GROUP: AS2571A TEAM:

Members :

No Student ID Name Mobile No.

1 2022621802 NURUL ANISSHA BINTI MOHD IDRUS 0177774221

2 2022836244 NUR ATHIRAH BINTI ABD AZIZ 0164314031

3 2022607976 SITI NURMAISARAH BINTI AHMAD SHUKRI 0132853257

LAB REPORT SUBMISSION

Practical #: 2

Title of Experiment: Observation of Microbial Cells

Date of Experiment: 25/10/2022

Submitted to: 1/11/2022

 (include this with every report)

Exceeds
Attribute Meet Expectations Below Expectations Marks
Expectations
Marks 5 3 1
1. Punctuality Submitted early Submitted on time Submitted late
Was the report submitted on time?
2. Organization of report Fully adhere to Mostly adhere to Not organized
Is the prescribed format used? format format

3. Originality of report Original, with creative Mostly written in own More than 50% of
Is the report original, or are most insights words, attempts to report was duplicated
parts duplicated from the manual? summarise verbatim
4. Introduction Good, concise OK. Materials included Inadequate summary
a. Did the students provide summary of but not summarised or /duplicated from
background materials? background too long. sources.
b. Is the objective well described? Clear and accurate Clear, but not accurate Not clear/ inaccurate

5. Methods Well written, clear Acceptable, some High number of

a. Was the methods rewritten in the and organised. organisation. mistakes/ simply
past tense? duplicated the
manual
b. Was the methods accurately Almost all steps were Acceptable, most steps Little attempt to
described? accurately described. were described. describe/ mostly
duplicated the
manual
6. Results
a. Is Figure in proper format? Well formatted, clear Proper format No.

b. Is a title provided? Title is in proper Title is adequate No.

format and accurate
c. Is a caption provided? Well described and Described. No caption or
informative irrelevant caption.
7. Discussion Data were well and Some attempts. mainly No attempt to
a) Analysis - are the data well creatively discussed. descriptive. discuss.
explained?
b) Interpretation - are the significance Significance well Some attempts. mainly No attempt to
of the data discussed? discussed. descriptive. discuss.

8. Conclusion Correct conclusion, Conclusion is provided, No conclusion/

Was a conclusion provided? related to the but not directly related conclusion not
objective to the objective related to objective(s)
9. Grammars, spellings Few, less than 3 Three to 10 mistakes More than 10
(per page) mistakes mistakes
10. References Used proper software Manually with mistakes No or inadequate
Is the report correctly cited? citation
SUM

TOTAL MARKS

Evaluated by : __________________________________________ Date : ________________________

Objectives

i. To prepare a mount of microorganism in a proper way

ii. To operate and examine on a microscope in a proper way
iii. To find features of each microorganism and sketch it

Introduction

Most types of cells do not have much natural pigment and are difficult to see
under the light microscope unless they are stained. Several types of stains are used to
make bacterial cells more visible. It was originally devised by Hans Christian Joachim
Gram, a Danish doctor. One type of staining procedure that can be used is the simple
stain, in which only one stain is used and all types of bacteria appear as the color of that
stain when viewed under the microscope. Some stains commonly used for simple staining
included methylene blue. Apart from Gram staining technique, the identification of bacteria
can also be based on shapes. The three most common shapes are spheres, rods and
spirals.

Methods

A. Direct observation

Bacterial and yeast cells

i. Using a loop, a drop of bacterial broth was transferred onto a clean glass slide.
ii. A smear and air dry were made.
iii. The slide was heated and fixed quickly by passing it and gently through the flame
for a few times.
iv. The slide was covered with methylene blue and was left for 30 seconds.
v. The stain was rinsed off gently under slow running tap water.
vi. The slide was blotted dry carefully with a paper towel.
vii. The slide was observed under a microscope. Low power was started first and then
it was changed to the high power. If necessary, use an oil immersion lens.
viii. The shape of the cells was drawn.
ix. The yeast cells were repeated above.
Mould

i. A drop of methylene blue was added onto a clean glass slide.

ii. The cellophane tape was cutted into about 2 cm long.
iii. The mould culture was touched using the cellophane tape.
iv. The tape was placed onto the prepared glass slide. The sticky side down.
v. The slide was observed under a microscope.

