Basic Microbiological Techniques and Ubiquity of Microorganisms

INTRODUCTION

Microorganisms are explained as the smallest organisms that can not be seen with the naked
eye (Willey,2017). The environment is full of microorganism. They can be found in the air
one breath, food one and water one drinks. Microorganisms contaminate every surface of all
objects except when a microbiological technique, like aseptic technique is performed
(Willey,2017). Aseptic technique is explained as a process that is used to decrease or
completely destroy the chance of contamination during the collection of cultures (Lab
Manual,2019). Aseptic techniques are sterile work area, cleaning each an every disinfected
lab benches and using open flame to destroy microorganism. Failure to practise that may lead
to undesired results especially in biologically contamination. This is because microorganisms
only can spread and live on the normal temperature. That is why it is needed to culture to a
new environment that will prevent microorganisms to affect the experiments. Nutrients used
to keep a favourable environment for microorganisms are culture media and there are three of
them in different forms (Lab Manual, 2019). There is a solid, broth and a semisolid. The
broth does not contain agar and it used to collect large numbers of bacteria. The semisolid is
used to determine the motility of the microorganism along with anaerobic growth. The solid
is used to observe the growth of microorganism and to determine the appearance of the
colony and isolation (Lab Manual 2019).
Pure cultures contain microorganisms of the same species such as two bacteria’s being use in
the lab, Serratia and Micrococcus (Lab Manual 2019). A mixed culture is an opposite of a
pure culture it contains a variety of species like M. luteus .( Lab Manual, 2019).Pure cultures
are formed through mixed culture which are diluted in order to lower that concentration of
the bacterial cells. Inoculating techniques is where depositing of the microorganisms and the
bacterium can occur aseptically onto the agar plate. The main aim of the streak plate is to
produce isolated colonies from high concentration of the bacterial cells. Simple staining can
be observed in variety of ways like observing the living, unstained organisms or by observing
dead cells stained with dyes. Most dyes are different but they are all composed of the benzene
ring, a chromophore an auxochrome The overall objective of this experiment is to prove that
microorganisms are everywhere and the use of aseptic technique and the other
microbiological techniques

Materials and Methods

-Ethanol for disinfection
-Loops and Needles for transferring
-Agar plates for nutrients of the bacteria
-pipettes for the transfer of the liquid
- test tubes and petri dishes
-Mixed culture of S.marcescens, E.coli and M.luteus
-streak plates and pour plates
-ubiquity plates
During the experiment 1 the lab bench was disinfected by using ethanol to ensure that there
are no microorganisms. All the loops were also sterilized by flaming until it was red and
then it was cooled. The loop was used to take the inoculum from the broth culture. The E.coli
culture was obtained. The inoculum is by shaking culture test tube. The culture was picked up
by using the inoculum liquid. The film of the culture of broth culture can be seen across the
loop as it was removed. The new media was also inoculated by inserting a loop containing
inoculum into sterile broth. The inoculum was then knocked around in the broth. The mouth
of the newly inoculated tube was flamed and the cap was replaced. The loop was flame so
that it can be sterile. The tubes was then incubated at a proper temperature. The sterile tubes
were then obtained. The sterile loop was inserted into the inoculum and the liquid was
picked. The loop with the bacteria was placed into the slant tube to the bottom of the slant.
The zig zag pattern was inoculated starting from the bottom. This increases the surface area
of the culture. The loop again was sterilised. The tubes were then incubated under standard
temperature
The loop full of inoculum was picked up from a broth. The lid of the petri dish was lifted to
insert the inoculation loop. The loopful of culture was placed on the surface of the agar
plants. The four way streak was done. The petri dishes were labelled. The tubes of molten
nutrient agar was cooled to 45 degrees. The aseptic technique was used after it the plates
were allowed to solidify and incubated at 30 degrees for 2 days. The streak plates were then
evaluated. During experiment 2 the plate with the well isolated colonies were picked. The
morphological characteristics were observed. A dissecting microscope was used to view the
colonies. The small drop was poured onto the slide using the loop. The small amount of
inoculum was transferred into a drop of water. They were both spread and airdried. The
loopful were placed on the centre of the slide passed over the Bunsen burner. After it was
cooled the staining begun . the heated smear was flooded with the crystal violet and washed
with distilled water. The smear was then viewed through the microscope.

