LABORATORY REPORT

MIC 125
GENERAL MICROBIOLOGY

PRACTICAL 2
EXAMINATION OF LIVING BACTERIAS

Group Members 1. NUR FARAH WAHEEDA BINTI JAIS ANUAR

(2023300871)
2. WAN SYAZAQISTINA BINTI W AB GHAFAR
(2023300959)
3. NURUL WAHEEDA BINTI SAIDI
(2023173357)
4. NUR HUSNA AFIQAH BINTI ROSLAN
(2023184477)
5. NOR ASMIERA EZZATY BINTI MOHD ASWANDY
(2023168065)
Group N3AS1142A1
Lecture MADAM SITI NORAZURA BINTI JAMAL
Date of Experiment 19th OCTOBER 2023
Date of Submission 3rd NOVEMBER 2023
OBJECTIVES
1. Prepare and observe wet-mount slides and hanging -drop slides.
2. Distinguish between true motility and Brownian movement.

INTRODUCTION
Bacteria are a species of fungi coming under the division of the fission fungi. Besides the
fission there are two other varieties the mold and budding fungi. Bacteria live all around us and
inside us. Brightfield microscopy is a widely used technique where objects appear dark against a
bright background. It is suitable for unstained samples but lacks contrast. In contrast, In phase-
contrast microscopy enhances contrast by highlighting differences in refractive index, making
structures within cells more visible without staining. It is a valuable tool for observing live cells
and their internal details.
In this experiment, wet-mount techniques will be used to observe different environments
to help aware of the numbers and varieties of microbes found in nature. The microbes will exhibit
either Brownian movement or true motility. Brownian movement is not true motility but rather
movement caused by the molecules in the liquid striking an object and causing the object to shake
or bounce. Brownian movement and occasionally the cells may spin or roll.
Many microbes such as bacteria, fungi, algae and protozoa can be found in pod water and
in infusions of organic matter. Direct examination of living microorganisms is very useful in
determining size, shape and movement. Using a wet mount is a fast way to observe bacteria, but
motility is difficult to determine because of the movement of the water. The historical context
provided about Anton van Leeuwenhoek's work with microorganisms is fascinating. Direct
examination of living microorganisms can indeed provide important information about their size,
shape, and movement. However, determining motility in a wet mount can be challenging due to
the movement of the water medium. This is where more advanced microscopy techniques like
phase-contrast or dark-field microscopy can be helpful in studying motile microorganisms. (The
microscopes of Antoni van Leeuwenhoek.)
 MATERIALS
 Compound microscope
 Cavity glass slides
 Cover slips
 Inoculating loop
 Petroleum Jelly
 Paper towels
 Applicator sticks
 Disposable pipettes
 24 hours nutrient broth cultures of :
1. Staphylococcus aureus
2. E.coli

PROCEDURE

A. Wet Mount Techniques

I. The nutrient broth where that contained bacteria was suction up using disposable pipetted
and small drop of the nutrient broth was transferred to the center of a glass slide
II. A coverslip placed carefully on top of the drop of the fluid.
III. The slide has been placed on the stage of the microscope and it was been observed with a
lowest power.
IV. The iris diaphragm was adjusted truthly so that only a small amount of light was admitted.
V. Concentrated your observations on the larger, more rapidly moving organisms. At this
magnification, bacteria were barely discernible as tiny dots.
VI. All magnifications were observed and has been recorded
VII. The slide that was observed with used the oil immersion lens.
VIII. The observed has been recorded including the relative size and shape of the organism.
IX. The wet mount was made from the other bacterial broth and was observed it. while it using
the low and high power objectives. The observations were recorded.
 B. Hanging Drops Techniques