B. Observation of microorganism movement

i. A hanging drop slide of Vibrio broth culture was prepared using a cavity glass slide.
The preparation was demonstrated by the lecturer in charge.
ii. The slide was observed under microscope.
iii. How the bacteria move around was taken note.
iv. Experiment was repeated with B.subtilis broth culture.
v. How it differs from previous bacteria was taken note.
vi. The same procedure was done to sample lake water.
vii. The microorganisms that can be seen under the microscope were sketched as
many as possible.
 Results

A. Direct Observation

Title: Structure of Bacillus subtilis Title: Structure of Aspergillus sp.

FIGURE 1: Bacillus subtilis under FIGURE 2 : Aspergillus sp.under

400X magnification 100X magnification

B.Observation of microorganism movement

Title:Structure of Bacillus subtilis

FIGURE 3: Bacillus subtilis under

400X magnification
Discussion

In activity (A), we are able to know and practice the proper way to prepare a mount
of microorganisms. One safety step that we always need to be aware of is to heat the fix
slides containing Bacillus subtilis and immediately sterilize the loop used to transfer the
Bacillus subtilis using the Bunsen burner flame to avoid microbial contamination from air,
glassware, hands, etc. We are also able to operate and examine the microscope properly
such as use the oil immersion lens with suitable magnitude to enhance the image of the
bacteria. Lastly, we are able to find features of each microorganism that we observed
under the microscope which is Bacillus subtilis and Aspergillus sp. We do use methylene
blue to stain the bacteria to enhance visualization of the Bacillus subtilis and Aspergillus
sp. image under a microscope because unstained bacteria are practically transparent
when viewed using the light microscope and thus are difficult to see.

When we observe Bacillus subtilis by using Gram stain method under 400X
magnification, we can see that it is a rod-shaped bacterium that typically forms small
clumps, short chains, or single cells. By measuring the thickness of the peptidoglycan
layer (in the cell wall), bacteria can be distinguished. The thick layer of peptidoglycan in
the cell wall of Bacillus subtilis causes its cells to look purple when stained with the Gram
method. Next, when we observe Aspergillus sp. (mould) we can see velvety colonies with
a flat surface; an anverse that was greenish-gray and reverse, colorless mycelia, and no
presence of exudates and soluble pigments.

We also observed that the image of Aspergillus sp. (mould) is much clearer than
Bacillus subtilis (bacteria) because Bacillus subtilis is much smaller in size rather than
Aspergillus sp. That is why even if we observe Bacillus subtilis under 400X magnification,
the image is not as clear as Aspergillus sp. under 100X magnification.
 Since bacteria’s image is hard to see under a microscope, we need to put extra
care in doing the experiment. First, we start with purified water and a clean slide. We do
ensure our dropper is entirely clean before releasing the water onto the slide because a
dirty dropper can easily skew our results and make it much more difficult to find what
we’re looking for. Gram staining also can benefit us enabling our bacteria to be seen more
readily through the microscope. Next, regardless of the highest magnification we want to
use, we always start small and focus before we move up to the next level of magnification
as skipping steps will make it considerably difficult for us to focus at higher numbers.
Lastly, we always wear gloves when handling bacteria because it is good practice to wear
gloves any time we use a microscope to prevent dirt and body oils from clouding up our
view.

We also had the chance to observe the movement of Bacillus subtilis under 400X
magnification by applying wet mount method (hanging drop method). Wet mount method
does have its own advantages and disadvantages. Some of the advantages are the
preparation time for a wet mount is minimal. It also enables the observation of living,
moving organisms. Wet mounts also preserve the architecture of the cells so that one
may observe the cellular arrangements of chains or pairs without affecting their
appearance due to heat or staining. The cells actual dimensions and shapes are visible.
On the other hand, wet mounts may be more challenging to photograph or draw on
because of the organisms movability. Additionally, certain species may swim vertically in
the water, moving into and out of the area of focus as a result.

