Domain: Healthcare — Basic Immunology
Target concept: Basophil Detection (Toluidine Blue Staining)

Video generation prompt: Prepare a peripheral blood smear on a glass slide, air-dry, then fix with methanol. Briefly apply toluidine blue stain, gently rinse with water, air-dry the slide, and examine under an oil-immersion light microscope.

Evaluation guide:
(i) Target concept: Metachromatic staining of basophil granules via toluidine blue requires strict procedural sequence (fixation before staining) to preserve morphology and enable selective dye aggregation.
Minimum mechanism: Blood must be fixed to prevent lysis; toluidine blue must contact intact granules; aqueous rinse must remove unbound dye without disrupting bound aggregates; oil immersion must be used to resolve sub-micron granules.
(ii)Storyline: [Shot 1] Hand places blood drop on slide → [Shot 2] Spreading motion creates thin film → [Shot 3] Slide held still, surface transitions from wet-shiny to dry-matte → [Shot 4] Methanol applied, spreads, then evaporates to leave transparent film → [Shot 5] Toluidine blue droplet contacts film and develops immediate purple-red foci → [Shot 6] Water stream rinses background but purple-red foci persist → [Shot 7] Slide placed on microscope stage, oil drop applied, objective rotated into place, focus adjusted → [Final frame] High-magnification field reveals sharp, punctate, purple-red granules exclusively inside basophil cytoplasm. All shots maintain fixed overhead/slight-angle viewpoint; no cuts break continuity of slide position or lighting; visibility constraints: granules must be resolvable (no defocus), oil ring must be optically evident, and no obstructing fingers/tools in final view.

Manual:
Experimental Procedure
1. Place one drop of peripheral blood near end of clean glass slide.
2. Spread blood into thin monolayer using second slide; air-dry completely.
3. Flood slide with methanol for fixation; air-dry again.
4. Apply toluidine blue stain for 30–60 sec.
5. Gently rinse with distilled water.
6. Air-dry slide thoroughly.
7. Examine under oil-immersion (100×) light microscope.
Observation of Experimental Phenomena
Basophils show distinct, densely packed, purple-red metachromatic granules against pale violet background; other WBCs lack this color.
Summary of Experimental Principles
Toluidine blue binds to acidic proteoglycans in basophil granules, inducing metachromasia — a spectral shift from blue to purple-red due to dye aggregation.

Prompt Grounding rubric:
level_1: None of the required items (the glass slide, blood drop, methanol, toluidine blue stain, water rinse, and oil-immersion microscope) appear in the video, or they appear but no step of the smear-and-stain procedure is ever carried out.
level_2: At least one required item appears (for example the slide or the toluidine blue bottle), but the procedure is not recognizable as blood-smear staining: most steps are missing, the few steps shown are in no sensible order, or the microscope is shown with no smear or stain step ever preceding it.
level_3: The slide, blood drop, methanol, toluidine blue, water rinse, and microscope are all shown, and at least three of the seven steps (blood placement, air-drying, methanol fixation, stain application, water rinse, second air-dry, oil-immersion viewing) appear in their correct order, but two or more of the remaining steps are missing, out of order, or show no visible change on the slide (for example stain is applied but no color ever appears).
level_4: All seven steps appear in the correct order (blood placed on the slide, air-dried, methanol-fixed, toluidine blue applied, water-rinsed, air-dried again, then examined under oil immersion), with the right tool used at each step (a dropper for the stain rather than a finger, methanol applied before the stain), and at most one small flaw remains, such as an unclear rinse-water direction or the focusing motion not being shown.
level_5: All seven steps appear in the correct order with no flaw: the blood drop is placed and spread into a thin film, the slide dries to a matte finish, methanol is applied and evaporates without leaving residue, toluidine blue is dropped on and spreads evenly, the water rinse visibly clears excess stain without disturbing the smear, the second air-dry leaves a uniform matte surface, and the final shot shows the oil-immersion objective in place and focused on the smear.

Scientific and Causal Correctness rubric:
level_1: No scientifically correct color change appears anywhere: the blood never leaves its original red color, the toluidine blue looks blue-green rather than blue-violet, or the final microscope view shows no purple-red granules in any cell.
level_2: A color or state mismatch appears in two or more key steps, for example the toluidine blue is applied but the slide stays uncolored, the methanol looks like a thick syrupy liquid instead of a thin fixative, the water rinse washes the smear away entirely, or the final field shows only lymphocytes with no purple-red granules anywhere.
level_3: The blood is the correct bright red before fixation and the toluidine blue reagent is the correct blue-violet color, and the final microscope field contains at least one cell with localized purple-red granules, but one causal step is wrong, for example the stain is applied before fixation and the smear visibly washes out, no color develops despite both fixation and staining being shown, or the purple-red color spreads diffusely through the cell rather than sitting in discrete granules.
level_4: Every color transition matches the real staining chemistry: red blood on the slide, a clear film after methanol fixation, a pale violet background with distinct purple-red granules inside at least three basophils after toluidine blue staining, and retained granule color with a clean background after the water rinse, with at most one small blemish, such as slightly uneven background tinting or one granule cluster with weaker contrast than the rest.
level_5: Every color and state change is correct and happens in the right causal order with no blemish: the film loses its liquid sheen only once methanol is applied, the toluidine blue produces a pale violet background together with a distinct purple-red shift confined to at least five intact basophils' granules, the water rinse clears the background tint while leaving the granule color untouched, and no other leukocyte type in the field shows this purple-red granule color.

Spatiotemporal Consistency rubric:
level_1: A step's result appears with no shot of the action that caused it: the slide is suddenly dry with no drying shot ever shown, the microscope view appears with no shot of the slide being placed on the stage, fixative appears on the slide with no bottle or dropper ever shown applying it, or the slide or a tool instantly jumps to a different position on screen between frames with no motion connecting them.
level_2: Two or more abrupt breaks occur between shots: the camera angle or lighting jumps inconsistently, the dropper or slide vanishes between one moment and the next with no motion carrying it away, the slide flips front-to-back with no visible turning motion, or the setting changes from the lab bench to a clearly different location.
level_3: The slide, dropper, and microscope stay in the same relative position and the lighting direction stays the same across every shot, and motions such as the dropper's descent onto the slide play out continuously, but one small break occurs, for instance a cut during the rinse step where the water stream's angle differs before and after, or the slide looks slightly rotated between the fixation and staining shots with no hand shown turning it.
level_4: No item disappears or breaks apart between shots, every motion (spreading, dropping, rinsing, focusing) plays out smoothly and at a believable pace, and the camera keeps the same distance and angle throughout, with at most one small mismatch such as the water droplet looking a slightly different size at the start and end of the rinse.
level_5: The slide never leaves the center of frame, every tool (dropper, rinse stream, objective lens) approaches from the same direction in every shot, each action is shown starting and finishing on screen rather than happening off-camera, the lighting direction and brightness never change, and no item flickers, jumps, or changes shape between one frame and the next anywhere in the sequence.