Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The overall goal of this procedure is to quantify fungal formm papillae in a consistent manner between scores and laboratories through the use of a dichotomous key. This is accomplished by first staining the subject's tongue blue with a food dye and adhering a filter paper with a one centimeter cutout to the tongue. The second step is to capture a high quality closeup image of the subject's tongue.

Next, the scorer uses the dichotomous key to examine the candidate papilla's shape, size, color, and elevation. A papilla that is not amorphous is at least 0.5 millimeters pinker than the background and is not recessed, is scored as a fungi form papilla. The final step is to confer counts between two scores and ensure counts are within 10%of each other.

Ultimately, the Denver Papillae protocol is used to define and prioritize the characteristics of fungi formm papillae to ensure consistent counting across scores and laboratories. The main advantage of this technique over existing methods, such as the Miller and Read protocol, is that it reduces the subjectivity of scores when quantifying fungi form papillae. It does this through clear definition of each characteristic and through use of a dichotomous key to ensure that all structures classified as fungi form papillae meet each of the criteria.

This method help answer key questions in the taste field, such as defining the role, if any, the fungi Formm papillae density plays in taste perception. We first had the idea for this method when we had our citizen scientists count fungi formm papillae using the Miller and Reading method, but we noticed that the counts varied wildly for the same tongue image. Talking to the scores revealed that they had prioritized the characteristics differently and varied on how they counted papillae that met some, but not all of the requirements.

Demonstrating the procedure would be Rudy Torres, a volunteer citizen scientist in the genetics of taste laboratory. To prepare subjects for this procedure, show them a photograph of how to pose themselves and what the captured image will look like. Direct the subject to dry her tongue with a paper hand towel and leave the tongue protruding from her mouth.

Using a sterile rayon applicator with a one inch tip, apply approximately three milliliters of blue food dye at a one to 36 concentration to the apex of the tongue. Instruct the subject to return her tongue to her mouth and swallow to remove excess dye. Next, instruct the subject to pose herself as shown in the photograph with her chin on her hands and elbows propped on a table for stability.

Direct the subject to extend her tongue a comfortable distance and secure it gently between her teeth. Adhere a 2.5 centimeter piece of filter paper with a 10 millimeter diameter circular cutout punched in it to the tip of the interior of the left side of the tongue. Next to the midline, attach a high quality digital point and shoot camera to a tripod for stability.

Using the macro setting, take at least three closeup images of the tongue, capturing the entire 10 millimeter circular cutout to ensure visualization of all fungi, formm, papillae, or FP within that area. Zoom in to the extent that the entire cutout is photographed while still within the camera's recommended zoom range. Ensure that the plane of the camera lens is parallel to the plane of the tongue and that the cutout appears circular in the photo.

Instead of oblong review photos to ensure they are of high quality and useful for counting. Begin this procedure by uploading the photos to the computer of the images taken of the same subject's tongue. Select only one for further analysis based on a clear definition between structures on the tongue at the highest zoom and the least distorted angle, so the tongue is broad and flat toward the camera.

Open the selected raw image in the open source software image J.Click on plugins, and in the dropdown menu, scroll to analyze and click cell counter. When the cell counter opens, click initialize to link the image to the cell counter. Use a standard magnification of 50%for consistency between scores and for use in subsequent steps.

To score fp, move this image to the left side of the screen next to the cell counter. This image will be referred to as Copy a Open a second copy of the raw image for measuring the diameters of the papillae being quantified. Move this copy to the right side of the screen so the two copies are not accidentally confused.

The second image will be referred to as copy B Zoom in, end out as needed to the preference of the individual score throughout the scoring process. Click on the line tool using copy B.Zoom in as much as necessary to accurately draw a diameter across the 10 millimeter inner circle of the filter paper at any angle and click analyze and set scale. Fill in the known distance as 10.

Verify the scale is correct by measuring an alternate angle and ensure the diameter range is between 9.8 to 10.2 millimeters. The Denver PAPALE protocol or DPP considers the shape, color, size, and recession of each candidate papilla in determining if it is an fp. The procedure for training scores to use the dichotomous key will not be shown in this video, but is available in the accompanying protocol text to begin the scoring.

Determine if the candidate papilla is amorphous or shapeless at 50%zoom. See if there is a commonly recognized geometric shape, such as oval, cuboidal, or round. If there is no geometric shape, go to the cell counter window and click type one on.

Copy A.Click the candidate papilla to mark it as amorphous and not an fp. Clicking on types one through four in this and subsequent steps allow scores to know they have addressed all candidate papillae and categorizes which rule was violated when rejecting a structure. If the candidate papilla has a geometric shape, proceed to the next step for examining its color, refer to copy A and determine if there is any color differentiation over the surface of the tongue.

If there is color differentiation, but the candidate papilla is blue and the surrounding papilla are lighter, go to the cell counter window and click type two on the cell counter on copy A.Click the candidate to mark as two blue and not an fp. If there is no color differentiation, or if any part of the candidate papilla has remained pink or stained lighter, proceed to the next step for size determination. Using copy B, zoom into the distance where one can comfortably see the outline of the candidate papilla.

Click on the line tool and measure across the longest dimension of the candidate papilla. Click, analyze and then measure if the measured length is 0.499 millimeters or less. Measure once more to ensure accuracy.

If it is still 0.499 millimeters or less, click type three on the cell counter window and onco a click the candidate pela to mark it as too small and not an fp. If the measured length is 0.5 millimeters or greater, proceed to examine its recession using copy a assess if the candidate papilla is either of uniform height with the rest of the tongue or elevated. If the papilla is in a crevice, determine its height compared to the other structures in the crevice, not the surface of the tongue.

If the papilla is lower than the papis surrounding it, click type four on the cell counter window and on copy A, click the candidate papilla to mark it as recessed and not an fp. If the candidate papilla is either of uniform height with the rest of the tongue or elevated, click type five and mark that this is an fp. This type five total is the raw FP score.

Write down the raw FP score in a notebook to save copy, a click export image on the cell counter window and save with the desired naming system of the laboratory. This will allow the opening of copy A in the future to retain the colored marks from types one through five. It'll not save the raw FP score for quality control.

Each photo must be scored by two individual scores and the score compared. If the higher FP raw score is within 10%of the lower FP raw score average, the two scores together for a consensus FP score. If the two FP raw scores differ by more than 10%pull both scored photos onto the screen.

Use counter types one through four to aid in discussion of discrepancies and confer on a final consensus score. If a consensus cannot be reached, pull in a third score to score the image and then dialogue with the other two scores. A representative photograph was first scored by two individual scores using the commonly used Miller and 3D methodology for FP identification.

The same photograph was then scored by two scores using the DPP. Red is scorer one, yellow is scorer two, and green is scores one and two. The counts shown a representative of the high variance observed using the original method compared to the lower variance using the DPPA comparison of score of variants for 11 individual scores.

Pre DPP training shown in gray and post DPP training shown in black revealed a significant decrease in score of variance post to DPP training. Once mastered, this technique can be done in under five minutes per image. While attempting this procedure, it's important to take frequent breaks to ensure fresh eyes on each photo and make sure that each candidate papilla is seen.

After watching this video, you should have a good understanding of how to quantify fungi form papilla in a consistent manner using a dichotomous key.