LABORATORY REPORT 2

BASIC LABORATORY TECHNIQUES FOR MICROSCOPIC EXAMINATION OF

STAINED CELL PREPARATIONS, MICROSCOPIC EXAMINATION OF LIVING
MICROORGANISMS USING A HANGING-DROP PREPARATION OR A WET
MOUNT AND THE MICROSCOPIC MEASUREMENT OF MICROORGANISMS

LECTURER : DR. SAILA BINTI ISMAIL

FACULTY : FACULTY OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES

COURSE : BACHELOR OF SCIENCE IN BIOCHEMISTRY WITH HONOURS

SUBJECT : BMY 3201- BASIC MICROBIOLOGY TECHNIQUES

BENCH NO. : 6

GROUP PAIRS MEMBERS:

NAME MATRIC NUMBER

1 AINA SYAKIRAH BINTI HASMAIZAL 210159

2 NUR FATIHAH NABILAH BINTI MOHD FADZIL 212652

3 RANI ANAK MAT 212111

1.0 ABSTRACT

Under the heading Microscopy, three experiments were carried out which are The Microscopic
Measurement of Microorganisms, The Microscopic Examination of Stained Cell Preparations, and
The Microscopic Examination of Living Microorganisms Using a Hanging-Drop Preparation or
Wet Mount. The first experiment examines the theoretical background, components, use,
maintenance, and practical use of the compound microscope for visualizing cellular morphology
from stained slide preparations. Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and
Saccharomyces cerevisiae were the microorganisms that were seen under a compound microscope
using high-power and oil immersion objective lenses. When microorganisms are observed, their
morphologies can be seen more clearly with an oil immersion magnifier than through a high-power
objective lens. The method for making hanging drops and wet mounts was then learned in the
second experiment. Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, hay
infusion, and pond water were examined using a compound microscope to observe the cell activity,
sizes, and shapes. P. aeruginosa, B. Cereus, and P. vulgaris were shown to have true motility and
to be rod-shaped. S. aureus, in contrast, has cocci shape and Brownian movement. Additionally, it
was noted that Protozoa was present in the sample of hay infusion and algae present in the pond
water. The method for measuring the microorganisms was examined in the previous experiment.
To determine the actual length of the microorganisms in micrometers (µm), the width and length
of B. cereus, S. aureus, and S. cerevisiae were measured using an ocular micrometer and multiplied
by a calibration factor determined at the beginning of the experiment.

2.0 INTRODUCTION

2.1 Background

Hans and Zacharias Jansen, and Dutch lens grinders, created the first microscope in 1590. Robert
Hooke first described the microscopic structure of cork in 1667, referring to the compartments he

1
saw as cells. The first person to observe living cells under a microscope was Anton van
Leeuwenhoek in 1675 which described a wide variety of cells, including bacteria. Since then, more
advanced and potent scopes have been made that enable clearer images and higher magnification.
Scientists and medical professionals use microscopy for a variety of tasks, such as the diagnosis
of infectious diseases, the identification of microorganisms in environmental samples, such as food
and water, and the study of the impact of pathogenic microbes on human cells (Libretext, 2021).

To have a better understanding of microscopes, learning the components of the microscope, their
resolving power, the theoretical basis, and proper handling is a must to comprehend and use it
effectively. Magnification, resolution, numerical aperture, illumination, and focusing were the
foundation of a microscope. The magnifying power of an ocular lens and the magnifying power of
the objective lens is multiplied to obtain the magnification, which is used to enlarge the specimen.
Resolving power, which is estimated by knowing the wavelength of light and numerical aperture,
is crucial in determining how far away two adjacent objects must be before a particular lens
presents them as discrete entities in addition to its magnification power. Effective magnification
and resolving power require successful illumination, which is provided by a tungsten lamp inside
of a light microscope compound. The light compound microscope is a device with two lenses that
magnify and a number of knobs that focus (resolve) the image. It is frequently referred to as a
compound microscope in addition to being named a light since it employs multiple lenses.

Living things can be seen and examined using the hanging drop and wet mount techniques. These
techniques require the Aseptic techniques to be applied when preparing the slide to avoid any
contamination that might lead to the growth of other organisms. The hanging drop is a more
complicated methodology, but it allows for longer-term observation and is more reliable to
examine the motility of microorganisms than the wet mount since the wet mount has a tendency
to dry out fast under the heat of the microscope light. Since no stains are typically used in these
procedures, it can be difficult to see the organisms. Reduce the microscope's illumination to make
sure there is still sufficient light to see the creature. Using these techniques, it is crucial to
differentiate between true motility and Brownian motion. While true motility enables the cell to
move in many directions and across wider areas, Brownian movement is caused by the cell
colliding with water molecules.

