RU2793545C2 - Fixing solution and method for obtaining a biological sample for testing - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a fixing solution for histological fixation of biological samples, in particular, parasitological samples, and can be used for testing thereof, e.g., by microscopy. Aqueous fixing solution for biological samples contains C2-C6 carboxylic acid at a concentration of 0.9 M or more, an alkaline earth halide salt at a concentration of 0.02 M or more, and a biocidal essential oil component at a concentration of at least 50 mcg/ml. The invention also relates to a method for obtaining a biological sample for testing, including fixing the sample by means of said aqueous fixing solution.

EFFECT: invention reduces the time required for fixation, ensures rapid death of biological organisms, such as helminth eggs, without the need for a toxic additive, such as formaldehyde, while retaining the cellular structure, and also leads to fine staining properties.

15 cl, 1 tbl, 28 ex

Description

The field of technology to which the invention belongs

The present invention relates to a fixative solution for histological fixation of biological samples and to a method for obtaining biological samples for examination, for example, using microscopy.

Prior Art

The invention relates in particular, but not exclusively, to a fixing solution suitable for fixing living organisms, in particular parasitological specimens such as, for example, helminth eggs or Giardia coli.

For such applications, a fixative solution is required to kill the organism (which may be pathogenic) and to preserve its cellular structure so that it can then be examined microscopically, usually after staining with a dye such as trypan blue, Lugol's solution, or, for example, using trichrome staining. The microscopic appearance of a sample after staining is affected by the fixative solution, but it is difficult or impossible to predict the effect of a given fixative composition on staining properties.

Formaldehyde is widely used in a fixative solution (eg, formalin in water), but is toxic, carcinogenic, and generally hazardous to the environment.

Other fixatives are used, but in many cases they are also dangerous.

More recently, non-hazardous fixatives have been developed, such as those disclosed in GB 2528426A (Reardon) and WO 92/19951A (Streck Lab), but there remains a need for a fixative solution that will reliably kill parasitic organisms such as helminths, which, having a strong outer layer, have a high resistance to the penetration of the fixative. If highly toxic chemicals such as formaldehyde are excluded, it is difficult to achieve cell death in a short time.

Therefore, it is an object of the present invention to provide a relatively benign fixative which is particularly suitable for the histological fixation of parasitological specimens.

A further object of the invention is to provide a fixative and a method for obtaining biological samples that result in good staining properties.

The essence of the invention

Accordingly, the invention provides an aqueous fixative solution for biological specimens, comprising:

a) C ₂ -C ₆ carboxylic acid at a concentration of 0.5 M or more,

b) C ₂ -C ₆ carboxylic acid salt or halide salt at a concentration of 0.02 M or more, preferably 0.04 M or more.

Optionally, said C ₂ -C ₆ carboxylic acid is acetic acid.

Optionally, the concentration of C ₂ -C ₆ carboxylic acid is 0.9 M or more, more preferably 1.3 M or more. Optionally, the concentration of C ₂ -C ₆ carboxylic acid is not more than 3.5 M.

It has been found that such concentrations of carboxylic acid (especially acetic acid) lead to the rapid death of biological organisms such as helminth eggs, for example, without the need for a toxic additive such as formaldehyde.

In an optional but preferred embodiment, the concentration of acetic acid is less than 10% w/v. Concentrations below this limit are classified as non-corrosive.

The carboxylic acid salt or halide salt of component b) may, for example, be an alkali or alkaline earth metal or ammonium salt, another cation which imparts acceptable solubility.

Optionally, component b) is a calcium or magnesium halide salt.

More preferably component b) contains calcium chloride at a concentration of at least 0.06 M, preferably at least 0.08 M, preferably at least 0.1 M and/or calcium bromide at a concentration of at least 0.06 M , preferably at least 0.08 M, preferably at least 0.1 M.

Such halide salts have been found to help maintain cellular structure, possibly an osmoregulatory effect involving the halide salts counteracting the turgor effect of the carboxylic acid.

Optionally, the fixative further comprises: c) benzoic acid or sorbic acid, or salts thereof, at a bacteriostatic or fungistatic concentration.