I. A clean of cavity glass slide was obtained.

II. A small amount of petroleum jelly has been picked up by using an applicator stick.
III. By using the technique demonstrated by the instructor, the four edges of the coverslip
was smeared with petroleum jelly.
IV. The coverslip was placed on the paper towel with the petroleum jelly side-up.
V. Transferred a small drop of the bacterial broth into the coverslip by using an inoculating
loop.
VI. Placed the cavity glass slide over the drop and it was flipped over.
VII. The drop were examined under low power by locating the edge of the drop and moved
where to make sure the edge of the drop crossed the center of the field.
VIII. Reduced the light with the iris diaphragm and focus.
IX. Observed the different sizes, shapes and types of movement.
X. The microscope was turn switched to high power and been observation was recorded.
XI. By using a new coverslip, the procedure were repeated with the culture of another bacterial
broth. The observations from the experiment was recorded
XII. The microscope has been cleaned and returned it to the proper cupboard.
RESULTS

A. Wet Mount Techniques

E.coli, 100x objective lens

S.aureus, 100x objective lens

B. Hanging Drops Techniques

E.coli, 10x objective lens

DISCUSSIONS

In this task, we were required to prepare slides of live bacterial culture such as
Staphylococcus aureus and E.coli using the hanging drop technique and temporary wet mount
technique before observing them under the microscope.
When we observed the prepared slides under the microscope, none of the bacteria we
observed displayed any kind of motility. This is most likely due to errors occurring during the
experiment. One of the errors which we overlooked was the light intensity of the microscope,
which was too high and thus killed the bacteria when we were viewing them using the microscope.
Another error that occurred was that the inoculating loop was too hot when we used it to obtain
the bacterial culture from its tube, thus killing the bacteria.
Hypothetically, if the experiment was carried out precisely, motility of bacteria could be
observed. The bacteria Staphylococcus aureus would appear to be moving in a dendritic way, in a
process called sliding that resembles Brownian movement. The bacteria E.coli would display true
motility, or flagellar motility, which means it can move itself through the means of its peritrichous
flagella, propelling itself towards a particular direction.

QUESTIONS:
1. How would you describe in words the size and shape of the microorganisms that you see
down the microscope?
The size and shape of the microorganisms that has been was so amazing and looked great
which were the E-Coli shape was bacillus where there was likes something long or rods
but in same size for each other around 2 micro meter until 5 micro meter. But the shape of
S.Aureus was cocci where it was looked like a sphere and its sizes was very small. When
we observed the microorganisms using 100x , the shape looked clearly and amazing .

2. Do you think the microorganisms are alive?

Microorganism were alive this is because microorganisms maintain the basic functions of
life. For example, microorganisms were react to external stimuli.

3. The movement of bacteria towards nutrients is known as Motility.

CONCLUSION

In conclusion, the skills and techniques of wet-mount slides and hanging-drop slides have
been prepared and observed in this experiment. The wet-mount slides allow us to view microscopic
living things without them drying out and help humans to learn their movement and behaviour
under microscopic. The hanging-drop slides help to observe the general shape of living bacteria
and the characteristic arrangement or groupings of cells.

REFERENCES

1. Articles
i. NELSON SN. METHODS OF EXAMINATION OF BACTERIA FOR LABORATORY
PURPOSES. Based upon Notes taken in Prof. Koeh's Laboratory in Berlin. JAMA. 1888
ii. Dunn, R. R., Fierer, N., Henley, J. B., Leff, J. W., & Menninger, H. L. (2013). Home Life:
Factors Structuring the Bacterial Diversity Found within and between Homes. PLOS ONE,
8(5), e64133. https://doi.org/10.1371/journal.pone.0064133

2. Websites
i. Antonie van Leeuwenhoek - Wikipedia https://en.wikipedia.org/wiki/Antonie_van_Leeuwenhoek
ii. https://microscopeclarity.com/wet-mount-slide-a-complete-guide/
iii. https://www.sciencedirect.com/topics/medicine-and-dentistry/microbiological-examination
iv. https://www.ruf.rice.edu/~bioslabs/BIOC318/living_bacteria.asp