Domain: Healthcare — Digestive Physiology
Target concept: Colorimetric detection of pepsin-mediated hemoglobin digestion via TCA precipitation and the Folin-Ciocalteu (Folin's phenol) reagent

Video generation prompt: An appropriate amount of rabbit gastric mucosal fluid, pre-extracted in dilute hydrochloric acid to activate its native pepsin, was placed in a test tube, followed by the addition of hemoglobin solution. The mixture was incubated at 37°C for 45 minutes, after which trichloroacetic acid was added to stop the reaction and precipitate any undigested protein. Once the resulting precipitate had settled, Folin-Ciocalteu reagent was added to the clear supernatant.

Evaluation guide:
(i) Target concept: Demonstration of pepsin's proteolytic digestion of hemoglobin, detected by reacting the Folin-Ciocalteu reagent with the acid-soluble digestion products left in solution after undigested protein has been precipitated out.
Minimum mechanism: The rabbit gastric mucosal fluid is pre-extracted in dilute hydrochloric acid, which releases pepsinogen from the mucosal tissue and converts it into active pepsin, so the fluid already contains working enzyme before it ever meets the hemoglobin. During the 37°C, 45-minute incubation, this pepsin hydrolyzes peptide bonds inside the hemoglobin tetramer, releasing short peptides and free tyrosine/tryptophan-bearing fragments into solution. Adding trichloroacetic acid (TCA) denatures and precipitates any hemoglobin that remains undigested, while the small digestion products stay dissolved; once this precipitate has settled out, only the clear supernatant is reacted with the Folin-Ciocalteu reagent, which develops a blue color in proportion to the tyrosine/tryptophan content of the small peptides present. Because the intact, undigested hemoglobin has already been removed by the TCA precipitation step, the intensity of blue that develops in the supernatant reflects how much hemoglobin pepsin actually digested.
(ii) Storyline: [Frame 0-1] Empty test tube enters view -> [Frame 2-3] Tan, turbid gastric-extract suspension is dispensed into the tube -> [Frame 4-5] Red/burgundy hemoglobin solution is added and visibly mixes into the extract -> [Frame 6-8] The tube sits motionless in a labeled 37°C water bath for the full incubation, its red-brown color unchanged during this interval -> [Frame 9] Trichloroacetic acid is added, and the mixture turns cloudy as a precipitate forms and begins settling toward the bottom of the tube -> [Frame 10] Once a clear layer has separated above the settled precipitate, Folin-Ciocalteu reagent is added to that clear layer, which turns blue; filmed throughout from one fixed frontal angle under steady, unchanging lighting.

Manual:
Experimental Procedure
1. Place dilute-hydrochloric-acid-extracted rabbit gastric mucosal fluid (a tan/beige, turbid suspension containing activated pepsin) into a clean test tube.
2. Add hemoglobin solution (red/burgundy liquid) to the same test tube.
3. Incubate the mixture at 37°C for 45 minutes without agitation.
4. Add trichloroacetic acid (TCA) to stop the reaction; a cloudy precipitate forms and is left to settle, leaving a clear supernatant above it.
5. Add Folin-Ciocalteu reagent to the clear supernatant.
Observation of Experimental Phenomena
After TCA is added, the red-brown mixture becomes cloudy as undigested hemoglobin precipitates out; once this precipitate has settled, the clear liquid above it turns blue after the Folin-Ciocalteu reagent is added.
Summary of Experimental Principles
The rabbit gastric mucosal fluid is prepared by extraction in dilute hydrochloric acid, which both releases pepsinogen from the mucosal tissue and converts it into active pepsin, so the fluid already contains working enzyme when the experiment begins. During the 37°C incubation, this pepsin hydrolyzes peptide bonds inside the hemoglobin tetramer, releasing short peptides and free tyrosine/tryptophan residues into solution. Trichloroacetic acid denatures and precipitates any hemoglobin that remains undigested, so only the small digestion products stay dissolved in the resulting clear supernatant. The Folin-Ciocalteu reagent then develops a blue color in proportion to the tyrosine/tryptophan content of these small peptides — so the intensity of blue in the supernatant reflects how much of the hemoglobin was digested by pepsin.

