Laboratory Activity No.

7
Title Bacterial Smearing and Gram Staining Technique (Virtual
Laboratory)

Name: Vigel Ann F. Villenas Date conducted/answered: 12-05-24

Year and Section: BSED-Science III Date submitted: 12-06-24

Objectives
At the end of this laboratory experiment, the students must be able to:
1. Enumerate the materials and procedures in bacterial smearing.
2. Enumerate the materials and procedures in gram staining.
3. Explain the importance of bacterial smearing.
4. Discuss the importance of gram staining.
Overview
You are aware of the characteristics of microorganisms and their
potential benefits and risks. Aseptic techniques were discussed and
practiced to avoid contamination and infection. One of these measures is
by ensuring a sterile field of work and discarding used materials in their
respective containers. In connection to that, you prepared slides to be
studied under the microscope. However, those slides contain non-
pathogenic cells, meaning they have no potential to cause diseases.
However, to collect and compile literature about the microorganisms (such
as bacteria, fungi, viruses) is to study them further and closely under the
microscope. This practice is risky as these microbes are potential
pathogens. Hence, procedures must be done to produce microbial slides
while minimizing risk of infection or contamination.
In 1877, Robert Koch introduced air-drying, chemical fixation, and
staining to investigate microbial morphology while maintaining aseptic
measures. This virtual laboratory will orient you about bacterial smearing
and gram staining technique.
Engaging
Check out this video for the virtual laboratory:
Bacterial Smearing: https://youtu.be/on5-5oQNNqo
Gram Staining Technique: https://youtu.be/_s0KgMcB8xg

Bacterial Smearing
Materials used:
 Distilled Water
 Inoculating Loop
 Permanent Marker
 Glass Slide
 Staining Stray
 Bunsen Burner
 Slide Warmer
 Bacterial Culture
 Gram Staining
Materials used:
 Distilled Water
 Crystal Violet Stain
 Gram’s Iodine
 95% Ethanol (decolorizer)
 Safranin Stain
 Bibulous Paper
 Staining Tray
 Pipette
 Bacterial Smear
 Timer/Watch

Exploring
With the knowledge gained on bacterial smearing and gram staining, your
task is to create a flow chart summarizing the major steps taken in
preparing a bacterial smear. Make sure to be creative in creating your flow
chart and limit up to five steps (per topic) only with brief explanation.

Steps in Bacterial Smearing

 Distilled water, Innoculating loop, Permanent
Preparation Marker, Glass Slide, Staining stray, Bunsen burner,
of Materials Slide warmer and Bacterial culture.

Preparation  Take a clean slide and draw a circle in

of Slides the middle and label it.

 Using a sterile loop, add a

Apply
small drop of sterile water
Water Drop
to the slide.
  Using the loop, collect a small
Transfer amount of bacteria.
Bacteria  Mix it into the water (for solid
culture) or apply directly (for
broth culture).
 Heat and fix the
Air Dry and
slide through
Fix the Slide running it on the
flame three
times.

Explanation:
To conduct the bacterial smear experiment, first gather necessary
materials and clean the microscope slides with alcohol. Using a
permanent marker, draw a circle at the slide's center and apply two or
three bacterial smears within this circle. Label the slide accordingly. If
using a solid agar culture, add water to the slide; if using a liquid nutrient
broth culture, no water is needed. Next, sterilize the inoculating loop by
running it through flame, allowing it to cool. The optimal water amount to
use for the smear is the volume that fits on the loop's end. After cooling,
run water down the loop until it accumulates. Flame the loop again to
maintain sterility before collecting the bacterial culture. Open the slant
tube while flaring its mouth to prevent contamination, then gently take a
small amount of the agar culture. Mix this with the water drop on the
slide, sterilize the loop to remove excess bacteria, and allow the slide to
air dry on a slide warmer. Finally, heat-fix the slide by passing it through
a flame three times.

