Gram’s Serendipitous Stain
In spite of his “defective” method, his name lives on …
hile working with which acted as a mordant to fix infected lung tissue the dye. Then he poured ethanol Wfrom cadavers, a over the slide to wash away the Danish pathologist stumbled dye. Certain bacteria upon a remarkable technique that (pneumococci) retained the color that is still in common usage (gram-positive), while other over 100 years later. How did species became bleached or de- this come about? colorized by the alcohol (gram- negative). His initial work with Hans Christian Gram (born in this staining process was
Jay Hardy, CLS, SM (ASCP) 1853) studied botany at the performed on Streptococcus University of Copenhagen in pneumoniae and Klebsiella Denmark. His study of plants pneumoniae. introduced him to the basis of Jay Hardy is the founder and pharmacology and the use of the CEO of Hardy Diagnostics. microscope. He began his career in microbiology as a Medical In 1883, he graduated from
Technologist in Santa medical school and after Barbara, California. traveling throughout Europe, he In 1980, he began settled in Berlin. His initial work manufacturing culture media concerned the study of human for the local hospitals. red blood cells. He was among Today, Hardy Diagnostics is the first to recognize that the third largest media macrocytes were characteristic manufacturer in the US. of pernicious anemia.
To ensure rapid and reliable In 1884, while examining lung
turn around time, Hardy tissue from patients who had Diagnostics maintains six distribution centers, and died of pneumonia, Gram had produces over 2,700 discovered that certain stains products used in clinical and were preferentially industrial microbiology taken up and “I am aware that as yet it is laboratories throughout the retained by world. bacterial cells. In very defective and imperfect” the first step, he Hans Christian Gram dried a fluid smear on a glass slide over a burner
flame and poured Gentian Gram was a modest man, and in his initial publication he (crystal) violet solution over it. remarked, "I have therefore After rinsing with water, he added Lugol’s solution - published the method, although I potassium triiodide solution - am aware that as yet it is very defective and imperfect; but it is classification in medical hoped that also in the hands of microbiology today. other investigators it will turn out to be useful." The Gram Stain Gram did not use a counterstain Mechanism in his procedure. It was a few years later, that the German What makes some cells retain pathologist Carl Weigert (1845- crystal violet (Gram positive) 1904) from Frankfurt, added a while others readily release the final step of staining with stain when alcohol is added safranin. Gram himself never (Gram negative)? used the red counterstaining in order to visualize the gram- Gram-positive bacteria have a Crystal violet (CV) dissociates in negative bacteria. thick mesh-like cell wall made of aqueous solutions into CV+ and
peptidoglycan (50-90% of cell chloride (Cl – ) ions. These ions In 1891, Gram became a lecturer wall), which stain purple and penetrate through the cell wall in pharmacology, and later that Gram-negative bacteria have a and cell membrane of both year was appointed professor at thinner layer (10% of cell wall), Gram-positive and Gram- the University of Copenhagen. In which stain pink. Gram-negative negative cells. The CV+ ion 1900 he resigned his Chair in bacteria also have an additional interacts with negatively charged Pharmacology to become outer membrane which contains components of bacterial cells Professor of Medicine. The king lipids, and is separated from the and stains the cells purple. of Denmark awarded him the cell wall by the periplasmic Iodine (I – or I3 – ) interacts Dannebrog Commander’s Cross, space. with CV+ and forms large first-class in 1912, and the complexes of crystal violet and Golden Medal of Merit in 1924. iodine (CV – I) within the inner He retired in 1923 and died in and outer layers of the cell. 1938.
When a decolorizer such as The staining technique he alcohol or acetone is added, it developed is still the most interacts with the lipids of the important method for cell membrane. A Gram- distinguishing between two negative cell will lose its outer major classes of bacteria. His membrane and the peptidoglycan discovery in 1884 occurred layer is left exposed. The CV – I during the golden years of complexes are washed from the clinical microbiology. This is Gram-negative cell along with when Petri plates were invented the outer membrane. In contrast, (1887), Agar was discovered a Gram-positive cell becomes (1881), and Pasteur and Koch There are four basic steps of the dehydrated from an ethanol were at their peak of Gram stain, which include treatment. The large CV – I performance, discovering the applying a primary stain (crystal complexes become trapped etiologic agents of many of the violet) to a heat-fixed smear of a within the Gram-positive cell vexing diseases of that day. bacterial culture, followed by the due to the multilayered nature of
addition of a mordant (Gram's its peptidoglycan. In spite of his modesty and iodine), rapid decolorization doubts of success, this technique, with alcohol or acetone, and The decolorization step is critical the Gram Stain, continues to be a counterstaining with safranin or and must be timed correctly; the standard procedure for bacterial basic fuchsin. crystal violet stain will be removed from both Gram- positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).
After decolorization, the Gram- positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively- charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color. Bacillus spp showing ambiguous staining results. Older cells readily decolorize and will appear gram negative, while the younger cells retain their gram positive nature.
An “Imperfect” procedure, and caution must be Method exercised.
Some bacteria, after staining In addition, for all bacteria with the Gram stain, yield a stained using the Gram stain, the Gram-variable pattern: a mix of age of the culture may influence pink and purple cells are seen. the results of the stain as seen in the photo above. The genera Actinomyces, Arthobacter, Corynebacterium, Using improved formulations, Mycobacterium, and such as Gram Stain Advanced Propionibacterium have cell will help in overcoming some of walls particularly sensitive to these problems. breakage during cell division, resulting in Gram-negative Jay Hardy, CLS, SM (ASCP) staining of these Gram-positive Santa Maria, CA cells. The staining of these organisms result in an uneven or granular appearance.
In cultures of the Gram-positive Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative. For this reason, these organisms are very prone to over- decolorization in the acetone- alcohol rinse step of the