If you search for “the blue biology stain”, the answer is usually methylene blue. It’s cheap, easy to use and forgiving. It’s the stain behind the classic school lab where you scrape cells from your cheek and see their nuclei turn blue. It’s also useful for bacteria, yeast and, at very low concentrations, live protozoa.
What methylene blue is and why it stains blue
Methylene blue is a synthetic dye first made in the 1870s. In solution, the coloured part of the molecule carries a positive charge, which makes it a basic (cationic) dye. Positively charged dyes are attracted to negatively charged structures in cells, above all nucleic acids: the DNA in the nucleus and the RNA in the cytoplasm and nucleolus.
That is why methylene blue:
- stains nuclei a strong blue, while the cytoplasm takes on a lighter tint;
- stains bacteria well, since their cells are rich in RNA and have negatively charged surfaces;
- highlights granules and other acidic structures in some cells.
Other blue biology stains
Methylene blue isn’t the only blue stain you’ll come across:
| Stain | Typical use |
|---|---|
| Methylene blue | General nuclear and bacterial stain; counterstain in some methods |
| Toluidine blue | Similar basic dye; can stain some structures purple instead of blue (metachromasia) |
| Alcian blue | Acidic mucins and cartilage matrix in histology |
| Trypan blue | Cell viability: dead cells take it up, living cells keep it out |
| Nile blue | Lipids and nucleic acids |
| Crystal violet | Purple rather than blue; the first stain in the Gram stain |
| Lactophenol cotton blue | Fungal structures in mycology |
Methylene blue also appears in mixtures. Romanowsky-type blood stains such as Wright’s, Giemsa and Leishman stains combine methylene blue (or its breakdown products) with eosin. And methylene blue is the counterstain in the Ziehl–Neelsen method for acid-fast bacteria.
What methylene blue is good for on a slide
- Animal cells: cheek cells, other epithelial scrapings, and cells in smears.
- Bacteria and yeast: simple stains to show shape and arrangement.
- Protozoa: nuclei and food vacuoles, and very dilute “vital” staining of live cells.
- Plant cells: it works, but iodine often gives more useful contrast in plant tissue. See how to prepare and stain an onion cell slide with iodine.
Buying and diluting it
Methylene blue for microscopy is usually sold as a ready-made aqueous solution, often at around 1%. Stronger stock solutions and powder are also available. For most amateur uses:
- Cheek cells and fixed smears: a ready-made ~1% solution can be used as is, or diluted with distilled water. A shorter staining time often matters more than the exact concentration.
- Live protozoa: use a very dilute solution, a faint pale blue. Higher concentrations kill the cells quickly.
Methylene blue sold as an aquarium treatment is the same dye, but concentrations and added ingredients vary by brand. It can work as a stain in a pinch, but you’ll have to experiment with dilution. Check the label and use a product that lists methylene blue as its only active ingredient.
Store the solution tightly capped, out of light, and away from children. Label it clearly.
Method 1: cheek cells (wet mount)
- Gently scrape the inside of your cheek with the flat end of a clean toothpick or a cotton bud. Don’t press hard: you only need the loose surface cells.
- Smear the material thinly in the centre of a clean slide.
- Add one small drop of methylene blue.
- Lower a coverslip at an angle onto the drop, letting the liquid spread to avoid bubbles. Our guide to wet mounts without air bubbles covers the technique.
- Wick away any excess stain from the edge with a piece of paper towel.
- View at 100x, then 400x. Look for flat, irregular cells, each with a dark blue nucleus in the middle. You’ll often see tiny dark dots too: bacteria from your mouth.
Cheek cells are a type of stratified squamous epithelium. The 7 types of epithelial tissue explains what that means.
Method 2: staining under the coverslip (irrigation)
This lets you watch the stain arrive and stop when the contrast looks right.
- Make an ordinary wet mount of your specimen in water.
- Put a drop of methylene blue against one edge of the coverslip.
- Hold a small piece of paper towel against the opposite edge. Capillary action pulls the stain under the coverslip and across the specimen.
- Watch through the microscope and stop when the contrast looks right. If it’s too strong, draw water through the same way to rinse.
Method 3: fixed smears of bacteria or yeast
- Spread a very thin film of sample on a clean slide. Good samples include yoghurt with live cultures, a little dental plaque, or a drop of bread yeast suspended in water. Let it air dry completely.
- Fix the smear so it doesn’t wash off. Traditionally this is done by passing the slide quickly through a flame two or three times, smear side up. Alternatively, cover the smear with a few drops of methanol or strong alcohol and let it evaporate. Both methods involve heat or flammable liquid, so take care.
- Cover the smear with methylene blue for roughly 30 seconds to 2 minutes.
- Rinse gently with a slow stream of water, blot dry with paper (don’t rub), and let it dry fully.
- Bacteria are best viewed under oil immersion at 1000x. Yeast cells are big enough to see at 400x.
Vital staining of live protozoa
Very dilute methylene blue can tint living protozoa enough to show the nucleus and food vacuoles before the cells are harmed. Add the faintest tint of stain to a drop of pond water or hay infusion culture and watch. Expect the stain to slowly build up in the cells, and expect them to stop moving eventually. A methylene blue preparation can also loosely show which cells are dying: in some uses, living cells can chemically reduce the dye to its colourless form, so they stay paler than dead cells. Treat this as an approximate observation, not a reliable viability test.
Troubleshooting
| Problem | Likely cause | Fix |
|---|---|---|
| Everything is dark blue | Too much stain, or left too long | Dilute the stain, shorten the time, rinse or irrigate with water |
| Hardly any colour | Too dilute, or too short | Use a stronger solution or leave it longer |
| Blue crystals or specks | Dried or old stain, or evaporation | Filter the stain, keep the coverslip edge wet, use fresh solution |
| Smear washed off | Not fully dried or fixed | Air dry completely and fix before staining |
| Bubbles everywhere | Coverslip dropped flat | Lower the coverslip from one edge |
| Cells clumped and thick | Too much material | Use less sample and spread it thinner |
Stain deposits are a common source of confusion; our article on microscopy artifacts shows how to tell them from real structures.
Safety, stains and clean-up
- It stains everything. Skin, clothes, benches and grout all turn blue. Wear gloves and old clothes, and work on paper towels. Skin stains fade over a day or two.
- Don’t swallow it or use stain solutions on your body. Methylene blue does have medical uses, but only as a pharmaceutical-grade drug given under medical supervision. Lab stains and aquarium products aren’t intended for that. Our article on what methylene blue does to your body covers its medical uses, risks and who should avoid it.
- Keep it away from eyes, and follow the safety information supplied with your product.
- Clean slides that have held cheek cells or plaque with disinfectant before reuse.
- Dispose of small amounts according to local guidance. Small amounts of dilute stain are commonly rinsed away with plenty of water, but check the product’s safety data sheet and local rules.