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

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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

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

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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, 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.)