Optionally, component c) is a salt of benzoic acid at a concentration of at least 5 mM, more preferably at least 10 mM.

Component c) as defined above can help prevent sample degradation.

Optionally, the fixative further comprises: d) a biocidal ester oil component at a concentration of at least 50 μg/ml.

The biocidal essential oil may be, for example, bacteriostatic or bactericidal or fungistatic or fungicidal.

Optionally, said biocidal essential oil component d) is one or more of: menthol, thymol, pulegol, dihydrocarveol, piperitol, menthone, menthene, o-cymene, p-cymene, citronellal, carvacrol, eugenol, 4-carvo-menthol ( termpinen-4-ol), allicin or camphor. Optionally, the essential oil component is present in its natural stereoisomeric or enantiomeric form.

Desirably, said biocidal ester oil component d) contains menthol and thymol.

These essential oils have been found to facilitate cell death and thus shorten the time required for fixation.

In preferred but optional embodiments:

i) said carboxylic acid component a) is acetic acid at a concentration of 0.8-1.75 M,

ii) said halide salt component b) is calcium chloride at a concentration of 70-100 mM plus calcium bromide at a concentration of 30-50 mM, and

iii) said component of the salt of benzoic acid d) is benzoate at a concentration of 5-15 mM.

In particularly preferred, but still optional, embodiments:

i) said carboxylic acid component a) is acetic acid at a concentration of 1.2-1.75 M,

ii) said halide salt component b) is calcium chloride at a concentration of 70-100 mM plus calcium bromide at a concentration of 30-50 mM, and

iii) said component of the salt of benzoic acid d) is benzoate at a concentration of 5-15 mM.

The above embodiments can advantageously lead to particularly good coloring properties.

In a further preferred embodiment:

i) said carboxylic acid component a) is acetic acid at a concentration of 0.8-1.75 M,

ii) said halide salt component b) is calcium chloride and/or calcium bromide in a total concentration of 1.0-4.5 M, and

iii) said benzoic acid salt component d) is benzoate at a concentration of 5-125 mM.

In some preferred embodiments, component b) is a halide salt at a concentration of 0.3-6M, preferably 1-5M, most preferably 1-4M.

Such embodiments have been found to be suitable for use as a flotation solution having a density large enough for cysts or eggs or other organisms of interest to float, thereby facilitating their separation and identification.

In such embodiments, the fixative optionally contains a non-polar solute which increases the density of the fixative solution to greater than 1.1 g/ml, preferably greater than 1.2 g/ml, most preferably 1.2-1.4 g/ml. In one embodiment, the density of the aqueous fixative solution is sufficient to float eggs or larvae or cysts or oocysts in the parasitological sample and is preferably in the range of 1.2-1.4 g/ml, more preferably 1.25-1. 35 g/ml.

Preferably, the non-polar solute is a soluble carbohydrate, preferably a sugar, preferably sucrose or glucose, or fructose, or maltose, or any combination thereof.

In a further aspect of the invention that requires protection, there is provided a method for obtaining a biological sample for examination, comprising fixing the sample with an aqueous fixative solution as defined above.

Optionally, the biological sample is a living organism. Optionally, the biological sample is a parasitological sample.

In a preferred embodiment, the biological sample is stained after fixation, for example, with an azo dye, or with iodine, or with a trichrome dye. You can also use other dyes.

Other preferred features are defined in the claims.

It is expressly intended within the scope of this application that the various aspects, embodiments, examples and alternatives set forth in the preceding paragraphs, in the claims and/or in the following description, and in particular their individual features, may be taken independently or in any combination. . That is, all embodiments and/or features of any embodiment or example may be combined in any suitable manner and/or in any combination, as long as such features are not incompatible. Applicant reserves the right to change any claim originally filed or file any new claim, as appropriate, including the right to change any claim originally filed to depend on and/or include any feature of any other claim, though not so originally claimed.

Detailed description of the invention

A study was conducted to test the effectiveness of a number of fixative formulations of the invention, to determine both their effectiveness in destroying the cell (i.e. making it inert in terms of infectivity) and in maintaining cell morphology over an extended period of time.

