Domain: Natural Science — Environmental Science
Target concept: Fluorescent Staining Assay for Bacterial Viability

Video generation prompt: Add the FDA fluorescent dye to the bacterial suspension and stain for 10 minutes in the dark, then examine the sample under a fluorescence microscope to compare the fluorescence of live and dead cells.

Evaluation guide:
(i) Target concept: Viability-dependent enzymatic conversion of non-fluorescent FDA to fluorescent fluorescein in intact bacterial cells.
Minimum mechanism: Live cells must possess functional esterases AND intact cytoplasmic membranes to retain fluorescein; dead cells must lack either enzyme activity or membrane integrity, resulting in binary green/dark phenotypic contrast under identical illumination.
(ii)Storyline: [Phase 1: Setup] Clear tube with turbid bacterial suspension visible; FDA dye bottle and dropper present nearby. [Phase 2: Action] Dropper approaches tube; dye dispensed as cohesive droplet contacting suspension surface, with no splashing or floating. [Phase 3: Incubation] Tube covered (lid/foil) or placed in darkened environment, ambient light visibly absent. [Phase 4: Observation] Microscope view replaces scene: fixed focal plane showing clustered bacterial cells; subset emits localized green fluorescence, adjacent morphologically identical cells emit zero green signal; background is uniformly non-fluorescent. [Perspective constraint] All phases use consistent macro/micro viewpoint, with no unrealistic zoom jumps; microscope view must show cells at resolution sufficient to resolve individual rods/cocci and assign fluorescence per cell. [Visibility constraint] No occlusion of critical events: dye contact, cover application, and final fluorescence contrast must be fully visible and unambiguous.

Manual:
Experimental Procedure
1. Place bacterial suspension in clear tube.
2. Add FDA fluorescent dye dropwise into suspension.
3. Cover tube and incubate in darkness for 10 minutes.
4. Transfer stained sample to microscope slide and observe under fluorescence microscope.
Observation of Experimental Phenomena
Live bacterial cells emit bright green fluorescence; dead cells appear non-fluorescent (dark) against same background — both coexist in same field of view.
Summary of Experimental Principles
FDA is a non-fluorescent esterase substrate that enters live cells, where intracellular esterases cleave it to release fluorescein — a green-fluorescing compound retained in intact membranes. Dead cells lack esterase activity and/or membrane integrity, so no green signal accumulates.

Prompt Grounding rubric:
level_1: No bacterial suspension is shown; no FDA dye bottle or dropper appears; there is no cue that the sample sits in darkness; no fluorescence microscope view is shown; the staining step never occurs.
level_2: At least one of the following appears (the bacterial suspension, the FDA dye container, or a microscope field), but there is no visible transfer of dye into the suspension, no dark/covered-container cue, and no clear order connecting dye addition to what follows.
level_3: The bacterial suspension and the FDA dye dropper are both shown; a drop of dye visibly meets the suspension's surface; the tube or container appears covered or placed in darkness; a fluorescence microscope field of view appears afterward, but the microscope frame is blurry, poorly lit, or too zoomed out/in to make out individual cells.
level_4: All prompt-specified elements are present and in the right order (suspension, dye added via dropper into the suspension, tube covered or placed in darkness, then a microscope view), and the microscope view is in sharp focus with individual cells resolvable as distinct rod/coccus shapes, but the shot includes a distracting extra element (e.g., stray light on the slide, an out-of-place object in frame, or only a partial view of the sample edge).
level_5: All score 4 conditions hold AND the microscope field is cleanly framed with no extraneous objects, uniform illumination across the field, and the visible sample area in sharp focus throughout: the shot is staged as a clean, unobstructed observation of the stained sample, independent of whether the fluorescence pattern itself is scientifically correct (that judgment belongs to the science rubric).

Scientific and Causal Correctness rubric:
level_1: The fluorescence pattern shown is not biologically plausible for this assay: e.g., the whole field glows uniformly green, dead cells glow green while live cells stay dark, the FDA dye itself is shown as a red or blue liquid, or green fluorescence is visible before the dye is even added.
level_2: Green fluorescence appears, but it violates the assay's basic cause-and-effect order: e.g., fluorescence shows up before the dye touches the suspension, fluorescence is still visible after the dye/sample is removed from view, or the only green signal sits on debris/clumps rather than on identifiable bacterial cells.
level_3: Green fluorescence appears only after the dye has been added and the dark incubation step has occurred, and the signal sits on cell-shaped structures, but there is no visible difference between individual cells: every visible cell is equally green, or every visible cell is dark, or the green glow is a diffuse haze with no cell outlines to attach it to.
level_4: Green fluorescence appears specifically on cells with clear rod or coccus outlines after the dark incubation step, and the space between cells stays dark, but no cell in the field is shown lacking the green signal (there is no dark cell sitting among the green ones to represent a dead cell).
level_5: A subset of intact, cell-shaped structures glows green while other intact, similarly shaped structures in the same field stay dark; both populations share a consistent size and shape typical of the bacterium, and no green signal appears on broken cell debris or material outside a cell outline.

Spatiotemporal Consistency rubric:
level_1: The bacterial suspension tube jumps from one location or appearance to another between frames; the microscope field appears abruptly with no transition from the bench scene; dye seems to appear inside the suspension with no visible drop or motion leading to it; the room lighting flips from light to dark with no transition; or the tube spins a half-turn or more with no frames showing the motion in between.
level_2: The dye droplet vanishes in mid-air before it reaches the suspension's surface; the microscope field appears with no zoom-in or focus-racking motion connecting it to the bench scene; the dark/covered stage is shown starting only after green fluorescence is already visible; or the bacterial cells blink in and out of the field between frames with no smooth motion linking their positions.
level_3: A brief, unexplained light flash appears during the dark incubation stage, or there is slight camera shake as the microscope view settles, or the tube is briefly blocked from view while the dye is added, but aside from these small blips, the suspension tube, dropper, and cells all keep consistent positions, orientation, and size across frames.
level_4: The dye's motion from the dropper tip down to the suspension surface is shown as one continuous path; the tube does not move during the dark incubation stage; the transition into the microscope view happens through a visible zoom or focus change rather than a hard cut; the room lights dim gradually or cut cleanly to black before the dark stage, rather than flickering; no object stretches, warps, or changes size unexpectedly between frames.
level_5: The dye's path from the dropper to the suspension surface is shown as one unbroken stream with no gap or jump; the tube's cover/lid is shown closing fully before the dark stage begins and stays closed until the microscope view starts; the microscope view holds the same focus depth and zoom level from its first frame to its last; there is no repeated or skipped frame, and every object's edges stay crisp and steady across the whole clip.