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Antiseptic Technique

Group 1/Wed. 1-4 PM


Alyssa Ables
Jennifer Jossart
Jackyline Mitchell
Eugen Tutunaru
Biology 300
Instructor: Dr. Conrad Valdez

Introduction:
Aseptic technique is a crucial experimental condition in a
microbiology lab. 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 while handling
the cultures.
The experiment introduced a challenging condition to the sterile
technique. The experimental procedures were performed on benchtop.
The myriad of microbes floating 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 contamination of growth medium with


foreign microbes from the environment.
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 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 L of E. coli culture was dispensed in the growth medium.
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The lid from plate J was partially opened to ensure minimal exposure to
atmosphere and 50 L 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 Lbroth 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.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.

The visual references of the results are presented in Illustration


1. Plate C presented a moderate cloudy growth medium that indicated
presence of bacterial contamination while Plate D showed a clear liquid
indicating no presence of 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 contamination for the
experiment. Plate H presented no bacterial contamination. Plate I
presented a cloudy medium showing moderate bacterial growth while
plate J showed no presence of bacterial growth. The bottle containing
L-Broth showed no bacterial 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 exposed to various conditions
of contamination would exhibit bacterial growth. Dishes 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 that showed no bacterial growth, and therefore no
contamination, were plates D, F, H, 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 reference to the plates that were not contaminated. Since all
conditions of the sterile technique were closely followed, the results
confirmed the hypothesis.
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

contamination could be minimalized applying proper hood sanitation,


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careful handling 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 remote
places, making water drinkable, etc.)