Domain: Healthcare — Digestive Physiology
Target concept: Sucrase Activity Assay (Sucrose Hydrolysis Method)

Video generation prompt: Solutions of sucrose are added to two test tubes separately. To one test tube, an active sucrase solution is added, while the control test tube receives a heat-inactivated sucrase solution. Both test tubes are incubated in a 37°C water bath. After incubation, Benedict's reagent is added to each test tube, followed by mild heating. Clearly demonstrate the experimental procedure, with labeled test tubes, reagents, water bath, and heating process. Keep the camera stable and perform the operation in a clear and concise manner.

Evaluation guide:
Target concept: Sucrase enzymatic activity is defined by its capacity to hydrolyze sucrose into reducing monosaccharides, a process strictly dependent on intact tertiary structure (lost upon heat denaturation). Minimum mechanistic assumptions: (1) Sucrose is non-reducing; (2) Glucose and fructose are reducing; (3) Benedict’s reagent reacts specifically with reducing sugars under heat to form Cu₂O precipitate; (4) Heat-inactivation disrupts sucrase conformation irreversibly. Expected phenomena storyline: Phase 1 (Setup): Two labeled tubes receive identical sucrose; differential enzyme addition establishes causal variable. Phase 2 (Incubation): 37°C enables active sucrase catalysis; control remains inert. Phase 3 (Detection): Benedict’s + heat reveals outcome. Brick-red precipitate *only* where hydrolysis occurred. Visibility constraints: Precipitate must be macroscopically visible, settled, and sharply contrasted against the blue supernatant; tube labels and the water-bath temperature indicator must be visibly present and legible at a glance, without requiring the viewer to zoom in or pause the video; all steps must unfold in strict chronological order with no temporal compression violating causality (e.g., heating before reagent addition).

Manual:
I. Basic Demonstration Information
Demonstration Title: Sucrase Activity Assay (Sucrose Hydrolysis Method) Demonstration
Subject: Healthcare - Digestive Physiology - Enzyme Function
Demonstration Purpose: To visually demonstrate that sucrase catalyzes hydrolysis of sucrose into glucose and fructose — reducing sugars detectable by Benedict’s reagent — and that heat-inactivation abolishes this activity; to reinforce the principle that enzyme function is temperature-sensitive and structure-dependent; to train observation of biochemical colorimetric endpoints.

II. Demonstration Carrier/Equipment
Core Carrier: Two identical glass test tubes (15×125 mm), sucrose solution (5% w/v), active sucrase solution (e.g., yeast or intestinal extract, kept on ice), heat-inactivated sucrase (boiled 10 min, cooled), Benedict’s qualitative reagent, 37°C water bath with digital thermometer, boiling water bath or hot plate, waterproof labels, calibrated droppers.

III. Demonstration Content Description
Layout: Test tubes placed upright in rack on clean lab bench. Left tube labeled ‘Active Sucrase + Sucrose’, right tube ‘Control (Heat-Inactivated) + Sucrose’. Both receive 2 mL sucrose solution first. Then 0.5 mL active sucrase added to left; 0.5 mL heat-inactivated sucrase added to right — using separate droppers. Tubes placed simultaneously into 37°C water bath for 30 min (timer visible). After incubation, tubes removed, 2 mL Benedict’s reagent added to each. Tubes then placed into boiling water bath for 3–5 min with gentle agitation.

IV. Observation of Core Phenomena
Active Tube Phenomenon: After heating, develops progressive color change: turquoise → green → yellow → brick-red precipitate (Cu₂O) at bottom — indicating reducing sugars present.
Control Tube Phenomenon: Remains homogeneous deep blue throughout — no precipitate — confirming absence of reducing sugars due to inactive enzyme.
Visual Feature Recognition: Brick-red precipitate is coarse, granular, and settles; blue color is translucent and uniform. Color contrast must be sharp and unambiguous under standard lab lighting.
Causal Verification: Only the tube receiving *active* sucrase + *incubation* + *Benedict’s* + *heating* yields precipitate — establishing necessary and sufficient conditions for detection.

V. Summary of Key Knowledge Points and Principles
Core Concept Definition: Sucrase is a brush-border disaccharidase that hydrolyzes sucrose into glucose and fructose. Its activity is abolished by heat-induced denaturation. Benedict’s test detects reducing sugars via reduction of alkaline Cu²⁺ to insoluble Cu₂O.
Physiological Significance: Confirms enzymatic specificity, thermal lability, and clinical relevance — e.g., congenital sucrase-isomaltase deficiency presents with osmotic diarrhea upon sucrose ingestion due to unhydrolyzed disaccharide.
Further Knowledge: Other disaccharidases (lactase, maltase) follow same assay logic; false positives may arise from reducing contaminants; controls must include substrate-only and enzyme-only tubes for full validation.

