Fixative solution and method of preparation of biological sample

Abstract

A fixative contains carboxylic acid, preferably acetic acid, for parasitological samples. An exemplary aqueous fixative solution for parasitological samples comprises: a) a C2 to C6 carboxylic acid (preferably acetic acid) at a concentration of 0.5M or greater; and b) a C2 to C6 carboxylate salt or halide salt at a concentration of 0.02M or greater, preferably 0.04M or greater. An optional composition comprises 1.3M acetic acid, 12 mM sodium benzoate, 0.9% calcium chloride, 0.9% calcium bromide, 100 g/ml menthol and 350 g/ml thymol. The fixative is free of formaldehyde or other hazardous compounds, and exhibits good killing and staining properties when used with parasitological samples such as ova and helminths.

Claims

1. An aqueous fixative solution for biological samples comprising: a) a C.sub.2 to C.sub.6 carboxylic acid at a concentration of 0.9M or greater; b) an alkaline earth halide salt at a concentration of 0.02M or greater; and c) a biocidal essential oil component in a concentration of at least 50 g/ml, wherein the aqueous fixative solution is alcohol free.

2. An aqueous fixative solution for biological samples consisting of: a) a C.sub.2 to C.sub.6 carboxylic acid at a concentration of 0.9M or greater; b) an alkaline earth halide salt at a concentration of 0.02M or greater; c) a biocidal essential oil component in a concentration of at least 50 g/ml; and optionally one or more of: d) a bacteriostatic or fungistatic concentration of benzoic acid or sorbic acid or a salt thereof; e) a soluble carbohydrate; f) citric acid; and g) a surfactant.

3. An aqueous fixative solution for biological samples comprising: a) a C.sub.2 to C.sub.6 carboxylic acid at a concentration of 0.9M or greater; b) an alkaline earth halide salt at a concentration of 0.02M or greater; c) a biocidal essential oil component in a concentration of at least 50 g/ml; and d) a bacteriostatic or fungistatic concentration of benzoic acid or sorbic acid or a salt thereof, wherein: i) said carboxylic acid component a) is acetic acid at a concentration of 0.9M to 1.75M; ii) said alkaline earth halide salt component b) is calcium chloride at a concentration of 70 mM to 100 mM plus calcium bromide at a concentration of 30 mM to 50 mM; and iii said component (d) is a benzoic acid salt at a concentration of 5 mM to 15 mM benzoate.

4. An aqueous fixative solution for biological samples comprising: a) a C.sub.2 to C.sub.6 carboxylic acid at a concentration of 0.9M or greater; b) an alkaline earth halide salt at a concentration of 0.02M or greater; c) a biocidal essential oil component in a concentration of at least 50 g/ml; and d) a bacteriostatic or fungistatic concentration of benzoic acid or sorbic acid or a salt thereof, wherein: i) said carboxylic acid component a) is acetic acid at a concentration of 1.2M to 1.75M; ii) said alkaline earth halide salt component b) is calcium chloride at a concentration of 70 mM to 100 mM plus calcium bromide at a concentration of 30 mM to 50 mM; and iii) said component (d) is a benzoic acid salt at a concentration of 5 mM to 15 mM benzoate.

5. An aqueous fixative solution for biological samples comprising: a) a C.sub.2 to C.sub.6 carboxylic acid at a concentration of 0.9M or greater; b) an alkaline earth halide salt at a concentration of 0.02M or greater; c) a biocidal essential oil component in a concentration of at least 50 g/ml; and d) a bacteriostatic or fungistatic concentration of benzoic acid or sorbic acid or a salt thereof, wherein: i) said carboxylic acid component a) is acetic acid at a concentration of 0.9M to 1.75M; ii) said alkaline earth halide salt component b) is calcium chloride and/or calcium bromide at a total concentration of from 1.0M to 4.5M; and iii) said component (d) is a benzoic acid salt at a concentration of 5 mM to 125 mM benzoate.

