DIFFERENTIAL STAINING

This week we will use differential staining techniques. 2 types of stains -at least- are used.
The stain first used is called the primary stain. The second stain is called counterstain.
Because cells or parts of cells color differentially, this method is used for identification or
structure examination of bacteria in addition to general morphological examination. So we
will be able to compare cells and see different cellular structures this week.

EXERCISE 8. GRAM STAINING

Gram staining is an essential technique in diagnostic procedures that require bacterial
identification. Microbiologists of this high-tech age are still indebted to Danish physician
Christian Gram, who invented the gram-staining method in 1884. Gram staining enables
bacteria to be grouped either as Gram (+) or as Gram (-) according to their cell wall structure
and composition.

Performing Gram staining when a specimen is first obtained often gives valuable information
that cannot be obtained from just culturing the sample or by clinical checks in diagnosis.
Properly Gram-stained preparations can quickly give you considerable information that can be
applied immediately to patient care and about further tests to be done. It also helps
interpretation of results of other tests.The Gram stain is particularly useful in the presumptive
diagnosis of bacterial meningitis, bacterial pneumonia, bacteriuria, gonorrhea, and pyogenic
infections of the brain, lung, abdomen, pelvis and wounds.

There are 4 chemicals used: First crystal violet (CV) is applied to the smear. Then Grams
iodine (I) is applied to increase affinity of dye to specimen. After that Ethanol (EtOH) is used
as decolorizing agent and finally safranin is used as counterstain. Lets write their functions
in detail.
1. Crystal Violet is the primary stain in Gram staining..
It is applied at the beginning of staining procedure in a way
similar to simple staining with CV. This causes both Gram
(+) and Gram (-) cells appear in purple. After applications of
other chemicals to smear, purple color remains in Gram (+)
cells. But in Gram (-) cells it is washed away. Cells get the
color of safranin applied later.
2. Grams iodine functions as mordant. Mordant can be
defined generally as a substance, usually a metallic
compound, binds to dye and makes an insoluble colored
compound. By this way it increases intensity of dye in cell,
making it not easily-removable . It forms crystal violet-iodine
Complex (CV-I) in cell wall. This prevents purple color to be
washed away by Ethanol.
3. Ethanol (decolorizing agent) application causes
Gram(-) cells to lose purple color by washing away of CV-I
complex. But Gram (+) cells retain their color. Ethanol-
acetone mixture can also be used as decolorizing agent.
4. Safranin (counterstain) stains colorless Gram (-)
cells to pink to make them visible. Counterstains generally
colors the the unstained part of the specimen i.e. not stained
by primary stain for ease of differentiation. Since the gram-positive bacteria are already
stained purple, they are not affected by the counterstain.

If the steps of the procedure are performed properly Gram (+) cells will be in purple color and
Gram (-) cells will be in pink.

This staining difference is due to differences in structure and composition of cell walls of
Gram (+) and Gram (-) cells. You learned cell wall properties of bacteria in your
Microbiology course. Gram staining is based on the ability of the bacterial cell wall to retain
crystal violet dye during decolorizing treatment with a decolorizing agent. The cell walls of
gram-positive bacteria have a higher peptidoglycan and lower lipid content than Gram-
negative bacteria. Decolorizer dissolves the lipid layer from the gram-negative cells. The
removal of the lipid layer allows the leaching of the crystal violet stain from the cells. In
contrast, the ethanol dehydrates the thicker gram-positive cell walls, closing the pores as the
cell wall shrinks. As a result, the violet-iodine complex is retained and the cells remain
purple. http://www.arrowscientific.com.au/new/mainarticle2.phtml?articleid=926c53f0ae

Gram staining can be applied to cells other than bacterial cells. Most living cells and animal
cells are Gram (-). Some bacteria, yeasts and a few molds are Gram (+). There are also some
bacterial species that Gram staining is not applicable: Mycoplasma species have no cell wall
and also have a different type of cell membrane that is not stained by the procedure. So this
group of bacteria is called as Gram-nonreactives. Also Mycobacteria have different cell wall
composition that only acid-fast staining is applicable.

If steps of Gram staining procedure are followed properly the results will be satisfactory. At
the end of the staining, Gram (-) cell are always seen as Gram (-) but Gram (+) cells can be
seen as Gram (-) due to some experimental errors. This unexpected result is called Gram-
variable reaction. Reasons for Gram-variable reaction are listed below.

1. Age of the culture stained with Gram staining should be smaller than 24 h. It should be
not damaged by chemicals or not treated by antibiotics.
2. Too thick smears may cause improper decolorization. So smear thickness should be
proper.
3. Too much heat fixation causes cells to rupture. In this way Gram (+) cells may lose
CV-I and can take Safranin color.
4. Fresh chemicals should be used in proper concentrations. Applications of low amount
or concentrations of Crystal violet, iodine solution or safranin may cause Gram (+)
cells stained as Gram (-).
5. Nature, concentration and amount of decolorizing agent is very important. If you
apply too much, you cause Gram (+) cells to lose CV-I complex and appear in pink..
Also prolonged washing between each step may cause over-decolorization.

So if you follow the steps careful enough youll have the expected result on your image.
There are other ways to identify the bacteria you are searching for. Growth characteristics,
biochemical activities, immunological-serological tests or genetic information are some of
them. Next week you will see different growth characteristics of the bacteria. The following
two weeks you will see how different biochemical activities are used to group bacteria.
 Procedure

Materials and instruments

Bacterial cultures of 24h Grams iodine solution

Microscope slides Safranin
Marking pencil 95% ethanol
Inoculating loop Tisssue paper
Staining rack Immersion oil
Cystal violet Gloves
Distilled water bottle

You will use your information na experiences from last week.Before staining, you will
prepare smears of cultures given to you. Also during staining procedure you will perform
staining, washing and blotting techniques like you did in simple staining.
To the smears you prepared before:
1. Apply few drops of crystal violet to cover the smear, allow it stand for 1 min.
2. Pour off excess dye. Rinse with dH2O as you did in simple staining.
3. Wash the tilted slide with Grams Iodine. Then cover the smear with Grams Iodine
and leave it 1 min.
4. Rinse with dH2O.
5. Wash with 95% Ethanol (decolorizing agent) via dropper as you do with dH2O. You
will do it till fluid flows off the smear is colorless,not more. Do not apply it directly
or harshly. Let it flow from the upper part of the slide.
6. Rinse immediately with dH2O.
7. Apply safranin to smear and wait 1 min.
8. Wash with dH2O.
9. Perform blot dry as you did in simple staining and then air-dry if necessary.
Your slide is ready to observe. Please do your observations with 100x objective and note
colors, shapes,sizes and staining properties of cells.