Prompt Grounding rubric:
level_1: No test tubes are visible; nothing distinguishes an active-enzyme condition from a control condition; no sucrose solution, sucrase solutions, Benedict's reagent, water bath, or heating step is shown; the camera framing excludes the core equipment or the operation sequence is unrecognizable.
level_2: At least two test tubes are present but look identical with no way to tell which received active sucrase versus heat-inactivated sucrase; a water bath or heating step is missing entirely, or no temperature cue of any kind (dial, thermometer, display) appears near it; reagent additions, if shown, are not clearly differentiated between the two tubes.
level_3: Two test tubes are visibly distinguished from each other (e.g. by tags, marker color, or separate containers) as the active-enzyme tube and the control tube; sucrose solution is added to both; a water bath appears with a visible reading at or near 37°C; Benedict's reagent is added to both followed by a heating step, but the distinguishing marks are hard to make out, the same dropper appears to serve both tubes with no visible switch, or incubation and detection run together with no visible pause between them.
level_4: Two test tubes are clearly and durably marked to distinguish 'active sucrase' from 'control / heat-inactivated'; sucrose solution is added to both first; active sucrase is added from one container and heat-inactivated (previously boiled) sucrase from a visibly separate container; both tubes sit in a water bath with a visible temperature indicator reading at or near 37°C; after an implied incubation period, Benedict's reagent is added to each tube with separate droppers, followed by a shared heating step, with only one small shortfall, e.g. the marking is a little small or the color development isn't shown before the clip ends.
level_5: All of the 4-point criteria are met, and additionally: the markings on each tube are large, high-contrast, and readable at a glance without pausing the video; the active-sucrase and heat-inactivated containers look visibly different from each other (different bottle, cap color, or label style); the water bath's temperature indicator is clearly visible and reads 37°C (or a value very close to it); Benedict's reagent is delivered through visually distinct droppers for each tube; the camera holds a steady, unobstructed view of both tubes so every step (sucrose addition, enzyme addition, incubation, Benedict's addition, heating) can be followed without a hand or object blocking the view; the active sucrase solution looks faintly cloudy/proteinaceous while the heat-inactivated sucrase shows a trace of flocculent haze consistent with a denatured protein.

Scientific and Causal Correctness rubric:
level_1: Neither tube ever changes color after Benedict's reagent and heating are applied (both stay blue), or both tubes turn brick-red/orange regardless of which sucrase was used, so the video shows no difference in outcome between the active-enzyme and heat-inactivated conditions.
level_2: A color change appears in only one tube, but the causal chain is broken, e.g. the color change happens before Benedict's reagent is even added, or before any heating occurs, or the control tube changes color too for no apparent reason, rather than showing a genuine contrast between active and inactivated enzyme.
level_3: After sucrose addition, incubation, Benedict's reagent, and heating, the active-sucrase tube visibly shifts from blue toward an orange/brick-red precipitate while the control tube stays blue (the correct outcome is shown), but the build-up feels thin, e.g. no incubation period is implied before Benedict's reagent is added, or heating is applied to only one of the two tubes.
level_4: The active-sucrase tube's transformation follows the full logical order: sucrose and active sucrase sit together for a period before Benedict's reagent is added to both tubes, both are then heated together, and only the active tube develops a brick-red/orange precipitate while the control stays blue: mix, wait, test, heat, and a distinct outcome all appear in sequence.
level_5: All of the 4-point criteria are met, and additionally: the active tube's color visibly passes through an intermediate shade (e.g. blue-green) on its way to orange/brick-red rather than jumping straight to the end color; the settled precipitate looks visibly different in texture from the surrounding liquid; the control tube's blue stays unchanged and even throughout; the video's own color sequence makes the causal story clear without needing any on-screen text, arrow, or equation overlay.

Spatiotemporal Consistency rubric:
level_1: The water bath appears to be steaming or bubbling in one shot and then still and cool the next with no heating event shown in between; a test tube's position or angle changes abruptly between shots with no hand visibly moving it; the sequence jumps backward in time, e.g. a tube already showing its final color is followed by a shot of Benedict's reagent being added to it as if for the first time; a reagent bottle or dropper floats and moves through the air with no hand holding it, defying gravity; or a hand introduces two visually identical, unlabeled vials and the video simply asserts one is heat-inactivated sucrase with nothing shown to actually distinguish them.
level_2: The water bath is implied to be warm (by a label or a dial) but shows no visible water, steam, or condensation; a reagent bottle looks full in one shot and empty in the very next with no pouring or dropping motion shown in between; a hot plate glows or a bath appears to be boiling before any tube has actually been placed in it.
level_3: The tubes, water bath, and reagent bottles stay in consistent positions across the sequence, and reagents are added with a visible dropper motion before any heating begins, but there is at least one gap, such as the incubation period being implied with no sense of elapsed time, or one tube going into the bath noticeably later than the other with no visible reason.
level_4: The bench, camera framing, and lighting stay consistent from the sucrose addition through the final heating step; each action (adding sucrose, adding sucrase, placing the tubes in the water bath, removing them, adding Benedict's reagent, heating) follows the last one in a continuous or cleanly cut sequence, and liquids behave under normal gravity (drops fall down, nothing floats); only one small hitch remains, such as a slight jump in camera angle between two steps.
level_5: The whole sequence reads as one continuous, physically coherent take or a seamlessly matched set of cuts: every liquid transfer, tube placement, and heating step happens in the right order with no jump in tube position, water level, or lighting; steam or condensation, if shown, behaves naturally; no hand, dropper, or tube appears or disappears without a visible entry or exit; the passage of time between incubation and detection is shown through a gradual, consistent visual cue (e.g. a still bath beforehand and a gently steaming one afterward) rather than an unexplained jump.