To achieve this, three types of staining were used, namely:

1. Staining with Lugol's solution - to determine the morphology.

2. Trypan blue staining - to determine viability.

3. Trichrome staining is a widely used laboratory procedure.

First, all substances were tested using a live sample of Giardia coli, which is a flagellated protozoan parasite that is widely distributed throughout the world. Its copious amounts and fragile internal structures make it a suitable candidate for testing. Samples of Trichuris suis (whiskerhead) were also tested with the most appropriate formulation (Example 8), mainly to see if the fixative was able to break down common helminth eggs in a reasonable time.

EXAMPLE 1

We received 200 ml of the fixative of the following composition:

• 185 ml distilled water

• 15 ml of glacial acetic acid (7.5% solution - 1.3 M)

• 0.06 g sodium benzoate - 2 mM

• 2.22 g calcium chloride - 100 mM

EXAMPLE 2

We received 200 ml of the fixative of the following composition:

• 181 ml distilled water

• 19 ml of glacial acetic acid (9.5% solution - 1.65 M)

• 0.36 g sodium benzoate - 12 mM

• 3.00 g calcium chloride - 100 mM

• 4.72 g calcium bromide

EXAMPLE 3

We received 200 ml of the fixative of the following composition:

• 181 ml distilled water

• 19 ml of glacial acetic acid (9.5% solution - 1.65 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

EXAMPLE 4

We received 200 ml of the fixative of the following composition:

• 190 ml distilled water

• 10 ml glacial acetic acid (5% solution - 0.87 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

EXAMPLE 5

We received 200 ml of the fixative of the following composition:

• 181 ml distilled water

• 19 ml of glacial acetic acid (9.5% solution - 1.65 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.04 g of menthol crystals - 200 mcg/ml (approximately half of the maximum solubility)

• 0.125 g thymol - 625 mcg / ml (approximately 70% of the maximum solubility)

EXAMPLE 6

We received 200 ml of the fixative of the following composition:

• 181 ml distilled water

• 19 ml of glacial acetic acid (9.5% solution - 1.65 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.02 g of menthol crystals - 100 mcg/ml (approximately a quarter of the maximum solubility)

• 0.07 g thymol - 350 mcg/ml (approximately 39% maximum solubility)

EXAMPLE 7

We received 200 ml of the fixative of the following composition:

• 190 ml distilled water

• 10 ml glacial acetic acid (5% solution - 0.87 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.02 g of menthol crystals - 100 mcg/ml (approximately a quarter of the maximum solubility)

• 0.07 g thymol - 350 mcg/ml (approximately 39% maximum solubility)

EXAMPLE 8

We received 200 ml of the fixative of the following composition:

• 185 ml distilled water

• 15 ml of glacial acetic acid (7.5% solution - 1.3 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.02 g of menthol crystals - 100 mcg/ml (approximately a quarter of the maximum solubility)

• 0.07 g thymol - 350 mcg/ml (approximately 39% maximum solubility)

EXAMPLE 9

We received 200 ml of the fixative of the following composition:

• 185 ml distilled water

• 15 ml of glacial acetic acid (7.5% solution - 1.3 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.04 g of menthol crystals - 200 mcg/ml (approximately a quarter of the maximum solubility)

EXAMPLE 10

We received 200 ml of the fixative of the following composition:

• 185 ml distilled water

• 15 ml of glacial acetic acid (7.5% solution - 1.3 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.07 g thymol - 350 mcg/ml (approximately 39% maximum solubility)

EXAMPLE 11

We received 200 ml of the fixative of the following composition:

• 185 ml distilled water

• 15 ml of glacial acetic acid (7.5% solution - 1.3 M)

• 0.36 g sodium benzoate - 12 mM

• 1.8 g of calcium chloride - 0.9%

• 1.8 g of calcium bromide - 0.9%

• 0.2 ml Triton X-100 - 0.001%

(The use of Triton® surfactant overrides the use of ELISA).

