Domain: Natural Science — Microbiology
Target concept: Gram-negative bacterial special staining (LPS staining)

Video generation prompt: Apply a smear of Gram-negative bacteria onto a clean glass slide, briefly heat-fix it, then apply a blocking buffer followed by a fluorescently labeled anti-LPS antibody probe, gently rinse away unbound antibody, apply a blue fluorescent nucleic-acid counterstain, rinse again, air-dry, and observe under an oil-immersion objective on a fluorescence microscope.

Evaluation guide:
(i) Target concept: Visual confirmation of a Gram-negative bacterium's surface-exposed LPS via non-permeabilized immunofluorescent labeling, observed under an oil-immersion objective on a fluorescence microscope.
Minimum mechanism: bacterial cells must be immobilized on the slide without permeabilizing the outer membrane; a fluorescently labeled anti-LPS antibody must bind surface-exposed LPS after blocking; unbound antibody must be rinsed away; a separate fluorescent counterstain must label the whole cell/nucleoid for contrast; because antibodies cannot cross an intact membrane, the anti-LPS signal is expected to concentrate at the cell periphery relative to the counterstain, rather than filling the whole cell evenly.
(ii)Storyline: [Frame 0–1] Clean glass slide enters view, centered; [1–2] Bacterial suspension deposited as a discrete oval smear near slide center; [2–3] Slide passed rapidly through a flame for heat-fixing (brief, non-charring passes); [3–4] Blocking buffer then a red/magenta-labeled anti-LPS antibody solution is dispensed onto the smear; [4–5] A rinse flows downward across the smear, visibly carrying away excess antibody solution; [5–6] A blue fluorescent counterstain is dispensed onto the same area; [6–7] A second rinse; [7–8] Slide rests undisturbed under ambient light (drying phase); [8–10] Camera smoothly moves in on the slide surface, an oil droplet appears, the objective lens engages, and the final shot shows a magnified fluorescence field where the red/magenta signal appears concentrated toward the edges of the rod-shaped bacteria relative to the blue counterstain, viewed from a fixed frontal perspective with no occlusion.

Manual:
Experimental Procedure
1. Place Gram-negative bacterial smear on a clean glass slide.
2. Heat-fix the smear with brief flame passes, then apply a mild fixative that preserves surface antigens without permeabilizing the outer membrane.
3. Apply a blocking buffer, then apply a fluorescently labeled anti-LPS antibody (red/magenta fluorophore).
4. Gently rinse with buffer to remove unbound antibody.
5. Apply a blue fluorescent nucleic-acid counterstain to visualize the whole cell/nucleoid.
6. Gently rinse again.
7. Air-dry, then observe under an oil-immersion objective on a fluorescence microscope.
Observation of Experimental Phenomena
The red/magenta antibody signal appears concentrated near the outer edge of each bacterial cell relative to its interior, because the antibody can only reach surface-exposed LPS on the intact outer membrane; the blue counterstain fills the whole cell body, so the combined image shows cells with a brighter red/magenta edge against a blue-filled body — a surface-labeling pattern rather than a resolved, separate membrane layer.
Summary of Experimental Principles
Lipopolysaccharide (LPS) is a surface-exposed component of the Gram-negative outer membrane. Because intact bacteria are not permeabilized in this protocol, an anti-LPS antibody can only bind LPS exposed at the cell surface, so its fluorescent signal concentrates at the cell periphery relative to a whole-cell counterstain. This peripheral-vs-whole-cell brightness difference — not a literal resolved membrane layer, which is well below the resolution limit of light microscopy — is what visually distinguishes LPS-specific labeling from the counterstain.