Description

DETAILED DESCRIPTION

(1) A study was conducted to validate the effectiveness of a number of formulations of fixative in accordance with the invention in order to determine both their effectiveness to denature a cell (i.e. make inert from an infectious perspective) and to preserve cellular morphology for a prolonged period of time.

(2) To achieve this, three types of stain were used, namely: 1. Lugol's Iodine staining to determine morphology 2. Trypan Blue staining to determine viability 3. Trichrome stain commonly used laboratory procedure.

(3) Initially all substances were tested using a live sample of Giardia lamblia, which is a flagellated protozoan parasite commonly found around the world. Its abundance in numbers and delicate internal structures made this a suitable candidate for testing. Samples of Trichuris suis (whipworm) were also tested with the most viable formulation (Example 8) to see mainly whether the fixative was able to denature a typical helminth ova in an acceptable amount of time.

Example 1

(4) 200 ml of fixative was prepared with the following formulation: 185 ml distilled water 15 ml of Glacial Acetic Acid (7.5% solution1.3M) 0.06 g Sodium Benzoate2 mM 2.22 g Calcium Chloride100 mM

Example 2

(5) 200 ml of fixative was prepared with the following formulation: 181 ml distilled water 19 ml of Glacial Acetic Acid (9.5% solution1.65M) 0.36 g Sodium Benzoate12 mM 3.00 g Calcium Chloride100 mM 4.72 g Calcium Bromide

Example 3

(6) 200 ml of fixative was prepared with the following formulation: 181 ml distilled water 19 ml of Glacial Acetic Acid (9.5% solution1.65M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9%

Example 4

(7) 200 ml of fixative was prepared with the following formulation: 190 ml distilled water 10 ml of Glacial Acetic Acid (5% solution0.87M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9%

Example 5

(8) 200 ml of fixative was prepared with the following formulation: 181 ml distilled water 19 ml of Glacial Acetic Acid (9.5% solution1.65M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.04 g Menthol crystals200 g/ml (around half of maximum solubility) 0.125 g Thymol625 g/ml (around 70% of maximum solubility)

Example 6

(9) 200 ml of fixative was prepared with the following formulation: 181 ml distilled water 19 ml of Glacial Acetic Acid (9.5% solution1.65M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.02 g Menthol crystals100 g/ml (around a quarter of max solubility) 0.07 Thymol350 g/ml (around 39% of maximum solubility)

Example 7

(10) 200 ml of fixative was prepared with the following formulation: 190 ml distilled water 10 ml of Glacial Acetic Acid (5% solution0.87M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.02 g Menthol crystals100 g/ml (around a quarter of max solubility) 0.07 Thymol350 g/ml (around 39% of maximum solubility)

Example 8

(11) 200 ml of fixative was prepared with the following formulation: 185 ml distilled water 15 ml of Glacial Acetic Acid (7.5% solution1.3M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.02 g Menthol crystals100 g/ml (around a quarter of max solubility) 0.07 Thymol350 g/ml (around 39% of maximum solubility)

Example 9

(12) 200 ml of fixative was prepared with the following formulation: 185 ml distilled water 15 ml of Glacial Acetic Acid (7.5% solution1.3M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.04 g Menthol crystals200 g/ml (around a quarter of max solubility)

Example 10

(13) 200 ml of fixative was prepared with the following formulation: 185 ml distilled water 15 ml of Glacial Acetic Acid (7.5% solution1.3M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.07 Thymol350 g/ml (around 39% of maximum solubility)

Example 11

(14) 200 ml of fixative was prepared with the following formulation: 185 ml distilled water 15 ml of Glacial Acetic Acid (7.5% solution1.3M) 0.36 g Sodium Benzoate12 mM 1.8 g Calcium Chloride0.9% 1.8 g Calcium Bromide 0.9% 0.2 ml Triton X-1000.001% (The use of Trition surfactant negates the use of ELISA).

Example 12

(15) Live Giardia samples were received and checked for morphology and to ensure correct cellular structure etc.