Wet mount method is usually used when we want to determine if the organism is
motile while gram staining is a common technique used to differentiate two large groups
of bacteria based on their different cell wall constituents. Plus, the wet mount method
does need extra care so that we do not kill the bacteria. We usually use depression slides
to complete the wet mount method (hanging drop method) to distinguish true motility of
the bacteria.
 The findings presented here demonstrate that B. subtilis colonies consist of
ordered groupings of cells that move in whirls and jets. Even though the individual
sub-elements were constantly changing, whirls and jets were arranged in relation to one
another to generate a greater design that endured. It was determined which regions were
around whirls and looked at how the motion patterns changed there. All underwent the
same temporal changes: Whirls disorganized, frequently into opposing jets, and then
reformed, typically into a whirl with the opposite direction from that which was initially
present. Jets also interacted with whirls, which, depending on the geometry of the
interaction, either reinforced or disorganized them. Marker particle addition indicated fluid
flows that were either whirls or jets that flowed in the same direction as the cells. Large
portions of the colony pattern were spanned by flows, which suggests that cells can move
over great distances by switching the cell groupings they interact with. When water was
given to dry colonies, sessile cells immediately began to swim and swiftly arranged
themselves into recognisable patterns. Therefore, in populations with high cell densities,
swimming itself seems to control motion patterns. Whirls and jets are located close to the
colony peripheral border, which shows that they have an impact on the boundary's
extension rather than just individual cells swimming motions. This implies that such
organization may be necessary for the formation of sophisticated colony forms.
Conclusion

From this experiment, we have learned proper ways to prepare the mount of
different microorganisms in a proper way to get the best images under a microscope. We
also learned to operate and examine all the microorganisms properly under the
microscope.

For the first experiment, to prepare the sample of Bacillus subtilis, we used a loop
to transfer a drop of bacterial broth on a clean glass slide. Heat the slide by passing it
quickly and gently through the flame to fix the bacteria. Cover the slide with methylene
blue then leave it for 30 seconds before observing it under a microscope. While for mold,
we need to add methylene blue onto the glass slide first. Cut cellophane tape then touch
the mold culture. Placed the tape onto the prepared glass slide before observing under
the microscope. While for experiment 2, we used the wet mount method to observe the
movement of Bacillus subtilis. We prepared the wet mount using the vaseline chamber. In
this method, we use a cavity glass slide instead of the ordinary one. We use the wet
mount method to increase the specimen’s translucency and to make it easier to stain.

In general, we can conclude that microorganisms have a variety of shapes and

features. For example, Bacillus subtilis is a typical germ, which is rod-shaped and
Gram-positive. While Aspergillus sp. often described as conidial fungi are characterized
by the formation of flask-shaped or cylindrical phialides.
References

Kaiser, G. (2020). 2.1: Sizes, Shapes, and Arrangements of Bacteria. Retrieved from
https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A Microbiology
(Kaiser)/Unit 1%3A Introduction to Microbiology and Prokaryotic Cell
Anatomy/2%3A The Prokaryotic Cell

Mendelson NH, Bourque A, Wilkening K, Anderson KR, Watkins JC. Organized cell
swimming motions in Bacillus subtilis colonies: patterns of short-lived whirls and
jets. J Bacteriol. 1999 Jan;181(2):600-9. doi: 10.1128/JB.181.2.600-609.1999.
PMID: 9882676; PMCID: PMC93416.

N. McClenny, Laboratory detection and identification of Aspergillus species by

microscopic observation and culture: the traditional approach, Medical Mycology,
Volume 43, Issue Supplement_1, January 2005, Pages S125–S128,
https://doi.org/10.1080/13693780500052222