Domain: Healthcare — Basic Immunology
Target concept: Lymphocyte Proliferation Assay (Simplified MTT Method)

Video generation prompt: The demonstration involves adding isolated lymphocytes to a culture plate, followed by the addition of a mitogen, and brief incubation. Subsequently, MTT reagent is added, and the plate is incubated again. Finally, a solubilization solution is added, and the mixture is gently mixed to prepare for measurement.

Evaluation guide:
(i) Target concept: Visual verification of causal chain linking mitogen-stimulated lymphocyte proliferation to MTT-derived formazan formation and solubilization.
Minimum mechanism: Lymphocytes must be present and metabolically active; mitogen must be co-delivered to trigger proliferation; MTT must be reduced intracellularly to form insoluble formazan; solubilization must dissolve crystals into measurable chromophore.
(ii)Storyline: [Frame 0–2] Lymphocyte suspension pipetted into plate wells → [Frame 3–5] Mitogen pipetted into same wells → [Frame 6–7] (off-screen incubation implied) → [Frame 8–10] MTT added → [Frame 11–12] (off-screen incubation implied) → [Frame 13–15] Blue-black granular crystals appear *only* in mitogen-treated wells → [Frame 16–18] Solubilization solution added → [Frame 19–21] Gentle orbital mixing applied → [Frame 22–25] Crystals fully disappear; homogeneous deep violet solution remains. All events occur from fixed frontal benchtop view; no camera movement; plate remains centered; hands enter frame only from bottom edge with natural motion arc.

Manual:
Experimental Procedure
1. Isolated lymphocytes added to wells of a sterile culture plate.
2. Mitogen (e.g., PHA) added to wells.
3. Plate incubated (briefly, not shown).
4. MTT reagent added to wells.
5. Plate incubated again (not shown).
6. Solubilization solution (e.g., DMSO) added.
7. Plate gently mixed to dissolve formazan crystals.
Observation of Experimental Phenomena
- After MTT addition and incubation: blue-black granular formazan crystals appear in wells containing lymphocytes + mitogen.
- After solubilization and mixing: crystals dissolve into a homogeneous deep violet solution.
Summary of Experimental Principles
Lymphocyte proliferation induced by mitogen increases mitochondrial activity → enhanced reduction of yellow MTT to insoluble blue-black formazan crystals → solubilization yields colored solution proportional to viable cell count.

Prompt Grounding rubric:
level_1: No lymphocyte suspension, culture plate, mitogen, MTT reagent, or solubilization solution appears anywhere in the video, and none of the sequential additions (lymphocytes into the plate, mitogen into the wells, MTT reagent into the wells, solubilization solution into the wells) is depicted.
level_2: At least one relevant object appears (for example a multiwell plate or a pipette), but the depicted actions do not follow the posted-prompt sequence: a single liquid is pipetted into the plate with no distinguishable mitogen, MTT, or solubilization steps, or the steps appear in the wrong order, such as solubilization solution being added before the MTT reagent.
level_3: A culture plate, a lymphocyte suspension, a mitogen vial, an MTT reagent vial, and a solubilization-solution vial are all visible, and the video shows, in order, lymphocytes pipetted into wells, mitogen pipetted into the same wells, MTT reagent pipetted in, and solubilization solution pipetted in followed by mixing; however at least one addition is ambiguous, for example the pipette tip does not visibly touch the well, or the mixing motion is not clearly directed at the plate.
level_4: All elements from the 3-point level are present, and each of the four additions (lymphocytes, mitogen, MTT reagent, solubilization solution) shows the pipette tip visibly entering the well and dispensing liquid, followed by a gentle orbital mixing motion clearly applied across the wells after the solubilization step; no addition is skipped, reordered, or swapped for an unrelated liquid.
level_5: All elements from the 4-point level are present, and the four vials/containers are visually distinguishable from each other by their liquid's appearance and container shape alone, without needing to read any printed text: for instance, the MTT reagent vial holds a pale-yellow liquid that is visibly different from the mitogen vial's clear liquid and from the solubilization-solution vial's liquid. The pipette moves smoothly between each vial and the wells for every addition, mixing is one continuous gentle orbital motion covering every well, and no step is skipped, duplicated, or shown out of order.

