碳酸锂胭脂红染色液为 Conn 改良 Weigert 革兰染色配套复染液，主要由胭脂红、碳酸锂、纯化水等配制而成，使用前需过滤处理，专用于改良法细菌革兰鉴别染色。
染色原理：经结晶紫初染、Weigert 碘液媒染后，革兰阳性菌细胞壁肽聚糖致密，结晶紫碘复合物难以被苯胺二甲苯脱色，保留蓝紫色；革兰阴性菌易被分化洗脱紫色，再经本染色液复染呈现红色，可清晰区分两类细菌，同时适用于组织切片内细菌观察，可区分细菌与钙盐等蓝色颗粒杂质。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、微生物涂片耗材：接种环、酒精灯、载玻片、光学显微镜、滤纸。
2、配套染色试剂：草酸铵结晶紫染色液、Weigert 碘液、分化液、透明液。
3、组织切片额外自备：10% 福尔马林固定液、中性树胶封片剂。
操作步骤 (仅供参考)
一、细菌涂片染色
1、涂片：载玻片中央滴无菌水，挑取新鲜菌苔混匀涂匀薄层。
2、干燥：室温自然晾干，或酒精灯低温快速烘干。
3、固定：玻片标本面朝上，火焰外侧来回移动 3-5 次固定，温度不超 60℃，冷却后染色。
4、初染：草酸铵结晶紫覆盖涂片染色 1-2min，清水轻洗。
5、媒染：滴加 Weigert 碘液覆盖 1-2min，水洗后滤纸吸干玻片水分。
6、分化：苯胺二甲苯溶液缓慢滴加分化 10-30s，至无紫色流出立即二甲苯冲洗终止。
7、复染 (本品使用步骤)：过滤后的碳酸锂胭脂红染色液滴染 30-60s，清水冲洗。
8、干燥、油镜镜检观察形态与染色区分。
二、石蜡组织切片染色
1、组织经 10% 福尔马林固定，常规脱水包埋，切片厚度 4μm，脱蜡至纯水；
2、草酸铵结晶紫染液浸染 5min，滤纸吸去切片周边残液；
3、Weigert 碘液媒染 1min，倾去碘液，滤纸彻底吸干切片水分；
4、苯胺二甲苯分化至无紫色析出，二甲苯快速冲洗；
5、过滤后的碳酸锂胭脂红染色液滴染切片 5min，无需水洗；
6、新鲜二甲苯多次漂洗，彻底去除苯胺残留；
7、中性树胶封片，显微镜观察。
注意事项
1、本品静置易产生沉淀，每次染色前必须过滤，否则沉淀附着菌体干扰镜检结果。
2、涂片背面提前标记圆圈，便于镜检定位标本区域。
3、无菌操作取菌，接种环使用前后火焰灭菌，试管口开盖 / 加盖均需过火。
4、加热固定严控温度，手背轻触玻片不发烫为宜，高温会破坏菌体细胞壁，造成染色假阴性。
5、染色核心控制点为苯胺二甲苯分化时间：分化过度阳性菌褪色呈红色；分化不足阴性菌残留紫色，判读错误。
6、待检细菌需新鲜培养物，老化、死亡、自溶革兰阳性菌易出现假阴性。
7、组织染色经 Weigert 碘液媒染后必须吸干水分，水分残留会导致分化不均；二甲苯漂洗务必彻底，残留苯胺会造成切片后期褪色。
8、操作全程穿戴实验服、一次性手套，避免染液沾染皮肤与衣物。
9、废液、废弃染片按实验室危废规范统一处理，不可直接倾倒下水道。
Lithium‑Carbonate Carmine Staining Solution is a counter‑stain for Conn‑modified Weigert Gram‑staining. It is mainly composed of carmine, lithium carbonate and purified water. Filtration treatment is required prior to use, and it is specially used for bacterial Gram differential staining by modified method.
Staining Principle: After primary staining with crystal violet and mordanting with Weigert’s iodine solution, Gram‑positive bacteria possess dense peptidoglycan in cell walls, so the crystal‑violet‑iodine complex cannot be decolorized by aniline‑xylene and retains blue‑purple color. Gram‑negative bacteria are readily differentiated and eluted of purple color, and then counter‑stained red by this staining solution. Thus the two groups of bacteria can be clearly distinguished. It is also applicable for observing bacteria in tissue sections and can tell bacteria apart from blue granular impurities such as calcium salts. This reagent is for research‑only purposes. It shall not be used for clinical diagnosis or other applications.
