MIC125 LABORATORY REPORT

PRACTICAL 2

TITLE:

EXAMINATION OF LIVING BACTERIA

BY:

1) NUR AKMA SHUHADA BT MOHD ZAMRO (2018269024)

2) NUR FATIHAH BT MUHAMMAD NAZIM (2018269248)

3) NOOR ARIFAH BT IBRAHIM (2018671246)

4) WAN NORHAKIMA BT WAN MOHD (2018214368)

GROUP: AS1162E2

DATE OF PRACTICAL: 20 MARCH 2019

DATE OF SUBMISSION: 27 MARCH 2019

TITLE: EXAMINATION OF LIVING BACTERIA (EXPERIMENT 2)

INTRODUCTION

In brightfield microscopy, objects are dark and the field is light. Brightfield
microcopy can be used to observe unstained microorganism. However, because the
optical properties of the organisms and of their aqueous environment are similar, very
little contrast can be seen. In phase-contrast microscope make the eye detects the
difference as contrast between the organisms and background and between structures
within a cell. The organisms appear as degrees of brightness against a darker
background. Phase-contrast microscopy allows study of structural detail within live cells.

In this experiment, wet-mount techniques will be used to observe different

environments to help aware of the numbers and vaieties 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. In Brownian movement, the particles
and microorganisms all vibrate at about the same rate and maintain their relative
positions. Motile microorganism move from one position to another. Their movement
appears more directed than Brownian movement and occasionally the cells may spin or
roll.

Many kinds of microbes such as protozoa, algae, fungi and bacteria canbe found in
pod water and in infusions of organic matter. Van Leeuwenhoek made some of his
discoveries using a peppercorn infusion similar to the one that the image that was be
seen in the experiment. 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.
OBJECTIVE

i. To prepare and observe wet-mount slides

ii. To distinguish between true motility and Brownian movemement.

MATERIAL
1. Compound microscope
2. Cavity glass slides
3. Cover slips
4. Inoculating loop
5. Petroleum jelly
6. Paper towels
7. Applicator sticks
8. Disposable pipettes
9. 24-hours nutrient broth cultures of:
a) Staphylococcus aureus
b) E. coli
PROCEDURE

A. Wet Mount Techniques

Some of the nutrient broth that contain bacteria was suctioned up using one of
the disposable pipettes and a small drop was transferred to the center of a glass
slide.

A coverslip was carefully placed on top of the fluid

The slide was placed on the stage of microscope and was observed with low
power.

The diaphragm was adjusted so that only a small amount of light was admitted.

0bservations was concentrated on the larger, more rapidly moving organisms. At

this magnification, bacteria were barely discernible as tiny dots.

The slide was examined with 40x objective. The light was increased using the iris
diaphragm as needed.

Observations was recorded.

 The slide was examined with the oil immersion lens.

The observations was recorded, noting the relative size and shape of the
organisms.

A wet mount was made from the other bacterial broth, and was observed, using
the low and high power objectives. Observation was recorded.

B. Hanging Drops Techniques

A clean cavity glass slide was obtained.

A small amount of petroleum jelly was picked up on an applicator stick.

The petroleum jelly was carefully touched to all four edges of a coverslip to get a
small rim of petroleum jelly around the entire coverslip.

The coverslip was placed on the paper towel, with the petroleum jelly-side up.

A small drop of the bacterial broth was transferred by using inoculating loop onto
the coverslip.

A cavity glass slide was a placed over the drop, the paper towel was scooted to
the edge of the table, and was carefully and quickly flipped the slide over so that
the drop of the fluid was suspended upside down from the coverslip into the
space provided by the cavityglass slide.
The drop was examined under low power by locating the edge of the drop and
the slide was moved so the edge of the drop crosses the center of the field.

The light was reduced with the iris diaphragm and was focused.

The different sizes, shapes, and types of movement was observed.

It was switched to high-power and the observations was recorded.

The procedure was repeated using a new coverslip with the culture of another
bacteria broth. The observations was recorded.
RESULTS

Magnification: 4X Magnification: 10X

Magnification: 40X

Figure 1: Image for E. coli under the microscope.

Magnification: 10X Magnification: 40X

Magnification: 150X

Figure 2: Image of E.coli under the microscope from the Internet.

