Medical Fun Facts Podcast

MFF0050: The Gram’s stain

If I’ve timed this correctly, tonight’s show is dropping on Thursday 23 March 2017.

As I mentioned in episode 49, my favourite test in microbiology is the Gram’s stain. This test is named after Hans Christian Gram. Gram was a Danish medical doctor who lived from 1853 to 1938. He developed his staining method in 1884.

He described a differential staining method which would separate bacteria based on the structure of the cell wall as well as the shape of the bacteria. Bacteria, in general, are either Gram-positive or Gram-negative. Some bacteria, like the mycobacteria do not stain well because of the waxy mycolic acid in their cell walls. Some bacteria are Gram-variable and do not stain predictably. A good example is Gardnerella vaginalis which is involved in the complex disease known as bacterial vaginosis. Gardnerella vaginalis is a small Gram-variable bacterium that attaches to vaginal epithelial cells.

The procedure consists of four steps after clinical material has been applied to the slide, dried and fixed. The first stain is crystal violet, all bacteria will stain with this but the stain is easily removed so it must be fixed with a mordant. The most commonly used agent is Lugol’s iodine. After allowing the iodine to trap the crystal violet-iodine complex, the slide is washed and then a decolourising agent is applied. Gram-positive bacteria will retain the crystal violet-iodine complex and stain which won’t be removed by the ethanol or acetone which are the commonly use decolourising agents. Gram-positive bacteria appear purple down the microscope. After the decolouriser is washed away, a counterstain is applied to the slide and after half a minute to a minute, it too is washed away. The commonly used counterstain is safranin but in some situations, especially when anaerobic bacteria may be present, carbol fuchsin is preferred. Gram-negative bacteria appear pink to red depending on the counterstain.

So, what sort of specimens do we accept for a Gram’s stain as part of a microbiological workup? Almost any specimen really. We tend not to stain urine, but in some circumstances, even a urine Gram’s stain is worthwhile. While on the subject of urine, for fluid specimens that we regard as normally sterile like urine and cerebrospinal fluid or joint fluid, we will perform a cell count on a direct specimen and then centrifuge some to try to concentrate any bacteria that may be present.

The common specimens that need Gram staining include swabs from wounds and other sites like genital specimens. Tissue specimens will also get a Gram’s stain as will sputum, although we tend not to do throat swabs, mostly because the pharynx is full of mixed bacteria and there isn’t a lot to be gained from a Gram’s stain in most circumstances.

So, what are the Gram stain reactions of common bacteria?

Common Gram-positive pathogens and commensals include: staphylococci, streptococci, enterococci, lactobacilli, clostridia, Bacillus species, and actinomycetes. Common Gram-negative pathogens and commensals include: gonococci and meningococci, all the species in the family Enterobactericeæ, pseudomonads and nonfermenting environmental bacilli, Bacteroides and other related anaerobic bacteria and oral spirochætes oddly enough stain Gram-negative.

In addition to seeing and describing bacteria, a Gram’s stain allows us to semiquantify the cellular component of a specimen. It’s important to know if there are a lot of pus cells or epithelial cells in a specimen. The presence of pus is a reasonable indicator for infection whereas a specimen with few pus cells and lots of squamous epithelial cells would suggest bacteria that grow may not be all that significant.

The other reason I love the Gram’s stain is because when you’re sitting at a bench looking at unusual or unfamiliar colonies on a plate, it is quick and easy to make a simple wet preparation, take a look and then convert it to a smear for a Gram’s stain. By just doing that, a lot of useful information can be gleaned.

Thank you, Hans Christian Gram, for my favourite test in microbiology.

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