细菌 L 型菌落染色液主要由美蓝、天青、苯甲酸、碳酸钠、麦芽糖等组分配制而成，专用于细菌 L 型菌落快速鉴别染色。
染色原理：滴加染液后，细菌 L 型菌落可在短时间内被美兰染成清晰蓝色；静置 15-30min 后，菌体新陈代谢会将美蓝还原为无色，菌落蓝色逐渐消退，因此染色完成后必须立刻镜检观察结果。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料
1、菌落操作工具：接种针、培养皿、载玻片、盖玻片、光学显微镜。
2、辅助耗材：无菌生理盐水、滤纸、一次性手套、实验服。
操作步骤 (仅供参考)
1、菌落取材：选取平板上典型细菌 L 型菌落，挑取少量菌落置于载玻片中央，滴加少量无菌生理盐水，轻柔涂成均匀薄层。
2、自然干燥：室温放置至涂片完全干燥，禁止高温加热，避免 L 型菌体破损变形。
3、染色：直接滴加足量细菌 L 型菌落染色液完全覆盖涂片，静置 30s-1min。
4、即时镜检：无需水洗，加盖盖玻片后立即置于显微镜下观察。
5、时限要求：染色完成后必须在 15min 内完成观察，超过 30min 菌落蓝色会消退，无法准确判读。
染色结果
| 样本 | 颜色结果 |
| 新鲜细菌 L 型菌落 | 深蓝色 |
| 放置 15‑30min 后的 L 型菌落 | 无色 (美蓝被菌体还原褪色) |
| 普通琼脂培养基背景 | 淡浅蓝色或近乎无色 |
注意事项
1、平板挑取菌落时做好无菌操作，接种针使用前后火焰灼烧灭菌，防止杂菌污染干扰观察。
2、涂片背面提前做好标记，方便快速定位菌落观察区域。
3、本试剂需 4℃ 避光储存，常温长时间放置会导致染色效力下降、提前褪色。
4、染色后观察时效性极强，超过 15min 菌体还原美蓝造成褪色，易造成漏检、误判；若需重复观察需重新制片染色。
5、禁止加热干燥、火焰固定涂片，高温会破坏细菌 L 型脆弱细胞壁，影响正常显色。
6、操作全程穿戴实验服、一次性手套，避免染液沾染皮肤、衣物。
7、废弃染液、染片按照实验室危险废弃物管理规范统一处理，不可直接排入下水道。
Bacterial L-form Colony Staining Solution is mainly composed of methylene blue, azure, benzoic acid, sodium carbonate, maltose and other components. It is specially used for rapid differential staining of bacterial L-form colonies.
Staining Principle: After the staining solution is added, bacterial L-form colonies can be clearly stained blue by methylene blue within a short period of time. After standing for 15-30 minutes, methylene blue will be reduced to a colourless state by the metabolism of bacterial cells, and the blue colour of colonies will gradually fade. Therefore, microscopic observation must be carried out immediately after staining. This reagent is intended for research only use and is not suitable for clinical diagnosis or other applications.
Materials to Be Prepared by Users
1. Colonyhandling tools: inoculating needle, petri dish, glass slide, coverslip, light microscope
2. Auxiliary consumables: sterile normal saline, filter paper, disposable gloves, lab coat
Operating Procedures (For Reference Only)
1. Colony Sampling: Select typical bacterial L-form colonies on the plate. Pick a small amount of colony and place it in the centre of a glass slide. Add a drop of sterile normal saline and gently spread it into an even thin layer.
2. Air-drying: Leave the smear at room temperature until completely dry. High-temperature heating is prohibited to prevent damage and deformation of L-form bacterial cells.
3. Staining: Add an adequate volume of Bacterial L-form Colony Staining Solution to fully cover the smear, and let it stand for 30 s-1 min.
4. Immediate Microscopic Examination: No water washing is required. Place a coverslip and observe under a microscope right away.
5. Time-limit Requirement: Observation shall be finished within 15 minutes after staining. The blue colour of colonies will fade after more than 30 minutes, leading to inaccurate interpretation.
Staining Results
| Sample | Color Result |
| Fresh bacterial L‑form colonies | Dark‑blue |
| L‑form colonies after standing for 15‑30 min | Colour‑less (Methylene‑blue is reduced and decolorized by bacterial cells) |
| Background of ordinary agar medium | Pale‑blue or nearly colour‑less |
Precautions
1. Perform aseptic operations when picking colonies from agar plates. Sterilize the inoculating needle by flaming before and after use to prevent miscellaneous‑bacterial contamination that interferes with observation.
2. Mark the reverse side of the smear slide in advance for quick location of the colony observation area.
3. Store this reagent at 4 °C protected from light. Prolonged storage at room temperature will reduce staining efficiency and cause premature fading.
4. Observation must be performed promptly after staining. Beyond 15 minutes, methylene‑blue will be reduced by bacterial cells and lead to fading, which may result in missed detection and misjudgment. Re‑prepare and re‑stain slides for repeated observation.
5. Do not heat‑dry or flame‑fix smears. High temperature will destroy the fragile cell walls of bacterial L‑forms and impair normal color development.
6. Wear a lab coat and disposable gloves throughout the whole operation to to prevent the staining solution from contacting skin and clothing.
7. Dispose of waste staining solution and stained slides in accordance with laboratory hazardous‑waste management regulations. Do not discharge directly into sewer drains.
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