Steps in Gram Staining

Explanation:
First, gather the necessary materials needed for this experiment. The
following step is to apply the crystal violet to the bacteria for staining.
Next apply the iodine referred to as mortin. Presently, the bacteria with
thick cell walls appear purple. Finally, apply the decolorizer for 12
seconds, specifically 95% ethanol. What this does is it will remove the
stain off the thin cell wall bacteria in 12 seconds effectively, but the thick
cell wall remains unaffected. At this point, after performing this step, the
thick cell wall bacteria will have a purple color while those with thin cell
walls remain colorless. Lastly, we have safranin, the pink staining dye
that would color any bacteria other than purple-stained ones, to be
referred to as gram-positive, which in contrast means they have thick
cell walls, while gram-negative represents those that appear pink on the
 slide as their walls are thin. The next step is to place the slide onto the
bibulous paper and gently press down. The final step is to examine the
specimen under the microscope. Bacteria with thick cell walls appear
purple and are gram positive, while those with thin cell walls are pink
and gram negative.

Explaining
Read the following questions carefully and answer in not more than three
sentences.

1. Why must only thin and even sheet of bacteria be smeared on the
glass slide?
A thin and even spread is necessary to stain properly and thus easily
visualize under the microscope; thick smears allow overlying cells that
cause impossibility in viewing their detailed morphology and arrangement;
as well, an irregularly spread smear can present varying staining that can
create poor identification.

2. It is advised to put only a small amount of water in the glass slide

for it to evaporate quickly. Why is it not advisable for the bacteria-
smeared glass slide to sit for longer period before being heat-fixed?
If left too long before heat-fixation, the smear will air-dry excessively
and may not adhere well to the slide, which might lead to the loss of
bacteria when stained and washed. Again, extended drying might cause
cell distortion, interfering with the correct observation of the bacterial
morphology.

3. A technique in heat-fixing is crucial. Why is it not advisable to heat-

fix the bacterial smear for a longer period?
Overheating during heat-fixing could damage bacterial cells, and this
can cause them to shrink or distort, which would interfere with the accurate
observation of their morphology. This could also lead to proteins getting
denatured excessively; thus, the smear cannot adhere well to the slide,
leading to uneven staining and potential loss of the smear during the
washing steps.

4. Why must the decolorizer be applied for only a specific amount of

time?
Decolorizer may be applied too long over the smear resulting in its
over-decolorization causing loss of color of stain for both Gram-positive
and Gram-negative. On applying for a period too small, Gram negative
bacteria do retain the principal stain that may lead to wrong
identification. Proper duration ensures the decolorisation of only Gram-
negative cells while leaving Gram-positive to retain principal stain for
distinction.

5. Differentiate gram-positive and gram-negative bacteria

Gram-positive bacteria have a thick peptidoglycan layer in the cell
walls that retains the crystal violet stain, so they appear purple under the
microscope. Gram-negative bacteria, on the other hand, have a thinner
 peptidoglycan layer and an outer membrane that results in losing the
crystal violet but taking up the safranin counterstain, making them pink.
This structural difference also impacts their susceptibility to antibiotics and
other treatments.

Expanding
To further your understanding on gram staining, fill-out the table below by
stating the function of the following materials and the time it stays on the
bacterial smear before rinsing:

Substance Function Time

Crystal Crystal violet is the principle primary
violet stain that is associated within the Gram
staining process for purposes of 1 minute
identifying Gram-negative vs. Gram-
positive.

Iodine Iodine acts as a mordant in the Gram

staining process. It forms a complex with
crystal violet, thereby increasing the
retention of the dye within gram-positive 1 minute
bacteria. This is to ensure that the stain
adheres more strongly to the thick
peptidoglycan layer.

Decolorizer The decolorizer, 95% ethanol, dissolves

the lipid layer of gram-negative
bacteria's outer membrane and
dehydrates the peptidoglycan layer, thus
letting the crystal violet-iodine complex 12 seconds
out. In gram-positive bacteria, the thick
peptidoglycan layer holds the complex,
so their color remains purple.

Safranin Safranin is a counterstain in the Gram

staining process used to color gram-
negative bacteria pink after losing the
primary crystal violet stain during 45 seconds
decolorization. This helps differentiate
gram-positive and gram-negative
bacteria in the microscope.