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An ocular micrometer, an eyepiece with a scale that will appear superimposed upon the focussed
specimen, is used to precisely measure cells as well as to determine the approximate size of a
microorganism. A stage micrometer, which is just a microscope slide with a finely divided scale
marked on the surface, will be used to measure the distance between graduations on an ocular
micrometer that is inserted into one of the microscopic eyepieces. The scale is used in the
calibration process for the ocular micrometer and has a known real length. The average number of
ocular divisions occupied (number of spaces occupied by microorganisms) and the calibration is
multiplied to determine the microorganism measurement.The calibration factor is calculated by
using formula;

1 division on ocular micrometer in mm = known distance between two lines on stage micrometer

Number of divisions on ocular micrometer

2.2 Objective

The objective of the fourth experiment which is Microscopic Examination of Stained Cell
Preparations is to understand the procedures and the components of light microscopy. Next, the
purpose of the fifth experiment which is Microscopic Examination of Living Microorganisms
using a Hanging-drop Preparation or a Wet mount is to be able to use compound microscopes for
visualisation of cellular morphology of viable microorganisms. Other than that, the experiment is
carried out to microscopically examine living microorganisms by using the hanging-drop and wet-
mount preparations. Last but not least, the objective of experiment A which is The Microscopic
Measurement of Microorganisms is to calibrate an ocular micrometer as well as to perform an
experimental procedure in measuring the microorganisms.

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3.0 MATERIALS AND METHODS

3.1 Materials and equipments

3.1.1 Experiment 4: Microscopic Examination of Stained Cell Preparations

The equipment used in this experiment were a brightfield microscope, paper lens and oil
immersion. Next, the slides were commercially prepared slides of Bacillus cereus, Staphylococcus
aureus, Saccharomyces cerevisiae and Bacillus subtilis.

The materials used in this experiment was alcohol where it was needed to wipe clean the lens after
using oil immersion.

3.1.2 Experiment 5: Microscopic Examination of Living Microorganisms using a Hanging-

drop Preparation or a Wet mount

The equipment used in this experiment were Bunser burner, inoculating loop, depression slides,
glass slides, coverslips, microscope, petroleum jelly, toothpick, cotton swabs and brightfield
microscope.

The materials used in this experiment were the cultures of twenty-four-broth cultures of P
.aeruginosa, B. cereus, S. aureus, P. vulgaris, hay infusion broth and pond water. Next, alcohol
was also being used in this experiment to wipe clean the lens after using the oil immersion.

3.1.3 Experiment A: The Microscopic Measurement of Microorganisms

The equipment used in this experiment were the prepared slides of Bacillus cereus, Staphylococcus
aureus and Saccharomyces cerevisiae. Next, ocular micrometer, stage micrometer, microscope,
immersion oil and lens paper were also being used.

The material used in this experiment was alcohol to wipe clean the lens after using the oil
immersion.

4
3.2 Methods

3.2.1 Experiment 4: Microscopic Examination of Stained Cell Preparations

Microscope was being placed on the laboratory bench and it was being observed. All unnecessary
materials were removed from the laboratory bench. The experiment was started by reviewing the
parts of the microscope by making sure the names and understanding of the function of each
microscope component was known. Next, instructions for the use of the microscope were reviewed
by giving special attention to the use of the oil-immersion objective. The first step, a microscope
slide with the specimen of S. aureus was placed within the stage clips on the fixed stage. Secondly,
the prepared slide of S. aureus was moved to the center of the specimen over the opening in the
stage directly over the light source. Thirdly, the microscope stage was raised as far as it could go
up. Fourthly, scanning lens or low-power lens (4x) was rotated into position. Fifthly, the body tube
was lowered with the coarse-adjustment knob to its lowest position. Sixthly, the fine-adjustment
knob was used while looking through the ocular lens in order to bring the specimen into sharp
focus. Seventhly, the substage condenser was adjusted to achieve optimal focus. The next step was
the light source and iris diaphragm adjusted for optimum illumination for each new slide as well
as when magnification was changed. Preparation can be made for visualizing the specimen under
oil immersion once the specimen was brought into sharp focus under oil with a low-powered lens.
The steps were repeated for the prepared slide of S. cerevisiae and B. cereus. All the observations
of each microorganism were recorded.