EXAMPLE 12

Samples of live giardia were obtained and checked for morphology to ensure correct cell structure and so on.

Into each of the test solutions (3.3 ml) contained in the Mini Parasep SF® Fecal Parasite Concentrator, 200 μl of live giardia were injected.

This was done in two repetitions with:

a. one sample containing only fixative (Sample 1); And

b. one sample containing fixative mixed with an appropriate sample of untreated faeces (Sample 2).

This was immediately followed by centrifugation at 400 g for 2 minutes. Then, 15 µl of the sample was taken from the bottom of each centrifuge tube, which was placed on a microscope slide with the addition of 15 µl of trypan blue dye. Since the main reason for this step was the viability of the cysts, each sample was centrifuged immediately after the addition of live giardia and placed directly after centrifugation.

Samples were left for a period of 24 hours, and then 15 µl was taken for morphology testing with Lugol's solution and trypan blue from sample 1.

Samples were left for a period of 48 hours, and then 15 µl was taken again for morphology testing with Lugol's solution from sample 1.

Samples were left for a period of 5 days, and then 15 μl was again taken for morphology testing with Lugol's solution from both samples.

All samples that showed good morphology were trichrome stained, with samples taken from Sample 1.

Additional morphology testing was performed on all samples that passed this point after a period of 2 months.

the results for the formulations of the above examples are shown in Table 1.0 below, along with comparative results for 10% formalin, commercially available SAF fixatives (formaldehyde-based, containing methanol as well as ≤ 2% acetic acid), Alcorfix (containing methanol, ethanol, isopropanol, zinc sulfate and 4.8% acetic acid), PVA (containing methanol, ethanol and zinc sulfate) and Safefix (containing 1-(cis-3-chloroallyl)-3,5,7-triaza-1-azoniadamantane chloride), and also distilled water as a control.

TABLE 1.0

Retainer Cell viability after 2 min
(trypan blue) Cell viability after 24 hours
(trypan blue) Cell structure after 24 hours
(Lugol's solution) Cell structure after 48 hours
(Lugol's solution) Cell structure after 1 week
(Lugol's solution) Cell structure after 1 week
(trichrome staining) Cell structure after 2 months
(Lugol's solution) 10% formalin Transparent (cross-linking - test invalid) good structure Fine structure good structure good clarity good structure SAF Transparent (cross-linking - test invalid) Fine structure Fine structure Fine structure Acceptable clarity Valid Structure Alcorfix Minimal blue in the cell wall (beginning of cell death) Dark blue penetration (cell death) Fine structure Fine structure Fine structure good clarity Fine structure PVA Transparent (Cell is viable) Dark blue penetration (cell death) Fine structure good structure Fine structure good clarity Fine structure SafeFix Transparent (Cell is viable) Transparent (Cell looks viable) Valid Structure Fine structure good structure Weak structure Distilled water Transparent (Cell is viable) Transparent (Cell is viable) Fine structure Valid Structure Weak structure Weak clarity Weak/complete cell breakdown Example 1 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure good clarity good structure Example 2 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure Acceptable clarity Fine structure Example 3 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure Acceptable clarity good structure Example 4 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure Weak clarity Fine structure Example 5 Dark blue in the cell wall (cell death) Dark blue in cell wall and dark blue cytoplasm (cell death) Fine structure Fine structure Fine structure Acceptable clarity Fine structure Example 6 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure Acceptable clarity Fine structure Example 7 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure good clarity good structure Example 8 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure Excellent clarity Fine structure Example 9 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure good clarity good structure Example 10 Dark blue in the cell wall (cell death) Dark blue in the cell wall and light blue in the cytoplasm (cell death) Fine structure Fine structure Fine structure good clarity good structure Example 11 Dark blue in the cell wall (cell death) Dark blue in cell wall and dark blue cytoplasm similar to Alcorfix (cell death) Fine structure Valid Structure Fine structure Weak clarity

The fixatives of Examples 5-11 showed particularly good results, the composition of Example 8 is currently considered the best.

The preferred aqueous fixative solution of the present invention can be used, for example, with faecal parasite concentrates.

