Practical Microbiology: Gram Stains

By Leonis | STEM stuff | 15 Sep 2019

Gram stains are some of the most basic microbiology tools in medicine. The idea is to give clinicians clues of the nature of the infection. They can give preliminary treatments on the patients as needed. Then, the lab can perform more detailed sensitivity testing.

         The color which bacteria stain depends on the level of peptidoglycan in their cell wall. Even though the stain is looking for bacteria, you can notice other organisms like yeast as well.

The gist of the method involves four steps:

  1. Primary stain of crystal violet
  2. Fixing stain of iodine
  3. Alcohol decolorizer
  4. Counter stain of safranin

Then, observe the slides under the light microscope at 100x power in oil.

         Where I work, acetone is the decolorizer of choice. Depending on the brand of stain you use, the time of each stain may vary on the inserts. But, the basic principles stay the same. Issues usually occur during the decolorizing step. Overdoing it results in everything looking like gram negative organisms. Another one would be confusing debris (stain crystals) as organisms.




         In addition to the stain, the organisms' shape and the way they aggregate also gives clue to what they could be. Here are some examples:


Gram-positive cocci in clusters



gpc clus.jpg
A Shutterstock image in case you can't tell.


Possible organisms: Staphylococcus species such as S. aureus


Gram-positive cocci in chains



gpc chns.jpg


Possible organisms: Streptococcus (including beta-hemolytic Strep.) or Enterococcus species


Gram-negative bacillus



Source This particular one is E. coli



Yeast and fungal elements



Also a stock photo, but from 123RF



Possible acid-fast organisms or Nocardia species





         This is also referred to as "gram-positive beaded bacilli".

         Of course, there are other forms and shapes one could see under the microscope. The point is, they give valuable clues to what the infectious agents could be. I have often used what I see under the scope to determine if the gram stains, done by hand or machine, are legitimate. Sometimes, you may not catch rare growth of bacteria as their numbers are few, but that is what the cultures are for. But in general, the gram stain should correlate with whatever growing on the plates.


Gram Stains in Practice


         The lab will read every STAT gram stains ordered. Every non-STAT gram stains sourced from sterile body sites receive the same treatment. Some specimens, such as sputum, needs examination to determine validity of specimen. The lab can reject sub-par specimen on the grounds of not suitable for testing.

         From my experience, gram stains from surface wounds and abscesses tend to be not read. They are often contaminated with various flora. Reporting junk-filled gram stains adds little value to clinicians treating patients. Time is better spent culturing and isolating the infectious agents.

         At large facilities, automatic stainers such as Aerospray® Gram Series 2 Slide Stainer/Cytocentrifuge perform most of the work. The manufacturer boasts how the product can produce up to 30 slides per cycle. In practice, that usually result in a sharp decrease in quality. It turns out that 12 to 16 are the safest batch size you'd want for clear reads.

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