Exercise 6: Bacterial Cell Wall

Good morning everyone! Today, we will conduct our post-laboratory discussion on Exercise 6:
Bacterial Cell Wall. Our discussion will start by introducing some concepts related to the aforementioned
exercise, the process we’ve performed (which we will not be focusing on kasi nadiscuss na ito nung
prelab), the results obtained after performing the exercise and the discussion of the principle behind those
results, as well as the errors we’ve might experienced upon the conduct of the exercise.

So, for the introduction, let’s focus on: (1) Definition and function of the bacterial cell wall, (2)
Gram + vs gram - cell wall, and (3) the methods of determining gram reaction.

INTRODUCTION

A. Bacterial Cell Wall (read slides)

Peptidoglycan, from the word itself, peptido + glycan, is a huge polymer of disaccharides
(glycan) cross-linked by short chains of identical amino acids (peptides) monomers. Its backbone
is composed of two derivatives of glucose: N-acetylglucosamine (NAG) and N-acetylmuramic
acid (NAM) with a pentapeptide coming off NAM and varying slightly among bacteria. The NAG
and NAM strands are synthesized in the cytosol of the bacteria. They are connected by
inter-peptide bridges. They are transported across the cytoplasmic membrane by a carrier
molecule called bactoprenol.
(read functions; simplify)
B. Gram + vs Gram -

Gram-Positive Cell Wall (parts unique)

● Teichoic acid - negatively charged polymers of glycerol or ribitol phosphate that are
covalently linked to peptidoglycan; contribute to the overall negative charge of the cell
surface (reason why gram + bacteria retains basic/positively-charged crystal violet)
● Lipoteichoic acid - anchored in the cytoplasmic membrane; have a role in cell wall
stability and adherence to surfaces.

Gram-Negative Cell Wall (parts unique)

● Lipopolysaccharide - LPS molecules are large, complex molecules located in the outer
leaflet of the outer membrane; plays a crucial role in protecting the bacterium from host
immune defenses and is a major component of the endotoxin responsible for the toxic
effects of Gram-negative bacterial infections.
● Porins - protein channels embedded in the outer membrane that allow the passage of
small hydrophilic molecules, including nutrients and ions, into the periplasmic space;
contribute to the selective permeability of the outer membrane.
 Gram-Positive VS. Gram-Negative Cell Wall (read while pictured of each cell walls
are flashed)

C. Methods of Determining Gram Reaction

Gram Staining Method (elaborate what’s indicated on the picture)

● Primary Stain (Crystal Violet): imparts a violet color to all bacterial cells
● Iodine Treatment (Mordant): enhances the retention of the crystal violet stain by
forming an insoluble complex, making it difficult for the stain to be washed out
during the decolorization step
● Decolorization: critical step that distinguishes between Gram-positive and
Gram-negative bacteria. Gram-positive bacteria have a thick peptidoglycan layer
that traps the crystal violet-iodine complex, making them resistant to
decolorization. Gram-negative bacteria, with their thinner peptidoglycan layer and
outer membrane, are unable to retain the stain and become colorless after
decolorization.
● Counterstain (Safranin): After decolorization, the colorless Gram-negative
bacteria need to be restained to be visible under the microscope. Safranin, a
contrasting red dye, is applied as a counterstain.

Gregersen’s Method
In the presence of potassium hydroxide, Gram negative cell walls are
broken down, releasing viscid chromosomal material which causes the bacterial
suspension to become thick and stringy. Gram positive organisms remain
unaffected. Hence the alternative name for this procedure, the “String Test”
The Potassium hydroxide test has its advantages; it is simple and easy
to use, rapid and inexpensive. In laboratories where large numbers of cultures
have to be processed, the above test may be used in addition to Gram stain for
preliminary differentiation.

METHODOLOGY
A. Bacterial Cultures, Culture Medium, Reagents and Stains
B. Schematic Diagrams
 RESULTS AND DISCUSSION
A. Gram Staining Method
B. Gregersen’s Method
C. Answer to Guide Questions

RECOMMENDATIONS
1. To obtain accurate Gram staining results, it is crucial to prepare a smear that provides a
thin, even layer of bacterial cells without overlapping. A well-prepared smear ensures
proper staining penetration, allowing for clear visualization of individual cells and accurate
differentiation between Gram-positive and Gram-negative bacteria.
2. If the primary stain is washed away, subsequent steps in the Gram staining process
(decolorization and counterstaining) will not yield meaningful results. Without the primary
stain, there won't be any stain left to be retained by Gram-positive bacteria, and the
counterstain won't be able to differentiate Gram-negative bacteria either.
3. The step that is most crucial in effecting the outcome of the stain is the decolorizing step.
Over-decolorizing will lead to an erroneous result where gram-positive cells may stain
pink to red indicating a gram-negative result, and under-decolorizing will lead to an
erroneous result where gram-negative cells may appear blue to purple indicating a
gram-positive result.
4. Already discussed
5. Outdated reagents may lose their potency, resulting in reduced staining intensity.
Gram-positive bacteria might appear Gram-negative or vice versa, leading to
misclassification.Changes in pH can impact the binding affinity of dyes to bacterial cells,
leading to inconsistent staining.
6. Various formulations of decolorizing agents may be used (acetone, acetone/ethanol,
ethanol). Acetone is the most rapid decolorizer followed by acetone/ethanol and then
ethanol. Ethanol is recommended for student use to prevent over-decolorization of
samples

REFERENCES:

American Society for Microbiology Conference for Undergraduate Educators (ASMCUE). 2005. [cited August 10, 2005] 2. Arthi K,
Appalaraju B, Parvathi S. Vancomycin sensitivity and KOH string test as an alternative to gram staining of bacteria. Indian
J Med Microbiol 2003;21:121-123

Cotter, P. D., & Hill, C. (2003). Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiology and molecular
biology reviews, 67(3), 429-453.

Dash, C., & Payyappilli, R. J. (2016). KOH string and Vancomycin susceptibility test as an alternative method to Gram staining.
Journal of International Medicine and Dentistry, 3(2), 88-90.

Halebian, S., Harris, B., Finegold, S. M., & Rolfe, R. D. (1981). Rapid method that aids in distinguishing Gram-positive from
Gram-negative anaerobic bacteria. Journal of clinical microbiology, 13(3), 444-448.

Stearn, E. W., & Stearn, A. E. (1924). THE CHEMICAL MECHANISM OF BACTERIAL BEHAVIOR I. BEHAVIOR TOWARD
DYES—FACTORS CONTROLLING THE GRAM REACTION. Journal of Bacteriology, 9(5), 463-477.

Talib, R. A., & Abeid Abiess, A. A. (2019). Application of a Rapid Method for Gram Differentiation of Human Pathogenic and
Non-Pathogenic Bacteria without Staining. Indian Journal of Public Health Research & Development, 10(4).