Differential Staining of Bacteria: APPENDIX 3I

Capsule Stain
Donald P. Breakwell,1 Rita B. Moyes,2 and Jackie Reynolds3
1
Brigham Young University, Provo, Utah
2
Texas A&M University, College Station, Texas
3
Richland College, Dallas, Texas

ABSTRACT
Bacterial capsules are composed of high-molecular-weight polysaccharides and/or
polypeptides, and are associated with virulence and biofilm formation. Unfortunately,
capsules do not stain well with crystal violet, methylene blue, or other simple stains. This
unit describes two methods of capsule staining. The first is a wet-mount method using
india ink; the capsule is visualized as a refractile zone surrounding a cell. The second is a
direct-staining dry-mount method that precipitates copper sulfate and leaves the capsule
as a pale blue zone. Both methods are easily performed within ∼5 min. Curr. Protoc.
Microbiol. 15:A.3I.1-A.3I.4.  C 2009 by John Wiley & Sons, Inc.

Keywords: differential stain r capsule stain r india ink r nigrosin r negative stain r
wet mount r dry mount

INTRODUCTION
Bacterial capsules are dense, viscous layers of high-molecular-weight polysaccharides
and/or polypeptides that are secreted by, and which adhere to, the surface of the cell. They
are associated with virulence (anti-phagocytosis or adherence) and formation of biofilms
(see Section 1B). Capsules stain very poorly with reagents used in simple staining (see
APPENDIX 3E) and a capsule stain can be, depending on the method, a misnomer because
the capsule may or may not be stained. 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. india ink is difficult to obtain nowadays; however,
nigrosin is easily acquired. A positive capsule stain requires a mordant that precipitates
the capsule. Because of this requirement, early capsule staining protocols were combined
with flagella staining (APPENDIX 3G) with the use of a methylene blue counterstain (Hiss,
1905; Bailey, 1930). 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. Many investigators use a negative stain because it is easier to do. A wet-
mount and a dry-mount protocol are described here. Encapsulated Klebsiella pneumoniae
or Streptococcus lactis are appropriate positive controls for these methods.

CAUTION: Follow all biosafety requirements relevant to the microorganism under in-
vestigation. Refer to UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for instructions
on safe handling of microorganisms.

DUGUID WET-MOUNT METHOD BASIC

PROTOCOL 1
This method requires suspending cells in nigrosin and making a film through which the
capsules are seen (Duguid, 1951). Because it is a relatively quick and easy procedure
with few reagents to prepare, its use is appealing.
Commonly Used
Techniques

Current Protocols in Microbiology A.3I.1-A.3I.4, November 2009 A.3I.1

Published online November 2009 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mca03is15 Supplement 15
Copyright C 2009 John Wiley & Sons, Inc.
 Materials
Nigrosin ampules (Becton Dickinson)
Bacterial culture
Clean glass microscope slides
Coverslips
Filter paper
Compound light microscope
Additional reagents and equipment for examining the slides using a light
microscope (UNIT 2A.1)
1. Open an ampule of nigrosin according to the manufacturer’s recommendation.
Fresh nigrosin is preferably used so that the dye is free of large particles.

2. Place a drop of nigrosin on a clean glass slide.

3. From either a culture grown on a solid medium, (such as 5% sheep blood agar) or a
broth culture, gently suspend a small amount of a colony or loopful of broth in the
nigrosin.
A large number of bacteria are required for satisfactory results.
Avoid diluting the nigrosin.

4. Place a clean coverslip on the dye-bacteria mixture.

5. Using a piece of filter paper, gently press down on the coverslip.
Practice is required to perform this step well because too much pressure will distort the
capsule, whereas too little will leave the cells obscured by the dye. A good rule of thumb
is that the slide should have the appearance of a photographic negative.

6. Examine the slide using the oil immersion objective of a compound light microscope
(UNIT 2A.1).

BASIC ANTHONY DIRECT–DRY STAINING METHOD

PROTOCOL 2
This is a direct method by which both the bacterium and its capsule are stained
(Anthony, 1931).

