A 47 year old female patient presents herself to you with symptoms of pneumonia.

Question

A 47 year old female patient presents herself to you with symptoms of pneumonia. All indications are that the patient is suffering from an infection of Streptococcus pneumoniae, a Gram-positive bacterium. Since the patient is not allergic to penicillin, you prescribe a dose of amoxicillin, a semi-synthetic penicillin. This drug works by blocking the peptide cross-links, between the muramic acid subunits, of peptidoglycan chains. This leads to a destabilization of the bacterial cell, leading to its rupture.

However, after the patient finished her course of amoxicillin, no improvement in symptoms were reported. Perplexed, you decide to culture and examine the bacteria infecting her lungs. The test you order is a Gram stain test, which comes back as Gram-negative. You feel like a failure as a health care provider due to your misdiagnosis of this bacterium.

The same day, you put the patient on an aminoglycoside class antibiotic. This antibiotic is Gram-negative specific and works by shutting down ribosomes. You are dismayed and confused when this treatment also fails.

To get a full read on the disease, you have a barcoding DNA test done on the causative bacterium. The test comes back and it clearly shows that the bacterium is within the group of Gram-positive bacteria. With the results of this test, you come up with the theory that the patient is infected with a strain of Drug Resistant Streptococcus pneumoniae (DRSP), resistant to amoxicillin. You decide to treat the DRSP by administering intravenous vancomycin, which is indicated for the treatment of serious, life-threatening infections by Gram-positive bacteria. This treatment also fails.

In the end, you figure out that you were wrong about everything, and that there was a simple solution to the treatment of the patient.

Now knowing that the patient was infected with Mycoplasma pneumoniae, what is the likely explanation for the false Gram-negative test?

Before you answer this question you may want to know how Gram staining works.  Briefly, the Gram stain is a differential stain meaning that you are using two dyes that stain different structures. Based on the structural differences of the bacteria, the dyes will interact differently with them, producing different results (colors). The Gram stain (Crystal Violet together with Gram's iodine) stains thick layers of peptidoglycan purple and thin layers of peptidoglycan very, very lightly purple. The second stain (or counterstain) is safranin. Safranin is a dye which interacts with and binds to lipid bilayers. Yes, safranin also stains the cytoplasmic membrane of Gram-positive bacteria, but the deep purple color of the Gram stain makes it impossible to see.

The Gram stain was able to penetrate the cell making it turn pink

The counterstain stained the cytoplasmic membrane, making it look like a Gram-negative bacterium

The counterstain stained the mycolic acid in the cellular envelope, thus making it look like a Gram-negative bacterium

The Gram stain lightly stained the present peptidoglycan (making it look pink rather than red).

The Gram stain was able to penetrate the cell making it turn pink

The counterstain stained the cytoplasmic membrane, making it look like a Gram-negative bacterium

The counterstain stained the mycolic acid in the cellular envelope, thus making it look like a Gram-negative bacterium

The Gram stain lightly stained the present peptidoglycan (making it look pink rather than red).

Explanation / Answer

One of the reason for the false Gram-negative test is due to the following reasons:

1. Excessive heat during fixation which alters the cell morphology and make the cells get easily decolourized.

2. very low concentration of crystal violet make the cells get easily decolourized. Upto 2% concentration of crystal violet is recommended.

3. excessive counterstaining (basic dye) which replace the crystal violet-iodine complex in gram positive cells to overstain with counterstain.

4. Sometimes insufficient mordant available which is necessary for the crystal violet-iodine complex formation. The lower the concentration, the easierto decolourize.

5. If the cells are prepared in hyper or hypotonic solution, the cell morphology can be disturbed.

6. The crystalviolet stain can be easily washed out while performing the washin step.Do not use more than 5 seconds at any stage of the procedure

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