Results
 Table 1: The Morphological characteristic of the ubiquity and mixed culture

Morphological ubiquity Mixed culture

characteristics
Form Circular Circular
Elevation Flat Raised
Margin Entire Entire
Surface Rugose Smooth
Optical characteristics Opaque Opaque
Consistency Butyrous Butyrous
Pigmentation growth Light yellow Light yellow

Table 1 demonstrates the characteristics of the ubiquity and the mixed culture viewed with
naked eye. The colonies share some of the characteristics like the form which is circular ,
optical characteristics which are opaque, pigmentation which is yellow and the margin which
is entire. But it does have some differences like the elevation for the ubiquity it is flat but for
the mixed culture which is raised

Table 2 : Growth results of the nutrient broth using none aseptic and aseptic technique

Test tube A (none aseptic Test tube B (aseptic

technique) technique)
Clarity Cloudy Clear

Table 2 demonstrate the outcomes of using a sterile loop and the none sterile loop. It is
visible that when the loop was sterile the was no growth of microorganism the nutrient broth
became clear which indicates that there is no contamination and that is in test tube B. In test
tube A, where the aseptic technique was not applied the growth of the microorganism was
visible because it was cloudy and that shows contamination.
Figure1: A streak plate S. marcescens (1000×)

Figure 2: A broth plate S.marcescen (1000×)

Figure 1 illustrates the results of the streak plate method performed with S. marcescen on
agar plate the specimen was viewed on microscope with the magnification of 1000×. Figure
2 shows a broth plate of S. marcescen also viewed with magnification of 1000×. The two
figures have no difference they look the same. They seem to be a streptococcus bacteria since
they have a line of small rings.
Discussion
Microorganisms can not be seen clearly with one’s naked eye, by the use of the nutrient agar
plates and incubation methods. The bacteria was able to grow and create colonies that are
visible with one’s naked eye. After observing the nutrient agar plates. The microorganisms on
table one showed not very much difference most of the characteristics are the same like the
pigmentation which is light yellow, form which is circular, and consistency which is
butyrous. There are similarities than differences thus proving that they were isolated and
single colonies. The second experiment in table 2 was done to the presence of
microorganisms using the aseptic and none aseptic techniques. In test tube A which was done
aseptically by heating the inoculating loop thus sterilising it and killing all the
microorganisms and it was found to be clear indicating no contamination. Test tube B was
done none aseptically the inoculating loop was not sterilised and after incubation it came out
cloudy thus showing contamination. This shows that the microorganisms are everywhere
especially when the aseptic technique is not performed.
The purpose of the streak plate method is to isolate colonies from highly concentrated
suspensions of cells. Therefore when the cultures are mixed it possible to separate the
bacteria. This technique is sterile or aseptic to prevent the microorganisms to move or be
transferred to the streak plate aside from the ones that are being used. The advantages of the
streak plate is that it is allows the mixed cultures to be isolated and to be individual colonies.
The disadvantage is that it makes it hard to count the colonies like the pour plate methods in
figure one and two the bacteria is completely visible thus showing that the microbiological
techniques were done properly. The streptococcus bacteria was clearly visible.
In conclusion the aim of this whole experiment was to understand microorganisms, how they
grow and that they are found almost everywhere. The experiment demonstrated that
microorganisms do affect some processes when the aseptic technique is not performed. There
these techniques are understood more than before. Aseptic are not only to be performed in the
lab but they can be also be performed in the health care setting at all times. The practical
demonstrated the ubiquity of microorganisms and how they can easily create contamination.
Therefore using proper techniques and basic skills for microbiological experiments, the
objective were obtained.

References

Discipline Of Microbiology.( 2019).Bacteriology Lab Manual. University of KwaZulu Natal.

Willey, J.M., Sherwood, L. M., Woolverton, C. J. (2017). Prescott's Microbiology. 10th Ed.

McGraw-Hill Education. New York.

Toba, F. (2015). The Advantages and Disadvantages of the Various Microbial Culture

Techniques. Retrieved October 07, 2016.