Gram Staining

Which step can be omitted without affecting the gram reaction?

safranin.

Bacteria stain differently because of chemical and physical differences in their cell walls:

-Crystal violet is picked up by the cell. -Iodine reacts with the dye in the cytoplasm to form CV-1. -In gram-negative cells, the decolorizing agent dissolves the outer lipopolysaccharide layer, and the CV-1 washes out through the thin peptidoglycan layer.

Explain the effects of the alcohol decolorizer on Gram+ and Gram- cells:

-Gram+: stays stained purple because there are lots of thick layers of peptidoglycan in the cell wall that traps the stain, making it hard for it to escape to the outside. -Gram-: has a small layer of peptidoglycan in the cell wall that allows for movement of the stain to the outside of the cell.

Steps of gram staining technique:

1. Apply primary stain (crystal violet). All bacteria are stained purple by this basic dye. 2. Apply mordant (Gram's iodine). The iodine combines with the crystal violet in the cell to form a crystal violet-iodine complex. (CV-1). 3. Apply decolorizing agent (ethyl alcohol or ethyl alcohol acetone).Decolorizers dissolve membranes. The primary stain is washed out (decolorized) of some bacteria, while others are unaffected. 4. Apply secondary stain or counterstain (safranin). This basic dye stains the decolorized bacteria red. Basic, charged stain= enters almost any cell.

List the steps of staining and the color of each in gram pos. and gram neg.:

1. Crystal violet: GP cells= purple, GN cells= purple. 2. Iodine: GP= purple, GN= purple. 3. Alcohol: GP= purple, GN= colorless. 4. Safranin: GP= purple, GN= pink.

What were the three species of bacteria observed? What was their morphology & arrangement? What was the final color, and what does that mean on whether it was gram (+) or gram (-)?

1. Staphylococcus epidermis: sphere, clustered; purple= gram positive. 2. Bacillus subtilis: chains of rods; purple= gram positive. 3. Escherichia coli: rods, no specific arrangement; pink= gram negative.

If you gram stained human cells, what would happen?

Human cells don't have cell walls, so the stain would not stay inside of the cell.

Suppose you are viewing a Gram stained field of red rods and purple cocci through the microscope. Why can this happen?

It is probably a mixed culture.

What is a gram stain?

It's a DIFFERENTIAL stain that allows for classification of a bacteria as either gram-positive or gram-negative. Discovered by Hans Christian Gram.

Can iodine be added before the primary stain in a Gram stain?

No. Iodine causes crystal violet to bond to the peptidoglycan in the cell walls, preventing the crystal violet from being washed off.

Why will old gram-positive cells stain gram-negative?

The cell can only retain primary stain for so long, so the cell wall degenerates and the stain can move to the outside of the cell wall, making it appear gram-negative.

Suppose you Gram stained a sample from a pure culture of bacteria and observed a field of red and purple cocci. Adjacent cells were not always the same color. WhY?

The culture may be old

The most important determining factor in this procedure is:

The fact that bacteria differ in their RATE of decolorization. -Gram (+) decolorize slowly and maintain the primary stain. -Gram (-) decolorize easily.

Since you can't identify bacteria from a Gram stain, why might a physician perform a Gram stain on a sample before prescribing an antibiotic?

They could use the gram stain to identify the cell wall type, which relates the the sensitivity to an antibiotic.