Magnification: 4X Magnification: 10X

Magnification: 40X

Figure 3: Image of S. aureus under the microscope

Magnification: 10X Magnification: 40X

Magnification: 150X

Figure 4: Image of S. aureus under the microscope from the Internet.

DISCUSSION

E. coli and S. aureus from the nutrient broth cultures are observed under the microscope.
E. coli (Escherichia coli) are bacteria found in environment, foods, and intestines of
people and animals. Meanwhile, S. aureus (Staphylococcus aureus) is a major bacterial
human pathogen that causes a wide variety of clinical manifestations. In Figure 1, the rod
shaped of E. coli cannot be seen under 4X and 10X magnification of microscope.
Meanwhile, under the 40X magnification of the microscope, the rod shaped of E. coli, only
a few can be seen. Besides, under the magnification of 10X and 40X in Figure 2, the rod
shaped of E. coli cannot be seen clearly but under the magnification of 150X, the rod
shaped can be seen clearly and the E. coli is moving too.

In Figure 3, the S. aureus cannot be seen under the magnification of 4X and 10X because
the student forgot to take the picture of S. aureus after putting the oil for oil immersion
magnification. Meanwhile, the coccus shaped of S. aureus can be seen clearly under the
40X magnification of the microscope but the picture is blurry because the camera cannot
focus on the S. aureus. In the Figure 4, the coccus shaped of S. aureus cannot be seen
clearly under 10X magnification but it can be seen but not clearly under the 40X
magnification. Meanwhile, the coccus shaped of S. aureus can be seen clearly and the
it is move under the magnification of 150X of the microscope.

In the beginning of the experiment, the wet mount technique is used to observed E. coli
and S. aureus but, these bacteria cannot be seen under the microscope and the hanging
drops technique is not used in this experiment too because of the cover slips. The benefit
of using the wet mount technique is because of the quick preparation and it is possible to
observe living and moving organisms by using this technique. Besides, there are little
artifacts and the specimens appear in their natural condition if there is no chemical and
physical processing of the specimens before observation. Wet mount technique also has
disadvantages which is it cannot be stored over a longer time and the heat of the lamp
causes the water to evaporate and dry more quickly, so more water must be added under
the cover glass from time to time. Besides, some organisms may swim vertically in the
water and therefore move in and out of focus. The advantages of using hanging drops
technique can preserves cell shape and arrangement. The Vaseline-sealed (petroleum
jelly) depression also slows down the drying-out process, so the organisms can be
observed for longer periods. The disadvantage of using the hanging drops technique is it
is not safe when one is dealing with extremely pathogenic organisms, as humans can
easily get an infection and the setting up the apparatus needed for the hanging drop
technique takes time.

CONCLUSION

As a conclusion, the skills and techniques of wet-mount slides and

hanging-drop slides has been prepared and observed in this experiment. The
wet mount can view microscopic organisms that grow in pond water and also
help human studying their movement and behaviour under microscopic. The
hanging drop slide help to observing the general shape of living bacteria and
the arrangement of bacterial cell when they associate together.
REFERENCES

1. Johnson.Ted R(2016). Laboratory Experiments in Microbiology(11 th edition). Pearson

Education. United States of America

2. E. coli (Escherichia coli),

https://www.cdc.gov/ecoli/index.html

3. Staphylococcus aureus,
https://www.ncbi.nlm.nih.gov/books/NBK441868/

4. Wet Mount Techniques,

http://www.microbehunter.com/making-a-wet-mount-microscope-slide/

5. Hanging Drops Techniques,

https://www.reference.com/science/disadvantages-hanging-drop-technique-
12aeb361e8e94c96
QUESTION

1) How would you describe in words the size and shape of the microorganisms that
you see down microscope?
The size and shape of the microorganisms that has been seen was so amazing
which were E. coli shape was bacillus where was likes something long or rods but
in same size each other around 2 micro meter until 5 micro meter. But, shape of
S. aureus was cocci where was likes sphere and it size was very small.

2) Do you think microorganisms are alive?

Microorganism are alive because microorganisms maintain the basic functions of

life. For example, microorganisms are reaction to external stimuli.

3) The movement of bacteria towards nutrients is known as _______

Motility