Indian Journal of Pathology and Microbiology

: 2012  |  Volume : 55  |  Issue : 2  |  Page : 154--157

In search of the rainbow: Colored inks in surgical pathology

Chandralekha Tampi 
 Lilavati Hospital and Research Centre, Mumbai 400 050, India

Correspondence Address:
Chandralekha Tampi
Consultant Histopathologist, Lilavati Hospital and Research Centre, Bandra West, Mumbai 400 050


Introduction: Although surgical pathologists are aware of the multiple advantages that coloured inks contribute to surgical pathology practice, these coloured inks are not available in India and importing them is not a viable proposition. A systematic search for locally available coloring agents was done, and resulted in identifying specific shades within a popular set of children«SQ»s hobby colors of a particular brand. They retain their bright distinct colors on paraffin blocks and sections. These paints are available all over India, and are cheap, safe, and easy to use. Coloring gross specimen excision margins with different colors, adds precision to margin examination. It allows three-dimensional microscopic reconstruction of the tumor vis-a-vis its various neighboring anatomic structures. It allows postoperative comparison of tissue planes predicted by preoperative imaging. It maintains orientation of grossed and dissected specimens, enabling the pathologist to re-visit the grossed specimen, if required, and confidently allows further sampling if necessary. Aim: A systematic search for indigenous coloring agents was carried out, which included the dyes used in the histopathology laboratory, gelatin, commercially available paints, including acrylic paints and inks. Results: The study identified specific shades within a brand of acrylic colors that are easily available and simple to use, with good results on microscopic examination. Conclusion: Colored inks lend precision to margin examination. A set of easily procurable colors are available in our country, which are easy to use, with distinct bright colors, safe, and reliable.

How to cite this article:
Tampi C. In search of the rainbow: Colored inks in surgical pathology.Indian J Pathol Microbiol 2012;55:154-157

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Tampi C. In search of the rainbow: Colored inks in surgical pathology. Indian J Pathol Microbiol [serial online] 2012 [cited 2022 Jan 27 ];55:154-157
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Tumor involvement of surgical margins of resected specimens has important therapeutic and prognostic implications. Margin examination by "marking sutures", separate submissions, radial, or shave sectioning techniques suffice only as rough guides. At present inking of the specimen surfaces prior to gross dissection serves the purpose most accurately [Figure 1]. This is so especially when tumor borders are infiltrative, irregular, and excision margins are close, as in organ sparing or surgery, postneoadjuvant chemotherapy. The traditional marking ink used by pathologists is India ink. However, when different surfaces/margins are to be studied or laterality of specimens needs to be identifiable even after specimen dissection, the lack of availability of differently colored inks is keenly felt. Differently colored inks also help when multiple samplings from different sites (e.g. prostate needle biopsies) or similar specimens of different patients are colored differently, to reduce identification error. A brief analysis of various indigenous and easily available coloring pigments that can be successfully used is hereby discussed. They are reliable, affordable, easy to obtain and use, safe, and stable.{Figure 1}

 Materials and Methods

Multiple types of dyes, paints, inks, and colored substances were evaluated, as tabulated in [Table 1]. These include:{Table 1}

The dyes routinely used in the Histopathology/Cytology laboratory, i.e. eosin, orange G-6, aniline blue, Alcian blue, Light green, etc. The powders were dissolved to full saturation in absolute alcohol and then painted on the specimen surface.Gelatin as described by Shinde et al. [1],[2] The solution wsas prepared by dissolving gelatin powder (edible gelatin/jellos of Crown food products), 4 g in 6 ml distilled water, and then heating in the microwave for 10 s.Commercially available marking inks, oil paints, and acrylic paints.

The following features were assessed:Easy to apply and quick to dry.Available in distinctly identifiable colors.Should withstand tissue processing and staining procedures, and be visible both on the paraffin blocks and microscopically on the sections.Should not stain/contaminate other tissues, processing fluids and solutions.Should not affect microscopic interpretation of nuclear and cellular detail, special stains or IHC studies.Have good shelf life, and be non-toxic.

Following precautions were taken to optimize the painting procedure. The surfaces of both fresh and fixed specimens were blotted dry with paper towels, before painting. The painted specimens were immersed in alcohol for few minutes or air dried, to fix the color on the surface. The specimen was then held in running water, to wash off excess color and then grossed routinely [Figure 1].


The most pertinent results are shown in [Table 1].


The results could be categorized into three groups.

