You receive a sputum sample from a patient who recently tested positive for exposure to tuberculosis with a Mantoux (TB) skin test. You perform a Ziehl-Neelsen acid-fast stain on the patient's sputum, but you are perplexed when your slide shows a mixture of unstained and weakly acid-fast staining bacteria. Assuming that the patient has an active tuberculosis infection, which of the following procedural errors could account for the unexpected staining result?
O The specimen may not have been heated sufficiently during primary stain application.
O The specimen was rinsed for too long with deionized water.
O Acid alcohol was omitted from the staining procedure.
O Methylene blue was kept on the sample for too long.
O The specimens were not sufficiently heat-fixed.