Question

A) You perform a gram stain on an unknown and two controls. The gram positive control organism appears pink when you observe it microscopically. Your unknown organism also appears pink. Is your unknown organism gram positive, gram negative, or can't tell?

B) what is the reason you could have the above result?

Answer 1

A) As your Gram-positive control appeared pink, you can not conclude on your test sample.

B) Reason for Gram-positive control appeared pink :

In Gram staining initially, bacteria are stained by crystal violet-iodine complex (which gives intense violet color). On decolorization using ethanol or acetone, this crystal violet-iodine complex can be retained or lost depending on the cell wall composition of bacteria. Gram-positive bacteria have a higher percentage of peptidoglycan and hence keeps the crystal violet-iodine complex and remains unaffected on applying counterstain (pink colored safranin). Gram-negative bacteria have low peptidoglycan percentage which gets removed on applying decolorizer (ethanol or acetone), and hence crystal violet-iodine complex is washed off which makes room for safranin to stain bacteria by pink color.

You may get false negative results in your Gram-positive sample because of the following reasons:

1. Excessive heat for fixation damages cell membrane and makes bacteria susceptible to decolorization.

2. Low concentration of crystal or iodine used for staining.

3. Prolonged decolorization can give you this type of result especially with acetone.

4. Excessive application of counterstain such as safranin can leach crystal violet-iodine complex, and you may get false negative results.

5. Certain conditions such as antibiotic treatment, cultures more than 48 hours old can damage cell membrane, and you may get false negative results.