Bacteria are nearly transparent, so to see them under a light microscope you first spread them thinly on a slide, attach them to the glass, and then color them with dyes. The stain you choose decides what you learn, from simple cell shape to whether a cell wall holds a dye after an alcohol rinse. The colors only tell the truth when the earlier steps go right, so the slide you prepare before any dye goes on matters as much as the staining itself. Most bad results trace back to a few controllable things: smears that are too thick, cultures that are too old, and alcohol applied for too long or too short a time.
How to Make a Thin, Even Smear
A smear is the thin film of bacteria you spread on the slide. With a liquid broth culture, you take a loopful with a sterile loop and spread it straight onto the glass.1General Microbiology Lab Manual. Preparing and Staining Bacterial Smears Techniques and Tips
A solid culture on a slant or plate starts differently. First place a single drop of distilled water on the slide.2General Microbiology Lab Manual. Preparing and Staining Bacterial Smears Techniques and Tips Then touch the loop to a small amount of growth and mix it into the drop, spreading it into a thin layer of cloudy water.3General Microbiology Lab Manual. Preparing and Staining Bacterial Smears Techniques and Tips The most common mistake with plates is taking too much growth. If you can see a blob on the loop, the smear will be too thick to read.
A good smear looks faintly cloudy or almost invisible once dry, not chalky white. A thick one stains unevenly, and the cells pile up so you can’t judge their shape. Spread it over roughly a coin-sized area rather than the whole slide, and circle its position on the underside with a marker so you can find it later. Let the smear dry completely in the air before fixing it. Heating a wet smear can boil it, which distorts or splatters the cells.
For Gram staining, pick from a single isolated colony so you’re looking at one kind of bacterium. Label the slide with the organism and date before you begin.
Keep your technique sterile throughout. Flame the loop until it’s red-hot before and after each transfer, and let it cool before touching growth so you don’t kill or splatter the cells. Keep lids open only briefly and hold tubes at an angle. Treat every culture as able to cause infection, and put used slides in the designated container.
Fixing the Smear With Heat or Methanol
Fixing makes the cells stick firmly to the glass so they aren’t washed away during staining and rinsing.4Microbiology Class. Preparation of Bacterial Smear & Heat-Fixing It also kills the bacteria, which makes the slide safer to handle. With heat fixing, you pass the dried slide, smear side up, quickly through a flame two or three times.
The slide should feel warm on the back of your hand, never too hot to touch. Holding it in the flame too long cooks the cells and changes their shape.
Methanol often works better. It keeps more cells stuck to the slide than heat does, for both Gram-positive and Gram-negative bacteria.5Bioscene: Journal of College Biology Teaching. A Comparison of Heat Versus Methanol Fixation for Gram Staining Bacteria Methanol-fixed Gram-positive cells stain the correct dark purple consistently, while heat-fixed ones range from purple to pink.6Bioscene: Journal of College Biology Teaching. A Comparison of Heat Versus Methanol Fixation for Gram Staining Bacteria Methanol also leaves cell shapes undistorted, with little background debris.7PubMed. Methanol fixation. An alternative to heat fixation of smears before staining
It isn’t perfect. Methanol can make some rod-shaped bacteria shrink slightly in length, by about half a micrometer in one rod species, so it’s a poor choice for precise size measurements.8PubMed Central. Effects of fixation on bacterial cellular dimensions and integrity For routine lab staining, though, it’s a good option if your instructor allows it.
To fix with methanol, flood the dried smear with a few drops. Let it sit about a minute, then drain it and let the slide air-dry. Methanol is flammable, so keep it well away from your burner, and follow your lab manual’s timing if it differs.
The Simple Stain for Shape and Arrangement
A simple stain uses just one dye, so all the bacteria appear that color.9General Microbiology Lab Manual. Preparing and Staining Bacterial Smears Techniques and Tips Common choices are crystal violet, safranin and methylene blue.10General Microbiology Lab Manual. Preparing and Staining Bacterial Smears Techniques and Tips That’s enough to show cell shape, size and arrangement, such as single cells, chains or clusters.11Microbe Online. Simple Staining: Principle, Procedure, Results, and Uses in Microbiology It can’t tell you Gram type.12General Microbiology Lab Manual. Preparing and Staining Bacterial Smears Techniques and Tips
Flood the fixed smear with the dye for about 30 to 60 seconds. Rinse gently with water from the edge of the slide, not straight onto the smear. Blot dry with bibulous paper without rubbing, then look at it under oil immersion, which means using the 100x lens with a drop of oil on the slide (covered at the end of this article).
