Domain: Natural Science — Microbiology
Target concept: Aflatoxin Detection (Czapek Medium - Sudan III Staining Method)

Video generation prompt: Prepare a Czapek agar plate, inoculate a sample of Aspergillus flavus at the center, incubate, then apply Sudan III stain to the surface of the colony and observe under a microscope.

Evaluation guide:
(i) Target concept: Visual confirmation of lipid-rich Aspergillus flavus via selective Sudan III staining on Czapek medium.
Minimum mechanism: Sudan III must dissolve in neutral lipids (not water or agar polysaccharides); lipids must be biosynthesized and stored intracellularly by metabolically active A. flavus; staining requires direct physical contact between dye and lipid-containing hyphae; microscopy must resolve subcellular granularity.
(ii)Storyline: [Phase 1: Setup] Static overhead view of poured Czapek agar plate (pale amber, semi-solid, circular). [Phase 2: Inoculation] Sterile loop deposits spore suspension at precise plate center, visible as small wet dot. [Phase 3: Incubation] Time-lapse implied by colony emergence and expansion to mature size/shape (no growth motion shown, but final colony is fully formed, centered, morphologically correct). [Phase 4: Staining] Dropper applies Sudan III solution exclusively onto colony surface: liquid wets and spreads over hyphae (no runoff to agar). [Phase 5: Observation] Seamless transition to microscope eyepiece view showing magnified hyphae with discrete, spherical red-orange granules inside cellular compartments, with zero red in background agar or extracellular space. All phases maintain fixed viewpoint on same plate/microscope system; no camera repositioning or object replacement.

Manual:
Experimental Procedure
1. Pour molten Czapek-Dox agar into sterile petri dish; cool to form pale amber semi-solid plate.
2. Inoculate Aspergillus flavus spore suspension at exact center.
3. Incubate 3–5 days at 25–30°C until colony matures (fluffy, yellow-green).
4. Flood colony surface with Sudan III stain solution; wait 1–2 min.
5. Blot excess stain; observe under microscope (400×) for red-orange intracellular lipid droplets.
Observation of Experimental Phenomena
- Mature Aspergillus flavus colony: dense, velvety, yellow-green aerial mycelium with conidial heads.
- Sudan III stains intracellular lipid droplets bright red-orange, visible as discrete granules inside hyphae.
- Unstained agar remains pale amber; no red coloration in medium.
Summary of Experimental Principles
Sudan III is a lysochrome dye that dissolves in and stains intracellular neutral lipid droplets red-orange; prominent lipid-body accumulation is common in actively growing Aspergillus flavus, making Sudan III staining a useful indicator of lipid storage in the colony. This staining pattern alone does not confirm aflatoxin production or toxigenicity — non-toxigenic A. flavus strains also accumulate lipid droplets — so confirming aflatoxin itself would still require a separate chemical assay (e.g., TLC or HPLC).

Prompt Grounding rubric:
level_1: No Czapek agar plate is shown, no fungal colony appears at its center, no Sudan III stain application is shown, and there is no microscope-view shot: none of these four required elements appears in the clip.
level_2: A petri dish or some colored liquid appears, but there is no plate that reads as a Czapek agar plate, no colony at the center, no stain being applied to a colony, and no microscope shot; if any step order is implied, it is reversed (e.g., stain shown before any colony has grown).
level_3: A circular petri dish with a pale amber/yellow semi-solid medium (Czapek agar) is shown; a single fungal colony sits at the center of the plate; a red-orange Sudan III liquid is shown contacting the colony surface; the clip cuts to a microscope-view shot: all four are present, but at least one causal step is missing (e.g., the stain touches the colony but no resulting color is ever shown, or the colony appears already stained with no prior unstained shot).
level_4: Everything in score 3 holds, and the steps run in the right order: plate shown, colony centered on it, stain applied directly onto the colony (not just the surrounding agar), then a microscope shot showing the stained colony's magnified field, with no jump from an unstained colony straight to a stained one without an application gesture in between.
level_5: Everything in score 4 holds, and additionally: the Sudan III liquid only ever touches the colony, never the bare agar around it; the microscope view shows a magnified field with visible red-orange granules inside fungal filaments rather than a flat red wash; the same plate stays recognizable across the whole clip (no swap to a different dish or colony); the camera stays on the plate/microscope subject without cutting to unrelated hands, labels, or equipment.

Scientific and Causal Correctness rubric:
level_1: The colony stays uncolored after the stain is applied, or the stain appears in a color other than red-orange (e.g., blue or green), or the fungal colony itself looks nothing like a mold (smooth, shiny, mucoid) rather than the fuzzy, filamentous growth expected of Aspergillus flavus.
level_2: The colony has plausible fuzzy, yellow-green mold texture, but no red-orange color develops after staining is shown; or the red-orange color spreads uniformly across the whole plate rather than concentrating on the colony; or the microscope shot shows only unstained fungal structures.
level_3: Red-orange color is visible on the colony and not on the surrounding agar, and the colony has recognizable fuzzy, dense mold texture; the microscope shot shows magnified fungal filaments, but the red-orange looks like a flat, even haze rather than showing any internal granularity.
level_4: Everything in score 3 holds, and additionally: the red-orange color stays confined to the fungal filaments and reproductive structures, with none of it bleeding onto the surrounding agar; under the microscope, distinct small red-orange spots are visible inside the filaments rather than one uniform wash of color, consistent with droplets of stained lipid rather than dye simply pooling on the surface.
level_5: Everything in score 4 holds, and additionally: the red-orange spots inside the filaments look enclosed within the cell outline rather than sitting loose on top of it; the staining reaches through the visible colony rather than leaving obvious untouched patches or margins; there is no stray red-orange precipitate or crystal sitting on the bare agar; the hue reads clearly as red-orange rather than pink, brown, or rust, staying consistent across the whole magnified field.

Spatiotemporal Consistency rubric:
level_1: The plate jumps between clearly different positions or framings from one shot to the next; the colony appears already fully grown before any inoculation gesture is shown; the stain appears already sitting on the colony with no application motion; the microscope image jumps straight from a wide view to full magnification with no zoom in between; the lighting swings from bright to dark between consecutive shots with no visible cause.
level_2: More than one editing break disrupts the flow: e.g., a hand enters frame to inoculate and the very next shot already shows a stained colony with nothing shown in between; the colony's position or angle changes between shots with no camera movement to explain it; the stain liquid sits motionless and then suddenly appears spread out with no visible flow; the microscope focus jumps from blurry to sharp with no visible adjustment.
level_3: One clear continuity slip is present: for example, the colony sits centered in the wide shot but appears off-center in the microscope view with no repositioning shown; or the stain-application gesture ends but the next shot shows no wetness or spreading on the colony surface; or the growth between inoculation and the mature colony is implied only by a label with no visible size change at all.
level_4: The plate stays in the same place and framing throughout; the inoculation motion moves continuously from off the plate to its center; the stain droplet visibly falls and spreads across the colony surface rather than snapping into place; the microscope focus shifts smoothly from the wide colony view to the magnified field; the overall lighting direction and brightness stay the same across shots, with nothing flickering, warping, or clipped by the frame edge.
level_5: Everything in score 4 holds, and additionally: no shot has a noticeable shake or wobble that breaks the sense of a steady camera; the plate's edges and the colony keep a believable relative depth as the camera moves; the timing of the stain droplet falling and spreading looks like it obeys normal gravity, neither sped up nor slowed down oddly; every transition, from setup to inoculation to staining to the microscope view, reads as a continuous action or a clean dissolve rather than a hard cut that skips a step.