Assessing gram stain error rates

Only a few reagents are required for staining

The gram stain, also known as the Gram stain for Hans Christian Gram, is one of the first techniques budding microbiology students learn in their introductory lab courses. It’s even a good exercise for younger students (with proper supervision, of course), due to its simplicity and the colorful, beautifully stained cells that result from the procedure. The protocol is often taught in tandem with lessons on bacterial structure, since the differential staining helps determine whether an isolate is a gram-positive or gram-negative bacterium.

Because of its simplicity and the rapid time-to-result turnaround, gram staining also plays an important role in clinical microbiology. Learning the cell structure helps eliminate potential disease etiologies: learning an isolate is a gram-negative rod doesn’t tell you what the diagnosis is, but it helps eliminate what it isn’t. This information can also inform clinicians about the best-choice antimicrobial drug, at least until the isolate can be cultured and its drug-resistance profile characterized. While relatively technologically simple, gram staining has played a big role in diagnostics for decades.

But what if one technician’s gram-positive stain is another’s gram-negative? That’s the predicament outlined in a Journal of Clinical Microbiology publication in the June issue but available online now. The interpretation of a gram-stain slide is inherently subjective, and many factors can play a role in a correctly stained sample, including specimen fixation, staining protocol, and slide analysis. The study compared gram-stain techniques and results at four major U.S. clinical microbiology centers.

Gram-positive cocci and gram-negative bacilli

Standardized criteria were used to compare the abilities of technicians in different labs to correctly identify respiratory, fluid, biopsy/tissue, and wound cultures. Each site had hundreds of specimens screened for those that were “discrepant” – if a corresponding culture analysis grew bacteria not matching the designated gram stain, the original stain was reviewed by a senior lab member to determine whether the stain result was an error. The error could be either an incorrect stain, or an inaccurate assessment of bacterial presence.

All clinical labs had around 5% of their examined specimens flagged as discrepant, and the first hurdle was identified when trying to find the original slides: only 87% could be found due to disorganization. In examining the available slides, the researchers found only around a quarter of discrepancies were due to misread stains (positive mischaracterized as negative or vice versa). Most discrepant results were either smear negative/culture positive or smear positive/culture negative – meaning that the most common mistake was in determining whether there was a bacterial infection at all.

Differences in gram staining interpretations have been found in previous studies, but most studies have concentrated on the person-to-person variability within a single lab. This multi-center study was designed to emphasize the need for stronger standards across all microbiology labs, since these errors can majorly impact patient outcome. A small specimen size, low bacterial cell to tissue, or inadequate fixation can all lead to the false negatives, which could delay treatment. Fixation type (methanol versus heating) didn’t play a major impact, as the one site that used methanol fixation had comparable error rates to the three sites using heat fixation. An automated gram stainer may standardize the protocol, but it didn’t prevent the one site with this machine from interpretation-based errors.

To correct these mistakes, the research team recommends education: both education of doctors, to order the correct tests (unordered anaerobic growth tests led to stain positive/culture negative results) and education of technicians, to better prepare and analyze specimen slides. Taking a page from anatomic pathologists, the researchers also recommend tracking error by type and individual, which can reveal addressable patterns of errors to address. Standardization of this cornerstone diagnostic procedure will help minimize preventable medical errors.

Do you have experience in clinical microbiology? What changes would you propose to eliminate misread gram stains? Does the procedure need to be automated? Leave your thoughts and experiences in the comments!

-- Julie Wolf

Photo credits: Gram stain materials, Gram stain micrograph,

Comments

[رجاء الحسن]

Good

[Carolina Macero]

In my experience as a bacteriologist, the most common errors are: 1) when inflammatory response and abundant leukocytes achieve an effect of pink background where it is difficult to distinguish the Gram negative bacteria, especially if they are scarce, and therefore not will report 2) when from a bacterial suspension problem, where there are no cells to guide us, we made the mistake of overstain or decolorear and 3) setting heat smear from liquid, which usually leads to a part of the sample is lost in the wash while she underwent Gram stain, which makes us report it wrongly. Recommendations: 1) make thin smears if the sample is purulent or revise the microscope the edges of the smear, where we see the separate leukocytes from each other, 2) in a sheet make three circles, and in the center circle to place the problem bacterial suspension, in the circle on the right side to place a Gram positive control strain, and left circle a Gram negative and thus proceed to color and 3) choose fixed with methanol samples from liquid as it prevents the smear off with water by withdrawing crystal violet.

[taoufik djerboua]

-for clinical specimen i prefer to start with methylen-blue staining i think it's easier to read. If its "positive" for bacteria i complete with a Gram staining.
-thick samples should be diluted in 0.9% NaCl water before smearing. Nevertheless fushin sticks a lot thats why methylene blue is preferable to start with for me.
-for cultured samples whether its a Gram or Ziehl staining i often use and advise others to make a "control smear". In Gram staining i mix a colony of a known Gram negative isolate with a colony of a known Gram positive isolate on the same smear. It helps verify the quality of the methode and of the reagents and makes the smears a lot easier to read.

[Dr Sourav Maiti]

What I do is using a mixture of a Gram positive cocci and a Gram negative rod on the same slide marked as control and at the same time both smears (control and test) are flooded with staining procedures as per protocol followed. As per control, the rods must stain pink and the cocci should stain purple. If it's not then staining is wrong.
However in clinical setting with experience we can get clues like pus cell nuclei should stain pink, any spore bearing bacillus should be Gram positive.

[Dr Sourav Maiti]

One must check quality of the batch of stains and the performance of the technician too to have good Gram stain. Smears must be of optimum thickness and proper timing should also be maintained. But most importantly the microscopist must have dedication and interpretative open minds to decode the mysteries....!!!!