. A kit for holding planar articles for sterilisation thereof, comprising a plurality of holders according to any one of the preceding claims, wherein the holders are adapted, in use, to be stacked together.

26. The kit of claim 25, further comprising a stand portion adapted to support the holders when in a stacked arrangement.

27. The kit of claim 26, wherein the stand portion comprises one or more fingers, and each holder comprises one or more corresponding stacking apertures, wherein, in the stacked arrangement, the or each finger extends through the corresponding stacking apertures of the holders.

28. A kit substantially as hereinbefore described with reference to any of Figures 1 to 11 or 13.

29. A kit substantially as hereinbefore described with reference to any of Figures 14 to 20.

30. A method of sterilising a slide or a slide coverslip using a holder according to any one of claims 1 to 24 or a kit according to any one of claims 25-29.