2015 Volume 48 Issue 1 Pages 9-14
We established a new “ecological” Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.
Periodic acid-Schiff (PAS) reaction [13], Gridley stain [5], and Grocott stain [6] represent histochemical techniques for demonstrating fungi in tissue sections. Of these three techniques, Grocott stain shows the highest sensitivity for detecting fungi and other polysaccharide-rich microorganisms in routinely prepared paraffin sections. Gomori’s methenamine-silver nitrate [4] and chromic acid comprise the major reagents used in conventional Grocott stain.
The staining sequence for Grocott stain has remained unchanged for more than half a century. Chromic acid (H2CrO4) contains hexavalent chromium and is capable of oxidizing many kinds of organic compounds, but it is highly corrosive, toxic, and carcinogenic to the lung [22]. At present, chromic acid oxidation is never used on an industrial scale. When it is disposed of, hexavalent chromium in the chromic acid solution must be reduced to non-hazardous trivalent chromium. It is actually impossible for medical laboratory staff to avoid the chemical hazard to environment and human body posed by this chemical, if we continue to use it. We believe that it is time to stop using this hazardous chemical for staining in order to immediately improve such environmentally compromising working conditions.
A strategy for curbing the environmental impact of waste is simple, and represented by the 4R principle of refusal, reduction, reuse, and recycle [17]. In order to develop an environmentally-friendly or “ecological” modification of Grocott stain, we firstly tried to reduce and reuse/recycle and finally to refuse chromic acid solution.
A total of nine fungal lesions (candidiasis 3, aspergillosis 3, and mucormycosis 3) were obtained from the autopsy file in Fujita Health University Hospital, Toyoake, Japan, and were used for the basic analysis. A variety of mycotic infections were further evaluated with our final modification protocol (Grocott stain without using chromic acid). These included pneumocystosis 3 lesions, cryptococcosis 3, histoplasmosis 2, penicilliosis marneffei 1, chromomycosis 2, dermatophytosis 2, chlorella infection 1, and actinomycosis 3. All the tissues were routinely fixed with 10% formalin and embedded in paraffin wax. The presence of fungi in each section was confirmed with hematoxylin and eosin stain, PAS reaction, and conventional Grocott stain.
Dilution of chromic acid for “reduction and reuse/recycle”Conventional Grocott stain employs 5% chromic acid solution, incubated at room temperature for 1 hr. The concentration of chromic acid was reduced to 1%. The incubation was set at 60°C for 1 hr, and the 1% chromic acid solution kept at room temperature was used repeatedly over the period tested.
Oxidization with periodic acid for “refusal” of chromic acidThe oxidization step was changed from chromic acid to 1% (ortho) periodic acid (H5IO6). Firstly, sections were oxidized in 1% periodic acid for 1 hr, 3 hr, 6 hr, 12 hr and 24 hr at room temperature, at 37°C or at 60°C. Secondly, sections were heat-treated by employing 4 different conditions: incubator warming at 60°C for 6 hr, electric jar boiling at 95°C for 30 min, 500 watt microwaving at 100°C for 10 min, and boiling in a pressure pan (T-fal, Rumily, France) at >120°C for 10 min. As soaking solutions, tap water, distilled water, 10 mM citrate buffer, pH 6, and 1 mM ethylenediamine tetraacetic acid (EDTA) solution, pH 8, were chosen. Subsequently, sections were oxidized with 1% periodic acid for 10 to 60 min at room temperature. In some experiments, sections were heated after periodate oxidation for comparison.
Ethical issueThe present study was approved by the institutional ethical review board for clinical and epidemiological investigations at Fujita Health University, Toyoake.
Oxidization with 60°C-warmed 1% chromic acid for 1 hr gave a satisfactory result comparable with conventional Grocott stain (Fig. 1). Nonspecific co-staining in the connective tissue component occasionally seen in conventional Grocott stain was suppressed by warm incubation with the diluted chromic acid. Repeated use (reuse and/or recycle) of 1% chromic acid solution, stored at room temperature, was possible up to 4 weeks. Actually, we reused/recycled the solution once a week for a total of four times.
Grocott stain with reduced concentration of chromic acid. (A & D) candidiasis, (B & E) aspergillosis, and (C & F) mucormycosis. (A, B, C): Conventional Grocott stain with 5% chromic acid oxidation at room temperature for 1 hr. (D, E, F): Modified Grocott stain with 1% chromic acid oxidation at 60°C for 1 hr. Modified Grocott stain with 1% chromic acid solution gave satisfactory staining comparable with the conventional method. Co-staining in the vascular connective tissue component seen in the conventional method in panel C (arrows) disappeared in the modified sequence (panel F). Bars=50 μm.
Treatment with 1% periodic acid solution at room temperature showed weaker staining even when the incubation period was prolonged for 24 hr. Staining intensity showed a peak at 6 hr oxidization in 1% periodic acid solution, but the condition was unsatisfactory and not so stable. Periodic acid solution preheated at 37°C or at 60°C failed to improve the stainability.