Materials
Bacterial culture
1% (w/v) crystal violet solution (see recipe)
20% (w/v) copper (II) sulfate (see recipe)
Microscope slide
Paper towel
Compound light microscope
Additional reagents and equipment for examining the slides using a light
microscope (UNIT 2A.1)
1. Prepare an air-dried smear of a bacterial culture.
If a broth culture is used, place a drop of the culture on a clean microscope slide.
Alternatively, if a plate culture is used, a suspension can be prepared by suspending
a small amount of the colony in a loopful of water. Using another clean slide placed
obliquely to the original slide, gently drag the oblique slide across the culture to form
a thin film. Then air-dry the preparation. Do not heat-fix the smear as it can distort or
destroy the capsule.
Capsule Stain

A.3I.2
Supplement 15 Current Protocols in Microbiology
 2. Flood the prepared slide with 1% (w/v) crystal violet solution for 2 min at room
temperature.
Do not rinse with water.

3. Holding the slide at an angle, gently rinse the slide with 20% (w/v) copper (II)
sulfate.
The copper (II) sulfate precipitates the capsule, which turns pale blue.

4. Blot the lower end of the slide on a paper towel to remove excess solution, and then
gently blot the smear.
5. Examine using the oil immersion objective of a compound light microscope
(UNIT 2A.1).

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Crystal violet, 1% (w/v)

Dissolve 1 g crystal violet in 100 ml distilled H2 O
Store up to 1 year at room temperature
Avoid using ethanol, as it can shrink capsules.

Copper (II) sulfate, 20% (w/v)

Dissolve 20 g CuSO4 ·7H2 O in 100 ml distilled H2 O
Store up to 1 year at room temperature

COMMENTARY
Background Information
The ability to identify the presence of a
capsule is important in the clinical laboratory
because the presence of a capsule is usually
associated with increased virulence.
Critical Parameter
Do not heat-fix the smear because this can
destroy the capsule and will also cause shrink-
age of the bacteria, making a clear area around
the cell that can mistakenly be identified as a
capsule.

Figure A.3I.1 Klebsiella pneumoniae stained using the Duguid wet-mount method. The capsule
can be seen as the clear, refractile area surrounding the cell against the dark background. Commonly Used
Techniques

A.3I.3
Current Protocols in Microbiology Supplement 15
 Anticipated Results
In the Duguid method, you will see capsules
as haloes surrounding a refractile cell using
bright-field microscopy. In the Anthony Direct
Method, bacteria stain purple, but the capsules
stain pale blue. Figure A.3I.1 shows Kleb-
siella pneumoniae stained using the Duguid
wet-mount method. The capsule can be seen
as the clear, refractile area surrounding the cell
against the dark background.
Time Considerations
These staining procedures take ∼5 min to
perform once the technique is mastered.
Literature Cited
Anthony, E.E. Jr. 1931. A note on capsule staining.
Science 73:319-320.
Bailey, H.D. 1930. A practical flagella and capsule
stain for bacteria. Science 72:95-96.
Capsule Stain
A.3I.4
Supplement 15
Duguid, J.P. 1951. The demonstration of bacte-
rial capsules and slime. J. Pathol. Bacteriol.
63:673.
Hiss, P.H. Jr. 1905. A contribution to the physiolog-
ical differentiation of Pneumococcus and Strep-
tococcus. J. Exp. Med. 6:317-345.
Key References
Hiss, 1905. See above.
One of the original capsule staining protocols.
Moat, A.G., Foster, J.W., and Spector, M.P. 2002.
In Microbial Physiology, 4th ed. pp. 315-323.
Wiley-Liss, Inc. New York.
A detailed description of capsule structure and
physiology, together with micrographs comparing
staining techniques.
Internet Resources
http://www.microbelibrary.org
Select the visual collection icon and search the free
image bank for your desired organism and stain.
Current Protocols in Microbiology