Jennifer Julizar

BIOL 351
Section 906

Staining Results and Conclusions

I. RESULTS

a. Acid Fast Stain

Table 1: Morphology of acid-fast stained microorganism viewed under 1000X magnification

Organism Cellular morphology Cell Dimensions (µm) Color Acid Fast

and arrangement Reaction (+/-)

Mycobacteriu Cocci 0.2 Reddish-purple +

m
In the acid-fast stain experiment (Table 1), a prepared slide of mycobacterium, an acid-fast

positive microorganism was observed using microscope under 1000X magnification with oil

immersion. The cells of mycobacterium measured were reddish-purple and spherical in shape

with a diameter of 0.2 µm.

b. Endospore Stain

Table 2: Morphology of endospore stained microorganism viewed under 1000X magnification

Organism Cellular morphology and Cell Dimensions (µm) Spores Spore Shape

arrangement and Position

B. cereus Elongated Rod-shape, Length: 4 Present Coccobacillus,

Clumps Width: 1 Central

In the endospore stain experiment (Table 2), a slide of Bacillus cereus was observed under

microscope under 1000X magnification using oil immersion. The Bacillus cereus measured

appeared as a red-colored elongated rod-shaped organism with a length of 4 µm and a width of 1

µm. Spores showed up as green-colored coccobacilli, which are located in the center of the cells.

c. Simple Stain

Table 3: Morphology of simple stained microorganisms in methylene blue viewed under 1000X

magnification

Organism Stain and Cellular Morphology Cell Dimensions (µm)

 Duration (min) and Arrangement
B. megaterium Methylene blue Bacillus, Elongated Length: 7

1 Rod-shape, Chaining Width: 1

M. luteus Methylene blue Coccus, Clustered, Diameter: 5

1 Chaining
E. coli Methylene blue Coccus, Clustered Diameter: 1

1 Tiny Dots
In the simple stains experiment (Table 3), three organisms were stained using methylene blue for

1 min, then were viewed under microscope using oil immersion. Bacillus megaterium was

observed as elongated rod shaped bacteria and appeared as clustered of chains with a cell length

of 7 µm and width of 1 µm. Micrococcus luteus was observed as spherical shaped bacteria and

appeared as clustered chains, each with a cell diameter of 5 µm. Escherichia coli was observed

as round shaped bacteria and appeared as groups of tiny dots of microorganisms with a cell

diameter of 1 µm.

d. Negative Stain

Table 4: Morphology of negative stained microorganisms in nigrosine viewed under 1000X

magnification

Organism Stain Cellular Morphology and Arrangement Cell Dimension

B. megaterium nigrosi Baccilus, clustered, white color with dark Length: 6

n background Width: 4
M. luteus nigrosi Cocci, dispersed, white color with dark Diameter: 5

n background
In the negative stains experiment (Table 4), two microorganisms were stained with nigrosine and

both showed white color with dark background. Bacillus megaterium appeared as rod-shaped

bacteria that were clustered together in group with a cell diameter of 1 µm. Micrococcus luteus

appeared as dispersed spherical-shaped organisms with a cell diameter of 5 µm.

e. Flagella Stain
 Table 5: Morphology of flagella stained microorganism viewed under 1000X magnification

Organism Cellular Morphology and Cell dimensions (µm) Flagella length (µm)

Flagella Arrangement
Flagella- Rod-shaped Cell, Length: 25 5-10

Peritrichious Peritrichious Flagella Width: 1

In the flagella stain experiment (Table 5), a slide of Flagella-peritrichious was observed under

microscope under 1000X magnification with oil immersion. The microorganism observed

appeared as rod shape with a cell length of 25 µm and width of 1 µm. Flagella is peritrichious

with various length about 5 to 10 µm.

f. Gram Stain

Table 6: Morphology of gram stained microorganisms viewed under 1000X magnification

Organisms Cellular Morphology Cell Dimensions (µm) Color Gram

and Arrangement Reaction (+/-)

E. coli Cocci, Clustered, Diameter: 0.1 Reddish -

Clumped (Very Dense) - pink

S. capitis Cocci, Clumped Diameter: 1 Purple +
C. xerosis Bacillus, Clustered Length: 3 Purple +

Width: 0.5
In the gram stain experiment (Table 6), two of three gram stained microorganisms were Gram-

positive. Escherichia coli, a Gram-negative bacteria appeared as a reddish-pink spherical shaped,

clumped together with a cell diameter of 0.1 µm. Staphylococcus capitis, a Gram-positive

bacteria appeared as purple spherical shaped with a cell diameter of 1 µm. Corynebacterium

xerosis, a Gram-positive bacteria that appeared as purple rod-shaped bacteria with a length of 3

µm and a width of 0.5 µm.

