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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 preparation: Add a drop of sterile water to the center of glass slide; pick fresh bacterial lawn and mix to spread a thin uniform smear.
2. Drying: Air‑dry at room temperature, or quickly dry with low‑temperature alcohol‑lamp heating.
3. Fixation: Place the slide specimen‑side upward; pass it back and forth 3‑5 times over the outer flame for fixation. The temperature shall not exceed 60 °C. Cool down before staining.
4. Primary staining: Cover smear with Ammonium Oxalate Crystal Violet for 1‑2 min; rinse gently with clean water.
5. Mordanting: Apply Weigert’s iodine solution and incubate 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 by xylene rinse once no purple outflow is observed.
7. Counter‑staining (procedure for this product): Apply filtered Lithium‑Carbonate Carmine Staining Solution for 30‑60 s; rinse with clean water.
8. Dry the slide and observe morphology and staining differentiation under 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.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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