Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

Papillae are the visible bumps on the tongue’s surface. There are four types of papillae: fungiform (FP), foliate, circumvallate, and filiform. While filiform papillae are spread over the surface of the tongue, circumvallate are localized only on the back of the tongue, and foliate are found on the sides. In contrast, FP are located toward the front of the tongue. FP, foliate, and circumvallate papillae are considered gustatory because they have the potential to contain taste buds while filiform do not. Taste buds are clusters of cells responsible for detecting chemical tastants and transducing the stimuli into a gustatory signal in the brain. Saliva and food molecules enter into the taste buds through openings called taste pores where taste molecules then have the potential of activating taste cells.

Interestingly, there are large individual differences in taste bud density, as first observed in a histological study of tongues from 18 cadavers (Miller, 1988)¹. Taste buds themselves are not visible on the tongue's surface, however, taste pores are visible when using proper magnification, lighting, and staining techniques which led Miller & Reedy (1990) to develop a method to identify taste pores and fungiform papillae using videomicroscopy in living human beings². A number of labs have modified their original videomicroscopy technique, using still photography and little or no magnification. However, since fungiform papillae are much easier to visualize than are pores using these latter techniques, labs turned to counting fungiform papillae in place of taste pores. In doing so, it is assumed that fungiform papilla density is a reasonable proxy measure for taste pore density, even though many fungiform papillae do not contain taste pores or taste buds³; not only are FP more visible and accessible than circumvallate and foliate papillae, but their image capture is less time consuming than taste pore analysis through the alternative method of videomicroscopy^2,4. In 1990, Miller & Reedy used this technique to find that taste pore densities are correlated with FP densities³ which was further corroborated in Bartoshuk et al.⁵ In addition to this anatomical relationship, Miller & Reedy also found that the 8 individuals in the upper half of the taste pore density distribution also rated sucrose, NaCl, and propylthiouracil (PROP) as significantly more intense than did the other 8 individuals³. Subsequent work by Bartoshuk et al. (1994) observed a correlation between suprathreshold PROP taste intensity and fungiform papilla density, as well as taste pore density, in 42 subjects⁵. This convenient method led to influential studies reporting that subjects whose tongues have many papillae elicit a stronger reaction to many tastants, including the bitter tastants phenylthiocarbamide (PTC) and PROP ^6,7.

PTC and PROP are well-researched tastants oft used due to the clear link between a person’s taste sensitivity phenotype and their genotype. Population studies have shown that people who have the homozygous recessive diplotype of the gene, TAS2R38, have a significantly lower sensitivity to PROP than carriers of the dominant or heterozygous variant of the gene^8-10. This heritable phenomenon was first reported in 1932 when Arthur L. Fox announced his discovery of “taste blindness” to PTC¹¹. Among people able to taste these bitter compounds, the intensity of taste reported can vary anywhere from slightly unpleasant to excruciatingly bitter⁶. To account for variance that cannot be explained by TAS2R38, the theory was put forth that it could be attributed to the density of FP on the tongue⁴.

As taste research progressed, the field was divided into two schools of thought around this theory concerning the role of FP and bitter taste sensitivity. Although many studies corroborated the original claim that FP density is a factor in PROP sensitivities^4,6,12,13, there has been a small, but important contingency that has found evidence to refute it, reporting an inability to replicate the role^10,14,15,16. Delwiche et al. (2001) warned that the trend is highly variable; FP density does not uniformly account for differences in bitterness sensitivity across subjects, and was only demonstrable for subjects with at least a moderate sensitivity to PROP⁷. In addition to the use of the less rigorously defined Miller & Reedy method on how to quantify FP, it is important to note that with exception of the Beaver Dam Offspring Study¹⁵ conducted by Fischer et al., most of the above studies had small sample sizes which may also contribute to inconsistent findings on the role of FP in taste.

Briefly, in Miller & Reedy’s seminal methodology paper (1990), the authors stained tongues blue to quantify FP because FP remain pink while filiform papillae absorb the dye and turn blue. They classified FP as “rounded pink structures about 0.5 mm in diameter”². While the authors advised that before using this method for further analytical work, FP characteristics should be more strictly defined and concluded that the variation already noted in the literature “might be attributable to different subject populations, different methods, and different investigators”², their research has led to the commonly accepted characteristics that FP are round^17,18, large², pink or stained lighter², and elevated⁴. Taken at face value, these characteristics appear very straight forward. In the Genetics of Taste Lab (GoT Lab or Lab), the use of the method was complicated by the scorers’ individual attempts at qualifying and prioritizing the importance of the above characteristics when some, but not all, of the characteristics were present. These complications included small papillae that appeared pink in the midst of much larger papillae; the entire tongue absorbing the blue dye thus eliminating classification based on color variation; oblong papillae instead of round; and tongues having no variation in elevation, all of which contributed to scorers reporting widely-varied FP counts of the same image.

