Qualitative Evaluation of Carbopol-940 Gel Used as a Preservative Medium Compared to Traditional Modified Kaiserling III Solution in Pathology Museum

Introduction: Body organs and tissue specimens have been preserved in human and veterinary anatomy, pathology museums for years in traditional modified Kaiserling III solution. Wax embedding, epoxy-resin embedding and Plastination techniques have also been tried. However, the use of Carbopol-940 gel as a preservative medium is not mentioned in the literature. Gel preservative medium can prevent leakage, toxic vapors, and irritation of eyes and skin. Aims: Qualitative evaluation of carbopol-940 gel as a preservative medium for specimens in pathology museums compared to modified Kaiserling III solution over one year. Objectives: 1) To qualitatively evaluate physical integrity of mounted tissue specimens stored in Kaiserling III solution and carbopol-940 gel for one year. 2) To qualitatively evaluate physical, chemical and microbiological parameters of Kaiserling III solution and carbopol-940 gel for one year. Materials and methods: In this qualitative cross-sectional study, different organs were mounted in jars containing modified Kaiserling III solution and carbopol-940 gel. The media were monitored for stability by physical tests, chemical test, and microbiological test, before and after the study. The organs were monitored for physical disintegration by observing for tissue shredding. Specimens were histologically assessed before and after the study for tissue integrity. Results showed good stability of the preservative media and good tissue integrity of the specimens over a period of one year.


Introduction
Normally, body tissue specimens are preserved in museums as mounted specimens, immersed in solution containing formalin reagent.This prevents tissue disintegration, increases tissue hardening and prevents contamination by micro-organisms.In 1897 (1) , Kaiserling developed a buffered solution which contained 400 ml of 40 % formalin, 30 g potassium nitrate, 60 g potassium acetate and 2000 ml tap water.This Kaiserling I solution, was used to fix the tissue for a period of up to two weeks.However, there was a loss of color contrast.He discovered that immersion in 80 % ethyl alcohol, Kaiserling II solution, restored the original color of the tissue.The specimen was then mounted in glass jars, containing a solution of 500 ml.glycerin, 200 ml of 1 % arsenious acid, potassium acetate 250 g and Thymol 2.5 g.The specimen mounted in this solution and sealed in glass jars could be stored for many years.This solution was named Kaiserling III solution.This solution was further modified and developed at Westminster Medical School (1) , and contained glycerin 30 %, Sodium acetate 10 %, and formalin 0.5 %, with pH adjusted to 8.0.
Usage of gel medium has few advantages.The gel en-sheathes the specimen, preventing leakage of contents on tilting the jar.The specimen will not get dislodged, and sloshing of the liquid medium will be prevented during rough handling and transport.Being jelly like, it will not leak through fine cracks in the glass jar; consequently, the examiner's hand, body or clothes will not be soiled.The gel itself is odorless, nontoxic, noninflammable and nonreactive to the skin, nose or eyes.Leaching of pigments from the tissue like bilirubin or blood will be localized due to the absence of conduction or convection currents of the fluid particles.
Gelatin embedding (1) with arsenious acid-gelatin was carried out, however gelatin tends to yellow with age and undergoes liquefaction.Embedding in solid plastic blocks is possible for hard tissues, certain insects and plants but difficult for normal pathological specimens.Cadaveric organs can be kept as dry specimens, for teaching and mounting in museum, by a new wax embedding technic (2,3 ) .Plastination was invented by Gunther Von Hagens (4,5) in 1977, wherein tissue was impregnated with synthetic resin.Insects have been embedded in resins (6) .Literature on use of carbopol-940 gel for preservation of tissue specimen is not available.

