Bacterial Staining Techniques
Bacteria are almost colorless to visualize bacteria, either dyes or stains, or an alternative
source of illumination (phase contrast or differential interference constrast) are used. Since
staining of bacterial cells is relatively fast, inexpensive, and simple, it is the most commonly
used technique to visualize bacterial cells. Staining not only makes bacteria more easily seen,
but it allows their morphology (e.g. size and shape) to be visualized more easily. In some
cases, specific stains can be used to visualize certain structures (flagella, capsules,
endospores, etc). of bacterial cells.
Staining techniques classified
• Simple stain: where only one stain is used and all bacteria are stained similarly. eg:
Methylene blue, dilute carbol fuchsin
• Differential staining: where different bacteria stain differently to a common staining
technique depending on their physiological properties. eg: Gram’s stain and Acid fast
staining
• Special stain: where structures of bacteria like spores, granules, capsule etc are
demonstrated Albert’s stain for demonstration of metachromatic granules.
Stains (dyes) are chemicals containing chromophores, groups that impart color. Their
specificity is determined by their chemical structure. Stains are generally salts in which
one of the ions is colored. (A salt is a compound composed of a positively charged ion and a
negatively charged ion.) For example, the dye methylene blue is actually the salt methylene
blue chloride which will dissociate in water into a positively charged methylene blue ion
which is blue in color and a negatively charged chloride ion which is colorless. Commonly
used microbiological stains generally fall into one of two categories - basic stains or acidic
stains ( although there are a few stains such as India Ink) which are neutral). A basic
dye is a stain that is cationic (positively charged) and will therefore react with material that is
negatively charged. The cytoplasm of all bacterial cells have a slight negative charge when
growing in a medium of near neutral pH and will therefore attract and bind with basic dyes.
Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue.
Acid dyes have negatively charged chromophores and are repelled by the bacterial surface
forming a deposit aroung the organism. They stain the background and leave the microbe
transparent. Nigrosine and congo red are examples of acid dyes.
If the culture is taken from solid (agar) medium.
1. Clean the slide using Bon Ami or other suitable cleaning material. Gently dry
the slide with a lint free cloth. (alternatively, it is possible to purchase precleaned slides)
2. Place a very small drop of distilled water on the surface of the slide.
3. Aseptically remove a small amount of the culture from the agar surface and
just touch it several times to the drop of water until it just turns cloudy.
4. Burn the remaining bacteria off of the loop. (If too much culture is added to
the water, you will not see clearly stained individual bacteria.).
5. Using the loop, spread the suspension over the entire slide to form a thin film.
6. Allow this thin suspension to completely air dry.
7. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4
times to heat-fix. Caution: Too much heat might distort the organism and, in
the case of the gram stain, may cause gram-positive organisms to stain gramnegatively. The slide should feel very warm but not too hot to hold.
If the organism is taken from a broth culture:
1. Clean the slide using Bon Ami or other suitable cleaning material. Gently dry
the slide with a lint free cloth. (alternatively, it is possible to purchase precleaned slides)
2. Aseptically place 2 or 3 loops of the culture on a clean slide. Do not use water.
3. Using the loop, spread the suspension over the entire slide to form a thin film.
4. Allow this thin suspension to completely air dry.
5. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4
times to heat-fix.
Simple (Direct) Staining with Methylene Blue
1. Prepare a heat fixed smear of the culture you wish to examine
2. Cover the smear with methylene blue
3. Allow the dye to remain on the smear for approximately 1 minute. (Note
staining time is not critical. Somewhere between 30 seconds and 2 minutes
should give you an acceptable stain. The longer you leave the dye on, in
general, the darker the stain.)
4. Wash the excess stain off the slide Pick up the slide by one end and hold it at
an angle over the staining tray. Using the distilled water wash bottle, gently
wash off the excess methylene blue from the slide by directing a gentle stream
of water over the surface of the slide.. Wash off any stain that got on the
bottom of the slide as well.
5. Blot off excess stain using bibulous paper. DO NOT rub the slide, rather place
the slide between two sheets of bibulous paper and press down gently. Paper
will absorb excess dye.
6. Examine the slide under the brightfield microscope.
7. Record the shape, arrangement, and approximate size of the organisms. You
should have data on two cultures which you have stained.
Gram Stain
Gram stain is a differential stain commonly used in the microbiology laboratory that
differentiates bacteria on the basis of their cell wall structure. Most bacteria can be
divided into two groups based on the composition of their cell wall:
1) Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma
membrane. Gram-positive cell walls stain blue/purple with the Gram stain.
2) Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and
an outer membrane beyond the plasma membrane.
Which are the theories of Gram staining?
• Cell wall theory: Cell wall of Gram positive bacteria are 40 times thicker than those of
Gram negative cells, hence they are thought to help retain the dye-iodine complex.
• Lipid Content Theory: Cell envelope of Gram negative bacteria contains an additional
membrane (outer membrane), hence containing more lipids than Gram positive
bacteria. Acetone or alcohol dissolves the lipid thus forming large pores in Gram
negative bacteria through which the dye-iodine complex leaks out. Alcohol/acetone
dehydrates Gram positive bacteria shrinking the cell wall and the closing the pores.
• Magnesium Ribonucleate Theory: A compound of magnesium ribonucleate and basic
protein concentrated at the cell membrane helps Gram positive bacteria retain the
primary dye. Gram negative bacteria do not possess this substance.
• Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are said to be more
acidic (2) than those of Gram negative ones (3). Hence the dye is said to bind with more
affinity to Gram positive cells.
The Gram staining procedure goes as follows:
1. Flood the slide with Crystal Violet (the primary stain).
2. After 1 minute, rinse the slide with water.
3. Flood the slide with Iodine (Iodine is a mordant that binds with Crystal violet and is then
unable to exit the Gram+ peptidoglycan cell wall.)
4. After 1 minute, rinse the slide with water.
5. Flood the slide with Acetone Alcohol. (Alcohol is a decolorizer that will remove the stain
from the Gram-negative cells.)
6. After 10 or 15 seconds, rinse the slide with water. (Do not leave the decolorizer on too long or
it may remove stain from the Gram-positive cells as well.)
7. Flood slide with Safrinin (the counterstain).
8. After 1 minute, rinse the slide with water.
9. Gently blot the slide dry. It is now ready to be viewed under oil immersion (1000x TM) with
a bright-field compound microscope.
QUESTIONS:
1. What is the difference between a simple and a differential stain?_________
___________________________________________________________________
___________________________________________________________________
2. Describe the function of each of the following in the Gram stain
Mordant: ________________________________________________
Primary stain: ____________________________________________________
Counterstain: _____________________________________________________
3. Which step in the Gram stain is most likely to cause poor results if done
incorrectly?__________________________________________________________
_________
4. Why must fresh bacterial cultures be used in a Gram stain?
___________________________________________________________________
____________
5. Briefly describe the mechanism of Gram staining.
___________________________________________________________________
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