(16) 200 l of Live Giardia were inculcated into each of the test solutions (3.3 ml) contained within a Mini Parasep SF faecal parasite concentrator device.

(17) This was carried out in duplicate, with: a. One sample containing just the fixative (Sample 1); and b. One sample containing the fixative mixed with a consistent sample of raw stool (Sample 2)

(18) This was immediately followed by centrifugation at 400 g for 2 minutes. A 15 l sample was then taken from the base of each sedimentation tube to be mounted on a microscope slide with 15 l of Trypan blue stain added. As viability of the cysts was the main reason for this step, each sample was centrifuged immediately after the addition of the Live Giardia, and mounted directly after centrifugation.

(19) Samples were left for a period of 24 hours and then 15 l was taken to check morphology with Lugol's iodine and Trypan Blue of Sample 1.

(20) Samples were left for a period of 48 hours and then 15 l was once again taken to check morphology with Lugol's iodine of Sample 1.

(21) Samples were left for a period of 5 days and then 15 l was once again taken to check morphology with Lugol's iodine of both samples.

(22) Trichrome stains were conducted for all samples which still showed good morphology, with samples being taken from Sample 1.

(23) Further morphology testing was carried out for all samples which passed this point after a period of 2 months.

(24) The results for the formulations of the above Examples are shown in the Table 1.0 below, together with comparative results for 10% formalin, the commercially available fixatives SAF (formaldehyde-based, containing methanol and also 2% acetic acid), a fixative solution containing methanol, ethanol, isopropanol, zinc sulphate and 4.8% acetic acid (sold under the trademark Alcorfix), PVA (containing methanol, ethanol and zinc sulphate) and a fixative solution containing 1-(cis-3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (sold under the trademark Safefix), and also distilled water, as a control.

(25) TABLE-US-00001 TABLE 1.0 Cell Cell Cell Cell Cell Cell Cell viability viability structure structure structure structure structure after 2 after 24 after 24 after 48 after 1 after 1 after 2 mins hours hours hours week week months (Trypan (Trypan (Lugol's (Lugol's (Lugol's (Trichrome (Lugol's Fixative blue) blue) Iodine) Iodine) Iodine) stain) Iodine) 10% Formalin Clear Good Excellent Good Good Good (cross- structure structure structure definition structure linking - test invalid) SAF Clear Excellent Excellent Excellent Reasonable Acceptable (cross- structure structure structure definition structure linking - test invalid) Alcorfix Minimal Dark blue Excellent Excellent Excellent Good Excellent blue in ingress structure structure structure definition structure cell wall (Cell (start of death) cell death) PVA Clear Dark blue Excellent Good Excellent Good Excellent (Cell ingress structure structure structure definition structure viable) (Cell death) SafeFix Clear Clear (Cell Acceptable Excellent Good Poor (Cell appears structure structure structure structure viable) viable) Distilled Clear Clear (Cell Excellent Acceptable Poor Poor Poor/Complete water (Cell viable) structure structure structure definition cellular viable) degeneration Example 1 Dark Dark blue Excellent Excellent Excellent Good Good blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 2 Dark Dark blue Excellent Excellent Excellent Reasonable Excellent blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 3 Dark Dark blue Excellent Excellent Excellent Reasonable Good blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 4 Dark Dark blue Excellent Excellent Excellent Poor Excellent blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 5 Dark Dark blue Excellent Excellent Excellent Reasonable Excellent blue in in cell wall structure structure structure definition structure cell wall and dark (Cell blue death) cytoplasm (Cell death) Example 6 Dark Dark blue Excellent Excellent Excellent Reasonable Excellent blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 7 Dark Dark blue Excellent Excellent Excellent Good Good blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 8 Dark Dark blue Excellent Excellent Excellent Excellent Excellent blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 9 Dark Dark blue Excellent Excellent Excellent Good Good blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 10 Dark Dark blue Excellent Excellent Excellent Good Good blue in in cell wall structure structure structure definition structure cell wall and light (Cell blue death) cytoplasm (Cell death) Example 11 Dark Dark blue Excellent Acceptable Excellent Poor blue in in cell wall structure structure structure definition cell wall and dark (Cell blue death) cytoplasm similar to Alcorfix (Cell death)

(26) The fixatives of Examples 5 to 11 gave particularly good results, with the Example 8 formulation currently being considered the best.

