MICROLAB: ORAL EXAMINATION REVIEWER GRAM STAINING
PART 1: Bacterial Smear Preparation
MATERIALS:
●​ Glass Slides
●​ Loop/Needle
●​ Distilled H2O
●​ Dropper
PROCEDURE:
a.​ Heat sterile glass slide
b.​ Place ONE DROP of distilled H2O on the slide
c.​ Place a loopful of bacteria onto the drop of H2O (create a H2O-bacteria mixture)
d.​ Air dry then heat fix
PART 2: Gram Staining
MATERIALS
●​ Bacterial smear
●​ Crystal Violet - 1st stain
●​ Gram's Iodine - mordant
●​ Ethanol (95%) - decolorizer
●​ Safranin - counterstain
PROCEDURE
a.​ Flood bacterial smear with crystal violet. Leave for 1 MINUTE. Then WASH. (V)
b.​ Flood bacterial smear with Gram’s Iodine. Leave for 1 MINUTE. Then WASH. (I)
c.​ Decolorize with Ethanol for 30 seconds (A)
d.​ Counterstain with Safranin for 30 SECONDS. Then WASH. (S)
e.​ AIR DRY, then OBSERVE UNDER MICROSCOPE
ALL ABOUT GRAM STAINING
Gram Staining
★​ procedure enables bacteria to retain color of the stains, based on the differences in
the chemical and physical properties of the cell wall.
★​ Gram Staining is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria
based on staining characteristics, enabling the bacteria to be examined using a
light microscope.
★​ The bacteria present in an unstained smear are invisible when viewed using a light
microscope. Once stained, the morphology and arrangement of the bacteria may be
observed as well.
★​ Gram positive (Gram+) bacteria are stained dark purple due to retaining the primary
dye called Crystal Violet in their cell wall.
○​ Examples of Gram+ bacteria are the Staphylococcus aureus which can cause
acne, Streptococcus pyogenes which can cause “Strep throat” and “Rheumatic
fever”, and Listeria monocytogenes which can cause the food-borne infection
“Listeriosis”.
★​ Gram negative (Gram-) bacteria are stained red or pink due to retaining the counter
staining dye called Safranin.
○​ Examples of Gram- bacteria are the Escherichia coli which is a normal
inhabitants of our intestines (some strains can cause food poisoning),
Treponema pallidum which causes the sexually transmitted disease “Syphilis”,
and Yersenia pestis which can cause the “Bubonic plague”
APPLICATIONS OF THE STAINS USED
★​ Crystal Violet dissociates in aqueous solutions into CV+ and Cl – ions.
○​ These two ions then penetrate through the cell wall and cell membrane of both
Gram-positive and Gram-negative cells.
○​ The CV+ ions later interact with negatively charged bacterial components and
stains the bacterial cells purple.
★​ Iodine acts as a mordant, meaning as a trapping agent for the dye to increase binding to
the cell component being stained.
○​ A mordant is a substance that increases the affinity of the cell wall for a stain by
binding to the primary stain, thus forming an insoluble complex which gets
trapped in the cell wall.
★​ In the Gram stain reaction, the crystal violet and iodine form an insoluble complex
(CV-I) which serves to turn the smear a dark purple color. At this stage, all cells will turn
purple. The underlying mechanism of Gram staining is rooted on the basic
morphological structure and properties of the bacteria’s cell wall.
UNDERLYING MECHANISM OF GRAM STAINING
★​ In a Gram-negative bacteria, the cell wall have a thinner layer of peptidoglycan (10% of
the cell wall) and lose the crystal violet-iodine complex during decolorization with the
alcohol rinse, but retain the counterstain Safranin, thus appearing reddish or pink.
They also have an additional outer membrane which contains lipids, which is separated
from the cell wall by means of periplasmic space.
★​ Meanwhile, Gram-positive bacteria have a thick mesh-like cell wall which is made up of
peptidoglycan (50-90% of cell wall), which stains purple. Peptidoglycan is mainly a
polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl
muramic acid. As adjacent layers of peptidoglycan are formed, they are cross linked by
short chains of peptides by means of a transpeptidase enzyme, resulting in the shape
and rigidity of the cell wall. The thick peptidoglycan layer of Gram-positive organisms
allows these organisms to retain the crystal violet-iodine complex and stains the cells as
purple.
★​ Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive
bacteria which is embedded in the peptidoglycan layer. It consists of teichoic acids which
are long chains of ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as
a regulator of autolytic wall enzymes (muramidases: Bacterial enzymes located in the
cell wall that cause disintegration of the cell following injury or death.).