Finally, all lenses were cleaned with alcohol being put on lens paper once the laboratory exercise
was completed. The low-power objective was placed in position and the body tube was completely
lowered. The electrical cord was coiled and both the microscope as well as the electric cord were
put at the original place before they were taken.

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3.2.2 Experiment 5: Microscopic Examination of Living Microorganisms using a Hanging-
drop Preparation or a Wet mount

For the procedure of hanging-drop preparation, the experiment was started by applying a ring of
petroleum jelly using a cotton swab. Next, a loopful of the culture was placed in the center of the
coverslip. After that, the depression slides were placed with the concavity surface facing down,
over the coverslip to cover the drop of culture. The slide was pressed gently to form a seal between
the slide and the coverslip. Then, the slide was quickly turned right side up. Lastly, the drop culture
was first focused under the low-power objective (10x) and the light source was reduced by
adjusting the condenser. The experiment was repeated using the high-power objective (40x). The
hanging-drop preparation was examined and the observations were recorded.

For the wet-mount procedure, the experiment was started by applying a thin layer of petroleum
jelly along the edge of the four sides of the coverslip using a cotton swab. Next, a loopful of the
culture was placed in the center of a clean coverslip. After that, a clean glass slide was placed over
the coverslip and the slide was pressed gently to form a seal between the slide and the coverslip.
Then, the slide was quickly turned right side up. Lastly, the drop culture was first focused under
the low-power objective (10x) and the light source was reduced by adjusting the condenser. The
experiment was repeated using the high-power objective (40x). The wet-mount preparation was
examined and the observations were recorded.

6
3.2.3 Experiment A: The Microscopic Measurement of Microorganisms

The ocular micrometer was gently placed into the eyepiece to begin the experiment. Second, the
stage micrometer was positioned over the illumination source on the microscope stage. Thirdly,
the eyepiece was slowly rotated to superimpose the ocular micrometer graduations over those of
the stage micrometer once the stage micrometer had been sharply focused beneath the low-power
objective. Fourth, the oil immersion was brought into focus by adding a drop of immersion oil to
the stage micrometer. Fifth, the mechanical stage was positioned so that, at one end, a line on the
stage micrometer and a line on the ocular micrometer coincide. A second line that matches a line
on the stage micrometer was observed on the ocular micrometer. The corresponding number of
divisions on the ocular micrometer was obtained after determining the distance on the stage
micrometer. Sixth, the calibration factor value for the oil immersion objective was determined.
The stage micrometer was removed from it in the seventh step. Eighth, under the oil-immersion
objective, the size of the S. aureus on the prepared slides was measured, and the number of ocular
divisions occupied by each of the three distinct S. aureus was computed. The data was all recorded.
The size was determined by multiplying the average value of the calculated calibration factor, and
the result was recorded. The average of the three measurements was then calculated and recorded.
After the calibration factor was obtained, the remaining prepared slides were observed under the
oil immersion as described in Step 8 to identify and record the size of the other microorganisms (
S. cerevisiae and B. cereus). It was necessary to measure both the length and the width. The
observations were examined and accomplished at the end.

7
4.0 RESULTS AND DISCUSSION

4.1 Results and Discussion

4.1.1 Experiment 4: Microscopic Examination of Stained Cell Preparations

The aim of this experiment is to understand the procedures and the components of light
microscopy. Students will master the use and care while handling the microscope to avoid any
accidents. There is a major limitation for this brightfield microscope, which is that it is difficult to
observe living cells (Richard M. Levenson, 2003). Therefore, most of this system observations are
performed on nonviable and stained preparations. In accordance with this experiment, we are also
familiarised with the practical use of the compound microscope for visualisation of cellular
morphology from stained slide preparations.

Based on the results in Table 4.1.1 shows the observations of S. aureus, B. cereus, S. cerevisiae
and B. subtilis under 40x and 100x objective lenses. From table 4.1.1, obvious observation on S.
aureus, where they are in clusters and arranged in a bunch of grapes under 400x magnification. A
clearer image form of S. aureus under 1000x magnification, where we can confirm that S. aureus
was spherical-shaped and purple in colour. Next, in 400x magnification showed that the shape of
the B. cereus is rod-shaped and also purple and violet in colour. In 1000x magnification can be
seen that the arrangements of the B. cereus were arranged in pairs and chains and most of them
are long-shaped rods. Furthermore, under 400x magnification showed that the arrangement of S.
cerevisiae were in rough type colonies and budded. Under 1000x magnification, a clearer view of
S. cerevisiae showed that S. cerevisiae appears spherical-shaped in purple colour. Lastly, shows
that under 400x magnification of B. subtilis where the image depicts chain-arrangement of B.
subtilis and were in rod-shaped cells. A clearer resemblance of B. subtilis under 1000x
magnification in where the rod-shaped cells obtained.

