Visualizing of Microorganisms and Isolating Microorganisms from Living Environments

Summary

The report is based on the experiment, which is divided into two parts. The first part is
visualizing the bacteria Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis,
Pseudomonas aeruginosa, Saccharomyces cerevisiae under the microscope. Before observing
the microbes under the microscope, the bacterial smear is prepared and dyed. The second part of
the experiment is collecting from the skin of hands and colonizing in agar plates. The growth of
these bacteria is also examined, and the morphologies are discussed.

Introduction

The objective of this experiment is to get familiarized with the basic techniques used in a
microbiology lab. The techniques practiced in this lab are preparation of bacterial smear, simple
stain, microscopy, and isolation of microorganisms from living environments. 
Since most microbes are too small to be seen with naked eyes, microscopes are necessary
to study microbiology. Using the compound microscope, the microbes can be identified and
visualized to study their physical characteristics. The objective lens and the ocular lens are used
to magnify the specimen. These objective lenses include the low power objective lens (10X), the
high dry objective lens (40X), and the oil-immersion objective lens of 100X magnification. The
coarse adjustment knob and fine adjustment knob are used to focus the images of the specimen.
The ocular lens of the compound microscope magnifies the image ten times resulting in a total
magnification of 100, 400, or 1000X. To obtain a clear image, high resolution, as well as
magnification, is required. The resolution is measured by measuring the smallest distance
between two points or lines that appear as two instead of one blurred image. For a better
resolution, the oil-immersion technique can be used by placing a drop of immersion oil between
the slide and the objective lens.
Preparing a bacterial smear is also an important technique. To get a good image under the
microscope, an appropriate concentration of bacterial cells should be placed on a slide and fixed.
A bacterial smear should be prepared before staining the bacteria and observing them under a
microscope. A small culture of bacteria is taken from an agar plate and mixed with distilled
water on a plate. A sterilized inoculation loop is used for rubbing the bacteria until evenly
emulsified. Then the smear should be fixed by passing the slide through the tip of the Bunsen
burner until all the water is evaporated. Lastly, the bacterial smear is stained with a dye for easier
observance under the microscope.
A properly prepared bacterial smear should have the following properties: it should be
thin, semi-transparent when observed through a microscope, and free from any contamination
(SAHIL BATRA). The bacteria should also be evenly spread so that a single bacteria can be
distinguished from a colony. The shape of the bacteria should be recognizable.
The last technique practiced in the experiment is the isolation of microbes from the living
environment. Using the saline-dipped sterilized cotton swab, the chosen sample's surface is
scraped and streaked in an LB agar plate.

Materials and Methods

Please refer to the lab manual.

Results and discussions

Figure 1: Escherichia coli under microscope with magnification 1000X

Figure 2: drawing of Escherichia coli

 The above figures show the bacteria Escherichia coli. E. coli can be found in the
environment, foods, and intestines of people and animals (CDC). While most E. coli are
harmless, some strains can cause sickness, most commonly diarrhea (CDC). By looking at the
picture, the basic morphology of E. coli can be determined. Escherichia coli can therefore be
concluded as being rod-shaped. The microbes are evenly spread on the slide, meaning that the
emulsification with distilled water was done well.

Figure 3: Bacillus subtilis under microscope with magnification 1000X

Figure 4: drawing of Bacillus subtilis

The second bacteria is Bacillus subtilis which is commonly found in the soil (P.J.
Piggot). B. sub is non-pathogenic and forms heat-resistant spores (P.J. Piggot). In the above
image, a big cluster of bacteria is filling about half of the slide. There are two possible reasons
for this: the portion of a bacteria colony transferred with the inoculation loop was too large, or
the rubbing was underdone, that the material is not evenly distributed in the slide. Although the
bacteria are not evenly spread in the slide, the rod shape of B. sib is recognizable from the
picture above.
Figure 5: Staphylococcus epidermidis under microscope with magnification 1000X

Figure 6: drawing of Staphylococcus epidermidis

The third bacteria viewed under the compound microscope is Staphylococcus

epidermidis. S. epi is a gram-positive cocci-shaped bacterium widely spread in the environment
(“Staphylococcus Epidermidis - an Overview | ScienceDirect Topics”). The sphere shape of S.
epi can be seen from the picture and the drawing above. They are organized as single cells, pairs,
tetrads, and clusters. Although a line of massive bacteria clusters aligns the middle of the slide,
the individual cells can still be distinguished.
Figure 7: Pseudomonas aeruginosa under microscope with magnification 1000X

