Adventist University of the Philippines

COLLEGE OF SCIENCE AND TECHNOLOGY

Department of Biology
ACTIVITY # 9
SPECIAL STAINING
SHAEFFER FULTON, NEGATIVE STAINING AND FLAGELLAR STAINING METHOD
By:
CLAIRE V. DURANTE Microbiology Monday 1:30 pm

OBJECTIVES:
1. Compare the Schaeffer Fulton method from Negative staining method
2. Identify specific bacteria that are used for these methods
3. Distinguish the bacterial results from using Schaeffer Fulton method and Negative
staining method
4. Locate the positions of endospores
5. Determine the procedure used for using Schaeffer Fulton method and Negative staining
method

INTRODUCTION:
The Schaeffer–Fulton stain is a technique designed to isolate endospores by staining any present
endospores green, and any other bacterial bodies red. The primary stain is malachite green, and the
counterstain is safranin, which dyes any other bacterial bodies red.

MATERIALS:
A. Schaeffer Fulton method
Reagents used for Endospore Staining

Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)

0.5 gm of malachite green
100 ml of distilled water

Decolorizing agent
Tap water or Distilled Water
Counter Stain: Safranin
Stock solution (2.5% (wt/vol) alcoholic solution)
2.5 gm of safranin O
100 ml of 95% ethanol

PROCEDURE:
1. Take a clean grease free slide and make smear using sterile technique.
 2. Air dry and heat fix the organism on a glass slide and cover with a square of
blotting paper or toweling cut to fit the slide.
3. Saturate the blotting paper with malachite green stain solution and steam for 5
minutes, keeping the paper moist and adding more dye as required.
Alternatively, the slide may be steamed over a container of boiling water.
4. Wash the slide in tap water.
5. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry.
6. Examine the slide under microscope for the presence of endospores. Endospores
are bright green and vegetative cells are brownish red to pink.

B. Negative Staining Method

CAPSULE
The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. A
capsule is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to
the cell wall. … The capsule stain employs an acidic stain and a basic stain to detect capsule
production

MATERIALS:
India ink
Nigrosin
Nigrosin 100 gm/L, Formalin 5 ml/L in water
Inoculating loop
Glass slide

PROCEDURE:
1. Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin)
on the slide.
Congo Red is easier to see, but it does not work well with some strains. India
Ink generally works, but it has tiny particles that display Brownian motion that
must be differentiated from your bacteria. Nigrosin may need to be kept very
thin or diluted.
2. Using sterile technique, add a loopful of bacterial culture to slide, smearing it in
the dye.
3. Use the other slide to drag the ink-cell mixture into a thin film along the first
slide and let stand for 5-7 minutes.
4. Allow to air dry (do not heat fix).
5. Flood the smear with crystal violet stain (this will stain the cells but not the
capsules) for about 1 minutes. Drain the crystal violet by tilting the slide at a 45
degree angle and let stain run off until it air dries .
6. Examine the smear microscopically (100X) for the presence of encapsulated cells
as indicated by clear zones surrounding the cells.

C. FLAGELAR STAINING METHOD

The main purpose of capsule stain is to distinguish capsular material from the
bacterial cell. A capsule is a gelatinous outer layer secreted by bacterial cell
and that surrounds and adheres to the cell wall. Most capsules are composed
of polysaccharides, but some are composed of
polypeptides. The capsule differs from the slime layer that most bacterial cells
produce in that it is a thick, detectable, discrete layer outside the cell wall. The
capsule stain employs an acidic stain and a basic stain to detect capsule
production

MATERIALS:
Reagents used for Capsule Staining

Crystal Violet (1%)

Crystal Violet (85% dye content) = 1 gm
Distilled Water = 100 ml
Nigrosin
Nigrosine, water soluble = 10 gm
Distilled Water = 100 ml
Glass slide
Wash bottle
Forceps
Proteus vulgaris