Heat pretreatment of deparaffinized sections was effective for enhancing the oxidizing effect in 1% periodic acid solution. All 4 types of soaking solution (tap water, distilled water, 10 mM citrate buffer, pH 6, and 1 mM EDTA, pH 8) provided comparable effects. Figure 2 demonstrates the effect of electric jar preheating in tap water at 95°C for 30 min. Heating after periodate oxidation was ineffective. After preheating sections, oxidation in 1% periodic acid at room temperature for 10 min gave a staining intensity comparable with conventional Grocott stain. Heat pretreatment in electric jar, microwave oven and pressure pan all gave satisfactory staining for candidiasis and aspergillosis (Fig. 3). Little tissue damage was observed after heating. When preheated in an incubator at 60°C for 6 hr, 1% periodic acid oxidation accelerated co-staining in connective tissue components. It should be noted that the staining intensity of hyphae in mucormycosis was significantly enhanced in our “ecological” modification of Grocott stain, while conventional Grocott stain resulted in weak staining (Fig. 3C).
Effect of preheating in demonstrating Aspergillus in lung. Periodic acid oxidation was performed before (A) and after (B) heating in tap water in an electric jar. Effect of heat pretreatment is evident. Bars=50 μm.
Effect of preheating in demonstrating fungi in lung with modified Grocott stain employing 1% periodic acid oxidation at room temperature for 10 min. (A & D) candidiasis, (B & E) aspergillosis, and (C & F) mucormycosis. (A–C): Conventional Grocott stain using 5% chromic acid solution. (D–F): Pressure pan heating in tap water for 10 min. Both conditions gave distinct staining for Candida albicans and Aspergillus fumigatus. Mucor hyphae showed enhanced staining in our modified technique (panel F), when compared with conventional Grocott stain (panel C). Bars=50 μm.
Figure 4 demonstrates the application of our modification without using chromic acid to a variety of mycotic pathogens in paraffin sections. Clear black staining was obtained in all the additionally evaluated fungal and related pathogens, such as Pneumocystis jiroveci, Cryptococcus neoformans, Histoplasma capsulatum, Penicillium marneffei, chromomycotic fungi, Dermatophytes, Chlorella, and Actinomyces israelii.
Demonstration of mycotic pathogens in biopsy specimens with our modified Grocott stain avoiding the use of chromic acid. (A) Pulmonary pneumocystosis in AIDS, (B) pulmonary cryptococcosis in AIDS, (C) pulmonary histoplasmosis, (D) cutaneous penicilliosis marneffei in AIDS, (E) cutaneous chromomycosis in hemodialysis, (F) dermatophytosis, (G) cutaneous chlorella infection in bharal (Himalayan blue sheep), and (H) pharyngeal actiomycosis. Both yeast type and hypha-forming fungi (A–F), as well as chlorella (G) and filamentous bacteria (H), are stained black with our ecological sequence. Bars=50 μm.
Table 1 summarizes the staining sequence of our new ecological modification of Grocott stain, employing preheating of sections in tap water and oxidation in 1% periodic acid solution. For preheating, either the electric jar, microwave oven or pressure pan is available, but we recommend pressure pan heating for 10 min for convenience sake.
| 1) Deparaffinize sections in xylene and soak them in alcohol. |
| 2) Rise sections in running tap water. |
| 3) Heat sections in tap water using either electric jar at 95°C for 30 min, 500 watt microwave for 10 min or pressure pan for 10 min. |
| 4) Cool down sections for 15 min on a table and then rinse them in running tap water. |
| 5) Keep methenamine-silver nitrate solution in a 60°C oven. |
| 6) Incubate sections in 1% periodic acid solution at room temperature for 10 min. |
| 7) Rinse sections in running tap water and then in distilled water (3 changes). |
| 8) Soak sections in the methenamine-silver nitrate solution pre-heated at 60°C for 40 to 60 min. |
| 9) Rinse sections in distilled water (3 changes). |
| 10) Replace silver with 0.1% gold chloride acid tetrahydrate (HAuCl4·4H2O) at room temperature for 3 min. |
| 11) Rinse sections briefly in distilled water. |
| 12) Fix sections in 2% sodium thiosulfate (Na2S2O3·5H2O) at room temperature for 3 min. |
| 13) Rise sections in running tap water. |
| 14) Counterstain sections in 0.04% light green solution at room temperature for 5 min. |
| 15) Rise sections in running tap water. |
| 16) Dehydrate sections in ethanol, change in xylene and mount with a cover glass. |
[Preparation of methenamine silver solution]
Prepare the methenamine-silver nitrate solution just before use. Add 5 ml of 5% silver nitrate stock solution (AgNO3) to 100 ml of 3% methenamine (hexamethylenetetramine) stock solution ((CH2)6N4). Then, add 25 ml of distilled water and 4 ml of 5% sodium borate stock solution (Na2B4O7·10H2O) to 25 ml of mixture solution. Finally, mix 2–3 drops of 5% gelatin to avoid the silver mirror reaction.