(27) Preferred aqueous fixative solutions of the present invention can be used with e.g. faecal parasite concentrator devices.

(28) Such devices are commercially available e.g. as the Parasep or ParaPRO devices or the devices shown in WO2017/187165A, in the name of the present applicants, for example.

(29) Such devices typically utilise a mixing chamber and a receiving chamber separated by a filter.

(30) In use, a fixative solution and a faecal sample are introduced into the mixing chamber and agitated in contact to liquefy the sample. The liquified sample is then filtered and the filtrate, (containing e.g. ova and cysts which pass through the filter) is collected in the receiving chamber.

(31) The fixative solution ensures that the filtrate is not biologically hazardous. The filtrate can then be examined under the microscope to identify e.g. the ova and cysts.

(32) For example, a mini Parasep device can be used, which will typically hold 3.3 ml of fixative solution within the mixing tube. A 0.5-1.0 g sample of stool is added to the mixing tube before the tube is sealed with a vertical filter attached to a sedimentation tube. The sample is then vortexed, inverted and typically centrifuged for 2 minutes at 400 g (dependent on filter version). The filter and mixing tube can then be removed and a sample taken from the pellet found at the base of the sedimentation tube. This is then mounted on a microscope slide, where a stain may be added before the sample is examined for the presence of parasitic organisms.

(33) In some cases, it is desirable for the fixative solution to float the ova and cysts so that they can be more readily identified and separated from the solution. Such solutions are known as flotation solutions.

(34) Such flotation solutions can for example be used in a mini Parasep device as follows:

(35) A 0.5-1.0 g sample of stool is added to the mixing tube before the tube is sealed with a vertical filter attached to a sedimentation tube. The sample is then vortexed, inverted and squeezed by hand to pressurise the vessel, forcing the filtration of the sample. The solution is left to rest for 3 minutes, before an inoculation loop is used to remove a layer from the top of the liquid (which will be holding the majority of the parasitic ova and cysts if present). This is then mounted on a microscope slide, where a stain may be added before the sample is examined for the presence of parasitic organisms.

(36) The following Examples relate to formulations of flotation solutions. The volumes of fixative are approximate whereas the masses and (in the case of glacial acetic acid) volume of the components are exact. The specific gravity values (density compared to pure water) are also exact.

(37) A series of high specific gravity fixative solutions were prepared as follows:

Example 13

(38) Approximately 500 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 250 g cane sugar 18.75 ml of Glacial Acetic Acid (3.75 v/v % solution0.66M) 0.45 g Sodium Benzoate0.09% (6.25 mM) 2.25 g Calcium Chloride0.45% (0.041M) 2.25 g Calcium Bromide hydrate0.41% (0.021M) 0.025 g Menthol0.005% 0.0875 g Thymol0.0175%

(39) The specific gravity of the resulting fixative was 1.265.

(40) This formulation was one of the most effective of the high density formulations in Examples 13 to 28. In a variant of this formulation (Example 28), a higher proportion of cane sugar was employed in order to raise the specific gravity of the fixative solution to about 1.29.

Example 14

(41) Approximately 500 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 250 g cane sugar 18.75 ml of Glacial Acetic Acid (3.75 v/v % solution0.66M) 1.8 g Sodium Benzoate0.36% (25 mM) 40 g Calcium Chloride8.0% (0.72M) 2.25 g Calcium Bromide hydrate0.41% (0.021M) 0.025 g Menthol0.005% 0.0875 g Thymol0.0175%

(42) The specific gravity of the resulting fixative was 1.245.

