CHESSIE R.

APLOD Microbiology Laboratory W 1:30-4:30 PM

COMPARISON
I. GRAM STAINING TECHNIQUE VS ACID FAST TECHNIQUE in terms of:

Gram Staining Technique Acid-Fast Technique

Primary Stain Crystal violet Carbolfuchsin

Mordant Iodine A mordant is not required.

Decolorizer Ethyl alcohol or acetone Acid-alcohol

Counter Stain Safranin Red Methylene Blue

Specific Gram-positive bacteria Mycobacteria

Bacteria
Gram-negative bacteria
Used/Tested

Diseases Venereal Disease Tuberculosis

Acquired
Bacterial Meningitis Leprosy
 Anthrax.
 Diphtheria.
 Enterococcal infections.
 Erysipelothricosis.
 Listeriosis.

Chemical The Gram-negative cell wall is The acid-fast cell wall consists of a
Component of composed of a thin, inner layer of thin, inner layer of peptidoglycan
the Cell Wall peptidoglycan and an linked to a layer of arabinogalactin,
outer membrane consisting of which in turn is linked to an outer
molecules of phospholipids, membrane containing
lipopolysaccharides (LPS), mycolic acids and overlaid with a
lipoproteins and surface proteins. variety of polypeptides and
Chemically, 60 to 90% of the Gram- glycolipids.
positive cell wall is peptidoglycan.

Color Reaction Gram positive cells appear as blue Acid-fast cells appear as red coloured
coloured while gram-negative cells while non-acid-fast cells appear as
appear as red coloured. blue coloured.
REFERENCES:
https://www.chegg.com/homework-help/compare-contrast-gram-stain-acid-fast-stain-chapter-
3-problem-10lo-solution-9780321802996-exc
Dixit, Praveena & Kotra, Lakshmi. (2011). Diseases Caused by Acid-Fast Bacteria. xPharm: The
Comprehensive Pharmacology Reference. 1-5. 10.1016/B978-008055232-3.60888-8.
http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/afcw.html
CHESSIE R. APLOD Microbiology Laboratory W 1:30-4:30 PM

II. ENDOSPORE STAINING VS CAPSULE STAINING

Endospore Staining Capsule Staining

Primary Stain Malachite green India ink/crystal violet

Mordant Heat A mordant is not required.

20% copper sulfate
Decolorizer Water
20% copper sulfate
Counter Stain Safranin Red
 Escherichia coli (in some strains)
Specific Endospores are dormant alternate life
Bacteria forms produced by 
the Neisseria meningitidis.
Used/Tested genus Bacillus, the
 Klebsiella pneumoniae.
genus Clostridium, and a number  Haemophilus influenzae.
other genera of bacteria, including
 Pseudomonas aeruginosa.
Desulfotomaculum, Sporosarcina,
 Salmonella.
Sporolactobacillus, Oscillospira, and
Thermoactinomyces.

Diseases Infectious diseases such Pneumonia

Acquired as anthrax, tetanus, gas
Otitis media
gangrene, botulism, and
pseudomembranous colitis are Meningitis
transmitted to humans by
endospores.

Chemical The endospore consists of the It usually consists of polysaccharides,

Component of bacterium's DNA, ribosomes and but can be composed of other
the Cell Wall large amounts of dipicolinic materials such as glycoprotein,
acid. Dipicolinic acid is a spore- polypeptide D-glutamic acid in B.
specific chemical that appears to help anthracis, and peptidoglycan and
in the ability for endospores to muramic acid found in E. coli
maintain dormancy. This chemical bacterial capsule.
accounts for up to 10% of the spore's
dry weight. Endospores can survive
without nutrients.
The background will be dark (color of
Color Reaction Whereas the counterstain (safranin)
india ink). The bacterial cells will be
is pink/reddish in color, the primary
 stain (malachite green) is green in
color. Therefore, endospores will
appear green in color while the
vegetative cells will pink/reddish in
color under the microscope.
stained purple (bacterial cells
takes crystal violet-basic dyes as
they are negatively charged). The
capsule (if present) will appear clear
against the dark background (capsule
does not take any stain).

HOW WILL YOU KNOW WHETHER THE ENDOSPORE IS STILL IN THE VEGETATIVE STAGE OR
MATURE STAGE?
Unlike vegetative cells, endospores are very dehydrated structures that are not actively carrying
out metabolism. Their composition makes them resistant to heat, radiation, acids, and many
chemicals. In fact, disinfectants that normally harm or kill vegetative cells cause no harm
to endospores.
ASIDE FROM USING INDIA INK STAIN FOR CAPSULE STAINING, WHAT ARE OTHER STAINS THAT
CAN BE USED? The primary stain can be crystal violet, and all parts of the cell take up the purple
crystal violet stain.

REFERENCES
https://microbeonline.com/bacterial-spores-structure-importance-and-examples-of-spore-
forming-bacteria/
https://microbeonline.com/capsule-stain-principle-procedure-results/
https://www2.hawaii.edu/~johnb/micro/m140/syllabus/week/handouts/m140.6.1.html
Anthony, E. E. Jr. 1931. A note on capsule staining. Science (New Series) 73(1890) :319–320.
Moat, G. A., and J. W. Foster. 1995. Microbial physiology, 3rd ed. Wiley-Liss Inc, New York, NY.