Assessing the effectiveness of 30% sodium chloride aqueous solution for the preservation of fixed anatomical specimens: a 5-year follow-up study

Abstract

Anatomical specimens used in human or veterinary anatomy laboratories are usually prepared with formaldehyde (a cancerous and teratogenic substance), glycerin (an expensive and viscous fluid), or ethanol (which is flammable). This research aimed to verify the viability of an aqueous 30% sodium chloride solution for preservation of anatomical specimens previously fixed with formaldehyde. Anatomical specimens of ruminant, carnivorous, equine, swine and birds were used. All were previously fixed with an aqueous 20% formaldehyde solution and held for 7 days in a 10% aqueous solution of the same active ingredient. During the first phase of the experiment, small specimens of animal tissue previously fixed in formaldehyde were distributed in vials with different concentrations of formaldehyde, with or without 30% sodium chloride solution, a group containing only 30% sodium chloride, and a control group containing only water. During this phase, no contamination was observed in any specimen containing 30% sodium chloride solution, whether alone or in combination with different concentrations of formaldehyde. In the second phase of the experiment, the 30% sodium chloride solution, found to be optimal in the first phase of the experiment, was tested for its long-term preservation properties. For a period of 5 years, the preserved specimens were evaluated three times a week for visual contamination, odors, and changes in color and texture. There was no visual contamination or decay found in any specimen. Furthermore, no strange odors, or changes in color or softness were noted. The 30% sodium chloride solution was determined to be effective in the preservation of anatomic specimens previously fixed in formaldehyde.

Introduction

Anatomical parts are fixed to prevent deterioration. Proper fixing is extremely important as it keeps the tissues firm and protected (Rodrigues, 2010). Good preservation also prevents the infestation and proliferation of pathogens that can cause illness in people who work in or around the lab (Corrêa, 2003). One such pathogen is fungal contamination. Fungal contamination in preserved anatomic specimens can trigger allergic reactions. When working with anatomic specimens, the fungal spores can be released and aerosolized, and then are suspended in the air, thereby causing the reaction. Exposure to certain fungi is also associated with disorders of the central nervous system, respiratory and immune responses (Johanning et al. 1996).

While dehydration can be successfully used for preserving anatomical specimens, thus eliminating the use of preservatives (Freitas et al. 2009), it is not ideal. Therefore, preservation in liquid fixing solutions is used. However, preservation in liquid media requires the use of substances to prevent decay and the proliferation of microorganisms. The most common are formaldehyde, glycerin, ethanol and phenol (Rodrigues, 2010).

Formaldehyde is the most commonly used fixative and preservative, usually in a 10% aqueous solution. Because it is cheap and penetrates tissues quickly (6 mm in 12 h), it is widely used in anatomy laboratories (Rodrigues, 2010). However, formaldehyde carries significant health risks: it was classified as a carcinogen and teratogen by the International Agency for Research on Cancer in 1995. Besides being unhealthy, it has a serious environmental risk, because the improper handling and disposal of carcasses and waste can contaminate the environment (WHO, 1991).

The resistance of different microorganisms to formaldehyde and its antimicrobial effectiveness was tested at various concentrations. Pseudomonas sp., Klebsiella sp. and Salmonella sp. were found to be more susceptible to formaldehyde than Staphylococcus sp. Fungi were more resistant than bacteria, and Aspergillus niger was more resistant than Candida albicans. This can be particularly problematic because the Aspergillus sp. fungus is found predominantly indoors, and clouds of its spores can penetrate structures and contaminate the indoor environment (Solomon, 1975).

Glycerin is a preservative with desiccant and antiseptic properties. It is effective against both fungi and bacteria (Alvarenga, 1992) and, unlike formaldehyde, does not have harmful fumes (Cury et al. 2013). It is, however, up to 10 times more expensive than formaldehyde (Krug et al. 2011).

