There are many amongst us who are unfortunate enough to be required to read Gram stains as part of our work, and usually lots of them. There will inevitably be occasion when one comes across a stain where it is uncertain whether bacteria are present or not, or whether it is just debris/stain deposit etc. And it usually doesn’t matter how much you look at it! You can scan the slide ad infinitum, then ask your colleague for their opinion, which usually turns out to be identical to yours…. “I’m not sure”
This happens to all of us regardless of our expertise or experience.
The first thing to do of course is to repeat the Gram stain, in the hope that it gives a clearer picture. However if you get a similar picture on repeat testing, how do you report it?
In superficial wound swabs it is of course of minor importance, which makes you wonder why we ever bother doing Gram stains on such samples (we are very selective now in this respect at my laboratory), but occasionally you will come across such a stain in a sterile site sample, such as a CSF or a joint aspirate.
My experience with such results is that when the Gram stain finding is uncertain like this, then it is rarely of help to the clinician, and does little to improve the patient’s outcome. In fact, on occasion, when an uncertain finding of bacteria are reported on Gram stains from joint aspirates, it can occasionally lead to an unnecessary procedure/operation….. I have seen this happen from time to time.
Depending on the level of uncertainty, one could telephone the clinician to relay this uncertainty. “I have had a look at the Gram stain and there might be a few things there which look like Gram positive cocci, but I cannot be certain….” The issue with this however is that uncertainty often becomes certainty by “Chinese Whispers”.
If it is unclear whether bacteria are present on the Gram stain, then depending on the clinical scenario, I think there is a very good argument for not reporting anything at all, as opposed to reporting something which is potentially misleading.
With the advent of automated bacteriology systems, it won’t be long before the majority of us are looking at digital images of agar plates as opposed to holding the plate in our hand.
But what about Gram stains, ZN stains etc?
A few weeks ago, whilst wandering around the exhibition hall at the ECCMID conference in Copenhagen, I stumbled across this piece of apparatus, called Metafer. This system can automatically feed stains onto the microscope platform, then create digital images at different magnifications and from different areas of the slide. It will even automatically add oil when required…
Not only that, but the company is also developing interpretative software for preliminary analysis of the stain. This seems to be most advanced at present for AFBs on ZN stains and the software will collate anything suspicious looking for an AFB on the slide and then display them as thumbnails on the screen for further checking/interpretation.
Such a system can now be interfaced with the advanced automation systems so that the digital images of the Gram stains can be viewed alongside the plate images.
The objectivity and standardisation of such a system is very appealing, along with the fact that the images can be stored indefinitely. Digital scanning and imaging of slides also has uses within the laboratory beyond microbiology.
I have no personal experience with using the Metafer platform or with the company that has developed it. There are probably other similar systems out there as well. However I imagine such systems will become commonplace over the next decade or so, and traditional “analogue” microscopes will soon die out…
Those who work in diagnostic bacteriology labs will hear his name mentioned every day, probably several times, but how many of us know about where the name “Gram” came from?
Hans Christian Gram was born in 1853 in Denmark. He initially studied Botany, then in 1883 he graduated from medical school and settled in Berlin. In 1884, he noticed that certain stains were preferentially taken up by bacteria from post mortem lung tissue samples. He used crystal violet as the initial stain and fixed the stain with potassium tri-iodide (Lugol’s solution). After this he used ethanol to wash the stain away. He did this with both Streptococcus pneumoniae and Klebsiella pneumoniae bacteria, observing that Streptococcus pneumoniae retained the stain after washing with alcohol (Gram positive) whereas Klebsiella pneumoniae did not. (Gram negative)
Hans Christian Gram only invented the first three parts of the Gram stain. The fourth part, counterstaining with safranin (or similar), did not happen for another few years, and was probably introduced by a German pathologist, Carl Weigert.
Hans Christian Gram never received the Nobel prize for his discovery, but received numerous other awards. He went on to become Professor of Medicine at the University of Copenhagen. His other main discovery was the link between macrocytic red cells and pernicious anaemia.
He died in 1938.
For a really nice article on Hans Christian Gram’s life and also the mechanism of action of the Gram stain along with it’s limitations, click here.
It is actually quite extraordinary that a method invented 130 years ago is still a mainstay of diagnostic microbiology today. Personally I think it’s really important that we know a little about the history behind the microbiology methods we currently use. It just adds that extra little bit of meaning to them….