Figure 8: drawing of Pseudomonas aeruginosa

The above shows Pseudomonas aeruginosa under the 1000X magnification of the

compound microscope. P. aer is widely found in the environment, like in soil and in water
(CDC, “Pseudomonas Aeruginosa in Healthcare Settings”). P. aer can cause infection in
humans, most commonly in blood and lungs, but also in other parts of the body (CDC,
“Pseudomonas Aeruginosa in Healthcare Settings”). Most infections are caused in hospitals after
surgery (CDC, “Pseudomonas Aeruginosa in Healthcare Settings”). The shape of the bacteria, as
seen from the figure above, is rod-shaped. From the above figures, two lines of bacterial clusters
can be seen. The material may not be evenly emulsified due to insufficient rubbing.
Figure 9: Saccharomyces cerevisiae under microscope with magnification 1000X

Figure 10: drawing of Saccharomyces cerevisiae

The last bacteria examined under the microscope is Saccharomyces cerevisiae. S. cer is
widely used in the field of biotechnology due to its unique biological characteristics such as its
fermentation capacity in the production of alcohol and CO2 (Min Wu, Xuefeng Li). S. cer is
cocci, in other words, sphere-shaped. There are only a few numbers of bacterial cells shown in
the slides. Transferring a
greater portion from a
bacterial colony is
necessarily better to
determine the
morphologies and
organizations of S. cer.
Figure 11: bacteria isolated from skin of hand

The source of the microbes is the skin of a hand. Microbes were collected from all over
the hands, from the lobes to the palm, nail, and under the nails. From the above figure, the
morphologies can be determined. The bacteria seem to be either punctiform or circular. The
elevation looks to be slightly raised, and the margin is entire. The colonies are evenly spread
throughout the plate.
Most environments are full of microorganisms. The human skin, especially the hands we
use to touch objects, also contains hundreds of different microorganisms species. However, most
of these species are not isolated in the plate. This is due to the different growth environments of
the bacteria. Being living organisms, different types of bacteria require specific pH, temperature
range, oxygen, and nutrients. For example, E. coli requires a temperature range of 4- 45°C, with
an optimal temperature of 37°C for growth, and can survive at pH 3.6 (Albrecht). On the other
hand, S. cerevisiae  grows best at 30°C and pH 5,0 (Zaïd Paula & Farley Trubendorffer). Thus,
out of the originally collected bacteria, most could not survive and colonize due to the lack of
meeting growth requirements.

Conclusion and Limitations

 In this experiment, a bacterial smear was prepared, stained, and observed under the
microscope. The shapes of bacteria Escherichia coli, Bacillus subtilis, Staphylococcus
epidermidis, Pseudomonas aeruginosa, Saccharomyces cerevisiae were examined and discussed
in the report. E. coli., B. sub., and P. aer are rod-shaped, and S. epi and S. cer are cocci. In the
second part of the experiment, microbes were isolated from the surface of any environment, in
this case, the skin of the hands. The bacterial colonies are round (circular) and slightly raised. 
Some errors were made when transferring and emulsifying the bacteria. Thus the image
on the slides was clustered, making it hard to examine the shapes of the bacteria. Thus, a smaller
portion of the bacterial colony should be used, and the transferred bacteria should be rubbed
sufficiently to avoid clusters. 
 Works Cited

Albrecht, Julie. “Escherichia Coli O157:H7 (E Coli).” UNL Food, 13 Aug. 2015,

food.unl.edu/escherichinia-coli-o157h7-e-coli.

CDC. “Pseudomonas Aeruginosa in Healthcare Settings.” Centers for Disease Control and

Prevention, 13 Nov. 2019, www.cdc.gov/hai/organisms/pseudomonas.html.

---. “Questions and Answers.” Centers for Disease Control and Prevention, 2019,

www.cdc.gov/ecoli/general/index.html.

Min Wu, Xuefeng Li. “Pseudomonas Aeruginosa - an Overview | ScienceDirect Topics.”

Sciencedirect.com, 2009,

www.sciencedirect.com/topics/medicine-and-dentistry/pseudomonas-aeruginosa.

P.J. Piggot. “Bacillus Subtilis - an Overview | ScienceDirect Topics.” Sciencedirect.com, 2016,

www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bacillus-

subtilis.

SAHIL BATRA. “Preparation of an Ideal Bacterial Smear | Microbiology Practicals.”

Paramedics World, 21 Feb. 2018,

paramedicsworld.com/microbiology-practicals/preparation-of-an-ideal-bacterial-smear/

medical-paramedical-studynotes.

“Staphylococcus Epidermidis - an Overview | ScienceDirect Topics.” Sciencedirect.com, 2011,

www.sciencedirect.com/topics/medicine-and-dentistry/staphylococcus-epidermidis.

Zaïd Paula & Farley Trubendorffer. Optimal Growth Conditions of Saccharomyces Cerevisiae in

Bioreactors.