8
Table 4.1.1 : Observation of S. aureus, B. cereus, S. cerevisiae and B. subtilis under 400x
magnification and 1000x magnification of objective lens.

High Power Oil Immersion

S. aureus

Figure 1 Figure 2

magnification 400x 1000x

B. cereus

Figure 2 Figure 4

400x 1000x
magnification

9
 S. cerevisiae

Figure 5 Figure 6

magnification 400x 1000x

B. subtilis

Figure 7 Figure 8

400x 1000x
magnification

Figure 1, 3, 5, 7 showed S. aureus, B. cereus, S. cerevisiae and B. subtilis under 400x

magnification respectively while figure 2, 4, 6, 8 showed S. aureus, B. cereus, S. cerevisiae and
B. subtilis under 1000x magnification respectively.

10
 In this experiment, examination of stained cells, Staphylococcus aureus, Bacillus cereus,
Saccharomyces cerevisiae and Bacillus subtilis was observed under a brightfield microscope. This
microscope provides a two-lens system for magnifying specimens, which is an ocular lens in the
eyepiece and the objective lens in the nosepiece. The image resulted as dark against the bright for
the observed specimen. This brightfield microscope also has its limitations as it is hard to observe
living cells in the absence of contrast between the specimen and the surrounding medium (Dimitri
Van De Ville, 2008). The majority of brightfield observations are thus made on stained and
nonviable preparations.

Through this experiment, there are some procedures we should obey as the proper care of the
microscope is very important. One of the significant steps we must be aware of is the cleanliness
of the lens system. It is because only a small amount of dust or small particles will decrease the
efficiency of the microscope. Cleaning of ocular, scanning. low-power, and high power lenses
involve lens paper or soft cotton cloth and also xylol. The lens paper was dampened with a small
amount of xylol and wiped out oil immersion that surrounds the lens (M.D Sousa Neto, 2013). But
be cautious that consistent use of xylol may loosen the lens thus making the result appear on the
lens become less clearer than before.

There are some purposes we adjust the component of the microscope. The first purpose we
adjust the iris diaphragm is in order to change the amount of light entering the lens system. Next,
the coarse-adjustment knob is used to adjust and move the body tube towards the lids and to adjust
the focus of the specimen to have better and clearer images of the specimens. Fine-adjustment
knob is used to bring the image into a sharp focus, since the fine-knob raises and lowers the stage
in very small increments (Rafael Rodondo, 2012). Take note on why we also adjust the condenser,
it is because we can move the condenser closer towards the stage in order to achieve optimal focus
since the condenser is made of at least two lenses that focus the light passing through the specimen.
Therefore the image obtained can improve its sharpness. Adjustable mechanical stage control
enables us to move the slide around the stage to observe the different sanction of the slide.

However, during this experiment we tend to make some accidents and mistakes while observing
specimens using a microscope. If there is a situation where some students are unable to bring the
specimen into sharp focus, we can follow these useful steps. Firstly, take the slide out and wipe

11
off the oil immersion with xylol and lens paper and start to make new observations. Next step is,
start with the lowest power of the objective lens; 4x, 10x, 40x, 100x (oil immersion). Lastly, If
there are still no changes, replace the old slide with the new slide and start over.

When there is insufficient light while viewing the specimen, we can play with the field
diaphragm to increase the amount of light through the specimen. Next, if we face the situation of
artifacts in the microscopic field, we can minimize the artifact by using lower illumination and
shortening the exposure (Yin Z., Kanade T., 2011).