Example 15

(43) Approximately 500 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 230 g cane sugar 18.75 ml of Glacial Acetic Acid (3.75 v/v % solution0.66M) 0.5 g Sodium Benzoate0.10% (6.9 mM) 60 g Calcium Chloride12.0% (1.08M) 2.25 g Calcium Bromide hydrate0.41% (0.021M) 0.025 g Menthol0.005% 0.0875 g Thymol0.0175%

(44) The specific gravity of the resulting fixative was 1.272

Example 16

(45) Approximately 500 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 200 g cane sugar 18.75 ml of Glacial Acetic Acid (3.75 v/v % solution0.66M) 0.5 g Sodium Benzoate0.10% (6.9 mM) 60 g Calcium Chloride12.0% (1.08M) 2.25 g Calcium Bromide hydrate0.41% (0.021M) 0.025 g Menthol0.005% 0.0875 g Thymol0.0175%

(46) The specific gravity of the resulting fixative was 1.297.

Example 17

(47) Approximately 500 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 180 g cane sugar 18.75 ml of Glacial Acetic Acid (3.75 v/v % solution0.66M) 0.25 g Sodium Benzoate0.050% (3.5 mM) 80 g Calcium Chloride16.0% (1.44M) 2.25 g Calcium Bromide hydrate0.41% (0.021M) 0.025 g Menthol0.005% 0.0875 g Thymol0.0175%

(48) The specific gravity of the resulting fixative was 1.324.

Example 18

(49) Approximately 850 ml of fixative was prepared with the following formulation: 462.5 ml de-ionised water 200 g cane sugar 37.5 ml of Glacial Acetic Acid (4.4 v/v % solution0.66M) 0.50 g Sodium Benzoate0.059% (3.5 mM) 390 g Calcium Chloride dihydrate34.7% (3.12M) 4.5 g Calcium Bromide hydrate0.48% (0.024M) 0.050 g Menthol0.0059% 0.175 g Thymol0.021%

(50) The specific gravity of the resulting fixative was 1.310.

Example 19

(51) Approximately 400 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 50 g cane sugar 18.75 ml of Glacial Acetic Acid (4.7 v/v % solution0.82M) 0.25 g Sodium Benzoate0.063% (4.33 mM) 250 g Calcium Chloride dihydrate47.1% (4.25M) 2.25 g Calcium Bromide hydrate0.52% (0.026M) 0.025 g Menthol0.0063% 0.0875 g Thymol0.022%

(52) The specific gravity of the resulting fixative was 1.360.

Example 20

(53) Approximately 450 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 25 g cane sugar 21 ml of Glacial Acetic Acid (4.7 v/v % solution0.82M) 0.25 g Sodium Benzoate0.055% (3.89 mM) 250 g Calcium Chloride dihydrate42.0% (3.78M) 2.25 g Calcium Bromide hydrate0.46% (0.023M) 0.025 g Menthol0.0056% 0.0875 g Thymol0.019%

(54) The specific gravity of the resulting fixative was 1.330.

Example 21

(55) Approximately 850 ml of fixative was prepared with the following formulation: 462.5 ml de-ionised water 360 g cane sugar 37.5 ml of Glacial Acetic Acid (4.4 v/v % solution0.77M) 0.50 g Sodium Benzoate0.059% (4.08 mM) 200 g Calcium Chloride dihydrate17.8% (1.60M) 4.5 g Calcium Bromide hydrate0.49% (0.024M) 0.050 g Menthol0.0059% 0.175 g Thymol0.021%

(56) The specific gravity of the resulting fixative was 1.308.

Example 22

(57) Approximately 950 ml of fixative was prepared with the following formulation: 550 ml de-ionised water 360 g cane sugar 37.5 ml of Glacial Acetic Acid (3.9 v/v % solution0.69M) 200 g Calcium Chloride dihydrate15.9% (1.43M) 4.5 g Calcium Bromide hydrate0.44% (0.022M) 0.050 g Menthol0.0053% 0.175 g Thymol0.018%

(58) The specific gravity of the resulting fixative was 1.280.

