A new project has come up in the core center - a researcher wants to study bacterial biofilms and will need you to document the process of biofilm formation. Bacterial biofilms are colonies of different species of bacteria that interact and allow the bacteria to grow in a new environment. Since this will be a mixed culture, you will need to use a staining protocol that allows for differentiation between bacteria based on cell wall properties. The Gram staining procedure uses a series of stains and alcohol decolorization to differentially color different species of bacteria that may be present in the growing biofilm. This differential colorization will allow for determination of bacterial morphology and give some insight into the bacterial cell wall composition. Put the following descriptions in order for the staining reactions in the cells of a bacterial smear during the Gram staining procedure. View Available Hint(s) Hint 1. Gram stain components Hint 2. Overview of the Gram stain Reset Help Cell wall of Gram-negative cells is broken down; Gram-positive cells remain intact. | Clear cells are stained a red or pink whereas the color of other cells remains unchanged. Dve molecules are rendered insoluble in presence of mordant, dye is rinsed away. All cells are stained purple. Unstained cells (start of process) Stained cells (end of process)

Unstained cells (start of process)
All cells are stained purple.
Dye molecules are rendered insoluble in presence of mordant; dye is rinsed away.
Cell wall of Gram-negative cells is broken down; Gram-positive cells remain intact.
Clear cells are stained a red or pink whereas the color of other cells remains unchanged.
Stained cells (end of process)

Explanation:

Gram Staining:

Gram staining is an identification technique for the classification and characterization of bacteria on the basis of cell wall composition. Gram staining uses a primary dye i.e. crystal violet that stains Gram positive bacteria due to high peptidoglycan content in their cell walls. A mordant i.e. iodine fixes the dye to the cell wall by forming a crystal violet-iodine complex (CV-I). This mordant prevents the decolorization of Gram positve cells upon application of ethanol based decolorizer. After the cells have been decolorized, a secondary dye, i.e. safranin stains Gram negative bacteria.

Gram positive bacteria are stained purple due to crystal violet and Gram negative bacteria are stained pink due to safranin.