On this page
"Gram-positive" and "Gram-negative" are among the first words a biology or medical student learns in microbiology. Fewer know that Gram was a person, a young Danish doctor who was trying to see bacteria in lung tissue, and who soon left microbiology altogether. This post covers who he was, how the stain works, a worked example of reading one step by step, and what his way of working can teach you.
Who was Hans Christian Gram?
The Dansk Biografisk Leksikon, Denmark's national biographical dictionary, gives his full name as Hans Christian Joachim Gram, born in Copenhagen on 13 September 1853 and died there on 14 November 1938. He was first drawn to the natural sciences, especially botany, then turned to medicine and qualified as a doctor in 1878. In 1883 he earned his doctorate for a study of the size of human red blood cells.
How did he come to invent the stain?
The Danish encyclopedia entry by the microbiologist Niels Høiby fills in the story. In 1883 Gram was one of eleven people on Carl Julius Salomonsen's first university course in bacteriology; Salomonsen had brought microbiology to Denmark. With an introduction from Salomonsen he went to Berlin, where he worked in the laboratory of the pathologist Carl Friedländer from October 1883 to March 1884.
The stains of the time coloured both the patient's own cells and the bacteria, which made it hard to study the causes of pneumonia. Gram's method left the tissue pale and the bacteria dark. Høiby writes that it let Gram show that the bacteria in the cases he studied were the Gram-positive pneumococci, not the capsulated Gram-negative bacterium Friedländer favoured, though Gram did not dare tell Friedländer while he was in Berlin.
Gram described the method in a short paper in 1884 in Fortschritte der Medizin, and in a talk at the International Medical Congress in Copenhagen that year. The national dictionary notes that this method made his name famous around the world.
Why do some bacteria keep the stain?
For decades, nobody knew. A 1996 review, The Gram stain after more than a century, notes that its mechanism was understood only in the decade before. The cell wall of Gram-positive bacteria holds on to a complex of crystal violet and iodine. In Gram-negative cells the staining steps damage the cell surface and the dye is lost. The review adds that what matters is a relatively thick wall, not its exact chemistry.
A 2001 review, Use of the Gram stain in microbiology, describes the two kinds of wall. Gram-positive bacteria have a thick wall made of peptidoglycan and other polymers that resists decolourising. Gram-negative bacteria have a thin peptidoglycan layer plus an outer membrane of lipid and protein, which alcohol can disrupt. Some bacteria with walls of intermediate structure stain unevenly, and for the Archaea the stain is not a useful test.
Worked example: reading a Gram stain step by step
Høiby's entry gives the steps and timings of the modern method.
Crystal violet, 1 minute. Every bacterium turns blue-violet.
Iodine solution, 1 minute. The iodine acts as a mordant; all bacteria now look brownish-black.
Alcohol, half a minute. Gram-positive bacteria keep their colour. Gram-negative bacteria and the patient's cells lose it and become colourless.
Red counterstain, half a minute (carbol fuchsin, or safranin in many labs). The colourless Gram-negative cells turn red.
Total time: 1 + 1 + 0.5 + 0.5 = 3 minutes.
Reading the slide. Purple cells are Gram-positive. Red or pink cells are Gram-negative. Ask which step decides the result: it is step 3, the alcohol, because that is where the two kinds of wall behave differently.
Common mistakes:
Treating the alcohol step casually. It is the step that separates the two groups, and the shortest in Høiby's timings, so it is where timing matters most.
Skipping the iodine. The 1996 review explains that the result depends on the wall holding a crystal violet–iodine complex. Without iodine, that complex never forms.
Treating the result as a species name. The stain sorts bacteria into two broad groups by wall type, and some bacteria stain variably. It is a first clue, not a final answer.
What happened to Gram afterwards?
He did not become a microbiologist. The national dictionary records that he became professor of pharmacology in 1891 and professor of internal medicine in 1900, a post he held until 1924. He was a lively lecturer, served on the commission for the Danish pharmacopoeia, and declined to become rector of the university. Høiby adds that he also became physician to the royal household.
What can a student take from his way of working?
Gram set out to solve a practical problem, seeing bacteria clearly in tissue, and kept his method simple enough to repeat in three minutes. He also published it quickly and modestly, so others could test it. A simple, repeatable method that others can check is worth more than a clever one nobody can copy. His stain is usually done on bacteria grown in the flat dishes described in our post on Julius Petri, using methods from Robert Koch's laboratory.
If cell walls and membranes are still blurry for you, the Fermi Concept Library covers every concept in physics, chemistry, biology, mathematics and computing, so you can go back to the idea you need.
Try it now: write the four Gram stain steps from memory, then add what colour a Gram-positive and a Gram-negative cell would be after each one.