Based on the observations in table 4.1.1, we obtain the clearer result of S. aureus, B. cereus,
S. cerevisiae and B. subtilis under 100x objective lens with oil immersion compared to when using
the high power objective which is 40x objective lens. This is the reason why oil immersion
objectives have better magnification than high-power objectives; as the working distance, or the
distance between the objective lens and slide decreases, the lens power increases. The working
distance of a 40x objective lens is between 0.5 to 0.7 µm while the working distance of a 100x
objective lens is nearer which is between 0.13 to 0.18 µm. In addition, the diaphragm opening for
oil immersion objective is fully opened while the diaphragm opening for high power objective is
not fully opened. The addition of immersion oil in the oil immersion objectives resulted in a high
resolution and vivid images of S. aureus, B. cereus, S. cerevisiae and B. subtilis. This is due to
loss of refracted light that can be compensated for by interposing oil immersion, which has the
same refractive index as glass, between the slide and the objective lens. In this way, decreased
light refraction occurs and more light rays enter directly into the objective lens, producing more
clearer images (Cargille John J, 2008). The fine-adjustment knob for a precise focus can also be
adjusted to provide a clearer image of the slides in both 40x and 100x objective lenses.

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4.1.2 Experiment 5: Microscopic Examination of Living Microorganisms using a Hanging-
drop Preparation or a Wet mount

The purpose of conducting the experiment is to enable viewing and identifying the motility of
the microorganisms under light compound microscope. Two methods were used to observe the
living culture which are hanging-drop and wet-mount preparations. Based on Table 4.1.2, shows
S. aureus which viewed under 400x magnification appeared as round shape. The organism has a
bright and white transparent colour which makes it hard to distinguish between the organism and
the ambient or contaminant microorganism. Upon observing under the microscope, S. aureus only
vibrates in place and makes no large movement. This circumstance explains a brownian movement
caused by a collision of water molecules with the suspensions. Next, displays that an organism, P.
aeruginosa was rod-shaped. Many of the cells moved only a short distance but there were also
some moved a longer distance. Depicts that organism B. cereus in the form of a rod. It
demonstrated an actual movement in which the cells moved at a greater distance. Furthermore, P.
vulgaris shows true motility because the cells appear moveable under the microscope unlike S.
aureus.

Table 4.1.3 illustrates the unknown organisms collected from pond water and a prepared hay
infusion. From the table, Diatom (algae) displays a rod-shaped organism found in collected pond
water that has true motility while Vorticella convallaria (protozoa) is a elongated-ovoid of a
prepared hay infusion organism with single flagellum at the end or also called as amphitrichous.

13
Table 4.1.2: Observation of the representative field, shape and motility of S. aureus, P.
aeruginosa, B. cereus and P. vulgaris

Organisms Representative field Shape True Motility or

Brownian
Movement

S. aureus Cocci Brownian movement

Under 400x
magnification
Figure 1

P. aeruginosa Rod-shaped True motility

Under 400x
magnification
Figure 2

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 B. cereus Rod-shaped True motility

Under 400x
magnification
Figure 3

P. vulgaris Rod-shaped True motility

Under 400x
magnification
Figure 4

Figure 1 , 2, 3, 4 are S. aureus, P. aeruginosa, B. cereus and P. vulgaris respectively viewed

under 400x magnification via light compound microscope.

15
 Table 4.1.3: Observations of the representative field, shape, motility and organisms seen on
pond water and hay infusion

Pond Water Hay Infusion

Representative field

Under 100x magnification Under 40x magnification

Figure 5 Figure 6

Shape oblong Elongated-ovoid

True motility or Brownian True motility True motility

movement

Organism Navicula spp. (Diatoms) Vorticella convallaria

(protozoa)

Figure 5 and 6 illustrates the physical morphology of Diatoms and Vorticella in pond water and
hay infusion under 100x and 40x magnification respectively via light compound microscope.

16
 Hanging-drop involved in suspending the microbes suspension into several drops of distilled
water. Concaves well placed at the centre of the depression slide are used instead of the flat glass
slide. A drop of the microbe suspension will be placed on the cover slip with petroleum applied at
every edge to adhere or attach the cover slip and depression slide. The suspension drop is hung
from the depression slide in the hollow concavity of the slide when the cover slip is inverted and
placed on top. The petroleum should not be applied in excess, but only enough to retain it from
melting as the microscope's illumination may heat it up. As a result, petroleum will enter the
suspension and contaminate it. This will alter the observation of microbes motility and might
mistaken it for movement of water molecules. Inoculation process of transferring culture from the
stock to cover slip still required aseptic technique while handling a living organism to ensure the
whole processes are ambient microbes-free and sterilised. This whole hanging-drop method was
first identified by a scientist named Robert Koch in 1878. The method is primarily used to
determine the ability of the organism to move, however natural patterns or cell shape are also to
be gained. The advantage of drops does not dry quickly, hanging-drop preparations can be
observed for a long time.