II. DISCUSSION
a. Acid-Fast Stain

Acid-fast microorganisms have mycolic acids present in their cell walls which assists with

attaching a stain into these cell that will be resistant to decolorization. There are two different

kinds of acid-fast staining procedures: the Ziehl-Neelsen (ZN) method and the Kinyoun (K)

method. The ZN method requires the use of heat in the staining process while the K method does

not. The purpose of heating during the staining procedure of the ZN method was to melt the wax

within the cell wall to enable the stain enter into the cell. (4) Neither of these methods were used

in the lab, since a prepared slide was provided. Carbolfuchsin was the primary stain used while

methylene blue was the counterstain used after decolorization. (4) Mycobacterium observed with

the microscope under 1000X magnification showed a positive fast-acid reaction, as the color was

reddish-purple. This indicated that mycobacterium had mycolic acids present within their cell

walls since the stain carbolfuchsin had successfully entered these cells. (4)

b. Endospore Stain

The endospore stain Malachite green is used to dye spores produced by bacteria. When an older

bacteria culture undergoes nutrient depletion, its response will include production of spores. (5)

Endospore is a dormant form of bacterium that is resistant to adverse environmental conditions.

Steaming the bacterial emulsion on the slide while utilizing endospore staining procedure forced

Malachite green to enter the spore. (5) This steaming step was not performed in this lab since a

prepared slide was provided. Malachite green will only color the spores present on the slide,

since this dye has lower affinity for cellular materials. Vegetative cells and spore mother cells

other than the spores themselves will be colored by the counterstain safranin. (5) A prepared

slide of Bacillus cereus was observed with the microscope under 1000X magnification using oil

immersion. This organism had green dots located in the center of the cell which indicated that
 had nutrient depletion in the Bacillus cereus had caused it to produce coccobacillus shaped

spores in order to survive. (5)

c. Simple Stain

Simple stains are used to color bacteria which have been heat-fixed to the slide. The heat-fixing

step is required to kill bacteria and make them more visible after staining. (1) Methylene blue

was used as simple stain in Bacillus megaterium, Micrococcus luteus, and Escherichia coli.

These three microorganisms appeared blue because of color of the dye, proving that the negative

charges on their surfaces readily absorbed this positively charged basic stain. (1)

d. Negative Stain

Negative stain uses negatively charged dye which can repel the negatively charged surfaces of

bacteria resulting in the appearance of unstained bacteria cells with dark backgrounds. (2)

Nigrosin was used to stain Micrococcus luteus and Bacillus megaterium. Both microorganisms

remained unstained with dark backgrounds after staining with nigrosin. This indicates that both

Micrcoccus luteus and Bacillus megaterium have negatively charged surfaces which repel the

negative charged nigrosine stain. (2)

e. Flagella Stain

Flagella in bacteria are too thin to be observed using regular stains. Mordant can be used to

overcome this problem so that flagella will appear thick enough to be observed. (6) A single

flagellum has a monotrichous arrangement, while flagella emerging from both ends of the cell

have amphitrichous arrangements. Several flagella emerging on one end of the cell have

lophotichous arrangements, while flagella emerging from around the cell have peritrichous

arrangements. (6) The rod-shaped bacteria observed from the prepared slide showed a
 peritrichous arrangement of its flagella, which appear to emerge from all over the surface of

these bacteria. (6)

f. Gram Stain

Gram stain is used to differentiate Gram-positive bacteria from Gram-negative bacteria. Gram-

positive bacteria have a thicker peptidoglycan which makes up their cell walls. Meanwhile,

Gram-negative bacteria have thinner peptidoglycan which makes up their cell walls but have

greater lipid content since these bacteria have an outer membrane. (3) The differences in their

cell wall architecture cause them to look different from one another by performing Gram staining

procedures. E. coli appeared reddish-pink which indicates that these bacteria do not bind the

crystal violet stain. Therefore, it is determined to be a Gram-negative bacteria. The decolorizer

extracted the lipid from outer membrane of Gram-negative bacteria washed off the crystal violet-

iodine complex. Safranin then was used to counterstain E.coli leaving the color pink. (3)

Staphylococcus capitis and Corynebacterium xerosis absorbed the purple color from crystal

violet. This indicates that both microorganisms were Gram-positive bacteria which have thicker

peptidoglycans in their cell walls that trap the crystal violet-iodine complex more effectively,

making them resistant to the decolorizer. (3)

III. REFERENCES

1. Leboffe M, Pierce BE. 2010. Simple Stains, p.100-102. In Microbiology Laboratory

Theory and Application, 3rd ed., Morton Publishing Co., Englewood, CO.

2. Leboffe M, Pierce BE. 2010. Negative Stains, p.103-104. In Microbiology Laboratory

Theory and Application, 3rd ed., Morton Publishing Co., Englewood, CO.

3. Leboffe M, Pierce BE. 2010. Gram Stain, p.105-109. In Microbiology Laboratory

Theory and Application, 3rd ed., Morton Publishing Co., Englewood, CO.
4. Leboffe M, Pierce BE. 2010. Acid-Fast Stains, p.110-114. In Microbiology Laboratory

Theory and Application, 3rd ed., Morton Publishing Co., Englewood, CO.

5. Leboffe M, Pierce BE. 2010. Endospore Stain, p.117-120. In Microbiology Laboratory

Theory and Application, 3rd ed., Morton Publishing Co., Englewood, CO.

6. Leboffe M, Pierce BE. 2010. Flagella Stain, p.124-125. In Microbiology Laboratory

Theory and Application, 3rd ed., Morton Publishing Co., Englewood, CO.