Gram staining
Classify bacteria by cell-wall type under the microscope.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Two kinds of wall, one dye
Hans Christian Gram's stain, published in 1884, still sorts most bacteria into two groups in about four minutes. It works because bacterial envelopes come in two basic designs.
2The decolorizer is a timer, not a switch
Given long enough, alcohol strips a Gram-positive cell too, and given too little time it leaves a Gram-negative purple. The result is a race, which is why the step is the one that most often goes wrong and why the stain is never trusted without controls.
Gram-positive
Thick peptidoglycan retains the crystal violet–iodine complex. Staphylococci, streptococci, enterococci, Bacillus, Clostridium, Listeria.
Gram-negative
Outer membrane dissolved, thin wall cannot hold the dye, safranin shows through. Enterobacterales, Pseudomonas, Neisseria, Haemophilus.
Gram-variable
Some cells of one culture purple and some pink. Usually old or damaged Gram-positive cells; occasionally an organism with an unusual wall.
Poorly staining
Mycobacteria (waxy walls, need an acid-fast stain) and mycoplasmas (no wall at all) do not give a meaningful Gram reaction.
3How the stain lies
- Over-decolorizing: too long in the decolorizer, or acetone–alcohol timed like ethanol. Gram-positives turn pink; the positive control shows it.
- Under-decolorizing, or a thick smear: Gram-negatives stay purple, especially in dense patches; the negative control shows it.
- Old cultures: Gram-positive cells that are autolysing or forming spores lose wall integrity and turn Gram-variable. Stain from an 18–24 h culture.
- Overheated smears: cooking a wet smear over the flame bursts and distorts cells and damages walls.
- Exhausted iodine: a pale, oxidised mordant makes too little complex, so Gram-positives decolorize easily.
- Too little counterstain: cleared cells are so faint that Gram-negatives in a mixed sample are overlooked.
- Mordant
- A substance that binds a dye inside the cell; here iodine, which forms the crystal violet–iodine complex.
- Decolorizer
- The solvent that removes the dye from cells that cannot retain it: 95% ethanol (slower) or acetone–alcohol (faster).
- Counterstain
- The second, contrasting dye (safranin or basic fuchsin) that makes decolorized cells visible.
Key terms
Practise these as flashcards →The mesh of sugar chains cross-linked by short peptides that forms the bacterial cell wall; many layers thick in Gram-positive cells, one or two in Gram-negative ones.
The second, lipopolysaccharide-containing membrane of Gram-negative bacteria, which the decolorizer dissolves.
Passing a completely air-dried smear briefly through a flame so the cells stick to the slide and are killed.
A culture in which some cells stain purple and some pink, usually because older or damaged Gram-positive cells lose the dye.
Using a 100× objective with immersion oil between lens and slide, giving the resolution needed to see the shape and arrangement of bacteria.
A thin film of cells spread on a slide, dried and fixed before staining; it should look like a faint haze, not a cloud.
When to use it
- The first look at an unknown isolate or a clinical sample, to decide which media and identification tests come next.
- Checking whether a culture that should be pure contains organisms of a second Gram reaction or shape.
- Rapid information directly from a specimen, such as a positive blood culture, before any colony has grown.
Compared with alternatives
The next step after the Gram stain: catalase, oxidase, biochemical strips or MALDI-TOF identify the genus and species rather than just the wall type.
Lets growth do the sorting: MacConkey agar suppresses most Gram-positives and shows lactose fermentation, which complements a stain on a mixed sample.
Unstained phase-contrast shows live cells, their motility and spores without fixing them, but cannot tell Gram-positive from Gram-negative.
For identification by sequence (16S rRNA genes) when an organism stains or grows ambiguously, or for whole communities.