Staining techniques

Because microbial cytoplasm is usually transparent, it is necessary to stain

microorganisms before they can be viewed with the light microscope.

In some cases, staining is unnecessary, for example when microorganisms are very large or
when motility is to be studied, and a drop of the microorganisms can be placed directly on the
slide and observed. A preparation such as this is called a wet mount.

A wet mount can also be prepared by placing a drop of culture on a cover slip (a glass cover
for a slide) and then inverting it over a hollowed out slide. This procedure is called the
hanging drop.

In preparation for staining, a small sample of microorganisms is placed on a slide and

permitted to air dry. The smear is heat fixed by quickly passing it over a flame.

Heat fixing kills the organisms, makes them adhere to the slide, and permits them to accept
the stain.

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 (1) Simple staining techniques
Simple = only one dye is used during the staining procedure

Staining can be performed with basic dyes such as crystal violet or methylene blue,
positively charged dyes that are attracted to the negatively charged materials of the microbial
cytoplasm. Such a procedure is the simple positive staining.

An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged
(acidic) dyes. They are repelled by the negatively charged cytoplasm and gather around the
cells, leaving the cells clear and unstained. This technique is called the simple negative
staining technique.

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Simple positive staining procedure
1. Add one loopful of the sample (mixture of microorganisms) onto a glass slide.
2. Allow it to air-dry.
3. Heat-fix the specimen on the glass slide, unless the specimen is heat-fixed, the
bacterial smear will wash away during the staining procedure.
4. Flood slide with crystal violet and wait 1 min. or safranin and wait 3-4min.
5. Wash the smear with tap water to remove the excess of stain.
6. Blot dray, than add cederwood oil (immersion oil) and examine under a microscope.

Simple negative staining procedure –

1. Place a small drop of nigrosin at the end of the slide.
2. Place a loopful of sample ( mixture of microorganisms) and mix with drop of nigrosin.
3. Using the edge of another slide, spread the drop out
across the slide.
4. Allow to air dray.
5. Place one drop of immersion oil and and examine
under a microscope.

The results of simple staining procedures (look at the figure below):

Simple positive staining: all bacteria are colored,

Simple negative staining: background is dark, bacteria are without any color

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 (2) Differential staining techniques
The differential staining techniques are based on the application of a set of several
different dyes which react differently with different types of microorganism.

Hence, they can be used to distinguish among them.

An example is the Gram staining technique. This procedure separates bacteria into two
groups: Gram positive bacteria and Gram negative bacteria.

Crystal violet is first applied, followed by the mordant iodine, which fixes the stain.

Then the slide is washed with alcohol, and the Gram positive bacteria retain the crystal violet
iodine stain; however, the Gram negative bacteria lose the stain. The Gram negative bacteria
subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear
red under the oil immersion lens, while Gram positive bacteria appear blue or purple,
reflecting the crystal violet retained during the washing step.

Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal
violet-iodine complex that occurs during staining, while Gram-negative cells have only a thin
layer of peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol, and Gram-
negative cells do decolorize. This allows the Gram-negative cells to accept the counter stain
safranin.

Gram Stain Procedure

1. Place a drop of NaCl onto the glass slide.
2. Using a sterilized and cooled inoculation loop, obtain a very small sample of a
bacterial colony.
3. Gently mix the bacteria into the NaCl drop.
4. Let the bacterial sample air dry
5. Using slide holder, pass the dried slide through the flame of Bunsen burner 3 or 4
times, smear side facing up.
6. Apply primary stain: Flood slide with crystal violet stain.
7. Rinse: After 1 minute, rinse the slide with water.
8. Apply mordant: Flood the slide with iodine.
9. Rinse: After 1 minute, rinse the slide with water.
10. Apply decolorizer: Flood slide with acetone alcohol.
11. 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.)
12. Apply secondary stain (counterstain): Flood slide with safrinin.
13. Rinse: After 1 minute, rinse the slide with water.
14. Dry on air
15. Place the stained smear on the microscope stage smear side up, apply oil directly
to the smear and focus using the 100X objective.

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 Results of the Gram staining procedure:

Gram-positive bacteria appear blue or purple

Gram-negative cells will appear pink to red

Examples of Gram-positive and Gram-negative bacteria:

Gram-positive bacteria: Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium

diphteriae
Gram-negative bacteria: Neisseria meningitidis, Escherichia coli, Pseudomonas aeruginosa

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 Acid fast staining technique

Another differential stain technique is the acid fast staining technique.