Sodium chloride is a naturally occurring chemical substance. It is a principal component of seawater, occurring at a rate of about 35 g NaCl per kg water (Díaz et al. 2000). It is inexpensive, non-toxic and easily disposed of. In one study, canine pericardium was retained in a solution of 1.5 g of common salt (sodium chloride) per mL of water for a minimum of 90 days, so as to be used as a biological membrane for repair of rectus abdominis muscles in Wistar rats. The storage medium showed no contamination by bacteria and fungi, and maintained the structural characteristics of the pericardium (Brun et al. 2002). In another study, canine center phrenics that were preserved in a similar saline solution, as well as a solution of 98% glycerin, for use in the repair of muscle lesions in Wistar rats showed no contamination (Brun et al. 2004).

In the Dead Sea, located at the Syrio-African Rift Valley, there is a unique chemical environment (Nissenbaum, 1975), and its water has a salt concentration of 322–340 g L⁻¹; in order of decreasing concentration, its major cations are Mg²⁺, Na⁺, Ca²⁺ and K⁺ (Nissenbaum, 1975; Ma'or et al. 2005).

When in seawater, some bacteria are subjected to an osmotic upshock, and their ability to overcome this by means of several osmoregulatory systems could largely influence their subsequent survival in the marine environment (Gauthier et al. 1987; Munro et al. 1989).

The goal of the current study was to evaluate the efficiency of a non-toxic and extremely cheap solution (30% aqueous solution of sodium chloride) in the conservation of animal anatomical specimens previously fixed with a 10% formaldehyde solution.

Materials and methods

First phase

Ten groups with different formaldehyde concentrations (0.612–10%), with or without 30% sodium chloride solution were used. The sodium chloride was from Sal Leão, Parnamirim, Rio Grande do Norte, Brazil. In addition, we used a group that contained only a 30% solution, and a control group containing only water, for a total of 12 groups (Table 1). The whole process was carried out in triplicate. Thirty-six transparent 500-mL plastic bottles with covers were used to simulate the environment occurring in the containers that the anatomical parts of the Laboratory of Animal Anatomy are kept in.

Table 1.

Distribution of the groups (G) of different concentrations of formaldehyde (F), with or without sodium chloride solution (C) as a preservative of ovine tissues

G1	G2	G3	G4	G5	G6
Water	F10%	F10%	F5%	F5%	F2.5%
		C30%		C30%
G7	G8	G9	G10	G11	G12
F2.5%	F1.25%	F1.25%	F0.6125%	F0.6125%	C30%
C30%		C30%		C30%

Pieces of the pelvic muscle of adult sheep (biceps femoris) and a fragment of jejunum from the same animal were placed in a vial. Each piece of biceps femoris was 27 cm³ (3 cm length × width × height), while the jejunum was 3 cm. The specimens were kept at the Animal Anatomy Laboratory of the Veterinary Medicine School, State University of Maringa – Umuarama Campus, Umuarama, PR, Brazil. They were previously fixed and stored with 10% formaldehyde solution approximately 2 years prior. All specimens were examined for signs of contamination prior to the start of the experiment. The fragments were washed in running water for 48 h to remove the excess preservative, and placed in plastic bottles labeled with the solution to be analyzed.

The samples were visually examined every day for 12 months to detect changes in the turbidity of the solution and the presence of contaminants. In cases where contamination was suspected, 5 mL of the solution was collected in a sterile plastic specimen collection bottle. The bottle was tagged and sent to the institution's microbiology laboratory for culture and identification of possible bacteria and fungi. To identify fungi, sterile swabs were used to plate samples on Sabouraud agar and tryptone-soya agar, and incubated for 48 h at a temperature of 36 °C. Bacteria were identified by culturing in Petri dishes on ordinary nutrient medium.

Results of the first phase

Culture results of microbiological samples are shown in Tables 2 and 3.

Table 2.

Days of experiment and sample contamination in the control group (water) and groups with different concentrations of formaldehyde (F)

Day of experiment	Contaminated sample
12	Water (1 & 2)
16	Water (3)
33	F 0.6125% (3)
41	F 1.25% (2)
44	F 0.6125% (1)
63	F 0.6125% (2)
127	F 1.25% (1)
153	F 1.25% (3)

Table 3.

