VCLab 4: Gram stain and capsule stain

GRAM STAIN

This is the most common type of stain. It is the first step in identifying an organism. It does not identify the organism, but it is the first step. It is one of the differential types of stain. The reaction of stains is different because of differences in the chemistry of the cell wall.

The cell wall of a gram negative organism has many lipids in the cell wall:

LPS – lipopolysaccharide

LP – lipoproteins

PL – phospholipids

GRAM POSITIVE GRAM NEGATIVE

Purple pink

Thick peptidoglycan thin peptidoglycan

Very little lipid Lots of lipid (in the outer membrane)

The Gram stain is used to distinguish between Gram positive bacteria (will look violet because they are not decolorized) and Gram negative bacteria (will look pink from the safranin because they were decolorized). Since all bacteria are either Gram positive or Gram negative, this stain is the first thing used to determine what type of bacteria is present in the specimen. This helps us figure out what organism we are dealing with. The results are recorded as Gram positive or Gram negative.

NOTE: All differential types of stains have the following steps, but with variations in order and chemicals.

Gram Stain Procedure

First, prepare the sample on a slide, air dry and heat fix.

1.  PRIMARY STAIN (the stain used first)

  1. Crystal Violet
  2. Leave on for 1 minute
  3. Rinse gently with distilled water

2.  MORDANT (a mordant is something that causes the primary stain to form a complex with it to chemically react with the cell).

  1. Gram’s Iodine (not regular iodine)
  2. Leave on for 1 minute
  3. Rinse gently with distilled water.
  4. The iodine forms a complex with crystal violet which reacts with the peptidoglycan in the cell wall.
  5. Without iodine, all of the stain would wash away.

3.  DECOLORIZER

  1. 95% Ethyl alcohol (ETOH)
  2. As an alternative, you can use ETOH mixed with acetone.
  3. This is the most critical step because the alcohol must be left on for just the right amount of time.
  4. Apply the alcohol by dripping it onto the slide, while rocking and rolling the slide as you hold it.
  5. Keep adding the alcohol and WATCH THE WASH for when the purple wash becomes clear, then STOP.
  6. Rinse gently with distilled water.
  7. Decolorizing time should be about 5 seconds for a thin smear and 10 seconds for a thick smear.

4.  COUNTERSTAIN

  1. Safranin
  2. Leave on for 1 minute
  3. Rinse gently with distilled water
  4. Blot dry.

What are some reasons why you might get a Gram positive culture show up with both purple and pink cells that are all the same size?

1.  The culture is too old (more than 24 hours). Some of the cells have died, and the peptidoglycan breaks apart, so it appears Gram negative.

2.  The decolorizer was left on for too long.

3.  The sample smear was too thick and the stain did not get through to all the cells.

Suppose you look at a Gram stain of staphylococcus and you see staphylo, diplo, singles, and tetrads on your slide. They are all purple and they are all the same size. Is the culture contaminated? No, it is probably still pure because the cells are all the same size. The clusters just broke apart during the preparation, or you are seeing a new cell in the process of dividing into a cluster.

If you see both purple and pink cells of different shapes, that is not a pure culture. If the culture is supposed to be pure, it became contaminated.

Get four clean slides and prepare one slide of the following cultures that are less than 24 hours old:

1.  E. coli

2.  B. Megaterium

3.  Staphylococcus aureus

Then used your fourth slide to make one slide of an old culture to compare them.

Problems:

When you do a Gram stain:

What would happen if you forget to use Gram’s iodine? (cells would be pink)

Over-decolorize? (cells would be pink)

Use no alcohol? (cells would be purple)

Use an old culture? (Gram + cells would be purple and pink because some PG has broken down)

GRAM STAIN SUMMARY

1.  PRIMARY STAIN: Crystal violet. This is the first stain used.

2.  MORDANT: Iodine. The mordant is what allows the primary stain to react chemically with the cell. It forms a complex with crystal violet and peptidoglycan in the cell wall of bacteria. It keeps the crystal violet from being washed out by the alcohol.

3.  DECOLORIZER: Alcohol or acetone. This removes the primary stain from some of the cells (decolorizes some of the cells).

4.  COUNTERSTAIN: Safranin. This is a red color that stains the cells that became decolorized.

CAPSULE STAIN: Some bacteria have a capsule which resists phagocytosis (being eaten by our white blood cells). An example is Streptococcus pneumoniae. This stain colors the background but the capsule remains clear. This will reveal the presence of a capsule, assisting in the diagnosis.

COMPARISONS OF STAINS

G+ (PURPLE) / G – (PINK) / ACID FAST / CAPSULE STAIN
PRIMARY STAIN / Crystal violet / Crystal violet / Carbol Fuschia / Malachite green
MORDANT / Iodine / Iodine / Heat / Heat
DECOLORIZER / ETOH or acetone:
Cell is purple / ETOH or acetone:
Cell is clear / Acid alcohol / Water
COUNTER-STAIN / Safranin:
Cell is purple / Safranin:
Cell is pink / Methylene Blue / Safranin:
Spores are green
Cell is pink