LAVALLE, Jestin B.

BMLS Intern Microbiology

Gram Staining

1. Describe the peptidoglycan layer of the bacterial cell wall.

Peptidoglycan is a complex of polymer consisting of backbone, which is composed of alternating N-acetylglycosamine and
N-acetylmuramic acid connected by B-1,4 linkages; Set of identical tetrapeptide sidechains attached to N-acetyl muramic
acid and set of identical peptide cross bridge

Gram Pos (+) cell wall Gram Neg (-) cell wall
Peptidoglycan (20-40 layers) > Plasma membrane Outer membrane (Periplasmic space) > Peptidoglycan (2-4
laters) > Plasma membrane

Techoic Acid – antigenic/serologic determinant

2. Principles behind the difference in stain uptake of the Gram positive bacteria and Gram negative bacteria.

Gram Pos (+) Gram Neg (-)

Retains primary stain which is crystal violet iodine complex
even it undergo decolorization with alcohol or acetone
Dark Blue or Violet
Do not retain crystal violet-iodine complex. Removed with
alcohol or acetone
Gram negative cell wall opens when the decolorizer is
added, so it will washes out the stains
Red due to safranin

Position Gram Pos (+) Gram Neg (-)

1 L - alanine L - alanine
2 D - glutamate D - glutamate
3 L - lysine Diaminepimelic acid
4 D - alanine D - alanine
DAP – immediate precursor of lysine biosynthesis

3. Discuss the procedure in Gram staining.

4. Explain briefly the general rule in Gram staining in relation to bacterial morphology.

 All COCCI are gram POS (+) except: Neisseria spp, veilionella spp and Bramhamella spp, Acidaminococcus,
Megasphera

 All BACILLI are gram NEG (-) except: Mycobacterium, Corynebacterium, Clostridia, Bacillus, Listeria,
Erysipelothrix, Lactobacillus, Actinomyces, Eubacterium, Propiobacterium, Mobiluncus, Bifido-bacterium

 All SPIROCHETES are gram NEG (-)

LAVALLE, Jestin B. BMLS Intern Microbiology

5. What are gram variable bacteria?

Gram (+) bacteria becomes Gram (-) bacteria (vice versa)

o Ex. Gardnerella Vaginallis

o Mix of pink or purple cells: Actinomyces, Corynebacterium (+)  (-)
 Long period of hours leaks/damages cell wall leading to change in color (Neg)

6. Discuss the factors that contribute to the Gram variability of a bacteria.

 Autolysis
 Aging – decreased peptideglycan layer
 Temperature of incubation results to loss of gram (+)
 pH – change
 Technical errors
 Incomplete decolorization
 Thick smear
 Removal of MgRNA with bile salts