Simple Staining

• A smear is used to obtain a thin film of bacterial cells.

Fixing

• Fixing denatures bacterial enzymes, preventing autolysis and digestion of cell parts.

• Fixing can be achieved through heat or chemical methods.

• Chemical fixing involves using methanol for 1 minute.

• A chromophore is the ion that is colored.

• Basic stains have a positive chromophore.

• Acidic stains have a negative chromophore.

• Most bacteria are stained by basic stains, which permeate the cell wall (which is negative).

• Simple stains use only 1 stain and are direct stains.

• A negative stain (unstained bacteria) is an example of a simple stain on bacteria and is also an example of a simple stain for background.

• Simple stains can test for morphology, cell arrangement, and size.

• Methylene blue is a dye name.

• 95% methanol is used after fixing.

• A negative stain does not stain the bacteria, only the background, because it is repelled by charge.

• An example of a colloidal stain used in negative staining is India ink.

• Nigrosin, which is a colloidal suspension that doesn't enter the cell, with a negative charge and pH of 7, is used in negative staining.

• Fixing or strong chemicals are not used in negative staining to avoid distorting the bacteria.

• Negative staining is used when other staining techniques do not clearly show cell morphology/size and is especially useful for coccobacilli (short and oval bacilli).

• In negative staining, rod-shaped bacteria appear as short rods with small cocci.

• Gram stain classifies bacteria as gram-negative or gram-positive.

• Gram stain was created when Hanns saw colored cells losing color when washed off.

Steps of a Gram Stain

• Primary stain uses crystal violet basic dye.

• Mordant uses gram iodine.

• Decolorizing agent uses ethanol/acetone.

• Secondary stain/counterstain uses safranin.

• Crystal violet is picked up by the cell.

• Mordant combines with crystal violet to form a crystal violet iodine complex.

• Decolorizing agents remove the primary stain from some bacteria.

• Secondary stains use a basic dye to stain decolorized bacteria red.

• Gram-negative bacteria decolorize easily.

• Gram-positive bacteria decolorize slowly and retain the primary stain.

• The peptidoglycan component of the bacteria cell wall causes different staining results; positive has multiple layers, and negative has a thin layer with an outer layer of lipoproteins, phospholipids, and lipopolysaccharides

• Crystal violet is picked up by peptidoglycan to form a crystal violet competition complex (cv-I) that is insoluble in water.

• Crystal violet iodine is not washed out by alcohol.

• Stain washes out of gram-negative bacteria because the decolorizing agent dissolves the outer lipopolysaccharide layer, and the crystal violet iodine complex washes out of this peptidoglycan layer.