Wet-mount preparations also have the similar objective as hanging-drop preparation which are
to detect living microorganism motility rapidly by placing a few loopful of the organism on a clean
glass slide. The fluid film also is much thinner than that of hanging-drop as it was covered with
cover slip exactly on top of the suspension. However, even when sealed with petroleum jelly, this
technique dries out faster and is quite dangerous to the person handling the pathogenic viable
organism on the slide. The ideal way to examine the slide is indeed with a phase-contrast
microscope, utilising high-dry magnification. But since a light compound microscope is used, it is
necessary to reduce the lighting to a bare minimum to achieve contrast in order to see a clear
morphology of an organism despite a bright background. Once the specimen is already in focus
under high-power objective lens, immersion oil could then be placed on the cover-slip (Benson,
1981).

S. aureus is historically defined as a non-motile organism (Wiley, 1972) but can be passively
expanded by sliding or brownian motion with the aid of surfactant which in this case is distilled
water due to its lack of motility mechanism like flagella. S. aureus appears to simply vibrate,
indicating that it lacks true motility in comparison to other actively motile organisms. Next, B.

17
cereus is peritrichous, having flagella over the entire surface of its body and thus, it is obvious
seeing them slowly move in twitching manner. In research from O’May and Tufenkji, P.
aeruginosa, known as opportunistic pathogen, causing both chronic and acute infections in
susceptible populations such as people with cystic fibrosis or burn wounds, as well as patients in
intensive care units can undergo the flagellum-mediated swimming motility and surface-associated
swarming and twitching motilities due to the presence of hyper flagellation and type-IV pili (2011).
Furthermore, P. vulgaris is a member of the genus Proteus, which was first described in 1885 by
Gustav Hauser, who demonstrated Proteus' ability to swarm and proposed intestines as their
reservoir. P. vulgaris have exhibited true motility on the slide due to flagella present.

The organism discovered in hay infusion is V. convallaria is a peritrich ciliate that feeds in
suspension and exists in two forms: free swimming telotroch and stalked trophont, both of which
are represented in Figure 6. Vorticella consists of features like the zooid (inverted-bell shaped cell
body) and the stalk attaching to its body. The zooid contains most of a Vorticella’s organelles,
such as food vacuoles, endoplasmic reticulum and mitochondria. The anterior region of V.
convallaria consists of an oral region surrounded by cilia for feeding. The stalk is made up of
external sheath containing fluid and spasmoneme that contracts in the presence of calcium ions.
The stalk end will attach to a substrate , such as rocks, aquatic plants and even aquatic animals via
an adhesive pad (feature of V. convallaria). Next organism found in pond water is diatoms, one of
the main groups of algae. Diatoms are collected in benthic samples from lakewater, freshwater or
marine sites. They are highly sensitive indicators in aquatic environments, and their microfossils,
which are deposited in lake sediments, can be used to infer environmental changes (Smol, 2008).
Cell motility is one of the most noticeable characteristics of pennate diatoms that allows them to
anticipate changing environmental conditions. Some pennate diatoms are capable of "gliding,"
which allows them to move across surfaces by secreting adhesive mucilage through a structure
called raphe. Pennate diatoms are bilaterally symmetric like Navicula sp. in Figure 5.

The advantages of staining a bacterial preparation before observing it under microscope are to
enhance visualisation of the cell or their cellular components and to differentiate between live and
dead cells in a sample. There are several steps in conducting hanging-drop preparation. Firstly,
add only a fair amount of vaseline on every edge of the cover slip using a toothpick to avoid it
squeezed toward the centre and mixed with the suspension. Secondly, place the depression slide

18
(concavity above) over the drop on the cover slip and ensure that both cover slip and depression
slide are sealed together then turn the slide over so that the cover slip is now on the top. Place the
preparation in the microscope slide holder and align it using naked eye so that the concave well
is under the low power objective. Diaphragm is closed and slowly adjusts the coarse focus knob
accordingly until a view of half edge of drop and half concave depression is observed. Without
raising or lowering the stage, change to a high dry objective lens then adjust the fine adjustment
knob by observing the slide through the eyepiece, until the edge of the drop appears as a thick dark
line. Next, the diaphragm lever is adjusted depending on maximizing the visibility of the cells.
After all this set-up, note down the microscopic properties like the shape, colour and motility.
Brownian movement is visible but true motility may be possessed. At last, thoroughly clean the
depression slide by putting it into a lysol solution for about 10 to 15 minutes to kill the possible
pathogens. Bacterium Treponema pallidum determines the presence of syphilis in a person. T.
pallidum is detected using dark-field microscopy in material from suspected abnormalities or
regional lymph nodes (Creighton, 1990). Dark-field microscopy allows visualization of live
Treponema obtained from a variety of cutaneous (relating to skin) or mucous membrane lesions.

