Gram Staining
On this page
Direct answer
Gram staining, described by Christian Gram in 1884, is the fundamental differential stain of bacteriology, dividing bacteria into gram-positive organisms that stain violet and gram-negative organisms that stain pink. The procedure uses a primary stain (crystal violet), a mordant (Gram's iodine), a decolouriser (acetone or alcohol) and a counterstain (dilute carbol fuchsin or safranin). The reaction depends on the cell wall: the thick peptidoglycan of gram-positive bacteria retains the crystal violet–iodine complex, while it leaks out of gram-negative bacteria, which then take the counterstain.
What you must remember
- Steps and timings: crystal violet one minute, Gram's iodine one minute, decolourise with acetone or alcohol for a few seconds, counterstain with dilute carbol fuchsin for about thirty seconds, washing between steps.
- Principle: iodine forms a crystal violet–iodine complex; in gram-positive organisms the thick peptidoglycan dehydrates and traps the complex, while in gram-negative organisms the decolouriser extracts outer-membrane lipid and the dye washes out.
- Reading the film: gram-positive bacteria appear violet, gram-negative bacteria pink; examine under the oil immersion objective and report morphology and arrangement as well.
- Common organisms: gram-positive — staphylococci, streptococci, corynebacteria, bacilli and clostridia; gram-negative — Neisseria, Enterobacteriaceae, vibrios and Pseudomonas.
- Limitations: mycobacteria need acid-fast staining, and Treponema, Mycoplasma, Rickettsia and Chlamydia are not demonstrated; old cultures of gram-positive organisms decolourise easily, giving false gram-negative results.
- Uses: primary classification of isolates, direct microscopy of pus, urethral discharge and cerebrospinal fluid, guidance of empirical antibiotic choice, and checking culture purity.
Common confusion
Students often state that gram-positive bacteria "retain the stain" without explaining that the thick peptidoglycan physically traps the dye–iodine complex during decolourisation, which is the decisive step. Over-decolourisation turns a gram-positive organism gram-negative and under-decolourisation does the opposite, so both false reactions are possible. The counterstain only makes decolourised cells visible; it does not cause the difference.
Exam-focused takeaway
In theory, write the aim, reagents with order and timings, the principle, the result and applications, including the organisms that cannot be demonstrated. In viva, expect "why do gram-positive bacteria stay violet?", its converse, and the exceptions list. At the practical, you may gram-stain a sample and report it as, for example, gram-positive cocci in clusters — the arrangement narrows the organism at the bedside.
Frequently asked questions
Why is Gram's iodine used?
It is a mordant forming an insoluble crystal violet–iodine complex, which the thick peptidoglycan of gram-positive bacteria traps during decolourisation.
What happens if the film is over-decolourised?
Even gram-positive bacteria lose the violet complex and take the counterstain, giving a false gram-negative report; under-decolourisation gives false gram-positive results.
Which bacteria are not demonstrated by Gram staining?
Mycobacteria, which need acid-fast staining, thin spirochaetes such as Treponema, and Mycoplasma, Rickettsia and Chlamydia.
Why do old cultures give false gram-negative results?
Ageing gram-positive cells have damaged walls that cannot retain the complex, so films should be made from fresh cultures.
What is reported in a gram-stained film?
The reaction, the morphology and the arrangement with pus cells — for example, gram-negative diplococci within neutrophils in gonococcal urethritis.
Which counterstains are used?
Dilute carbol fuchsin or safranin for about half a minute, so decolourised gram-negative cells appear pink.