Microbiology USMLE and General Review

A staining method developed by Hans Christian Gram that allows for bacteria to be distinguished based on their cell wall, specifically the thickness of the peptidoglycan layer. Also called Gram Method.

Bacteria that retain crystal violet stain due to their thick peptidoglycan layer. Purple to blue color due to the crystal violet

Bacteria that can not retain crystal violet due to their thin peptidoglycan layer and therefore stain pink with the counterstain safranin

• There are four basic steps of the Gram stain:
• 1. Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixing kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure.
• 2. The addition of a mordant, which binds to crystal violet and traps it in the cell (Gram's iodine)
• 3. Rapid decolorization with alcohol or acetone, and
• 4. Counterstaining with safranin. Carbolfuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.

• It is the Lipopolysaccharides (LPS) that are found on only gram-negative bacteria that:
• - Activate macrophages
• - Release cytokines
• - cause fever, tissue damage
• - lead to Shock and DIC

• **The toxic part of LPS that leads to cell death is Lipid A
• *Called endotoxin because it is under the capsule of the bacteria
• 

• Can be secreted by gram-positive and gram-negative organisms. They are secreted proteins. They are A-B toxins, the B portions binds toxin to receptor allowing it to be taken up by the cell, and the A portion has the activity and attacks the cell.  The following are A-B toxins:
• - Enterotoxins
• - Neurotoxins
• - Cytotoxins

• There are also Cytolysins which lyse cells by damaging the membrane
• And there are Toxoids which are a chemically modified version