Living organisms can be seen and examined using the hanging drop and wet mount techniques.
The hanging drop is a more complicated method, but it allows for longer-term observation and is
more reliable to examine the motility of microorganisms than the wet mount since the wet mount
has a tendency to dry out fast under the heat of the microscope light. Since no stains are typically
used in these procedures, it can be difficult to see the organisms. This happens because bacteria
are so small and their refractive index is close to water so there is not much contrast between the
two leaving a clear image. Staining adds contrast and makes them easier to see. Also, some bacteria
can move around fast making it hard to see them under the microscope. Next, reduce the
illumination of the microscope so that there is still sufficient light to see the creature. In this
experiment, the Hanging drop method is being used to identify the type of motility because a quick
result can be obtained by using this method. During the microscopic observation of a drop of
stagnant pond water, the criteria needed to take note to distinguish viable organisms from
nonviable suspended debris was viable organisms will move around and most have intracellular
organelles. The precaution that students needed to be alert while doing this experiment was that
the pond water could contain disease-causing microbes so pre and post hygiene precautions should
be taken and if there are any cuts or abrasions do ensure to cover cuts or abrasions with waterproof

19
plasters or gloves. Students will need to wash their hands with appropriate cleansers before leaving
the laboratory.

4.1.3 Experiment A: The Microscopic Measurement of Microorganisms

The objective of experiment A is to calibrate an ocular micrometer as well as to perform an

experimental procedure in measuring the microorganisms. Based on the findings in table 4.1.4,
the average measurement for each organism (B. cereus, S. cerevisiae and S. aureus) was multiplied
with the calibration factor that has been calculated before. This is to ensure that the accurate
measurement for each organism was obtained for both width and length. From the table 4.1.4, the
length of each microorganism (B. cereus, S. cerevisiae and S. aureus) was calculated where B.
cereus has an average reading of 3.67 ocular divisions and a calibration factor of 1, respectively,
and its total length is 3.67 µm. The average reading for S. cerevisiae is the number of ocular
divisions and the calibration factor is 3.83 and 1, respectively, while the S. cerevisiae total length
is 3.83 µm. Last but not least, S. aureus has an average reading of 0.5 and 1 for the number of
ocular divisions and calibration factor, respectively, and 0.5 µm for the total length.

Based on the results in table 4.1.5 shows that the average reading of the number of ocular
divisions and calibration factor for B. cereus is 1.33 and 1 respectively, and the total width for B.
cereus is 1.33 µm. Next, the average reading of the number of ocular divisions and calibration
factor for S. cerevisiae is 2.17 and 1 respectively, and the total width for S. cerevisiae is 2.17 µm.
Lastly, the average reading of the number of ocular divisions and calibration factor for S. aureus
is 0.5 and 1 respectively, and the total width for S. aureus is 0.5 µm.

Calculation

Distance on the stage micrometer : (1 × 0.01 mm)

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Divisions on ocular micrometer : 10

Calibration factor for one ocular division : 0.001 mm = 1 µm

One division on ocular micrometer in mm for 10x objective lens = ( 5 × 0.01 mm) / 5

= 0.01 mm

One division on ocular micrometer in mm for 40x objective lens = ( 1 × 0.01 mm) / 4

= 0.0025 mm

One division on ocular micrometer in mm for 100x objective lens = ( 1× 0.01 mm) / 10

= 0.001 mm

Table 4.1.4: Observations measurement of length of microorganisms for B. cereus, S. cerevisiae

and S. aureus

LENGTH OF MICROORGANISMS IN MICROMETERS (μm)

Number of Ocular Divisions

Organism Readings Average x Calibration = Size
Factor
1 2 3

B. cereus 4 3.5 3.5 3.67 1 3.67

S. cerevisiae 4 4 3.5 3.83 1 3.83

S. aureus 0.5 0.5 0.5 0.5 1 0.5

Table 4.1.5: Observations measurement of width of microorganisms for B. cereus, S. cerevisiae

and S. aureus

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 WIDTH OF MICROORGANISMS IN MICROMETERS (μm)