We established a newly modified Grocott stain technique that does not use chromic acid. Our environmentally-friendly method consistently yielded clear and satisfactory results for black staining of fungi. So far, several modifications of Grocott stain have been reported [14–16, 19, 21]. To the best of our knowledge, no research was focused on the ecological point of view eliminating the use of chromic acid.
Periodic acid is a common oxidative reagent widely employed in PAS reaction [13] and periodic acid methenamine-silver (PAM) stain [11], and is also used for quenching endogenous peroxidase in immunostaining [9]. It is well known that periodic acid oxidizes vicinal diol hydroxyl (-OH) groups on the sugar moiety into aldehyde groups (-CHO) but the substitution to carboxyl groups (-COOH) never happens [10]. In contrast, chromic acid finally oxidizes hydroxyl groups to carboxyl groups.
Utilization of periodic acid instead of chromic acid in Grocott stain for fungi has been reported so far, but the researchers commented that the results were not fully applicable [2, 24]. In the present study, however, the combination of preheating and oxidation in 1% periodic acid solution for 10 min yielded satisfactory results for the consistent and stable visualization of a variety of fungi in pathological lesions. Carson et al. [1] reported the use of warm periodic acid as an oxidant, but satisfactory results were hardly obtained in our trial. Heat pretreatment is commonly used for epitope retrieval in immunostaining [20]. The heating process allegedly loosens and even removes formalin-induced methylene-bridges among tissue proteins [3]. The heat-induced change in the spatial structure of fungal proteins may thus contribute to the vulnerability to periodate oxidation. It should be noted that the stainability of Mucor hyphae was greatly enhanced in our modification. With conventional Grocott stain, Mucor hyphae are often stained weakly, probably due to the thinness of the cell wall [7, 23]. Kawabata et al. [12] briefly described in Japanese the enhanced staining of Mucor hyphae by double treating sections with heating and periodic acid oxidation. They simply aimed at enhancing the staining intensity of Mucor hyphae, so that chromic acid oxidation was preserved for demonstrating Candida spp. and Aspergillus spp.
Regarding the heating method, the electric jar, microwave oven, and pressure pan all gave satisfactory results, and all 4 types of soaking solution (tap water, distilled water, 10 mM citrate buffer, pH 6, and 1 mM EDTA, pH 8) provided comparable effects. Needless to say, tap water should be chosen as a soaking solution. The heating method can be selected, depending upon which one the laboratory technicians is most accustomed to use. We recommend pressure pan heating in tap water, because of its simplicity and convenience once one gets used to it.
During trial and error, we first succeeded in reducing the concentration of chromic acid from 5% to 1%. We discovered that warming at 60°C was needed, and we confirmed the repeated use (reuse and/or recycle) of the 1% chromic acid solution for a long period of time. Because of the high melting temperature of chromic acid at 196°C [22], warming at 60°C does not accelerate the risk of vaporization of the dangerous metal into air. It should be noted that the reduction in chromic acid from 5% to 1% had little effect on the perceived toxicity of the chemical. Referring to the Exposure Standards from the Hazardous Substances Information System (HSIS) [18], such risk phases still exist: R23 (toxic by inhalation), R36/37/38 (irritating to eyes, respiratory system and skin), R42/43 (may cause sensitization by inhalation and skin contact), R45 (may cause cancer), and R46 (may cause heritable genetic damage).
We also referred to the report by Hayashi et al. [8], who improved PAM stain by enhancing the silver reaction through the pretreatment with thiosemicarbazide after periodic acid oxidization. However, thiosemicarbazide belongs to the hazardous chemical category and so we abandoned using it.
Our new “ecological” Grocott stain is definitely friendly to both environment and human body. Total staining time is on a par with conventional Grocott stain. Since our modified technique requires no special equipment or additional reagents, we believe our simple and practical method is easily applicable worldwide.
The authors have no conflict of interests.
We wish to thank Mr. Naoyuki Okada, Teacher of Chemistry, St. Mary’s Junior and Senior High School, Gifu, and Mr. Sadayuki Hiroi, M.T., Department of Pathology and Laboratory Medicine, National Defense Medical College, Tokorozawa, for their valuable advice and suggestions. We also cordially acknowledge the technical assistance by Ms. Mika Maeshima and Ms. Sayaka Takeuchi, Department of Pathology, Fujita Health University School of Medicine, Toyoake.
A portion of the present work was poster-presented at the 54th meeting of the Japan Society of Histochemistry and Cytochemistry held in Tokyo, 2013, and supported by a Research Grant from Fujita Health University, 2012.