When you describe what you see, start with shape: round cells are cocci, rods are bacilli, and spirals are spirilla. Then add arrangement. Clusters like bunches of grapes suggest staphylococci, and chains suggest streptococci.
How to Do a Gram Stain and Why It Works
- Crystal violet: the first dye, which colors every cell purple.13PubMed. Gram staining
- Gram’s iodine: a mordant, meaning a chemical that locks the dye in. It forms a large crystal violet-iodine clump that won’t dissolve in water.14Microscopy. Gram Staining
- Alcohol rinse: the decolorizer, which pulls the purple out of some cells. It’s typically applied for 5 to 15 seconds with 95% ethanol or acetone-alcohol.15Preparing and Staining Bacterial Smears: Techniques and Tips. Common Gram Staining Errors and How to Fix Them
- Safranin: the counterstain, a second, pink dye that colors any cell that lost its purple.16PubMed. Gram staining
The dye-iodine clump forms inside both kinds of bacteria at first.17PubMed Central. Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain Gram-positive bacteria have a thick, tough wall that holds the clump during the alcohol rinse, so they stay purple.18PubMed. Use of the gram stain in microbiology Gram-negative bacteria have a thin wall wrapped in an outer fatty membrane that the alcohol breaks open, so they lose the purple and take up the pink.19PubMed. Use of the gram stain in microbiology
For timing, crystal violet and iodine each typically go on for about a minute, with a gentle water rinse between every step. Add the alcohol drop by drop and stop as soon as the runoff turns clear. Safranin usually goes on for 30 to 60 seconds before the final rinse and blot.
The alcohol step is the critical one, because it’s the only point where the two groups begin to look different. Keep a timer ready and have the water bottle in your hand before you add the alcohol.
Whenever you can, stain a known Gram-positive and a known Gram-negative alongside your unknown. If the controls come out wrong, your technique is the problem, not your organism. If they come out right, your technique is likely sound, though smear thickness and culture age can still affect your unknown’s result.
Fixing Wrong Gram Stain Colors
If every cell is purple, the alcohol step was too short, so lengthen it next time.20General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips If every cell is pink, the alcohol step was too long, so shorten it.21General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips Cut the alcohol time by about 5 seconds, and make sure the alcohol hasn’t concentrated through evaporation.22Preparing and Staining Bacterial Smears: Techniques and Tips. Common Gram Staining Errors and How to Fix Them Too short a time in crystal violet or iodine can also leave cells too pale.23CellaVision. What are the main problems you encounter when Gram staining?
Culture age is the other usual culprit. Older Gram-positive cultures can lose wall strength and stain falsely pink.24Preparing and Staining Bacterial Smears: Techniques and Tips. Common Gram Staining Errors and How to Fix Them In one Gram-positive species, cells kept for 3 days were classified as Gram-positive no more than about a quarter of the time.25PubMed. Incubation time and culture medium affect gram staining accuracy: A Cellpose image analysis study Use cultures 18 to 24 hours old.
Some results are real. Gram-variable species, those that show both pink and purple cells on the same slide, produce mixed colors naturally.26General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips
If both controls came out the same color, redo the stain. Thick areas often look falsely purple, so read the thin areas at the smear’s edge, and restain from a fresh culture when results are unclear.
A few artifacts can fool you. Dye crystals from unfiltered or old stain look like dark specks, not cells. Scratches and dust on the slide can mimic shapes. Rinsing straight onto the smear can wash patches away and leave you with bare glass where cells should be.