Materials And Methods
The specimens for mounting were selected purposively to include common lesions in different organs viz.spleen infarct, lipoma, gallbladder with chronic inflammation, kidney with chronic pyelonephritis, ovary with serous cyst and uterus with fibroid.The organs were well fixed in 10% formalin with easily recognized lesions.Unfixed and specimens undergoing autolysis were not included as also unidentifiable lesions.
The specimens were strung over a glass plate using needle and string.Appropriate two sets of similar organs were mounted in six jars, each containing modified Kaiserling III solution in one set and carbopol-940 gel in another set.Before sealing the mounted jars, the media and tissue specimens were subjected to various physical, chemical and histopathological tests and the results were recorded as pre study data.The mounted specimens were sealed by glass lids on the glass jars using a sealant (M-seal, Pidilite Industries, India) and stored for one year in room temperature.After a period of one year, the lids were reopened and the media and the specimens were again subjected to retesting, as done previously.The results were recorded as post study data.
The modified Kaiserling III solution was prepared as follows: 100 g sodium acetate was dissolved in 500 ml of distilled water.To this was added 5.0 ml of 40% formalin solution.Next 300 ml of glycerin was added to the mixture and the solution was made up to 1000 ml by distilled water.This was filtered and used as mounting medium.
The carbopol-940 gel was prepared by dissolving 15g of carbopol-940 powder (Manufacturer: Aquatios Gel, India), in 900 ml. of distilled water, using hand held electrically operated stirrer.To this mixture was added 5.0 ml.40 % formalin solution.The volume was made up to 1000 ml by addition of distilled water.Triethanolamine solution, 99%, (Manufacturer: Aquatios Gel, India), 5.0 ml was added drop wise to the solution and stirred continuously till it formed a gel.The carbopol-940 dispersion solution has a low pH 2.5-3.5.Triethanolamine (TEA) acts as a neutralizer, and its addition raises the pH to 7.0 resulting in gel formation.
The Kaiserling III solution, carbopol-940 gel and the tissue specimens were subjected to various physical tests, chemical test, microbiological test and histopathological tests as follows: Growth of organisms: The media were tested for organisms by gram staining.A loop full of the medium was taken and smeared on the clean slide.It was heat fixed on a flame.The smear was laid on a horizontal slide rack and covered with gram stain for one minute.Gram's iodine was poured over the smear to act as a mordant for a minute.The smear was washed and few drops of acetone alcohol were poured over it for de-colorization.The smear was counterstained with safranin stain for 30 seconds.The smear was washed, air dried and observed under oil immersion objective for presence of gram positive blue stained bacteria or gram-negative orange-stained bacteria.Fungal organisms can also be confirmed by the stain.

Specimen color:
The tissue may become purple to brown to black in color due to putrefaction.The tissue may show color changes due to presence of blood in congestion and hemorrhage.Also, presence of bilirubin and breakdown of blood may impart a golden brown to greenish pigmentation.
Specimen histopathology: Tissue slices, measuring 3-5 mms thick, are taken from the specimens and subjected to tissue processing.It is passed through beakers of ascending grades of alcohol 70 %, 80%, 90%, 100%, 100% for an hour each to dehydrate the tissue.Then it passes through two cycles of xylene reagent of an hour each, followed by two cycles of one hour each in molten paraffin wax and finally is embedded in paraffin block.The tissue paraffin block is sectioned by microtome (Leica Microsystems, India) and 3-4-micron thick sections are taken on a slide, fixed and stained by Haematoxylin and eosin stain.The prepared slide is observed under 100x magnification and cellular characteristics photographically recorded at 400x magnification.Disintegrated tissue shows loss of architecture, absence of nuclear staining, and fading of the stains with absence of blue and pink color differentiation.Well preserved tissue will show sharply stained blue nucleus, and pink cytoplasm and matrix, with regular tissue architecture.

