Domain: Natural Science — Microbiology
Target concept: The alcohol decolorization step results in the selective loss of stain from Gram-negative cells.

Video generation prompt: Observation of a mixed bacterial smear after crystal violet and iodine treatment under the optical microscope; during the decolorization step, clarified alcohol flows over the smear.

Evaluation guide:
(i) Target concept: Differential alcohol-mediated decolorization in Gram staining
Minimum mechanism: Crystal violet–iodine complex must be formed first; alcohol must act as selective solvent only on Gram-negative cell walls; cell morphology must remain physically intact post-decolorization to enable visual distinction.
(ii)Storyline: [Frame 0–2] Microscope view shows uniform purple mixed smear (post-iodine). [Frame 3–5] Clarified alcohol enters frame from top and flows continuously downward over smear surface, with no color change during flow. [Frame 6–8] Flow completes; same field now reveals adjacent purple cells (sharp, dense violet) and colorless cells (same size/shape, fully outlined, no stain, no transparency loss). [Frame 9] Static hold confirming coexistence. Perspective: Fixed high-magnification microscope view; no panning, zooming, or angle shift; all events occur within single continuous shot; smear and alcohol must share physical plane and lighting conditions.

Manual:
Experimental Procedure
1. Place mixed bacterial smear on glass slide.
2. Flood with crystal violet (30 sec), rinse.
3. Flood with iodine (30 sec), rinse.
4. Decolorize with clarified alcohol—flow continuously over smear.
5. Observe under 100x oil immersion microscope.
Observation of Experimental Phenomena
Pre-alcohol: Uniform purple bacterial smear. During alcohol flow: No immediate change. Post-alcohol: Two distinct populations in same field—intact purple cells (Gram-positive) and intact colorless-but-visible cells (Gram-negative).
Summary of Experimental Principles
Alcohol decolorization selectively removes crystal violet–iodine complex from Gram-negative cells due to thinner peptidoglycan layer and higher lipid content, leaving Gram-positive cells stained.

Prompt Grounding rubric:
level_1: No mixed bacterial smear is visible; crystal violet and iodine treatment is not shown; the alcohol decolorization step is not depicted; no optical microscope view is shown at all.
level_2: At least one of the required objects appears (bacterial smear, crystal violet stain, iodine, alcohol, or a microscope field), but they don't appear in the correct order or in the same scene; there's no visible link in time between the staining/alcohol step and the microscope observation.
level_3: The mixed bacterial smear is visible under the microscope; both crystal violet and iodine treatment are visually represented (the smear appears purple before alcohol is applied); alcohol is shown flowing over the smear, but it isn't clear from the video whether the alcohol's flow and any resulting change in the smear's appearance are spatially or temporally linked (e.g., the alcohol and the smear don't clearly share the same field of view at the same moment).
level_4: All required elements are present and in the same continuous scene: the smear sits under the microscope and looks uniformly purple before alcohol is added; the alcohol flows continuously across the smear within that same field of view with no cut to a different scene; the smear's appearance visibly changes after the flow, but the transition happens off-screen or is only shown in a single after-shot with no continuous coverage of the flow itself.
level_5: All required elements are present and fully continuous: the smear sits under the microscope and looks uniformly purple after crystal violet and iodine are applied; the alcohol flows steadily across the smear within that same field of view; the camera holds on this single continuous shot from before the flow begins through after it ends, with no cut and no gap in coverage, so that whatever change occurs in the smear's appearance is directly observable rather than inferred.

Scientific and Causal Correctness rubric:
level_1: The color change contradicts basic microbiology: e.g., the smear turns a different color entirely (not purple loss), or every cell keeps its purple color after alcohol, or the wrong population loses color (Gram-positive cells lose stain while Gram-negative cells keep it).
level_2: After alcohol, either every cell loses its purple color or every cell keeps it: no split between the two populations is visible; or alcohol touches the smear but no color change happens at all; or a color change occurs somewhere in the smear before the alcohol ever reaches it.
level_3: Some cells lose their purple color after alcohol while others keep it, but the pattern doesn't track real Gram-stain biology: for example the color loss looks patchy or randomly scattered rather than following distinct cell populations, or the fading looks gradual across the whole field rather than a clean per-cell split, or the remaining purple sits only in debris rather than in intact cells.
level_4: The differential loss is visible and cleanly split by cell population: purple-retaining cells and colorless cells appear together in the same field; the purple cells share one consistent shape (e.g., short rods or clustered cocci) and the colorless cells share a different, consistent shape, but the video doesn't clearly confirm that the colorless cells still have a visible outline (it only shows that they've lost the purple, without confirming they remain visible as intact cells).
level_5: The Gram-stain outcome is reproduced correctly: before alcohol the whole smear looks uniformly purple; during the alcohol flow no sudden color change occurs; after the flow, two clearly distinguishable groups of intact cells appear in the same field: one group of cells that are sharply outlined and still purple (Gram-positive), and a second group of cells that are just as clearly outlined but have lost all purple color (Gram-negative), with no ambiguity about which cells are still intact or where the stain has gone.

Spatiotemporal Consistency rubric:
level_1: The smear disappears and reappears; or the alcohol seems to flow in from off-screen and then reappear partway across the smear; or the microscope view jumps between different magnifications or angles; or the lighting suddenly changes in a way unrelated to anything happening in the scene.
level_2: Several continuity problems appear at once: the alcohol flow starts mid-motion with no visible origin; the smear's position shifts between the before-alcohol and after-alcohol views; the microscope's focus shifts so cells blur out and then sharpen again; or the video cuts between a wide pour of alcohol and a separate close-up view with no visual connection between them.
level_3: One clear continuity flaw remains: for example the alcohol is shown flowing but the smear looks completely unchanged for the whole duration of the flow; or the before/after alcohol views are shot at visibly different focus levels (one blurry, one sharp) with no smooth transition between them; or the cells' shapes change in ways they shouldn't (rods becoming round cells) after the alcohol step.
level_4: The smear, alcohol, and microscope view keep consistent positions and framing throughout; the alcohol flows continuously and stays in contact with the smear the whole time it's visible; the microscope's magnification, focus, and angle stay the same before and after; the cells' shapes and layout stay the same before and after alcohol, aside from the change in stain color. The only flaw is a small amount of camera shake, or a brief moment of soft focus during the transition that clears up quickly.
level_5: No spatiotemporal flaws are visible anywhere in the clip: the smear stays in the same part of the frame throughout; the alcohol flows as one continuous, unbroken stream across the smear with no splattering or pausing; the microscope view never drifts, zooms, or rotates; the cells' outlines, density, and arrangement look the same before and after the alcohol step, aside from the purple stain being present or absent.