Acid-Fast Staining for Waxy Bacteria
Acid-fast bacteria have waxy, nearly watertight walls rich in fats.27General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips Ordinary stains barely get in, so they need a special method.28General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips Once dyed, these bacteria resist being decolorized by acid-alcohol, which is what acid-fast means.29General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips Very few bacteria are acid-fast, and the main group is Mycobacterium, which includes the tuberculosis germ.30General Microbiology Lab Manual. Preparing and Staining Bacterial Smears: Techniques and Tips
The Ziehl-Neelsen method uses heat to drive red dye into the cells, while the Kinyoun method is a cold version with a stronger dye. In both, acid-fast bacteria show up red or pink against a blue background. In tuberculosis sputum testing, the two methods agree closely.31PubMed Central. Comparative Evaluation of Ziehl Neelsen Staining and Knowledge, Attitudes and Practices of Laboratory Personnel in Relation to Ziehl Nielsen32Journal of Nepal Health Research Council. Evaluation of Different Staining Techniques (Ziehl Neelsen Stain, Kinyoun Stain, Modified Cold Stain, Fluorochrome Stain) for The Diagnosis of Pulmonary Tuberculosis
For the hot method, steam the slide over a water bath rather than boiling the dye. Keep the smear covered with dye and add more if it starts to dry out. Let the slide cool before applying acid-alcohol.
Student labs usually use harmless relatives of Mycobacterium rather than the real tuberculosis germ. Real TB samples are handled only in labs with special precautions.33CDC Stacks. Tuberculosis Laboratory Biosafety Manual In class, avoid splashing and work slowly when steaming slides.
Endospore, Capsule and Flagella Stains
Endospores are tough, resting forms that some bacteria make, and the Schaeffer-Fulton method stains them.34PubMed Central. Preparing and Staining Bacterial Smears: Techniques and Tips Steaming forces green malachite green dye into the hard spore coat.35Microbiology Info.com. Endospore Staining- Principle, Reagents, Procedure and Result The dye holds poorly to ordinary cells, so a water rinse washes it out of them.36Microbiology Info.com. Endospore Staining- Principle, Reagents, Procedure and Result Spores stay green while the rest of the cells take a red counterstain.
When you read a spore slide, note whether the spore sits in the middle of the cell, near the end, or at the very tip. Spores that look free-floating have already been released from their cells.
A capsule stain uses a negative stain, a dye that colors the background instead of the cells.37LOUIS: The Louisiana Library Network. Preparing and Staining Bacterial Smears: Techniques and Tips The background dye is usually India ink, nigrosin or Congo red.38Microbiology Info.com. Capsule Staining- Principle, Reagents, Procedure and Result After drying, a second dye colors the cells.39LOUIS: The Louisiana Library Network. Preparing and Staining Bacterial Smears: Techniques and Tips The capsule stays unstained and shows as a clear halo around each cell.40LOUIS: The Louisiana Library Network. Preparing and Staining Bacterial Smears: Techniques and Tips Capsule smears are usually not heat-fixed, since heat can shrink the capsule.
Flagella, the whip-like tails, are too thin to see with a light microscope on their own.41PubMed. Differential staining of bacteria: flagella stain Flagella stains coat them with a mordant such as tannic acid to thicken them.42American Society for Microbiology. Bacterial Flagella Stain Protocol They become just thick enough to see under the oil immersion lens.43PubMed. Differential staining of bacteria: flagella stain
Flagella break off easily, so don’t vortex the sample or scrape hard. Let the sample drift across the slide rather than spreading it with a loop, and use clean, grease-free slides so the coating goes on evenly.
Viewing Your Slide Under Oil Immersion
Oil immersion means placing a drop of oil between the slide and the 100x lens. The 100x oil lens needs it to give a bright, sharp image.44POLTEKKES KEMENKES YOGYAKARTA. CASTOR OIL SEBAGAI ALTERNATIF IMMERSION OIL UNTUK PEMERIKSAAN BAKTERI MYCOBACTERIUM TUBERCULOSIS Oil keeps light from bending as it passes between the slide and lens.45POLTEKKES KEMENKES YOGYAKARTA. CASTOR OIL SEBAGAI ALTERNATIF IMMERSION OIL UNTUK PEMERIKSAAN BAKTERI MYCOBACTERIUM TUBERCULOSIS
Start at 10x and focus on the smear’s stained edge. Move to 40x and refocus with fine focus only. Then swing the lens aside, place one drop of oil on the smear, and rotate the 100x lens into the drop. At 100x, use only fine focus, since the coarse knob can crack the slide.
Never swing the 40x lens back through the oil. If pale pink cells are hard to see, there’s probably too little light, so open the diaphragm. When you’re finished, wipe the oil off the lens with lens paper. If you want to keep the slide, blot the oil off rather than wiping it, which would smear the cells.