Download VCLab 4 Gram stain and capsule stain

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
Purple
Thick peptidoglycan
Very little lipid
GRAM NEGATIVE
pink
thin peptidoglycan
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)
a. Crystal Violet
b. Leave on for 1 minute
c. 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).
a. Gram’s Iodine (not regular iodine)
b. Leave on for 1 minute
c. Rinse gently with distilled water.
d. The iodine forms a complex with crystal violet which reacts with the
peptidoglycan in the cell wall.
e. Without iodine, all of the stain would wash away.
3. DECOLORIZER
a. 95% Ethyl alcohol (ETOH)
b. As an alternative, you can use ETOH mixed with acetone.
c. This is the most critical step because the alcohol must be left on for just
the right amount of time.
d. Apply the alcohol by dripping it onto the slide, while rocking and rolling
the slide as you hold it.
e. Keep adding the alcohol and WATCH THE WASH for when the purple
wash becomes clear, then STOP.
f. Rinse gently with distilled water.
g. Decolorizing time should be about 5 seconds for a thin smear and 10
seconds for a thick smear.
4. COUNTERSTAIN
a. Safranin
b. Leave on for 1 minute
c. Rinse gently with distilled water
d. 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 – (PINK)
ACID FAST
Crystal violet
Crystal violet
Iodine
ETOH or
acetone:
Cell is purple
Safranin:
Cell is purple
Iodine
ETOH or
acetone:
Cell is clear
Safranin:
Cell is pink
Carbol
Fuschia
Heat
Acid alcohol
G+ (PURPLE)
PRIMARY
STAIN
MORDANT
DECOLORIZER
COUNTERSTAIN
Methylene
Blue
CAPSULE
STAIN
Malachite green
Heat
Water
Safranin:
Spores are
green
Cell is pink