SUMMARY OF RESULTS
A. STRUCTURE OF BACTERIAL CELL WALL
Feature
Gram-Positive
Gram-Negative
Peptidoglycan Layer
Thick (20-80 nm)
Thin (2–7 nm)
Outer Membrane
Absent
Present (LPS layer)
Teichoic Acids
Present
Absent
Retention of Stain
Retains crystal
violet–iodine complex
Does not retain it after
alcohol wash
Reagent Used
Purpose/Reaction
B. GRAM STAINING PROCEDURE
Step
1.​ Primary Stain
Crystal Violet (purple)
Stains all bacteria initially
2.​ Mordant
Gram’s Iodine
Forms CV-I complex;
larger molecules trap in
thick peptidoglycan
3.​ Decolorizer
Ethanol Alcohol/ Acetone
Gram-positive retains
stain; Gram-negative loses
it
4.​ Counterstation
Safranin (pink/red)
Stains Gram-negative
pink/red
C. RESULTS INTERPRETATION
Result
Color
Example Bacteria
Gram-Positive
Crystal Violet (purple)
Staphylococcus aureus,
Bacillus subtilis
Gram-Negative
Gram’s Iodine
Escherichia coli,
Pseudomonas aeruginosa
Q&A (POSSIBLE QUESTIONS)
1.​ What is the theory about the mechanism of the Gram-stain reaction?
➔​ Theory: The difference in cell wall structure between Gram-positive and
Gram-negative bacteria explains the reaction:
◆​ Gram-positive: Thick peptidoglycan traps the crystal violet-iodine (CV-I)
complex, keeping the cells purple.
◆​ Gram-negative: Thin peptidoglycan and an outer membrane → during
decolorization, the outer membrane dissolves, pores enlarge, and CV-I
complex leaks out → takes up counterstain (pink/red).
2.​ Which step is the most crucial or most likely to cause poor results in the Gram
stain? Why?
➔​ The decolorization step (with alcohol or acetone) is most crucial.
◆​ Over-decolorization → Gram-positive cells may lose stain → appear
Gram-negative (false negative).
◆​ Under-decolorization → Gram-negative cells retain the purple → appear
Gram-positive (false positive).
◆​ Reason: Alcohol disrupts membranes and permeability; timing and
technique are critical.
3.​ Why must young cultures be used when doing a Gram stain?
➔​ Older cultures of Gram-positive bacteria may have weakened or damaged cell
walls, causing them to lose the CV-I complex during decolorization → they may
appear Gram-negative (Gram-variable).
➔​ Young cultures (18–24 hours) give reliable results because their cell walls are
intact.
4.​ What part of the bacterial cell is most involved with Gram staining, and why?
➔​ The cell wall, specifically the peptidoglycan layer.
◆​ It determines whether the CV-I complex is retained (Gram-positive) or
washed out (Gram-negative) during alcohol treatment.
◆​ Thicker = retention; thinner = loss.
5.​ What is the function of the iodine solution in the Gram stain? If it were omitted,
how would staining results be affected?
➔​ Function: Iodine is a mordant, forming an insoluble complex with crystal violet to
help fix it inside the bacterial cell.
➔​ If omitted: Crystal violet would wash out from both Gram-positive and
Gram-negative cells during decolorization → poor or no differentiation.
6.​ What is the purpose of the alcohol solution in the Gram stain?
➔​ Purpose: Decolorizer that differentiates Gram-positive from Gram-negative.
➔​ Dissolves the outer membrane of Gram-negative bacteria.
➔​ Shrinks and tightens the thick peptidoglycan layer in Gram-positive bacteria,
trapping CV-I complex inside.
➔​ Without alcohol → both types stay purple → no differentiation.
7.​ What counterstain is used? Why is it necessary? Could colors other than red be
used?
➔​ Counterstain: Safranin (pink/red).
➔​ Necessity: To stain Gram-negative bacteria, which lost the primary stain → allows
clear visual contrast.
➔​ Other colors? Yes. Other stains like basic fuchsin (pink) or carbol fuchsin can be
used. Color doesn’t matter as long as it contrasts with purple.
8.​ What would be the function of heat-fixing in the preparation of smear?
➔​ Functions:
➔​ Kills bacteria → safer to handle.
➔​ Adheres bacteria to slide → prevents washing off during staining.
➔​ Slightly preserves cellular structure.
➔​ Overheating → can distort cells.
9.​ What do you think would be easier to prepare bacterial smear from, liquid or solid
culture?
➔​ Easier from liquid culture.
➔​ Liquid → already dispersed → ready for spreading.
➔​ Solid → needs to be mixed with a drop of water first to make an even smear.
➔​ However, solid cultures usually contain more concentrated bacteria, useful for
low-density cultures.
10.​Aside from bacteria, do you think that Gram staining can be used with microbes
such as fungi? Explain briefly your answer.
➔​ Possible, but limited use.
➔​ Fungi (like yeasts) have thick cell walls rich in polysaccharides (e.g., chitin,
glucans), which may retain crystal violet, but it’s not reliable for classification.
➔​ Other stains like lactophenol cotton blue, calcofluor white, or PAS are better for
fungi. Gram stain is primarily for bacteria due to its design for peptidoglycan
detection.