Such devices are on the market, for example, as Parasep® or ParaPRO® devices or devices shown, for example, in WO2017/187165A to the authors of the present application.

Such devices typically use a mixing chamber and a receiving chamber separated by a filter.

In use, the fixative solution and the fecal sample are introduced into the mixing chamber and agitated in contact to liquefy the sample. The liquefied sample is then filtered and the filtrate (containing, for example, eggs and cysts that pass through the filter) is collected in a receiving chamber.

The fixative solution ensures that the filtrate is not biohazardous. The filtrate can then be examined under a microscope to identify, for example, eggs and cysts.

For example, a Mini Parasep® device can be used, which will typically contain 3.3 ml of fixative solution inside a mixing tube. 0.5-1.0 g of a faecal sample is added to a mixing tube before capping the tube with a vertical filter attached to the centrifuge tube. The sample is then shaken, inverted and typically centrifuged for 2 minutes at 400 g (depending on the filter option). The filter and mixing tube can then be removed and the sample taken from the pellet at the bottom of the centrifuge tube. It is then placed on a microscope slide where dye can be added before the sample is examined for parasitic organisms.

In some cases, it is desirable to have eggs and cysts floating in the fixative solution so that they can be more easily identified and isolated from the solution. Such solutions are known as flotation solutions.

Such flotation solutions, for example, can be used in the Mini Parasep® device as follows:

a 0.5-1.0 g faecal sample is added to the mixing tube before capping the tube with a vertical filter attached to the centrifuge tube. The sample is then shaken, inverted, and squeezed by hand to pressurize the vial to help filter the sample. The solution is left for 3 minutes before using the inoculation loop to remove the layer from the top of the liquid (which will contain most of the eggs and parasite cysts, if any). It is then placed on a microscope slide where dye can be added before the sample is examined for parasitic organisms.

The following examples refer to flotation solution compositions, fixative volumes are approximate, while the weights and (in the case of glacial acetic acid) component volumes are exact. Specific gravity values (density compared to pure water) are also accurate.

A series of high specific gravity fixing solutions was prepared as follows.

EXAMPLE 13

Received approximately 500 ml fixative of the following composition:

• 231.25 ml deionized water

• 250 g cane sugar

• 18.75 ml glacial acetic acid (solution 3.75 v/v% - 0.66 M)

• 0.45 g sodium benzoate - 0.09% (6.25 mm)

• 2.25 g of calcium chloride - 0.45% (0.041 M)

• 2.25 g of calcium bromide hydrate - 0.41% (0.021 M)

• 0.025 g menthol - 0.005%

• 0.0875 g thymol - 0.0175%

the specific gravity of the resulting fixative was 1.265.

This formulation was one of the most effective high density formulations in Examples 13-28. In a variant of this formulation (Example 28), a higher proportion of cane sugar was used to increase the specific gravity of the fixer to approximately 1.29.

EXAMPLE 14

Received approximately 500 ml fixative of the following composition:

• 231.25 ml deionized water

• 250 g cane sugar

• 18.75 ml glacial acetic acid (solution 3.75 v/v% - 0.66 M)

• 1.8 g sodium benzoate - 0.36% (25 mm)

• 40 g calcium chloride - 8.0% (0.72 M)

• 2.25 g of calcium bromide hydrate - 0.41% (0.021 M)

• 0.025 g menthol - 0.005%

• 0.0875 g thymol - 0.0175%

the specific gravity of the resulting fixative was 1.245.

EXAMPLE 15

Received approximately 500 ml fixative of the following composition:

• 231.25 ml deionized water

• 230 g cane sugar

• 18.75 ml glacial acetic acid (solution 3.75 v/v% - 0.66 M)

• 0.5 g sodium benzoate - 0.10% (6.9 mm)

• 60 g calcium chloride - 12.0% (1.08 M)

• 2.25 g of calcium bromide hydrate - 0.41% (0.021 M)

• 0.025 g menthol - 0.005%

• 0.0875 g thymol - 0.0175%

the specific gravity of the resulting fixative was 1.272

EXAMPLE 16

Received approximately 500 ml fixative of the following composition:

• 231.25 ml deionized water

• 200 g cane sugar

• 18.75 ml glacial acetic acid (solution 3.75 v/v% - 0.66 M)

• 0.5 g sodium benzoate - 0.10% (6.9 mm)

• 60 g calcium chloride - 12.0% (1.08 M)

• 2.25 g of calcium bromide hydrate - 0.41% (0.021 M)

• 0.025 g menthol - 0.005%

• 0.0875 g thymol - 0.0175%

the specific gravity of the resulting fixative was 1.297.