Prompt Grounding rubric:
level_1: No slide, smear, heat-fixing, blocking/antibody step, counterstain, rinsing, or fluorescence-microscope shot appears anywhere in the clip.
level_2: A slide or a dropper appears and some liquid is dispensed or wiped, but none of the required step-pairs are shown together (heat-fixing never follows the smear, the antibody step never precedes the counterstain step), so the procedure's dependencies are missing.
level_3: All of the objects and actions appear somewhere in the clip (slide, smear, heat-fixing motion, antibody/blocking application, a rinse, counterstain application, a second rinse, drying, and a final fluorescence-microscope shot with an oil-immersion objective), but at least one is out of the required order (e.g., counterstain applied before the antibody, or a rinse shown before anything was applied to rinse away).
level_4: Everything in score 3 is present and occurs in the right order (smear on slide, heat-fix, antibody/blocking application, first rinse, counterstain, second rinse, drying, then the oil-immersion fluorescence view), but one small flaw remains (e.g., the slide is held at an odd angle during a rinse, or the final microscope shot shows an empty field instead of any visible cells).
level_5: Everything in score 4 is present, in order, and each step reads clearly: the smear is visibly a thin layer on the slide; heat-fixing is shown as brief flame proximity without scorching; the antibody solution is a distinct colored liquid landing on the smear; the first rinse visibly carries excess liquid away; the counterstain is a second, distinct-colored liquid; the second rinse likewise clears the excess; drying is shown as the slide sitting still; the final shot clearly shows the oil-immersion objective engaged with a magnified field containing visible bacterial shapes.

Scientific and Causal Correctness rubric:
level_1: No fluorescent color appears on any bacteria at all, or the red/magenta and blue signals are indistinguishable from each other or from the background, or neither signal shows any relationship to where the bacteria actually are.
level_2: One of the two fluorescent colors is visible somewhere in the field, but it doesn't track onto any bacterial shape: e.g., the red/magenta shows up as a diffuse glow with no cell-like structure, or the blue counterstain fills the whole field rather than concentrating where the bacteria are.
level_3: Both the red/magenta and blue signals are visible and each roughly follows the shape of the bacteria, but the two signals look evenly mixed across each cell with no visible difference between the cell's edge and its middle; there is no sign that the red/magenta signal favors the periphery over the interior.
level_4: The red/magenta signal is visibly brighter or more concentrated toward the outer edge of each bacterial shape than in its middle, while the blue counterstain fills the body more evenly, but the effect is inconsistent (present on some cells and not others, or with a fuzzy, ill-defined edge rather than one that follows the cell's outline).
level_5: On essentially every visible bacterial cell, the red/magenta signal is noticeably brighter toward the cell's edge than its middle, giving a rim-like impression around a more evenly blue-filled body; the pattern holds consistently across the visible field rather than only on a few cells; there is no red/magenta signal sitting on visibly non-bacterial specks or debris, and no cell shows blue exceeding red at its very edge in a way that contradicts the expected peripheral labeling.

Spatiotemporal Consistency rubric:
level_1: The slide, dropper, or flame disappears from one shot and reappears in another without any motion connecting them; rinse liquid appears on the slide with no visible source or flow; the shot cuts straight to the microscope view with a jarring lighting mismatch; brightness swings drastically (more than roughly 50%) between consecutive shots with no cause.
level_2: At least three clear continuity breaks appear: e.g., the slide rotates a large amount with no hand visibly turning it; a wet, just-treated slide is suddenly shown dry with nothing in between; the flame is present in one shot and simply gone in the next with no pulling-away motion; the final microscope view shows a different zoom level than the setup shot implied, with no zoom shown happening.
level_3: The slide stays centered and upright, the dropper always approaches from the same side, the flame stays at a consistent position relative to the slide, rinse liquid flows downward in a consistent direction, and the microscope view keeps the same orientation as the prior slide shot, apart from one small slip, like a single flickering frame of lighting or a brief wobble of the slide that doesn't actually change its position.
level_4: No object disappears and reappears, nothing looks physically broken or deformed, lighting stays close to constant shot to shot (no brightness shift greater than roughly 20%), the camera's angle and distance don't jump around, and nothing stretches or passes through another object; the dropper lowers steadily, the rinse flows continuously downward, and the flame moves slowly and smoothly relative to the slide edge, aside from a minor artifact like a touch of motion blur on a moving edge, or slightly uneven brightness appearing only in the microscope shot.
level_5: Everything in score 4 holds, with no visible camera shake at any point; the antibody and counterstain applications show a continuous, unbroken descent and contact with the slide rather than snapping into place; the rinse shows an unbroken downward flow with a consistent liquid edge; the heat-fixing shows the flame staying close to the slide in a steady, continuous motion; the transition into the microscope view reads as a smooth, continuous zoom rather than a hard cut, ending at the same centering and framing implied by the prior shot; overall brightness and light direction stay constant throughout.