Microorganisms identified in contaminated samples

Sample	Microorganism
Water 1	Pseudomonas sp. e Aspergillus flavus
Water 2	E. coli e Penicillium sp.
Water 3	E. coli
F – 0.6125% (1)	Pseudomonas sp.
F – 0.6125% (2)	Pseudomonas sp.
F – 0.6125% (3)	Pseudomonas sp.
F – 1.25% (1)	Pseudomonas sp.
F – 1.25% (2)	Pseudomonas sp.

There was no contamination in the samples containing 30% sodium chloride, either alone or in combination with different concentrations of formaldehyde. Thus, in this phase of the experiment, we determined that 30% sodium chloride appears to be ideal as a preservative.

Second phase

The aqueous solution of 30% sodium chloride found to be an optimal preservative in the first phase of the experiment was tested for conservation of anatomic specimens and animals.

Approximately 500 anatomical specimens of dogs, cats, equines, cattle, sheep, goats, pigs and birds were previously fixed with an aqueous 20% formaldehyde solution and held for 7 days in a 10% aqueous solution of the same active ingredient. Then, the specimens were washed in running water for 48 h to remove the excess preservative, and were distributed in 20 plastic boxes of 50 L (smaller parts, such as lungs, hearts, tongues, stomachs, intestines, eyes, spleens and brains) and four boxes of 1000 L (larger pieces and small, undissected animals) of this solution. The specimens were assessed three times a week for 5 years for visual contamination, the presence of malodors, and changes in color and texture. Floating specimens were slightly turned over in every inspection.

Care was taken not to overcrowd the boxes with anatomical parts, so that all the pieces were free inside the boxes. As the pieces tended to float in the saline (except those very dense, like forelimbs and hind limbs of large animals), a thin cotton cloth was used to cover them in order to always keep them wet.

The specimens were used weekly in the Veterinary Medicine Institution's ‘Animal Anatomy’ class. Because of this, the specimens were removed and taken directly to the tables for demonstration during the classes. At the end of the class, the specimens were returned directly to the saline. No washing was necessary. All materials used in class, such as metal clamps and stainless steel tables, were washed with soap and water after use to prevent rusting.

Results of the second phase

All specimens were successfully used during practical sessions for the entirety of the 5-year experiment. No visual contamination or decay was found on any specimen, nor were there strange odors or changes in color or softness. Because pieces could be taken directly from the preservative solution without washing and returned to the solution after use, there was considerable water savings throughout the experiment. However, due to the corrosive nature of the solution, care was taken to wash the tables and any materials used during the lessons with soap and water to prevent rusting.

Discussion

Contaminated tissues were kept in solutions classified as not suitable for conservation, as suggested in the literature (Corrêa, 2003). The bacteria and fungi most commonly observed in these solutions were Pseudomonas sp. and Aspergillus, respectively (Spicher & Peters, 1976).

The 30% aqueous solution of sodium chloride proved to be extremely effective in preserving tissues fixed by formaldehyde, as there was no contamination observed during the study period. This was similar to what was described in the studies of canine pericardium stored for a minimum of 90 days in a supersaturated solution of sodium chloride (Brun et al. 2002) and canine center phrenics stored in glycerin (Brun et al. 2004), both of which were preserved for surgical purposes.

There are other benefits of using saline as well. Unlike formaldehyde, saline does not generate or release contaminated effluents (WHO, 1991) or toxic fumes (Cury et al. 2013). Finally, the cost of the saline solution is approximately 10% of the cost of formaldehyde, it is much lower than other conservation solutions such as Thiel's (Wolff et al. 2008), which is composed of sorts of substances like boric acid, ethylenglycol, nitrates and methylphenol, and used to provide realistic conditions similar to the living body.

Evidence for the successful use of 30% sodium chloride solution may be suggested by the difficulty microorganisms have surviving in an environment requiring an enormous capacity for osmoregulation, such as in oceans (Gauthier et al. 1987; Munro et al. 1989) and the Dead Sea (Nissenbaum, 1975).

Conclusion

The aqueous solution of 30% sodium chloride was effective in the preservation of anatomic specimens previously fixed with formaldehyde, and is non-toxic, non-flammable and very inexpensive, which enables its use as a preservative in anatomy laboratories.