Washed away during formalin fixation itself.Survived fixation and processing and were seen on the paraffin block, but not on the sections. This category can be used to color small biopsies for identification and orientation in the paraffin block. [Figure 2]d, to ensure the full representation in the sections prepared. Eosin, methylene blue, orange G-6, light green, and aniline fell in this category.{Figure 2}Withstood processing and staining procedures and were seen on both the paraffin block and the sections, as indicated in [Table 1]. These could be used as true marking inks. Some specific shades of Fevicryl Acrylic Hobby colors were seen in this group, with bright distinct colors on microscopy [Figure 3] and [Figure 4]. Primary colors like red, blue, green, and black were identified.{Figure 3}{Figure 4}


Pigments are a motley bunch of widely differing substances, whose only common feature is their display of color. As they are all different compounds, it is difficult to predict which of these colors would survive interaction with chemicals and solvents.

India ink, a colloidal suspension of inert carbon black, has been in use in India since fourth century BC, where it was called "Mashi" (Tamil). Jain sutras and several documents written with this ink have been unearthed intact as far as China. The carbon black was obtained by burning bones, tar pitch, etc., then mixed with gum/shellac/resin for binding and with water-ethylene glycol as a medium for the emulsion.

India ink fulfills most of the qualities required in a surgical marking ink. It survives tissue processing and staining procedures and ultimately when viewed under the microscope, the black ink line is seen, faithfully following the edges of the tissue.

Acrylic paints are also particulate pigments suspended in acrylic polymer emulsions, the colored pigment hanging in a mixture of multiple chemicals as a thick emulsion. First created around 1950, acrylic paint is increasingly popular and finding multiple uses, as these paints are easy to apply, quick to dry, and once dry they are water and solvent insoluble [Figure 4], and long lasting. However , it was seen that all acrylic colors do not suffice as grossing inks, and do not retain their color when immersed in alcohol and xylene. There are some specific shades of the Fevicryl acrylic Hobby color group, which do fulfill the requirements of a surgical marking ink [3] They fix on fresh and formalin fixed tissue easily, survive the overnight processing, and the staining procedures and the colors are seen clearly on both the paraffin blocks and microscopically on the sections. They do not unduly color the processing fluids, or spread within the tissue sections. These colors are available in most stationery shops, can be bought as single units, have long shelf life, are nontoxic and the cost works out to <1 ps per section. The other shades of acrylic paints lose their distinct pigment on processing, and only brownish particulate matter adheres on the surfaces marked.

Gelatin initially seemed to serve well, in that it adhered to the tissue surface, dried quickly, survived tissue processing and on microscopy it showed as a smooth purplish-pink (mix of eosin and hematoxylin) colored layer over the surgical margin. It, however, was not easily visible on the paraffin block. Colored pigments were then added into the gelatin prior to painting to get colored gelatins. The differently colored gelatins were clearly visible on the paraffin block, but after cutting and staining, on viewing the sections under the microscope the color in the gelatin had leached out, and the microscopic sections showed the same "purplish-pink" border irrespective of the different colors visible on the paraffin block [Figure 2]A and B. Although this can still serve the purpose by referring to the color in the paraffin block for identification, it was not an ideal situation. Also, the process was tedious in that required quantity of liquid gelatin had to be prepared just prior to use, due to its rapid congealing on cooling and so shelf life of prepared gelatin was next to nil.

There have been reports [4] of some marking inks mimicking microcalcifications on specimen radiography of breast lumpectomies. These colors should be avoided on specimens, where microcalcifications need to be identified after excision, by checking for radio opacity of the paint prior to use.

Painting specimen surfaces in the laparoscopic retrieval bag prior to morcellation of the mass has been reported, [5] to enable pathological evaluation of surgical margin status.

At present, in our Laboratory, these specific acrylic hobby colors are in regular use to mark the different surfaces in pancreaticoduodenectomy specimens, radical prostatectomy specimens, thyroidectomy specimens, breast lumpectomies, wide excision specimens of soft tissue tumors, further specimen types being added on as the need arises.

Coloring the margins with different colors adds precision to margin examination. It allows three-dimensional microscopic reconstruction of the tumor vis-a-vis its various neighboring anatomic structures. It allows postoperative comparison of tissue planes predicted by preoperative imaging. It maintains orientation of grossed and dissected specimens, enabling the pathologist to re-visit the grossed specimen, if required, and confidently allows further sampling if necessary. These colors do not interfere with special stains or IHC studies.


Easily available indigenous colored paints are available for surgical marking purposes. The colors in use in our laboratory are black (Camel special waterproof drawing ink), green (Fevicryl hobby colors, nos. 12, 22, 32), red (Fevicryl hobby colors no. 04), blue (Fevicryl hobby colors nos. 23, 19, and Alcian blue), and Mauve (Fevicryl hobby colors no. 15). These colors more than serve their purpose when differently colored inks are required for marking surgical surfaces. They are reliable, affordable, easy to obtain and use, safe and stable. Additional acrylic colors will no doubt be available after trial and error of the multiple shades available in the market, but at the moment these colors more than serve the purpose.


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