Example 23

(59) Approximately 820 ml of fixative was prepared with the following formulation: 500 ml de-ionised water 280 g cane sugar 37.5 ml of Glacial Acetic Acid (4.5 v/v % solution0.80M) 200 g Calcium Chloride dihydrate18.4% (1.66M) 4.5 g Calcium Bromide hydrate0.51% (0.025M) 0.050 g Menthol0.0061% 0.175 g Thymol0.021%

(60) The specific gravity of the resulting fixative was 1.270.

Example 24

(61) Approximately 800 ml of fixative was prepared with the following formulation: 500 ml de-ionised water 200 g cane sugar 37.5 ml of Glacial Acetic Acid (4.9 v/v % solution0.82M) 240 g Calcium Chloride30.9% (2.70M) 4.5 g Calcium Bromide hydrate0.52% (0.027M) 0.050 g Menthol0.0063% 0.175 g Thymol0.022%

(62) The specific gravity of the resulting fixative was 1.330.

Example 25

(63) Approximately 420 ml of fixative was prepared with the following formulation: 231.25 ml de-ionised water 100 g cane sugar 18.75 ml of Glacial Acetic Acid (4.5 v/v % solution0.78M) 0.45 g Sodium Benzoate0.11% (7.44 mM) 15 g Calcium Chloride3.57% (0.32M) 2.25 g Calcium Bromide hydrate0.49% (0.025M) 0.025 g Menthol0.0059% 0.0875 g Thymol0.021% 30 g Citric acid7.1% (0.037M)

(64) The specific gravity of the resulting fixative was 1.288.

Example 26

(65) Approximately 400 ml of fixative was prepared with the following formulation: 250 ml de-ionised water 100 g cane sugar 20.0 ml of Glacial Acetic Acid (5.0 v/v % solution0.87M) 0.45 g Sodium Benzoate0.11% (7.44 mM) 90 g Calcium Chloride22.5% (2.03M) 2.25 g Calcium Bromide hydrate0.52% (0.026M) 0.025 g Menthol0.0063% 0.0875 g Thymol0.022% 60 g Citric acid15.0% (0.78M)

(66) The specific gravity of the resulting fixative was 1.296.

Example 27

(67) Approximately 420 ml of fixative was prepared with the following formulation: 250 ml de-ionised water 50 g cane sugar 20.0 ml of Glacial Acetic Acid (4.8 v/v % solution0.83M) 0.45 g Sodium Benzoate0.11% (7.44 mM) 200 g Calcium Chloride dihydrate 36.0% (3.24M) 2.25 g Calcium Bromide hydrate 0.49% (0.025M) 0.025 g Menthol0.0059% 0.0875 g Thymol0.021% 60 g Citric acid14.3% (0.74M)

(68) The specific gravity of the resulting fixative was 1.337.

Example 28

(69) Approximately 5100 ml of fixative was prepared with the following formulation: 2300 ml de-ionised water 3750 g cane sugar 375 ml of Glacial Acetic Acid (7.4 v/v % solution1.3M) 4.5 g Sodium Benzoate0.88% (6.1 mM) 60 g Calcium Chloride1.2% (0.11M) 30 g Calcium Bromide hydrate 0.59% (0.027M) 0.25 g Menthol0.049% 0.875 g Thymol0.017%

(70) The specific gravity of the resulting fixative was approximately 1.29.

(71) This SG formulation is the most preferred of Examples 14 to 28.

(72) Magnesium halides may be substituted for the calcium halides in the Examples.

(73) It is considered that similar results would be obtained if the concentrations of the acetic acid in the Examples were varied by about 0.3M or more and that acceptable results would be obtained over broader ranges as defined in the claims. It is considered that similar results would be obtained if the concentrations of the remaining components in the Examples were varied by at least 10%, possibly 20% or more of their stated values and that acceptable results would be obtained over a wider range of concentrations.

(74) In general, the upper limit of halide salt concentration is set only by the solubility of each particular salt in the fixative solution.