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Biology 300
Instructor: Dr. Conrad Valdez
Introduction:
Preventing the contamination of a sample with foreign microbes from the environment
is the main focus of the present experiment. The participants were required to maintain
a sanitary environment isolating the samples to be contaminated with microbes that were
not intentionally introduced into the growth medium. Secondly, but equally important,
was to prevent the contamination, with possible pathogenic bacteria, of the lab members
in the atmosphere and on the benchtop, can constantly contaminate the samples,
therefore careful, fast and precise handling was necessary to achieve success in the
experiment.
It can be hypothesized that applying successful aseptic technique will prevent the
Methods:
Prior to touching the material provided, gloves were applied by all lab group
members and sanitized with 70% ethanol. Along with sanitation of gloves, the benchtop
was treated with 70% ethanol to prevent contamination from the tabletop. A set of 8 petri
dishes was labeled C-J, along with the date and group number. In each plate 8 mL of L-
broth (Casein enzymic hydrolysate or Tryptone, Yeast Extract, NaCl) was dispensed. The
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L-Broth was previously autoclaved to ensure that no microbes are present in the growth
medium. The lid of the plates was only partially opened when the growth medium was
dispensed, to ensure minimal exposure to atmosphere. The plates were placed, treated
and handled in various conditions. The lid from Plate C was removed and the growth
medium was exposed to the atmosphere for 10 minutes. Plate D was kept closed. The
growth medium from Plate E was placed the fume hood for 10 minutes and the plate was
exposed to the air inside the hood. Plate F was kept closed. The lid from Plate G was
partially opened to minimize exposure to the environmental air and the growth medium
was touched with the finger that was dirty (finger touched hair, saliva and the surface of
the sink). Plate H lid was partially removed to prevent air from atmosphere to
contaminate the sample and growth medium was touched with a clean finger (the finger
was cleaned with 70% ethanol). The lid from Plate I was partially opened to ensure
minimal exposure to the environment air and 50 μL of E. coli culture was dispensed in the
growth medium. The lid from plate J was partially opened to ensure minimal exposure to
atmosphere and 50 μL of L-Broth was dispensed in the growth medium. The plates were
later stored in an incubator at 37 ℃ and they incubated for 48 hours. Along with the plates
the bottle containing L-broth that was used was stored in the incubator for 48 hours to
ensure that growth medium was not contaminated with any microbes. After 48 hours the
results were documented and the data recorded was used to determine the validity of
the hypothesis.
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Results:
Illustration 1: Visual references of each sample used in the experiment. Illustration 1: Visual
references of each sample used in the experiment: Plate C presented medium contamination, Plate D
showed no contamination, Plate E displayed minimal contamination, Plate F presented no contamination,
Plate G showed the highest level of contamination in experiment, Plate H displayed no contamination, Plate
I showed moderate contamination, Plate J presented no contamination. The L-Broth bottle showed no
contamination.
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The visual references of the results are presented in Illustration 1. Plate C
contamination. Plate E presented a cloudy growth medium that indicated the presence
of bacterial contamination that was less noticeable than the other plates. Plate F showed
no bacterial growth. Plate G displayed a cloudy growth medium that was noticeable more
dense and, in addition, white aggregates were observed, indicating the highest level of
presented a cloudy medium showing moderate bacterial growth while plate J showed no
contamination.
Discussion:
It was expected that the Petri dishes unexposed to the atmosphere and other
conditions of contamination would not present bacterial growth, while plates that were
that displayed bacterial growth were plates C, E, G, and I. The plates were exposed to
contaminated mediums that allowed microbes to contaminate the samples. The dishes
and J. Plate I was used as a positive control and it was used as reference in comparison
with the other contaminated plates. Plate J was used as negative control being used as
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reference to the plates that were not contaminated. Since all conditions of the sterile
Plate E was exposed to a medium that minimized the contact of the sample with
atmosphere. Plate E presented a cloudy growth medium but with less intensity than other
plates exposed to other conditions. Since plate E was placed in the fume hood, the
number microbes that contaminated the sample was smaller compared with the other
contaminated plates. The airflow inside the hood partially prevented the infestation of
the sample. Observing how the members of the lab handled the samples that were placed
in the fume hood, it can be predicted that, for future experiments, the level of
the samples, checking the direction and the amount of airflow inside the hood.
Conclusion
Analyzing the recorded data, it can be concluded that the experiment was
successful and the aseptic technique used was properly and thoroughly applied. The
present experiment proves that sterile technique can be achieved even in more
challenging condition when the procedures are completed on an open surfaces such as
bench top. Further studies can be address more challenging conditions for aseptic
techniques than the settings in the present experiment. These studies can provide
valuable information and procedures that can be used when a sterile technique is
required in harsh conditions outside a microbiology lab (surgeries in the field, first aid in