Materials to Be Prepared by User
1. Supplies for microbial smear: inoculating loop, alcohol lamp, glass slide, light microscope, filter paper.
2. Matching staining reagents: Ammonium Oxalate Crystal Violet Staining Solution, Weigert’s Iodine Solution, Differentiation Solution, Clearing Solution.
3. Additional supplies for tissue sections: 10 % Formalin fixative, Neutral Balsam mounting medium.
Operating Procedures (For Reference Only)
I. Bacterial Smear Staining
1. Smear: Place a drop of sterile water in the center of a glass slide. Pick a fresh bacterial lawn, mix well and spread into a thin even film.
2. Drying: Air‑dry at room temperature, or quickly dry over the low‑temperature flame of an alcohol lamp.
3. Fixation: Keep the specimen‑side of the slide facing upward; pass it back and forth 3‑5 times over the outer flame. Ensure the temperature does not exceed 60 ℃. Cool down before staining.
4. Primary staining: Cover the smear with ammonium oxalate crystal violet and stain for 1‑2 min, then rinse gently with clean water.
5. Mordanting: Apply Weigert’s iodine solution and cover the smear for 1‑2 min. Rinse with water and blot slide moisture with filter paper.
6. Differentiation: Slowly add aniline‑xylene solution for differentiation for 10‑30 s. Stop immediately with xylene rinse once no purple eluate flows out.
7. Counterstaining (Procedure for this product): Apply filtered lithium‑carbonate carmine staining solution and counterstain for 30‑60 s, then rinse with clean water.
8. Drying and microscopic examination: Dry the slide and observe cell morphology and staining differentiation under an oil‑immersion objective.
II. Paraffin Tissue Section Staining:
1. Fix tissues in 10 % formalin, perform routine dehydration and embedding; prepare sections at 4 μm thickness, and de‑wax down to pure water.
2. Immerse sections in Ammonium Oxalate Crystal Violet Staining Solution for 5 min; blot residual liquid around sections with filter paper.
3. Mordant with Weigert’s iodine solution for 1 min; decant iodine solution and thoroughly blot section moisture with filter paper.
4. Differentiate with aniline‑xylene until no purple leaches out; rinse rapidly with xylene.
5. Apply filtered Lithium‑Carbonate Carmine Staining Solution to sections for 5 min. No water rinse is needed.
6. Rinse repeatedly with fresh xylene to completely remove aniline residues.
7. Mount with neutral balsam and observe under microscope.
Precautions
1. This product tends to form precipitates upon standing. Filtration is mandatory before each staining run; otherwise, precipitates may adhere to bacterial cells and interfere with microscopic observation results.
2. Mark a circle on the reverse side of the glass slide in advance to facilitate locating the specimen area during microscopy.
3. Perform aseptic techniques for bacterial sampling. Sterilize the inoculating loop over flame before and after use. Pass the test‑tube mouth over flame both when opening and closing the cap.
4. Strictly control the temperature during heat‑fixation. The slide should feel lukewarm when touched lightly with the back of your hand. Excessive heat will damage the bacterial cell wall and cause false‑negative staining results.
5. The key control point of staining lies in the differentiation time of aniline‑xylene. Over‑differentiation will decolorize 6. Gram‑positive bacteria and turn them red; insufficient differentiation leaves Gram‑negative bacteria with residual purple color, leading to misinterpretation.
7. Fresh bacterial cultures are required for testing. Aged, dead or autolyzed Gram‑positive bacteria are prone to false‑negative results.
Thoroughly blot off water after mordanting with Weigert’s iodine solution for tissue staining; residual water will cause uneven differentiation. Xylene rinsing must be complete, since residual aniline will result in fading of sections over time.
8. Wear a lab coat and disposable gloves throughout the whole operation to prevent staining solution from contacting skin and clothing.
9. Waste liquid and used stained slides shall be disposed in accordance with laboratory hazardous‑waste regulations. Do not pour directly into sewer drains.
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