Domain: Natural Science — Biochemistry & Molecular Biology
Target concept: Succinate Dehydrogenase Catalyzed Reaction (a Key Reaction in the TCA Cycle and the Electron Transport Chain)

Video generation prompt: Add succinate and methylene blue solution to a mitochondrial suspension containing succinate dehydrogenase, mix gently, and incubate in the dark for about 3 minutes.

Evaluation guide:
(i) Target concept: Enzyme-catalyzed electron transfer from succinate to methylene blue via succinate dehydrogenase in intact mitochondria.
Minimum mechanism: Mitochondrial suspension must contain functional succinate dehydrogenase; succinate must be present as substrate; methylene blue must be present as the redox indicator; respiring mitochondria in the suspension rapidly consume dissolved oxygen, and it is this local oxygen depletion, not the darkness itself, that keeps the reduced (colorless) methylene blue from being re-oxidized by air; the dark setting mainly guards against light-driven side reactions of the dye rather than being what blocks re-oxidation; reduction must occur within the suspension matrix (not merely at the surface).
(ii)Storyline: [Frame 0–2] Tube with turbid mitochondrial suspension centered; [Frame 3–4] Colorless succinate solution dispensed into tube; [Frame 5–6] Deep blue methylene blue solution dispensed into same tube; [Frame 7–8] Tube gently swirled: liquid rotates cohesively, meniscus deforms smoothly; [Frame 9–10] Tube held still under uniform dim light for about 3 minutes: deep blue color visibly fades across entire liquid column to near-colorless by final frame. Fixed frontal view; no cutaways; no hands visible after dispensing; all actions occur within same continuous optical plane.

Manual:
Experimental Procedure
1. Place mitochondrial suspension in transparent tube.
2. Add succinate solution dropwise.
3. Add methylene blue solution dropwise.
4. Gently mix by swirling or inverting tube once.
5. Incubate in dark for about 3 minutes (no light exposure).
Observation of Experimental Phenomena
Deep blue color of methylene blue fades to colorless during dark incubation.
Summary of Experimental Principles
Succinate dehydrogenase oxidizes succinate to fumarate, reducing FAD to FADH₂; FADH₂ donates electrons to methylene blue (artificial electron acceptor), reducing it from blue (oxidized) to colorless (leuco form).

Prompt Grounding rubric:
level_1: No mitochondrial suspension is shown; no succinate solution or methylene blue solution container appears; no mixing action is depicted; no dark incubation setting is shown; the sequence gives no sense that the tube sits still in low light for an extended period.
level_2: Only one of the three key items (the mitochondrial suspension, the succinate solution, or the methylene blue solution) appears on screen, and they are never brought together in one tube; no swirling or inversion is shown; the background stays brightly lit throughout with no darkened setting; nothing in the sequence conveys a multi-minute incubation period.
level_3: The turbid mitochondrial suspension, the colorless succinate solution, and the deep-blue methylene blue solution all appear in the same tube; succinate is added first, methylene blue second; the tube is gently swirled or inverted to mix; the tube then sits in a dim or darkened setting; the sequence conveys addition, mixing, and a settled multi-minute incubation period (an exact time label is not required).
level_4: All conditions for score 3 are met, and mixing happens only after both succinate and methylene blue have been added (not beforehand or mid-addition); the suspension stays visibly turbid/cloudy throughout; no unrelated reagents, tools, or on-screen labels appear; the camera stays fixed on the reaction tube the entire time.
level_5: All conditions for score 4 are met, and before mixing the succinate solution is visibly colorless/clear while the methylene blue solution is visibly deep blue; the mitochondrial suspension is shown in a transparent tube with a visible cloudy or slightly sedimented layer before anything is added; the whole sequence plays as one uninterrupted shot with no cuts, zooms, or viewpoint changes.

Scientific and Causal Correctness rubric:
level_1: The methylene blue stays deep blue in the tube for the entire video with no fading at all; or the suspension and dye show no visible interaction; or nothing links the succinate/methylene blue addition to any later color change.
level_2: The blue color changes, but not in a way consistent with succinate dehydrogenase activity: for example it fades before succinate has been added, fades during the mixing motion itself rather than during the still incubation period, or briefly fades and then visibly re-blues before the video ends (a spurious reversal inconsistent with continued enzymatic reduction).
level_3: After succinate and methylene blue are both added to the mitochondrial suspension and the tube is mixed, the deep blue color visibly lightens (toward pale blue or colorless) during the still incubation period, with the lightening clearly apparent by the end of the video; no other dye or reagent is present to explain the change.
level_4: All conditions for score 3 are met, and the fading is gradual (deep blue moves through an intermediate lighter-blue stage before reaching its final shade) rather than an abrupt jump; the fading occurs only after mixing has stopped, during the still incubation, and the final color holds steady with no flickering back toward blue.
level_5: All conditions for score 4 are met, and by the end of the video the liquid is essentially colorless (not merely pale blue); the suspension keeps its original cloudy/turbid appearance with no separate clearing or clumping unrelated to the dye; the color change looks uniform across the liquid column rather than patchy or streaked.

Spatiotemporal Consistency rubric:
level_1: The tube vanishes from the frame and reappears elsewhere, or its position/size jumps without any motion; the room lighting swings from bright to dark (or back) with no transition; the tube's glass appears to bend, crack, or change shape; the swirling motion skips or jitters between poses; the liquid surface floats or curves in a way that ignores gravity.
level_2: The tube rotates by a large angle (well over 90 degrees) with no visible hand or motion causing it; liquid appears to splash or jump without any visible arc or trajectory; the swirling motion starts or stops with no smooth speeding-up or slowing-down; the dim/dark setting switches on or off instantly with no dimming transition.
level_3: The tube stays the same size and in the same place relative to the bench throughout; the liquid level stays steady while succinate and methylene blue are added (no overflow, no sudden suck-back); the swirling or inversion motion looks smooth and continuous with the liquid following along naturally; the lighting stays evenly dim with no sudden bright flashes or jumping shadows.
level_4: All conditions for score 3 are met, and the tube does not slide, rock, or drift on the surface while being swirled; the liquid's curved surface (meniscus) bends the correct way as the tube rotates; there is no flickering or strobing between frames; nothing (like a hand) enters the frame and then fails to leave it in a visible way.
level_5: All conditions for score 4 are met, and the liquid surface shows a natural gentle ripple during swirling rather than moving as one rigid rotating block; the fading blue color stays clearly visible with no blurring or streaking that hides its true shade; the tube wall and the liquid inside it move together with no visible sliding or offset between them; there is no doubled or mirrored reflection of the tube or liquid.