WORLD OF MICROBIOLOGY AND IMMUNOLOGY Gram staining253•
bacterial infection; Haemophilus; Infection control;
Lipopolysaccharide and its constituents; Meningitis, bacterial
and viral; Microbiology, clinical; Serology; Streptococci and
streptococcal infections; VaccineGRAM, HANSCHRISTIANJOACHIM
(1853-1938)Gram, Hans Christian Joachim
Danish pharmacologistHans Christian Joachim Gram was a Danish physician and
bacteriologist who developed the most widely used method of
staining bacterial cells for microscopic study.
Gram was born in Copenhagen, Denmark, on
September 13, 1853. He received a B.A. in the natural sci-
ences from the Copenhagen Metropolitan School in 1871 and
served as an assistant to the zoologist Japetus Steenstrup from
1873 to 1874. He subsequently became interested in medicine
and earned a medical degree from the University of
Copenhagen in 1878. Gram, who worked in several areas of
science and medicine, earned a gold medal in 1882 for a study
on human erythrocytes. The following year he received a doc-
toral degree for his work in this field.
After obtaining his degree, Gram pursued post-doctoral
studies in Berlin, focusing on bacteriology and pharmacology.
It was in Berlin in 1884 that he published his work on the tech-
nique of staining cells, a procedure that became widely known
as Gram staining.
At that time, the method of staining cells was not
entirely new to scientific research and several methods were
already being used. Gram borrowed from a procedure initially
devised by Paul Ehrlich, who used alkaline aniline solutions to
stain bacteriacells. Experimenting with pneumococci bacte-
ria, Gram first applied Gentian violet, which stained the cells
purple, and then washed the cells with Lugol’s solution
(iodine), which served as a mordant to fix the dye. He fol-
lowed those steps by applying alcohol, which washed away
any dye that was not permanently fixed. Gram found that
some cells remained purple (Gram positive), while others
stayed essentially unstained (Gram negative). Gram’s method
aided microscopic study of bacteria, as well as provided a
means of differentiating and classifying bacteria cells. Several
years later, the pathologist Carl Weigart improved upon
Gram’s method by adding another staining step, which con-
sisted in dyeing the Gram-negative cells with saffranine.
Gram remained in Berlin working as an assistant in a
hospital until 1891, when he was appointed as a professor of
pharmacology at the University of Copenhagen. In 1889,
Gram married Louise I. C. Lohse, and in 1892, advanced to
the position of chief of internal medicine at the Royal
Frederiks Hospital. Extremely active in the field of medical
education, Gram also maintained a large internal medicine
practice. From 1901 to 1921, Gram served as chairman of the
Pharmacopoeia Commission, during which time he abolished
the use of many useless and obsolete therapeutic treatments. In
addition, he published a four-volume book on the importance
of rational pharmacology in clinical science. After his retire-ment in 1923, he returned to an earlier interest: the history of
medicine. During his career, Gram received several honors
including the Danneborg Commander’s Cross, the Golden
Medal of Merit, and an honorary M.D. Gram died in
Copenhagen, on November 14, 1938.See alsoLaboratory techniques in microbiologyGGram stainingRAM STAINING
In the second half of nineteenth century, scientists proved that
specific bacterial organisms caused specific diseases, and the
field of microbiology was on its way to becoming a distinct
science. The microscopewas also further developed during
that time, and scientists were concerned with identifying and
classifying bacteria. Most bacteria are difficult to see with a
compound microscope, but can be seen when there is obvious
contrast between the bacteria cells and their surrounding
medium. Various dyes are used to stain cells so that they are
more easily seen. As early as the late eighteenth century, sci-
entists had developed some basic methods of staining cells to
aid in their study and used natural substances such as saffron,
which stained some parts of a cell. The discovery of synthetic
dyes in the mid-1800s enabled scientists to utilize more colors
to stain cells.
In 1884, the Danish physician Hans Christian Joachim
Gramfurther developed a method of staining bacteria origi-
nally developed by the German biologist Paul Ehrlich. Ehrlich
used aniline water and gentian violet (a cationic dye) to stain
cells, and the cell walls would appear purple after staining.
Gram added a potassium triiodide solution, which acted as a
mordant for the gentian violet dye, and then poured ethanol
over the cells to wash away the unfixed dye. Gram found that
some of the cells remained purple, while others did not.
Bacteria that remained purple were termed positive and those
that did not remain purple were called negative. A few years
later, Carl Weigert, director of the Senckberg Foundation in
Frankfurt, Germany, added another step to the staining
method. Weigert followed Gram’s procedure with a final stain-
ing using saffranine (an anionic dye), which subsequently
stained the negative bacteria red. The Gram stain is still con-
sidered the definitive, differential test to determine the chemi-
cal make-up of a bacterium cell wall. On the basis of a cell’s
reaction to the Gram stain, bacteria are divided into two
groups, Gram positive and Gram negative.
The distinguishing feature between Gram positive and
negative bacteria is the difference in the structure of the cell
walls. The cell wall of a Gram positive is a thick, single layer
of a cross-linked polysaccharide that is easily stained by gen-
tian violet, while the cell wall of a Gram negative bacterium
consists of a thin layer of polysaccharide and is covered by a
lipid layer that resists the gentian violet, but can be stained by
saffranine. Many dyes, which are organic compounds, are pos-
itively charged and easily combine with the negatively
charged, acidic polysaccharide wall. Other dyes are negatively
charged and combine with protein-based cell constituents.womi_G 5/6/03 3:18 PM Page 253