EXAMPLE 17

Received approximately 500 ml fixative of the following composition:

• 231.25 ml deionized water

• 180 g cane sugar

• 18.75 ml glacial acetic acid (solution 3.75 v/v% - 0.66 M)

• 0.25 g sodium benzoate - 0.050% (3.5 mm)

• 80 g calcium chloride - 16.0% (1.44 M)

• 2.25 g of calcium bromide hydrate - 0.41% (0.021 M)

• 0.025 g menthol - 0.005%

• 0.0875 g thymol - 0.0175%

the specific gravity of the resulting fixative was 1.324.

EXAMPLE 18

Received approximately 850 ml fixative of the following composition:

• 462.5 ml deionized water

• 200 g cane sugar

• 37.5 ml glacial acetic acid (solution 4.4 v/v% - 0.66 M)

• 0.50 g sodium benzoate - 0.059% (3.5 mm)

• 390 g calcium chloride dihydrate - 34.7% (3.12 M)

• 4.5 g calcium bromide hydrate - 0.48% (0.024 M)

• 0.050 g menthol - 0.0059%

• 0.175 g thymol - 0.021%

the specific gravity of the resulting fixative was 1.310.

EXAMPLE 19

Received approximately 400 ml fixative of the following composition:

• 231.25 ml deionized water

• 50 g cane sugar

• 18.75 ml glacial acetic acid (solution 4.7 v/v% - 0.82 M)

• 0.25 g sodium benzoate - 0.063% (4.33 mm)

• 250 g calcium chloride dihydrate - 47.1% (4.25 M)

• 2.25 g calcium bromide hydrate - 0.52% (0.026 M)

• 0.025 g menthol - 0.0063%

• 0.0875 g thymol - 0.022%

the specific gravity of the resulting fixative was 1.360.

EXAMPLE 20

Received approximately 450 ml fixative of the following composition:

• 231.25 ml deionized water

• 25 g cane sugar

• 21 ml glacial acetic acid (solution 4.7 v/v% - 0.82 M)

• 0.25 g sodium benzoate - 0.055% (3.89 mm)

• 250 g calcium chloride dihydrate - 42.0% (3.78 M)

• 2.25 g calcium bromide hydrate - 0.46% (0.023 M)

• 0.025 g menthol - 0.0056%

• 0.0875 g thymol - 0.019%

the specific gravity of the resulting fixative was 1.330.

EXAMPLE 21

Received approximately 850 ml fixative of the following composition:

• 462.5 ml deionized water

• 360 g cane sugar

• 37.5 ml glacial acetic acid (solution 4.4 v/v% - 0.77 M)

• 0.50 g sodium benzoate - 0.059% (4.08 mm)

• 200 g calcium chloride dihydrate - 17.8% (1.60 M)

• 4.5 g calcium bromide hydrate - 0.49% (0.024 M)

• 0.050 g menthol - 0.0059%

• 0.175 g thymol - 0.021%

the specific gravity of the resulting fixative was 1.308.

EXAMPLE 22

Received approximately 950 ml fixative of the following composition:

• 550 ml deionized water

• 360 g cane sugar

• 37.5 ml glacial acetic acid (solution 3.9 v/v% - 0.69 M)

• 200 g calcium chloride dihydrate - 15.9% (1.43 M)

• 4.5 g calcium bromide hydrate - 0.44% (0.022 M)

• 0.050 g menthol - 0.0053%

• 0.175 g thymol - 0.018%

the specific gravity of the resulting fixative was 1.280.