This technique differentiates species of Mycobacterium from other bacteria. Because
the cell wall is resistant to water-based stains, acid-fast organisms require a special staining
technique.
Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells.
Then the cells are washed with a diluted acid alcohol solution.
Mycobacterium species resist the effect of the acid alcohol and retain the carbolfuchsin
stain (they appear bright red under the microscope). Other bacteria lose the stain and take on
the subsequent methylene blue stain (they appear blue under the microscope).

The Ziehl-Neelsen staining procedure with Kinyoun modification (acid fast

technique)

1. Place a drop of NaCl onto the glass slide.

2. Using a sterilized and cooled inoculation loop, obtain a very small sample of a
bacterial colony.
3. Gently mix the bacteria into the NaCl drop.
4. Let the bacterial sample air dry
5. Using slide holder, pass the dried slide through the flame of Bunsen burner 3 or 4
times, smear side facing up.
6. Flood slides with Kinyoun carbolfuchsin for 5 minutes.
7. Rinse gently with water until the water flows off clear.
8. Flood slides with acid-alcohol (3% HCl in ethanol) for 3~5 seconds.
9. Rinse gently with water until the water flows off clear.
10. Flood slides with methylene blue for 1 minutes.
11. Rinse gently with water until the water flows off clear.
12. Allow slides to air dry before viewing.

Results of the Ziehl-Neelsen staining procedure:

acid fast bacteria appear bright red
non-acid fast bacteria appear blue

Examples of acid fast and non-acid fast bacteria:

Acid fast bacteria: Mycobacterium tuberculosis, Mycobacterium avium
Non-acid fast bacteria: Escherichia coli, Staphylococcus aureus, etc.

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 Other staining techniques are designed to identify various bacterial structures of
importance. For instance, a special staining technique highlights the flagella of bacteria by
coating the flagella with dyes or metals to increase their width. Flagella so stained can then be
observed. A special staining technique is also used to examine bacterial spores. Malachite
green is used with heat to force the stain into the cells and give them color. A counterstain,
safranin, is then used to give color to the nonsporeforming bacteria. At the end of the
procedure, spores stain green and other cells stain red.

Capsule staining

Bacterial capsules play a role in adherence, protection (they inhibit ingestion and killing
by phagocytes), securing nutrients, and cell-to-cell recognition.

PRINCIPLE: Chemically, the capsular material is a polysaccharide, a glycoprotein or a

polypeptide. Capsule staining is more difficult than other types of differential staining
procedures because the capsular materials are water soluble and may be dislodged and
removed with vigorous washing. Bacterial smears should not be heated, because the resulting
cell shrinkage may create a clear zone around the organism, an artifact that can be mistaken
for a capsule. The capsule is non-ionic, so that the dyes commonly used will not bind to it.
Two dyes, one acidic and one basic, are used to stain the background and the cell wall,
respectively.

Negative staining methods contrast a translucent, darker colored, background with stained
cells but an unstained capsule. The background is formed with india ink or nigrosin or
congo red.

A positive capsule stain requires a mordant that precipitates the capsule. By

counterstaining with dyes like crystal violet, methylene blue or carbolfuchsin, bacterial cell
wall takes up the dye. Capsules appear colourless with stained cells against dark background.

Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating.
A drop of serum can be used during smearing to enhance the size of the capsule and make it
more easily observed with a typical compound light microscope.

Procedure of the capsule staining

1. Using sterile technique, add a loopful of bacterial culture to tube with 1 ml NaCl.
2. Add one drop of carbolfuchsin into the tube and mix gently.
3. Heat the mixture under flame 1min.
4. Place a drop of mixture onto the glass slide.
5. Place a drop of nigrosin onto the same glass slide next to drop of mixture of bacteria
and the dye.
6. Use the other slide to drag the nigrosin-cell mixture into a thin film along the first
slide.
7. Allow to air dry for 5-7 minutes (do not heat fix).
8. Examine the smear microscopically (100X) for the presence of encapsulated cells as
indicated by clear zones surrounding the cells.

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Results of the capsule staining procedure:
Encapsulated bacteria: Clear halos (capsules) are observed around pink bacteria against
dark background
Bacteria without capsules: pink bacteria with no clear halos

Examples of encapsulated and non-encapsulated bacteria:

Encapsulated bacteria:

Bacillus anthracis, Klebsiella pneumoniae, Streptococcus pneumoniae,

Neisseria meningitidis, Clostridium perfringens;

Non-encapsulated bacteria:
Neisseria gonorrhoreae, etc.

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Summary of the staining techniques: