STAINING TECHNIQUES FOR BACTERIA

Gram Staining

1. Stain fixed microbial smear with ammonium oxalate crystal violet for 1 minute.
2. Wash thoroughly but gently with tap water. Shake off excess water.
3. Cover smear with Gram’s iodine solution for 1 minute.
4. Wash with tap water and shake off excess water.
5. Decolorize with 95% ethyl alcohol for 15-20 seconds.
6. Wash with water.
7. Counterstain with safranin solution for 30 seconds.
8. Wash with water and blot dry.

Negative Staining

Negative or background staining involves mixing the microorganisms in a small amount of India Ink or
nigrosine and spreading the mixture over a clean slide. These two pigments are not really bacterial
stains because they do not penetrate the microorganisms; instead, they obliterate the background,
leaving the organisms transparent and visible in a darkened field. This technique can be useful for
determining cell morphology and size. Since no heat is applied to the slide, there is less shrinkage of the
cells and consequently, more accurate cell size determination results than with other methods.

The source of organisms in this exercise will be the normal flora that exist between the teeth. Success
of this method depends on the following considerations (1) the slide must be absolutely clean: the
presence of any residual grease or dirt on the slide will produce an uneven smear, (2) the amount of
India ink or nigrosine is critical; most students tend to use too much. If you keep these in mind, you
should be able to produce a perfect slide the first time. Proceed as follows :

1. Add a very small drop (not over 1/8" diameter) of nigrosine or India ink near the right end of one
slide.
2. Remove a small amount of material from between your teeth with a toothpick and mix it into the
drop on the slide. Complete emulsification of the organisms is important at this stage. If necessary,
use a flame-sterilized inoculating loop to help break the clumps of bacteria.
3. Place the edge of another clean glass slide to the left of the suspension. This shall serve as your
spreader slide. Move the spreader slide toward the suspension until contact is made. It should be
noted that the suspension drop spreads along the back edge of the spreader slide.
4. Drag the spreader slide away from the suspension. Observe that the smear will be thick where it
begins and feather out into a very thin smear at the end of the spreading stroke. Somewhere
between the thickest end and the thinnest end will be an ideal thickness of suspension.
5. Allow the slide to dry at room temperature. Do not apply heat to the slide.
6. Examine under oil immersion. Select that part of the smear that shows the cells distinctly with a
dark background.
Capsular Staining

Some bacterial cells are surrounded by a pronounced gelatinous structure called capsule. There is
considerable evidence to support the view that all bacteria have some amount of slime material
surrounding the cells. In most instances, however, the layer is not of sufficient magnitude to be readily
discernible. Although some capsules appear to be made of glycoprotein, others contain polypeptide. All
appear to be water-soluble.

Staining the bacterial capsule cannot be accomplished by ordinary simple staining procedures. The
problem with trying to stain capsules is that if you prepare a heat-fixed smear of the organisms by
ordinary methods, you will destroy the capsule and if you do not heat-fix the slide, the organism will
slide off the slide during washing. In most of our bacteriological studies, our principal concern is simply
to demonstrate the presence or absence of a capsule. This can be easily achieved by combining
negative and simple staining techniques.

Procedure

1. Mix a loopful of Bacillus subtilis grown in nutrient agar with 20% sucrose with a small drop of India
ink on a clean glass slide.
2. The ink suspension of bacteria is spread over the slide by dragging a spreader slide. Allow air drying
of the suspension at room temperature.
3. Gently heat dry the slide to fix the organisms to the slide.
4. Stain the smear with crystal violet for one minute.
5. Wash off the crystal violet stain.
6. Blot dry with clean tissue paper and examine under oil immersion objective.

Spore Staining (Schaeffer-Fulton Method)

Bacteria species belonging to the genera Bacillus and Clostridium produce extremely heat-resistant
structures called endospores. In addition to being heat resistant, they are also very resistant to many
chemical disinfectant, dessication and radiation. Among other factors, such resistance is due to a thick,
tough spore coat.

Gram staining will not stain endospores. Only when considerable heat is applied to a suitable stain can
stain penetrate the spore coat. Once the stain has entered the spore however, it is not easily removed.

The method to be performed in this activity is the Schaeffer-Fulton Method. This method utilizes
malachite green to stain the endospores and safranin to stain the vegetative portion of the cell. Utilizing
this technique, a properly stained spore-former will have a green endospore contained in pink cells.
Proceed as follows:

1. Prepare a bacterial smear of Bacillus subtilis on a clean glass slide. Fix the smear.
2. Cover the smear with malachite green for 20 minutes.
3. After the slide has cooled sufficiently, rinse with water for 30 seconds.
4. Counterstain with safranin for about 30 seconds.
5. Rinse briefly with water to remove safranin.
6. Blot dry with clean tissue paper and examine under oil immersion.

Reference

Benson, Harold. 1990. Microbiological Applications. Laboratory Manual in General Microbiology. 5th
edition. WCB Publishers.