Number of Ocular Divisions

Organism Readings Average x Calibration = Size
Factor
1 2 3

B. cereus 1 1.5 1.5 1.33 1 1.33

S. cerevisiae 2 2 2.5 2.17 1 2.17

S. aureus 0.5 0.5 0.5 0.5 1 0.5

Determination of microbial size is not as simple as one might assume. Before an accurate
measurement of cells can be made, the diameter of the microscopic field must be established by
means of optic devices, namely, an ocular micrometer and a stage micrometer. The ocular
micrometer, which is placed on a circular shelf inside the eyepiece, is a glass disc with graduations
etched on its surface. The distance between these graduations will vary depending on the objective
being used, which determines the size of the field (Nicole G., 2017). This distance is determined
by using a stage micrometer, which is a special glass slide with etched graduations that are 0.1 mm
or 10 µm apart, since there are 1000 micrometers in 1 millimeter. The calibration procedure for
the ocular micrometer first requires that the graduations on both micrometers be superimposed on
each other, which is accomplished by rotating the ocular lens. A determination is then made of the
number of ocular divisions per known distance on the stage micrometer. Finally, the calibration
factor for one ocular division (OD) is calculated as follows;

1 division on ocular micrometer in mm = known distance between two lines on stage micrometer

Number of divisions on ocular micrometer

In this experiment, it is important to understand the concept of microscope calibration. This is

because Microscope Calibration can help ensure that the same sample, when assessed with
different microscopes, will yield the same results. Even two identical microscopes can have

22
slightly different magnification factors when not calibrated (Madsen, A., 2018). Take note that the
same calibration factor cannot be used to determine the size of a microorganism under all
objectives. This is because with each objective, the calibration must be recalibrated because the
calibration only applies to its calibrated objective. Next, it is crucial to master the conversion of
units as this experiment involved calculations that need the conversion of units. In this experiment
it is also important to master in calculation the distance between 2 lines on the ocular micrometer.

Expectation of the measurements for given organisms also have been taken note either it would
be the same or if a size determination is made from a stained and unstained preparation. Our
expectation is it would be different in size because before specific staining can occur, tissue
samples must undergo preparation through the following stages of Fixation, processing,
embedding, sectioning, and sometimes antigen retrieval. The process of sectioning involves
mounting the specimen on a microtome and cutting it into sections. The preferred thickness is 4-5
micrometers so that it can be stained and put on a microscope slide for examination. Also when it
comes to dehydration.

Based on the results in Table 4.1.4 and Table 4.1.5, the reason why the microscope is
calibrated is that even though two identical microscopes are used for example each with 10x
eyepieces and a 40x objective, it can still have slightly different magnification factors. Therefore,
it is important to calibrate the eyepiece lens with a stage micrometer before making measurements
with the eyepiece lens. This ensures that accurate measurements will be achieved with the
microscope. The higher the magnification, the lower the calibration factor. S. aureus has the
shortest width and length compared to S. cerevisiae and B. cereus. S. aureus as it is one of the
smallest bacteria which ranges from 0.1 to 0.25 μm in width and roughly 1 to 1.5 μm in length
(Madsen, A. M., 2018). From table 4.1.4 and table 4.1.5, S. cerevisiae has the longest length
which is 3.83 and has the thickest width which is 2.17.

5.0 CONCLUSION

Based on experiment 4, it can be concluded that the objective is successfully achieved.

Students were able to use the compound microscope for visualization of cellular morphology from

23
stained slide preparations by observing the slides of microorganisms of Bacillus cereus,
Staphylococcus aureus, Saccharomyces cerevisiae and Bacillus subtilis under 400x and 1000x
objective lens.

Based on experiment 5, it is possible to conclude that the objective has been successfully
delivered. Students used a microscope to see clearly through the organism in the mixture of water
and suspensions such as S. aureus, B. cereus, P. vulgaris, P. aeruginosa, pond water and hay
infusion. These hanging-drop and wet-mount experiments allow students to identify the type,
shape, and movement of living microorganisms without the use of staining.

By the end of experiment A, it can be concluded that the objective of this experiment is
successfully achieved where the students were able to measure the microorganisms by operating
the ocular micrometer and stage micrometer using the formula of the average numbers of ocular
divisions multiplied by calibration factors to determine the length and width of the S. cerevisiae,
B. cereus, and S. aureus.

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