Prompt Grounding rubric:
level_1: The test tube is empty or holds an unidentifiable liquid; none of the required substances or steps (gastric extract, hemoglobin solution, a precipitate-forming step, and a final blue-developing reagent) can be told apart or recognized, and no incubation cue (water bath, heater, or on-screen temperature/timer label) is present.
level_2: The tube contains liquid, but the extract, hemoglobin, precipitating reagent, and final color reagent all look like one indistinct fluid rather than separable substances added in sequence; steps are missing or reordered (for example no visible precipitate ever forms, or the final reagent is added with no prior precipitation step shown), and the 37°C, 45-minute incubation has no accompanying thermal or timing cue.
level_3: The tan, turbid gastric extract and the red/burgundy hemoglobin solution are each clearly recognizable and enter the tube in the correct order, a 37°C incubation cue is visible, and adding the next reagent visibly produces a cloudy precipitate, but the final blue-developing reagent is added before that precipitate has visibly settled into a distinct clear layer.
level_4: All required substances and steps are visually distinct and occur in the correct order (tan/beige extract first, red/burgundy hemoglobin second); the tube then holds still through a depicted 37°C, 45-minute incubation, a precipitating reagent is added afterward producing a visible cloudy layer that is allowed to settle into a clear upper layer, and only then does the final blue-developing reagent enter that clear layer.
level_5: Every procedural element matches the stated setup precisely: the tube starts holding only the tan gastric extract, the red hemoglobin solution is poured or dropped in with visible mixing into that same extract, the combined red-brown mixture sits undisturbed through the complete 37°C, 45-minute incubation with a thermal cue present throughout, the precipitating reagent is added afterward producing a distinct cloudy precipitate that settles fully into a clearly separated clear supernatant, and the final blue-developing reagent is added only to that clear supernatant, with a clear break between the settling step and the reagent-adding action.

Scientific and Causal Correctness rubric:
level_1: No blue ever appears in the tube, or a blue color is already present before the final reagent is shown entering the clear supernatant; i.e., the video shows no dye-triggered color event tied to the depicted procedure at all.
level_2: Blue coloration appears, but its timing is scientifically wrong for the depicted setup, e.g., it appears before the gastric extract and hemoglobin have been combined, or before any incubation or precipitation step is shown, or the tube turns a hue other than blue (such as staying red-brown, or turning an unrelated color) even after the final reagent is fully mixed in.
level_3: A cloudy precipitate visibly forms only after the precipitating reagent is added following the incubation, and blue develops only after the final reagent contacts the settled supernatant, but the result is uneven, e.g., the precipitate and clear layer never fully separate, or the blue appears patchy within the clear layer rather than filling it uniformly.
level_4: The full sequence plays out cleanly and in the correct order: the extract and hemoglobin combine and sit through incubation with no color change; the precipitating reagent then produces a visible cloudy layer that settles into a clearly separated clear supernatant; the final reagent is added only to this clear layer and produces a uniform blue there, while the settled precipitate at the bottom keeps its own separate off-white/tan color rather than turning blue.
level_5: All 4-point criteria are met, and the depicted result reads as biologically calibrated: a visible amount of precipitate remains at the bottom of the tube (consistent with only partial digestion occurring in a fixed 45-minute window, rather than an implausibly precipitate-free tube suggesting complete digestion); the blue that develops in the supernatant is a single even hue with no patches of a different color; and no red or brown tint remains within the clear supernatant layer itself once the final reagent has fully mixed in.

Spatiotemporal Consistency rubric:
level_1: The test tube's presence, size, or position changes abruptly between shots (e.g., it disappears and a differently shaped or placed tube appears next), one of the liquid volumes jumps to a different level with no pouring shown, or the mixture switches straight from red-brown to a settled precipitate with a blue supernatant across a cut with no reagent-adding action ever shown; the lighting abruptly flickers or shifts in brightness or color temperature between frames with no cause, or the camera view jumps or rotates with no motion shown to justify it.
level_2: The tube stays visible but its position or the camera framing shifts noticeably between the extract-adding, hemoglobin-adding, precipitating-reagent-adding, and final-reagent-adding moments; at least one of the liquids or reagents is already inside the tube without its addition ever being shown; or the incubation or settling segment is presented as a hard cut rather than one continuous still shot.
level_3: The tube stays in a consistent spot throughout; the gastric extract, hemoglobin solution, precipitating reagent, and final blue-developing reagent are each shown entering the tube on screen; the incubation and settling periods each play as one continuous shot with no cuts; brief camera shake or soft focus may occur but the tube and its contents are never lost, duplicated, or swapped.
level_4: The tube stays fixed in frame and in focus for the entire sequence; each liquid or reagent addition is one smooth, continuous motion into the tube with no jump in the dispensing hand's position between frames; the incubation and settling segments each show uninterrupted stillness with steady framing; the mixture holds its red-brown color until the precipitating reagent visibly enters it, at which point the cloudy precipitate begins forming and settling; and the supernatant stays clear until the final reagent visibly touches it, at which point the blue begins to spread.
level_5: All 4-point criteria hold, and additionally: the tube's outline, tilt, and distance from the camera stay visually unchanged from the first frame to the last; each liquid's fill level rises smoothly only during its own addition step, with no unexplained jump in volume between neighboring frames; the precipitate settles gradually toward the bottom of the tube rather than snapping instantly into its final settled position; and the clear-to-blue transition in the supernatant spreads gradually through that layer rather than switching everywhere at once in a single frame.