Certification Board for Sterile Processing and Distribution (CBSPD) Sterile Processing Technician Practice Exam

Which property helps differentiate between Gram-Positive and Gram-Negative bacteria?

1. Color of the stain

2. Shape of the bacteria

3. Size of the microorganisms

4. Presence of spores

The correct answer is: Color of the stain

The ability to differentiate between Gram-Positive and Gram-Negative bacteria primarily hinges on the color of the stain obtained during the Gram staining procedure. In this staining process, Gram-Positive bacteria retain the crystal violet stain due to their thick peptidoglycan layer, resulting in a purple color. Conversely, Gram-Negative bacteria have a thinner peptidoglycan layer and will not retain the crystal violet stain after washing with alcohol or acetone. Instead, they take up the counterstain, usually safranin, appearing pink. This fundamental difference in staining characteristics is key to identifying and categorizing bacterial species based on their cell wall structure. The other properties listed—shape of the bacteria, size of the microorganisms, and presence of spores—are not reliable indicators for distinguishing between Gram-Positive and Gram-Negative bacteria. While bacteria can be categorized by shape (cocci, bacilli, etc.) and size can vary, these characteristics are not exclusive and are common among many bacterial species. The presence of spores is specific to certain genera, such as Bacillus and Clostridium, and does not play a role in the Gram classification. Therefore, the color of the stain in the Gram staining procedure is the definitive property used