Scientific and Causal Correctness rubric:
level_1: No mitogen-linked color change occurs anywhere in the video: the MTT reagent stays pale yellow in every well regardless of whether mitogen was added, no blue-black formazan crystals ever appear, and adding the solubilization solution produces no color change at all, so there is no visible link between the mitogen/MTT steps and any later color.
level_2: Only one biologically grounded event happens correctly: for example, blue-black granular crystals do form after the MTT reagent is added, but they appear identically in wells that never received mitogen; or the MTT reagent is added and incubated but no crystals ever form in any well; or the solubilization solution is added but the crystals never dissolve and remain as visible solid granules in the liquid.
level_3: Wells that received both lymphocytes and mitogen show blue-black granular crystals forming after the MTT reagent is added; adding the solubilization solution afterward is followed by these crystals dissolving into a uniform violet liquid; however the timing is somewhat off, for example the crystals appear to form before the MTT reagent is shown entering the well, or the liquid turns violet before the solubilization solution is shown being added.
level_4: Blue-black granular crystals appear only in wells that contain both lymphocytes and mitogen, and only after the MTT reagent has been added; the solubilization solution's addition is immediately followed by these crystals dissolving into a homogeneous deep-violet liquid, with no yellow, orange, or red hues left over in any well.
level_5: Crystal formation is visibly confined to wells with both lymphocytes and mitogen present, while any well shown without mitogen stays pale yellow and free of crystals; the MTT reagent's own color before reduction is pale yellow, never purple or violet; the crystals look granular and unevenly distributed just before the solubilization solution is added; after solubilization the liquid is fully uniform with no floating particles or patchy coloring, so the color sequence of pale-yellow MTT reagent, to blue-black crystals, to a uniform deep-violet solubilized liquid plays out exactly, with no stray hue shift that looks like a lighting artifact rather than a real chemical change.

Spatiotemporal Consistency rubric:
level_1: The culture plate visibly jumps to a different position between one frame and the next; the pipette is present in one frame and simply missing from the shot in the next with no hand motion carrying it away; the number or arrangement of wells changes partway through; the orbital mixing motion starts or stops abruptly with no visible transition into or out of it.
level_2: The plate drifts or rotates without any hand or support visibly moving it; the pipette tip appears at a well in one frame and is gone the next with no motion blur or visible withdrawal; the solubilization solution appears to pour in from off-screen with no vial or hand ever shown bringing it into frame; the mixing motion visibly reverses direction partway through a stroke.
level_3: The culture plate stays in the same place on the bench for the whole video; the pipette moves along a continuous, visible path from each vial to the wells for every addition, with no jump cuts; the orbital mixing motion is applied smoothly by the same hand to the same plate; no vial, pipette, or plate appears or disappears unexpectedly between frames.
level_4: All elements from the 3-point level are present, and additionally: the plate never visibly shifts or rotates, even slightly, across the whole video; the pipette's path between vial and well is smooth and consistently timed rather than jumping between positions; the mixing motion is continuous, low in amplitude, and visibly reaches every well rather than only part of the plate; a hand never blocks a well in one frame and reveals it in the next without a visible movement explaining the change.
level_5: All elements from the 4-point level are present, and additionally: the plate's position and orientation look the same from the first pipetting step through the final mixing step; the pipette's speed changes smoothly, slowing naturally as it nears the well rather than moving at a constant mechanical rate; the mixing motion keeps a steady, unbroken circular path centered on the plate for its full duration; every transition between the addition-and-incubation steps happens through continuous, visible motion rather than a hidden cut, fade, or camera trick.