PROCEDURE:
1. Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin)
on the slide.
Congo Red is easier to see, but it does not work well with some strains. India
Ink generally works, but it has tiny particles that display Brownian motion that
must be differentiated from your bacteria. Nigrosin may need to be kept very
thin or diluted.
2. Using sterile technique, add a loopful of bacterial culture to slide, smearing it in
the dye.
3. Use the other slide to drag the ink-cell mixture into a thin film along the first
slide and let stand for 5-7 minutes.
4. Allow to air dry (do not heat fix).
5. Flood the smear with crystal violet stain (this will stain the cells but not the
capsules) for about 1 minutes. Drain the crystal violet by tilting the slide at a 45
degree angle and let stain run off until it air dries .
6. Examine the smear microscopically (100X) for the presence of encapsulated cells
as indicated by clear zones surrounding the cells.
PICTURES:
Obtain pictures from the internet and answer the following:
Schaeffer – Fulton method Capsule Stanning Method

Pictures for positions of endospores

CENTRAL TERMINAL SUBTERMINAL

TYPES OF FLAGELLAR ARRANGEMENT IN BACTERIA (IDENTIFICATION)

MONOTRICHOUS AMPHITRICHOUS LOPHOTRICHOUS PERITRICHOUS
GUIDE QUESTIONS:
1. How does the endospore appear in the bacterial cell?
Answer: Endospores can arise in many parts of the vegetative cell. They are
classified as central, subterminal, or terminal.

2. Describe the importance of the endospore in the bacterial organism?

Answer: It allows the bacterium to produce a dormant, incredibly resistant cell in
order to preserve the cell's genetic material during times of great
stress. Endospores may be able to endure environmental threats that
would normally kill the bacteria.

3. List at least 3 species of endospore – forming bacteria?

Answer: Desulfotomaculum, Sporosarcina, Sporolactobacillus,

4. How does the capsule appear in the bacterial cell?

Answer: On a dark backdrop, it appears as a brilliant halo around the cell. There
appear to be amorphous gelatinous patches surrounding the cell as
well.

5. Describe the importance of capsule to the bacterial organisms?

Answer: The capsule prevents bacteria from desiccation, serves as a food
reserve, shields anaerobes from oxygen toxicity, and aids in the
attachment of bacteria to surfaces.

6. What other staining technique would demonstrate bacterial capsule?

Answer: India ink and Gram crystal violet.

7. List 3 examples of capsule – forming bacteria?

Answer: Haemophilus influenzae, Pseudomonas aeruginosa, Salmonella

8. Why are heat fixing and blotting with absorbent paper no longer required in this
process?
Answer: Heat fixing and blotting with absorbent paper are no longer required
since they can damage the capsule and cause the bacteria to shrink,
resulting in a clear zone surrounding the cell that might be
misinterpreted as a capsule.

9. State the function of the flagella?

Answer: Flagella has a role in cell motility. Flagella are whip-like features that
help a cell move through liquid. Few species employ unique flagella as
sensory organs to detect pH and temperature changes.

10. List four bacteria with their types of flagellar arrangement?

Answer: (1) Pseudomonas - monotrichious, (2) Campylobacter - amphitrichous,
(3) Helicobacter – lophotrichous, and (4) Escherichia coli - peritrichous
References used at least three:
1. Keenleyside, W. (2019). 2.4 staining microscopic specimens. In Microbiology: Canadian
Edition. Pressbooks.

2. Tankeshwar, A. (2013, April 28). Bacterial capsule: Importance, capsulated bacteria.

Retrieved March 24, 2022, from Microbe Online website:
https://microbeonline.com/bacterial-capsule-structure-and-importance-and-examples-
of-capsulated-bacteria/

3. Libretexts. (2017, May 7). 4.5A: Endospores. Biology LibreTexts; Libretexts.

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundles
s)/4%3A_Cell_Structure_of_Bacteria_Archaea_and_Eukaryotes/4.5%3A_Specialized_Ext
ernal_Structures_of_Prokaryotes/4.5A%3A_Endospores

4. Bacterial endospores. (n.d.). Cornell.Edu. Retrieved March 21, 2022, from

https://micro.cornell.edu/research/epulopiscium/bacterial-endospores/
5.