EXAMPLE 23

Received approximately 820 ml fixative of the following composition:

• 500 ml deionized water

• 280 g cane sugar

• 37.5 ml glacial acetic acid (4.5 v/v% solution - 0.80 M)

• 200 g calcium chloride dihydrate - 18.4% (1.66 M)

• 4.5 g calcium bromide hydrate - 0.51% (0.025 M)

• 0.050 g menthol - 0.0061%

• 0.175 g thymol - 0.021%

the specific gravity of the resulting fixative was 1.270.

EXAMPLE 24

Received approximately 800 ml fixative of the following composition:

• 500 ml deionized water

• 200 g cane sugar

• 37.5 ml glacial acetic acid (solution 4.9 v/v% - 0.82 M)

• 240 g calcium chloride - 30.9% (2.70 M)

• 4.5 g calcium bromide hydrate-0.52% (0.027 M)

• 0.050 g menthol - 0.0063%

• 0.175 g thymol - 0.022%

the specific gravity of the resulting fixative was 1.330.

EXAMPLE 25

Received approximately 420 ml fixative of the following composition:

• 231.25 ml deionized water

• 100 g cane sugar

• 18.75 ml glacial acetic acid (solution 4.5 v/v% - 0.78 M)

• 0.45 g sodium benzoate - 0.11% (7.44 mm)

• 15 g calcium chloride - 3.57% (0.32 M)

• 2.25 g calcium bromide hydrate - 0.49% (0.025 M)

• 0.025 g menthol - 0.0059%

• 0.0875 g thymol - 0.021%

• 30 g citric acid - 7.1% (0.037 M)

the specific gravity of the resulting fixative was 1.288.

EXAMPLE 26

Received approximately 400 ml fixative of the following composition:

• 250 ml deionized water

• 100 g cane sugar

• 20.0 ml glacial acetic acid (solution 5.0 v/v% - 0.87 M)

• 0.45 g sodium benzoate - 0.11% (7.44 mm)

• 90 g calcium chloride - 22.5% (2.03 M)

• 2.25 g calcium bromide hydrate - 0.52% (0.026 M)

• 0.025 g menthol - 0.0063%

• 0.0875 g thymol - 0.022%

• 60 g citric acid - 15.0% (0.78 M)

the specific gravity of the resulting fixative was 1.296.

EXAMPLE 27

Received approximately 420 ml fixative of the following composition:

• 250 ml deionized water

• 50 g cane sugar

• 20.0 ml glacial acetic acid (solution 4.8 v/v% - 0.83 M)

• 0.45 g sodium benzoate - 0.11% (7.44 mm)

• 200 g calcium chloride dihydrate - 36.0% (3.24 M)

• 2.25 g calcium bromide hydrate - 0.49% (0.025 M)

• 0.025 g menthol - 0.0059%

• 0.0875 g thymol - 0.021%

• 60 g citric acid - 14.3% (0.74 M)

the specific gravity of the resulting fixative was 1.337.

EXAMPLE 28

Received approximately 5100 ml fixative of the following composition:

• 2300 ml deionized water

• 3750 g cane sugar

• 375 ml glacial acetic acid (solution 7.4 v/v% - 1.3 M)

• 4.5 g sodium benzoate - 0.88% (6.1 mm)

• 60 g of calcium chloride - 1.2% (0.11 M)

• 30 g calcium bromide hydrate - 0.59% (0.027 M)

• 0.25 g menthol - 0.049%

• 0.875 g thymol - 0.017%

the specific gravity of the resulting fixative was approximately 1.29.

Most preferred is the SG composition of Examples 14-28.

In the examples, magnesium halides can be replaced by calcium halides.

It is believed that similar results would be obtained if the concentrations of acetic acid in the examples varied by about ± 0.3 M or more, and that acceptable results would be obtained in the wider ranges that are defined in the claims. It is believed that similar results would be obtained if the concentrations of the other components in the examples varied by at least ±10%, possibly ±20% or more, from their stated values, and that acceptable results would be obtained over a wider range of concentrations.

