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Professor Manuel Varela: Who was Christian Gram?

Mar 20, 2017 by

An Interview with Professor Manuel Varela: Who was Christian Gram?

Michael F. Shaughnessy –

1) Dr. Varela, we often hear the term “Gram staining technique” in science and microbiology and the like. What exactly does this mean and why is it important?

The Gram stain technique has been around since Dr. Hans Christian Gram developed it during the mid-1880s. No doubt you have heard about it often because Gram’s technique is routinely taught in all Microbiology laboratory courses and even in good freshmen Biology lab courses. Furthermore, the Gram stain is regularly used in clinical medicine and medical diagnostic laboratories. It is a staple procedure in each of the infectious disease specialties. In fact, the Gram stain is frequently the very first laboratory test that’s conducted when a new species of bacteria is discovered and characterized further. The Gram status of all bacteria is used in taxonomic and systematics circles to classify all extant and newly discovered bacteria. Many bacteria are classified as Gram-positive or Gram-negative.

If a bacterium is denoted as Gram-positive, it means that the chemical stain, called crystal violet, stays with the bacterium when washed; on the other hand if a bacterium is Gram-negative, it means that the stain comes off the cell after washing. Depending on the nature of the bacterial species being studied, the cell walls that cover a Gram-positive versus a Gram-negative bacterial cell will have distinctive biochemical and cellular structures within their cell wall coverings. These different biological properties of bacteria are used to classify them and to differentiate between various bacterial species, and these properties are often focuses of both basic and applied scientific research areas.

2) So, who exactly was Christian Gram, and where was he born and what do we know about his early life?

Hans Christian Joachim Gram was born in Copenhagen, Denmark, on September 13, 1853, to parents Frederick Terkel Julius Gram, a law professor, and Louise Christiane Roulund.

3) Where did he attend university and how did he get involved in science and research?

Gram was an undergraduate at the University of Copenhagen, in Denmark, where he primarily focused his studies in the field of botany, which is the study of plants. Gram reportedly took his undergraduate degree in 1871. This educational activity introduced Gram to the important scientific fields of microscopy, as well as in pharmacology. He then became a botany assistant under the famous Danish professor of zoology, Japetus Steenstrup. Gram went onto medical school and took his M.D. degree in 1878; he then earned his doctorate in 1883 studying erythrocytes under the supervision of Prof. Steenstrup. While travelling about Europe, Dr. Gram took up a new position in 1883 with Dr. Carl Friedländer, a bacteriological pathologist housed in Berlin, Germany. It is in Friedländer’s laboratory that Dr. Gram developed his now famous Gram staining procedure.

4) This “gram staining technique for bacteria”- how did it come about and what is its relevance in terms of bacteria? And does it apply to viruses also?

The story of Gram’s discovery began with a controversy. At the time, there had been a disagreement between Gram’s supervisor, Dr. Friedländer, and Dr. Albert Fraenkel, a German physician. Dr. Friedländer had isolated a bacterium from the lung of a patient who had died of pneumonia. While Dr. Fraenkel had also isolated a bacterium, but from another patient, this one having died from lobar pneumonia. Fraenkel was convinced that his bacterium and that of Friedländer’s were one and the same, while Friedländer was equally convinced that the two bacterial isolates were completely different from each other.

To settle the controversy, Friedländer used Gram’s staining technique in 1883, even before Gram had published it, to visualize both bacteria. Friedländer found that his bacterium was Gram-negative while Fraenkel’s bacterium was Gram-positive. Thus the two bacteria were different ones, settling the matter once and for all.

Friedländer’s bacterium later came to be called Klebsiella pneumoniae while Fraenkel’s bacterium was later called Streptococcus pneumoniae, both of which are serious pathogens in their own right.

While in Friedländer’s lab, Gram was aware of a stain used by Prof. Paul Ehrlich composed of a mixture of analine, water, and gentian violet. After adding iodine mixed with potassium iodide (known as Lugol’s solution) to make Ehrlich’s stain bind bacteria better, Gram noticed that the stain sometimes came off some bacteria after washing with an alcohol solution, making these Gram-negative cells difficult to see under the microscope—because there was no stain. Bacteria are actually colorless. So Gram formulated a method to visualize these colorless cells that lost the crystal violet using a so-called counter-stain, called Bismarck brown or vesuvin. The Gram-negative bacteria could be seen under the microscope. This Gram staining procedure was published in 1884.

The Gram staining procedure today commonly consists of the following steps performed on bacteria that are heat-fixed on a microscope slide:

  1. Addition of a primary stain, crystal violet—bacteria turn purple or blue.
  2. Addition of Gram’s iodine—a mordant that fixes the crystal violet to the bacteria—bacteria are purple or black.
  3. Decolorization with ethanol—Gram-positive cells keep the crystal violet (remaining purple) while Gram-negative cells lose the stain (turning colorless) upon the alcohol washing.
  4. Addition of a counterstain—usually safranin, which turns Gram-negative bacteria red or pink.

On the microscope the Gram-positive bacteria appear blue or purple, and the Gram-negative bacteria appear pink or red.

Incidentally, two additional types of bacteria later came to be known. One type was called acid fast, which retained Gram’s stain but was actually neither Gram-positive nor Gram-negative in its cell wall structure. The other type of bacterium lacked cell walls completely. Thus, most bacteria can be categorized into these four distinctive groups: Gram-positive, Gram-negative, acid-fast, and cell wall less.

With respect to the viruses, they are neither bacteriological nor cellular in the way that you and I know about. The viruses merely consist of a protein coat that surrounds an internal genome, sometimes harboring a membrane envelope.  Thus, the Gram staining procedure is not used to characterize viruses.

5) What other inventions or discoveries was Christian Gram known for?

Dr. Gram had conducted experiments demonstrating how red blood cells became enlarged during a condition known as pernicious anemia, which occurs in patients with a deficiency in vitamin B12 and results in a lowering of the red blood cell count.

In 1890, Dr. Gram went to the University of Copenhagen and became a professor of clinical medicine and pharmacology.  During this time he took great interest in teaching, and his course lectures were eventually published into several large volumes, which became popular for teaching clinical medicine by other faculty throughout Denmark.

6) What have I neglected to ask about his life and work?

During Dr. Gram’s ensuing years as a clinical faculty, after his great discovery, he formed a thriving medical practice. During this time, 1901 – 1921, he was the chairperson of the so-called Pharmacopoeia Commission and medical director of his department at Frederick’s Hospital, until his retirement in 1923.

Dr. Gram was celebrated widely, garnering many accolades and honors.  In retirement, he had a keen interest in the history of medicine. In 1938, Dr. Gram died on November 14.

It may be of interest to your readers to consider briefly what subsequent studies showed regarding the biological mechanism that explains why Gram-positive bacteria retain the crystal violet stain while Gram-negative cells lose it.

The first hint emerged in 1929 when Victor Burke and Mildred Winchester Barnes proposed that Gram-negative bacteria had an increased permeability in their cell walls, meaning that the crystal violet stain simply readily exited through the cell wall while such permeability did not occur in Gram-positive cells.

In 1963, this permeability notion was confirmed experimentally when Prof. Milton R. J. Salton definitively showed that alcohol treatment created small pockets in a protein-sugar complex called peptidoglycan that trapped the crystal violet in cell walls of Gram-positive bacteria; while the  peptidoglycan of Gram-negative apparently bacteria did not make these pocket traps, thereby allowing the crystal violet to escape.

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