Result and Discussion
The mounted spleen, lipoma, gallbladder, kidney, ovarian cyst and uterus fibroid specimens in the two different media, both pre study and post study, showed good stability of the media as well as the preservation of the tissue The specimen jars of spleen (Figure 1.01 to 1.04), gallbladder (Figure 3.01 to 3.04), and ovary cyst (Figure 5.01 to 5.04) display excellent features of preservation of tissue and stability of the mounting media Kaiserling III solution and Carbopol-940 gel.The specimen of Lipoma (Figure 2.04) in carbopol-940 gel, post study, shows minimal haziness on the surface.The specimen of kidney (Figure 4.04) in carbopol-940, post study, shows minimal focal haziness at the hilum of the kidney.The uterus specimen (Figure 6.03, 6.04) in carbopol-940 gel, pre study and post study, show similar minimal, diffuse smokiness of the medium.In all the above cases, no organism was detected by gram staining of the media samples.
The physical parameters of the Kaiserling III solution and carbopol-940 gel (Table1 to Table 6) suggest good stability of the mounting media.Physical disintegration of the tissues is absent.Globules of fat released in the above cases of lipoma (Figure 2.04), and kidney (Figure 4.04) are related to mechanical manipulation of the tissue.The histopathological appearance shows well stained blue nuclei of the cells of various tissues in a background of pink eosinophilic matrix, which is indicative of good preservation of the tissues, post study of a period of one year, in both the media Kaiserling III solution and carbopol-940 gel.The various parameters assessed for the media are color, turbidity, specific gravity, pH, gram staining of bacterial and fungal organism, and for the tissue organs were color, gross disintegration and histological changes seen in tissue disintegration.
Color: The color of the Kaiserling III solution and carbopol-940 gel were colorless.If the tissue specimens were hemorrhagic or bilious the media would be colored blackish, brownish, yellowish or greenish because of leaching of the pigment into the media.Similarly, contamination with pigment producing bacteria or fungi would turn the media blackish, brownish or greenish.When the pigment leaching occurs in liquid media, it would diffusely spread throughout the media, whereas in gel media it would remain localized due to absence of conduction or convection of the fluid particles.

Turbidity:
The media may show turbidity due to impurities in the medium and faulty polymerization during preparation, presence of contaminating micro-organisms like fungi and bacteria or disintegration of the tissue following autolysis.Separation of the shredded tissue particulate matter within the media would cause turbidity and opacity.Fat particulate matter would float on the surface of the liquid and gel media.Solid tissue particles will sink in the liquid media, but float in the gel media due to its high        viscosity compared to that of the liquid media.The specimen of Lipoma (Figure 2.4) in carbopol-940 gel, post study, shows minimal haziness on the surface suggestive of minimal fatty globules released by the sliced lipoma tissue.The specimen of kidney (Figure 4.4) in carbopol-940, post study, shows minimal focal haziness at the hilum of the kidney which could be due to the sliced perirenal fat.Globules of fat released in the above cases of lipoma (Figure 2.4), and kidney (Figure 4.4) are related to mechanical manipulation of the tissue.The uterus specimen (Figure 6.3, 6.4) in carbopol-940 gel, pre study and post study, show similar minimal, diffuse smokiness of the medium.This could be attributed to polymerization defect during preparation of the carbopol-940 gel medium due to improper dispersal and mixing in water.In all the above cases, no organism was detected by gram staining of the media samples.
The pH: The pH of the Kaiserling solution was high.This was because the sodium acetate imparted an alkaline pH to the medium.The pH was carried out in undiluted Kaiserling III solution.The carbopol-940 powder dispersion in distilled water gives a low pH from 2.5 to 3.5.Addition of a neutralizing reagent like sodium hydroxide or tri-ethanolamine raises the pH to 7.0 to 7.4, which is important for polymerization and to form gel. Hence pH reading of the gel is essential.Lowering pH will cause liquefaction of the gel.

Specific gravity:
The Kaiserling III solution had to be diluted to get necessary reading on the scale because of high salt concentration.The specific gravity was beyond 1.070, hence not readable on the visual scale.Dilution 1:3 with distilled water showed a specific gravity 1.060, which could be read on the scale.

Growth of organisms:
Contaminants introduced within the media may be bacterial or fungal organisms.They may also be introduced by the specimens harboring them, in case the specimens are not properly fixed by 10 % formalin.Alternatively, addition of phenoxyethylene in concentration less than 1% in the gel may prevent growth of bacteria or fungal organisms.

