When I receive a sputum sample from a patient, my first step that I would take would be to prepare the specimen to be viewed under a microscope. Knowing that it would be Bacillus, Escherichia or Mycoplama, I would start ruling them out one by one.
I would start with Bacillus. They are rod shaped bacterium which form endospores. Their movement is propelled by flagella. Bacilli are gram positive, but because if forms endospores, I would do the Endospore staining. Endospores are resistant to stain and relatively uncommon in bacterial cells. You start with a heat fixed smear. Fixing a slide kills the microorganism. A thin film of the microorganism is put on the slid and allowed to dry. Once dried, it is then fixed by either being passed through the flame of a Bunson burner several times or covered with methyl alcohol for 1 minute. Malachite green is applied and the smear is heated to steaming for 5 minutes, helping the stain penetrate the endospore wall. It is then washed with water for 30 seconds. This removes the stain from the cell except for the endospore wall. Safranin is applied as a counterstain to stain portions of the cell other than the endospore. In the smear, the endospores appear green with the rest of the cell red or pink. If there no endospores in the smear, it would eliminate bacillus as a contributing factor.
Escherichia, commonly called E-coli, are also rod shaped microorganisms, and are gram negative. A gram stain is a differential stain developed Danish bacteriologist Hans Christian Gram. You start with a heat fixed smear using the procedure stated above. After they are fixed, it is then covered with crystal violet (or basic purple dye). After a short time, the dye is washed off and the smear is covered with iodine, which is a mordant. A mordant is a chemical additive to intensify the stain. The iodine is then washed off. At this point, both