Usually the upper limit of the concentration of the halide salt is determined only by the solubility of each particular salt in the fixing solution.

Claims (29)

1. Aqueous fixative solution for biological samples, containing: a) C ₂ -C ₆ carboxylic acid at a concentration of 0.9 M or more; b) an alkaline earth halide salt at a concentration of 0.02 M or more; And c) a biocidal essential oil component at a concentration of at least 50 µg/ml. 2. Aqueous fixative solution according to claim 1, in which the specified C ₂ -C ₆ carboxylic acid is acetic acid, and/or in which the concentration of C ₂ -C ₆ carboxylic acid is 1.3 M or more; and/or in which the specified C ₂ -C ₆ carboxylic acid is acetic acid, and the concentration of acetic acid is less than 10% wt./about. 3. Aqueous fixative solution according to claim 1 or 2, in which the concentration of alkaline earth halide salt is 0.04 M or more; and/or component b) is a calcium or magnesium halide salt. 4. An aqueous fixative solution according to claim 1, wherein component b) contains calcium chloride at a concentration of at least 0.06 M and/or calcium bromide at a concentration of at least 0.06 M. 5. An aqueous fixative solution according to any of the preceding claims, further comprising a non-polar solute which increases the density of the fixative solution to more than 1.1 g/ml, preferably more than 1.2 g/ml, most preferably 1.2-1.4 g/ ml. 6. The aqueous fixative solution of claim 5, wherein the non-polar solute is a soluble carbohydrate, preferably sugar, preferably sucrose or glucose, or fructose, or maltose, or any combination thereof. 7. An aqueous fixative solution according to any preceding claim, wherein said biocidal ester oil component c) is one or more of: menthol, thymol, pulegol, dihydrocarveol, piperitol, menthone, menthene, o-cymene, p-cymene, citronellal, carvacrol, eugenol, 4-carvo-menthol (termpinen-4-ol), allicin or camphor; or said biocidal essential oil component c) contains menthol and thymol. 8. An aqueous fixative solution according to any one of the preceding claims, further comprising: d) benzoic acid or sorbic acid or their salts in bacteriostatic or fungistatic concentration. 9. An aqueous fixative solution according to claim 8, in which component d) is a salt of benzoic acid, and at the same time i) said carboxylic acid component a) is acetic acid at a concentration of 0.9-1.75 M, ii) said alkaline earth halide salt component b) is calcium chloride at a concentration of 70-100 mM plus calcium bromide at a concentration of 30-50 mM, and iii) said component of the salt of benzoic acid d) is benzoate at a concentration of 5-15 mM. 10. An aqueous fixative solution according to claim 8, in which component d) is a salt of benzoic acid, and: i) said carboxylic acid component a) is acetic acid at a concentration of 1.2-1.75 M; ii) said alkaline earth halide salt component b) is calcium chloride at a concentration of 70-100 mM plus calcium bromide at a concentration of 30-50 mM; And iii) said component of the salt of benzoic acid d) is benzoate at a concentration of 5-15 mM. 11. An aqueous fixative solution according to claim 8, in which component d) is a salt of benzoic acid, and at the same time: i) said carboxylic acid component a) is acetic acid at a concentration of 0.9-1.75 M, ii) said halide salt component b) is calcium chloride and/or calcium bromide in a total concentration of 1.0-4.5 M, and iii) said benzoic acid salt component d) is benzoate at a concentration of 5-125 mM. 12. A method for obtaining a biological sample for research, including fixing the sample with an aqueous fixative solution according to any of the previous paragraphs. 13. The method according to p. 12, in which the biological sample is a living organism; and/or wherein the biological sample is a parasitological sample. 14. The method of claim 13 wherein the density of the aqueous fixative solution is sufficient to float eggs or larvae or cysts or oocysts in the parasitological sample and is preferably in the range of 1.2-1.4 g/ml, more preferably 1. 25-1.35 g/ml. 15. The method according to any one of paragraphs. 12-14, in which the biological sample is stained after fixation; and/or wherein the biological sample, after fixation, is stained with an azo dye, or with iodine, or with a trichrome dye.