Exercise 6
Post-Laboratory Discussion
LUNA, IRISH MAE P. | MCB 101 - E1L
TABLE OF CONTENTS
01 INTRODUCTION 02 METHODOLOGY
RESULTS AND
03 DISCUSSION 04 RECOMMENDATIONS
INTRODUCTION
01 Definition and Functions of a Bacterial Cell Wall
Differentiation of Gram-Positive and Gram-Negative Cell Wal
Methods of Determining Gram Reaction
INTRODUCTION: Bacterial Cell Wall
● Fairly rigid layer that lies outside the
plasma membrane
● Mainly composed of peptidoglycan
layer/murein
- Backbone: N-acetylglucosamine
(NAG) and N-acetylmuramic acid
(NAM)
- Linkage: inter-peptide bridges
- Carrier molecule: bactoprenol
INTRODUCTION: Bacterial Cell Wall
- Protective shield against hostile environment
(toxic substances and pathogens)
- Allows communication with its surroundings
- Confers to the cell shape
- Aids transport of molecules into and out of the cell
- Protects against osmotic lysis
INTRODUCTION: Gram-Positive Cell Wall
Image Source: [Link]
INTRODUCTION: Gram-Negative Cell Wall
Image Source: [Link]
INTRODUCTION: Gram + VS. Gram - Cell Walls
INTRODUCTION: Gram + VS. Gram - Cell Walls
INTRODUCTION: Gram Staining Method
- Developed by Hans
Christian Joachim Gram
(1884)
- Most useful and widely
used differential staining in
bacteriology (gram-positive
vs. gram-negative)
- Valued because of its
efficiency and availability of
reagents
INTRODUCTION: Gregersen’s Method
- Also called KOH string
test/rapid method for gram
reaction differentiation
- Advantages: simple, rapid,
inexpensive
- Disadvantages: older
cultures may appear + after
30 seconds; KOH should be
freshly prepared
- w/slime (gram -)
- w/o slime (gram +)
METHODOLOGY
02 Bacterial Cultures, Culture Medium, Reagents and Stains
Schematic Diagrams
METHODOLOGY: Materials Used
Bacterial Cultures:
Reagents and Stains:
- Bacillus megaterium
- Freshly prepared 3%
- Escherichia coli
KOH
- Unknown bacterium
- Gram staining set
(crystal violet, iodine, 95%
Culture Medium:
alcohol, safranin)
- Nutrient Agar (NA) slant
METHODOLOGY:
Gram Staining
Procedures
METHODOLOGY:
Gregerson’s Test
Procedures
RESULTS & DISCUSSION
03 Gram reactions and KOH reactions of the Bacterial Cultures
Answer to Guide Questions
RESULTS AND
DISCUSSION:
Bacillus megaterium (1000x)
Gram staining reaction color:
purple
Cell shape: rod-shaped
Cell arrangement: streptobacillus
KOH reaction: without slime
Inference: GRAM POSITIVE
RESULTS AND
DISCUSSION:
Escherichia coli (1000x)
Gram staining reaction color:
pink
Cell shape: rod-shaped
Cell arrangement: bacillus
KOH reaction: with slime
Inference: GRAM NEGATIVE
GQ 1: Can you vary the primary stain and
counterstain in the Gram staining procedure?
Explain.
Primary Stain (Crystal Violet)
- Stain color: violet/purple
- Retained by: peptidoglycan layer of gram + bacteria
- Alternatives: gentian violet or basic fuchsin
Counterstain (Safranin)
- Stain color: pink/red
- Retained by: gram - bacteria
- Alternatives: fuchsin, malachite green, or methyl green
GQ 2: What would be the effect of replacing iodine
with other oxidizing agents?
- Incomplete staining = poor color retention
- Overdecolorization = gram-positive and
gram-negative bacteria are both colorless
- Formation of unstable complexes = inconsistent
staining patterns and unreliable results.
- Altered affinity for counterstain = final coloration
of Gram-negative bacteria.
GQ 3: What is the importance of freshly prepared
KOH?
If the KOH solution is not freshly prepared and
its pH has increased due to the absorption of
carbon dioxide, it may not effectively lyse the
cells of Gram-negative bacteria, leading to
inaccurate results (Dash 2016; Talib, 2019).
GQ 4: What is the influence of pH and age of the
cultures on Gram reaction?
pH
- Excessively high = prevent Gram-negative
bacteria from taking up crystal violet
- Excessively low = washed-out staining; hardness
in distinguishing
Age
- Too old = cell walls are not intact (autolysis);errors
in staining
- Too young = insufficient cell wall development;
inaccuracy in results
-
GQ 5: What is the significance of using
known/reference cultures in Gram staining?
Microbiologists can make preliminary identifications.
This information is valuable for determining the
appropriate follow-up tests and analyses required for a
more precise identification.
RECOMMENDATIONS
04 Experimental Errors
Ways to Minimize Experimental Errors
EXPERIMENTAL ERRORS
Too heavy/too Too old bacterial
light smear cultures
Direct flooding
Too old
of water after
reagents
staining
Over-
Overdecolorizing
counterstaining
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
● Bartholomew, J. W. and Finkelstein, H. (1958). Relationship of cell wall staining to Gram differentiation. J. Bacteriol., 75(1): 77-84.
● 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.
● Giligan, P. H. 2013. Identification of Pathogens by Classical Clinical Tests. The Prokaryotes – Human Microbiology.4 th ed. Springer
Science+Business Media Inc., New York, NY.
● Gram stain | Principle | Steps | Interpretation | Tips ([Link])
● 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.
● Moat, A.G., Foster, J.W., Spector, M.P., 2002. Microbial Physiology. 4 th ed. Wiley-Liss, Inc. New York, NY.
● 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).
