Put the following substances in the order they are used in a Gram stain:
Counterstain
Decolorizing agent
Mordant
Primary stain

Verified step by step guidance

1. Understand the purpose of each substance in the Gram stain procedure: the primary stain colors all cells, the mordant fixes the stain, the decolorizing agent differentiates Gram-positive from Gram-negative cells, and the counterstain colors cells that lost the primary stain.
2. Identify the first step: the primary stain is applied first to stain all bacterial cells, typically using crystal violet.
3. Next, apply the mordant, which is usually iodine; it forms a complex with the primary stain to help it adhere better to the cell wall.
4. Then, use the decolorizing agent, often alcohol or acetone-alcohol, to wash out the stain from Gram-negative cells but not from Gram-positive cells.
5. Finally, apply the counterstain, such as safranin, which stains the decolorized Gram-negative cells to make them visible under the microscope.

Key Concepts

Gram Stain Procedure

The Gram stain is a differential staining technique used to classify bacteria into Gram-positive or Gram-negative based on their cell wall properties. It involves sequential application of specific reagents that highlight differences in bacterial cell wall structure.

Role of Mordant in Gram Staining

A mordant, typically iodine, is applied after the primary stain to form a complex with the dye, enhancing its retention in the bacterial cell wall. This step is crucial for differentiating bacteria by stabilizing the crystal violet stain in Gram-positive cells.

Decolorizing Agent and Counterstain Functions

The decolorizing agent, usually alcohol or acetone, selectively removes the primary stain from Gram-negative bacteria, while Gram-positive cells retain it. The counterstain, such as safranin, is then applied to stain the decolorized Gram-negative bacteria, providing contrast.