The present invention relates to a single use Wright's Stain packet.
Wright Stain is a well known mixture of eosin with methylene blue, available commercially in powder form. For use, the powder is dissolved in methanol, normally between 0.1 and 0.3 grams per 100ml methanol. Some formulations recommend inclusion of glycerine to enhance solubility of the dye. A typical practice is to age a freshly prepared solution at 37°C., then filter and store in dark bottles use. A separate buffer solution pH 6.4 -7.4, usually with a phosphate buffer, is employed as part of the slide staining procedure.
A suggested technique for Wright's Stain is to add the methanol stain solution to the blood smear slide, let it stand for about 1 to 3 minutes, then replace the methanol stain solution with the aqueous buffer solution, let the buffered solution stay on the slide for 3 to 5 minutes, whereafter the slide may be rinsed in tap or distilled water and dried.
Normally, such a procedure is carried out manually, requiring a certain degree of skill on the part of the laboratory technician. Automated systems which have been suggested require expensive machinery, special stain packaging and are relatively time consuming. A dry strip which has been proposed offers some advantages but requires immersion to activate the stain ingredients thereron.
A single use Wright's Stain packet would be of considerable value to the laboratory technician.
A principal object of the present invention is to provide a Wright's Stain packet of a single use nature adapted to the needs of the laboratory technician.
Briefly stated, the Wright's Stain packet of the present invention comprises a single use, air and liquid impermeable package which contains a slide sized pad impregnated with a buffered methanolic solution of Wright's Stain. When opened, the pad may be removed from the wrapper by tweezers and placed over the blood smear on a slide, whereupon it releases the stain solution in a controlled and uniform fashion. After about 30 seconds, the slide has become stained. The slide, with the pad still thereon, is rinsed in tap water. If the slide is held under a gentle stream of tap water, the pad floats away.
For further understanding of the inventors reference is made to the attached self-explanatory drawing wherein:
FIGS. 1, 2, 3, and 4 are diagrammatic views illustrating the structure and use of the Wright's stain packet.
As is apparent in the drawing, the packet 10 consists of a stain impregnated pad 14 sealed between upper and lower air and liquid impermeable wrapper sections 11 and 12, the sections being adhered together at 13 so as to enclose pad 14.
When wrapper sections 11, 12 are peeled apart, pad 14 may be removed by tweezer 15 and placed upon a slide 16. Subsequently the pad may be lifted off slide 16 by disposing slide 16 under the water stream 18 available from any convenient water tap 17.
A Wright's Stain single use packet, according to the present invention, offers many advantages. Thus, use of the stain is easier than staining by existing techniques, permitting less skilled personnel to Wright's Stain a slide. The pad applied staining solution eliminates the real, or at least the potential, mess of a bulk liquid staining technique. Working time for staining a slide is reduced. The need for a special buffer solution is eliminated altogether; existing water supplies may be employed for rinsing. The prepackaged Wright's Stain packet allows for a high degree of stain uniformity and reproduceability. Variations in staining characteristics attributable to differences solution to solution can be eliminated, through mass produced uniformity packet to packet. Moreover, the preferred formulation of the Wright's Stain solution herein described is relatively stable, thereby creating a high shelf life packet.
The packet wrapping itself forms no part of the present invention. Standard aluminum foil packing material commonly employed to package many products (such as for example, single servings of instant coffee) may be used to form a peelable, air-tight, liquid-tight wrapping for Wright's Stain impregnated pad. To repeat, the wrapping material for the packet must be both air impermeable and water impermeable.
In like fashion, the pad itself forms no part of the present invention, A conventional woven or non-woven thin fabric may be employed for purposes of the Wright's Stain impregnated pad. It is only necessary that the fibrous material be inert, nonreactive and capable of acting as a carrier with uniform impregnation. Its purpose is to cover the blood smeared slide, to spread the Wright Stain uniformly on the face of the slide. Thus, the pad should be about the size of the standard microscope slide employed for blood smear tests using Wright's Stain.
Although typical known Wright's Stain formulations may be employed for practice of the present invention, it has been found that a barbitol buffered Wright's Stain is particularly desirable for the single use packet of the present invention. It has also been found that inclusion of Giemsa Stain within the formulation produces superior slide results. Accordingly, a preferred mode of the invention is barbitol buffered Wright's Stain and Giemsa Stain in methanol formulation.
According to a preferred mode of the invention, such a formulation is prepared as follows:
Powdered certified Wright's Stain (5KG) is added slowly to hot methanol (350 liters) with constant stirring. The mixing is continued for 8 hours. Thereafter, the methanol solution is permitted to cool to room temperature and filtered (through Whatman No. 1 paper).
Separately, the powdered Giemsa stain (0.92KG) is similarly dissolved in hot methanol (125 liters), stirred, cooled and filtered as above.
The dye solutions are kept separate in sealed containers and aged for 2 weeks. During this period, occasional agitation is effected. At the end of the aging period, solution volumes are adjusted (by addition, appropriately, of methanol to 350 liters and 125 liters respectively), to make up any evaporation losses.
A buffer solution is prepared as follows:
I. Dissolve 0.340 kilograms of sodium chloride in 4.0 liters of water;
II. Prepare 0.1 normal hydrochloric acid (12.1);
III. Dissolve 0.353 kilograms of sodium barbitol and 0.233 kilograms of sodium acetate in 12.0 liters of water.
The actual buffer solution is prepared by mixing together 10.5 liters of the sodium barbitol containing solution, 10.5 liters of the 0.1 NHcl and the 4.0 liters of sodium chloride. The final pH of this solution should be pH 7.3 ± 0.1. If adjustment is necessary to achieve this pH, appropriate quantity of the remaining barbitol or acid solution is added.
The three solutions are now mixed together, stirred and the 500 liter total stored in polyethylene lined steel drums until use.
The mixed solutions are thereafter impregnated into 1 inch × 1.75 inches pads with 0.4ml per pad using conventional multi-needle automated injection and each impregnated pad is packaged in aluminum foil by conventional packaging machinery. The pad is a non-woven, mono-filament single layer fabric.