Specimen color:
Freshly preserved tissue may show varied color due to the native tissue which may be brownish white to whitish in color.Gall bladder may be greenish brown due to bile pigment.Congestion of the organ or presence of hemorrhage may impart a red to brownish black color.Presence of purulent exudate in inflammation may show greenish white to dark color.Autolysis due to improper fixation by 10% formalin, or presence of contamination by bacteria or fungus may result in disintegration of the tissue.The tissue may become purple to brown to black in color due to putrefaction.Few pigmented bacteria or fungal organisms may produce a dark, blackish pigment.
Specimen histopathology: Disintegrated tissue shows loss of architecture, absence of nuclear staining, and fading of the stains with absence of blue and pink differentiation.Well preserved tissue will show sharply stained blue nucleus, and pink cytoplasm and matrix, with regular tissue architecture.Hence histological examination of the tissues helps in determining whether the tissues are well preserved or not.
This study was for a period of one year only.The histological appearances remained consistently well preserved for this period of time.Kaiserling III mounted specimens have lasted for more than a decade.Stability of the medium carbopol-940 gel as well as the tissue mounted in it, have not been confirmed beyond one year.The consistency of the gel state of carbopol-940 is limited by temperature, pH and concentration of salts.Carbopol-940 gel requires a cooler temperature to remain in a gel state.Hence the room temperature of the museum should remain within 25 degrees centigrade.Also, the pH of the gel should be checked and not let it fall below 6.0.Addition of Triethanolamine will raise the pH and help to correct the liquefaction.

Conclusion
It In conclusion, the physical, chemical, microbiological and histopathological parameters revealed good correlation of qualitative stability of carbopol-940 gel and different mounted specimens with that of the traditional preservative modified Kaiserling III solution.Usage of carbopol-940 gel as preservative medium may be a good alternative to Kaiserling III solution.Further study of carbopol-940 usage as a mounting medium for specimen preservation for a longer period up to five years may be a possibility.

Figure- 1 Figure 1 . 1
Figure-1.0 (Appearance of mounted specimen and mounting media before study and after study)

Figure 1 . 2 Figure 1 . 3 Figure 1 . 4 Figure 1 . 5
Figure 1.2 Gross appearance of spleen infarct 7.5x8.0x1.5 cms.post study in Kaiserling -III.The organ is dark brown.Medium is colorless.Paper print easily read in the background Figure 1.3 Gross appearance of spleen infarct 7.5x8.0x1.5 cms.pre study in Carbopol-940 gel.The organ is dark brown.Medium is colorless.Paper print easily read in the background Figure 1.4 Gross appearance of spleen infarct 7.5x8.0x1.5 cms.post study in Carbopol-940 gel.The organ is dark brown.Medium is colorless.Paper print easily read in the background

Figure 2 . 1
Figure 2.1 Gross appearance of Lipoma 6.5x5.0x3.0 cms.pre study in Kaiserling III.The tissue is yellow Medium is clear, colorless.Paper print easily read in the background.

Figure 2 . 2
Figure 2.2 Gross appearance of Lipoma 6.5x5.0x3.0 cms.post study in Kaiserling III.The tissue is yellow Medium is clear, colorless.Paper print easily read in the background.

Figure 2 . 3
Figure 2.3 Gross appearance of Lipoma 7.0x5.0x3.0 cms.pre study in Carbopol-940.The tissue is yellow Medium is clear, colorless.Paper print easily read in the background.

Figure 2 . 4
Figure 2.4 Gross appearance of Lipoma 7.0x5.0x3.0 cms.post study in Carbopol-940.The tissue is yellow Medium is clear, colorless.Print light hazy up in the background.

Figure
Figure-2.5 Lipoma histology, H/E stain 400X magnification.It shows fat cells with clear cytoplasm and surrounding fibrocollagenous tissue Prestudy.Kaiserling III solution.

Figure 3 . 5
Figure 3.5 Gallbladder histology H/E stain 400X magnification.Papilla lined by columnar cells with visible blue nucleus.Pre study Kaiserling solution.

Figure 3 Figure- 4 (Figure 4 . 1
Figure 3.6 Gallbladder histology H/E stain 400X magnification.Papilla lined by columnar cells with visible blue nucleus.Post study Kaiserling solution Figure 3.7 Gallbladder histology H/E stain 400X magnification.Papilla lined by columnar cells with visible blue nucleus.Pre study Carbopol-940 gel Figure 3.8 Gallbladder histology H/E stain 400X magnification.Papilla lined by columnar cells with visible blue nucleus.Post study Carbopol-940 gel

Figure- 6 . 0 (Figure 6 . 1
Figure-6.0 (Appearance of mounted specimen and mounting media before study and after study)

Table -
Figure-2.0 (Appearance of mounted specimen and mounting media before study and after study)