These variations in analysis led us to review the literature in order to pinpoint the subjectivity, identify any exceptions to the commonly accepted FP characteristics and ultimately devise a protocol to accurately and objectively define and score FP.

First, FP shape is described in the literature as a rounded, mushroom-like structure^17,18. However, notable exceptions were also mentioned. Miller emphasized that the term “fungiform” is a misnomer and that many FP are not mushroom-shaped but can vary widely in size and morphology¹⁸. Melis et al. (2013) also described several papillae that were considered “distorted,” where the FP diameter in one direction was at least two standard deviations longer than in another direction¹⁹. Furthermore, Cheng & Robinson found that when indicating FP by staining, they ranged from flat-topped to elongated in appearance²⁰. Noting the above exceptions, the characterization of an FP being round and mushroom-shaped appears too narrow a definition.

Second, FP color is described as “pink circles against a blue background”⁷ after staining the tongue with blue dye. Though staining lighter was the most consistent criterion of FP throughout the literature it was still not a uniform qualifier. Cheng & Robinson observed that FP were not always lightly stained, making identification difficult and uncertain²⁰. It appears then that tongues absorb dye differently and while the contrast is apparent on many tongues, some become entirely blue with no distinction while others immediately lose any trace of blue dye²¹.

The third characteristic, FP size, was fairly consistent among papers, ranging from 0.5 mm to 0.97 mm^6,18. In the literature Miller noted that there were two size ranges of papillae on the dorsal anterior of the tongue. The papillae with larger diameters were primarily fungiform and conical papillae, while the papillae with a smaller diameter were mostly filiform¹⁸. However, with the exception of the Beaver Dam Offspring Study²¹, size did not appear to determine if a structure is a FP in the literature, but was provided for papillae already classified as “fungiform".

Finally, elevation is listed as the fourth FP characteristic⁴ though Miller pointed out that distinction between filiform and fungiform papillae is difficult when using this criteria near the margin of the tongue. He gave two examples of FP that varied greatly in elevation, one being 0.8 mm in height while another was less than 0.1 mm in height, but still had two taste pores that were clearly observed¹⁸. This observation was also followed up and confirmed by Shahbake et al.⁴

These definitions and their reported exceptions show that the taste field has long recognized the inconsistency in characterizing FP and the shortcomings of the commonly-used protocol from Miller & Reedy. Indeed, Miller & Reedy exhorted the field to further define the features of FP². We hypothesized that scorers were giving different weight to the characteristics and prioritizing their importance differently when papillae failed to meet every criterion, such as when a papilla was large and mushroom shaped, but stained blue. It was reasoned that investigators of the previously published studies likely did the same which, when combined with small sample sizes, may account for the varied results coming from use of this method.

The GoT Lab addressed this gap in FP methodology by developing a guideline that defines each characteristic using objective measurements and prioritizes the characteristics to indicate which takes precedence in analysis. This method is the Denver Papillae Protocol (DPP), a dichotomous key with clear and distinct characteristics of FP to ensure accurate and repeatable counts from digital photographs of the tongue. DPP is a proposed method to standardize the previously used Miller & Reedy method, thus removing individual interpretation and ensuring more consistent results when analyzing FP density. By doing so, DPP can be used to more confidently determine the role of FP in taste.

The tongue images taken of the subjects were analyzed by the citizen scientists (scorers) of the Lab who are the true subjects of this study. The Lab’s core of citizen scientists is comprised of members from the community ranging in age (years) from 16 to mid-80s. A summative evaluation report commissioned by the Museum in 2012 describes citizen scientists as having a strong interest in science and with two-thirds having earned a college degree in a scientific discipline²². Candidate citizen scientists are currently recruited via word of mouth, must successfully complete an interview process with veteran volunteers at the Museum, and undergo a trial period in the Museum’s permanent health exhibition, Expedition Health, before they have the opportunity to apply for a position in the Lab. Once accepted into the Lab, citizen scientists undergo a 12-week training to become certified to enroll human subjects. Following this certification, they are able to take training modules on various techniques in the Lab (e.g. papillae counting, DNA extraction) and following additional certifications and regular quality control measures, these citizen scientists have the opportunity to actively participate in data analysis. Citizen scientists volunteer their time and are not compensated for their contributions to the Museum.