How Long Does A Sterile Field Remain Sterile?
by Rebecca A. Burns, SUNY Canton, Canton New York
ABSTRACT
This experiment was conducted to show the likelihood of
contamination of a petri dish by bacteria when left exposed.
The number of bacterial colonies that grew on exposed petri
dishes were counted to determine the amount of contamination
over a period of 1, 15, and 30 minutes. The number of bacterial
colonies and molds that grew on each dish increased generally
with the amount of time exposed. Therefore, it was clear that
microbes are present in the air and can contaminate materials in
as little as one minute.
DISCUSSION
Figure 2: Solidified agar exposed to the
environment
Figure 1: Pouring melted nutrient agar
into a petri dish
PROCEDURE
The following procedure was carried out by students in Biology 209:
Introduction to Microbiology this spring 2014 semester. Each
student in five laboratory sections was randomly assigned to a
different time treatment.
INTRODUCTION
1. Melt and temper enough nutrient agar to pour all plates
from the same sample of agar.
2. Pour agar into petri dishes and allow dishes to solidify.
(See figure 1). Keep lids on to prevent contamination,
but leave space for excess moisture to escape.
3. Remove the lids from the first set of petri dishes, and
leave exposed to the air for one minute. (See figure 2).
4. Expose the second set of plates for 15 minutes.
5. Expose the third set of plates for 30 minutes.
6. Replace all petri dish lids after appropriate amount of
time.
7. Label petri dishes and incubate inverted at room
temperature (22°C) for 7 days.
8. Count the number of colonies grown on each plate.
When conducting experiments, it is important to maintain a sterile
field to prevent undesirable contamination. “Sterile field” has different
meanings in different situations, however. In medical situations, a
sterile field is established and strictly maintained for operations and
procedures. Individuals must face the operating table at all times, as
their back is not sterile, and only certain areas, such as disinfected,
gloved hands, on that person are considered sterile. In the
experimental lab, the “sterile field” is maintained by disinfecting the
countertop, working with clean instruments and materials, and always
having unsoiled hands. To reduce contamination when using petri
dishes, the lid is left on unless material is being placed in the dish.
When agar is poured or a lawn culture of microorganisms prepared,
the lid is only lifted slightly to prevent contaminants from entering the
culture. Are these precautions necessary? Would material really be
contaminated in a short space of time? Where would the
microorganisms come from?
Figure 5: 30 minutes of
exposure to the
environment and 7
days of incubation
Number of Colonies of Bacteria
Average Number of Microbial Colonies
Per Plate for Trials 1 & 2
Figure 4:15 minutes of
exposure to the
environment and 7 days of
incubation
Where did the contaminants come from, though? There are microbes,
including bacteria, viruses, molds, and other microorganisms in the
outdoor air all the time. Out-of-doors, microbes are blown around by
the wind, carried by dust particles, and are present on every organism
and on nonliving material. There are more than 1,800 different kinds
of bacteria present in the air at any one time (Krotz) and over a 1,000
species of bacteria living on our skin alone (Grice et al.). The majority
of these are harmless or beneficial, but many bacteria and other
microbes are pathogenic. Airborne microbes enter indoor
environments by blowing in through a window, carried by human
clothing and skin, and even by breathing. The dust in a room has been
found to contain the greatest amount of bacteria, in comparison with
indoor air, outdoor air, and dust from ventilation ducts (Gould).
Humans are the greatest culprits for microbial contamination. We
bring microbes into a room by entering, and in moving around, we stir
up the dust and cause more microbes to fly through the air. Crowded
conditions increase the spread of bacteria and viruses and the chance
of contamination (Bact.). A full lab could potentially increase the risk
of contamination because there are more people moving dust and air
around and contributing to the amount of microbes in the air.
RESULTS
The petri dishes exposed for 1 minute grew an average of two colonies (see figure 3).
The samples exposed for 15 minutes grew an average of 10 colonies on each plate (see
figure 4). The plates exposed for 30 minutes grew an average of 21 colonies of assorted
bacteria and molds (see figure 5).
Figure 3: After 1 minute of exposure to
the environment and 7 days of incubation
The results strongly suggest that the longer petri dishes are exposed to
the air, the more bacteria and mold spores fall on the dishes and grow
colonies. Clearly, more time exposed results in more contamination,
although it was noted that the amount of variation within each
treatment is large.
25
20
21
15
10
10
5
0
It is necessary, therefore, when conducting experiments, to follow
proper procedures for reducing contamination. The microbes flying
throughout the air must be kept out of mediums for accurate results of
many experiments and tests. W\hen working in the lab, avoid moving
quickly and turning your back to the lab bench. This could help reduce
the amount of air flow in the room and, consequently, decrease the
chances of contamination. In addition, it would be best to decrease the
number of people in a lab at one time. In any situation that requires the
absolute maintenance of a sterile field (e.g. a surgical room) it is
necessary to wear sterile gloves, gowns, face masks, head coverings,
and shoes coverings in order to minimize the likelihood of microbes
from our hair, skin, or clothing from entering the air and the sterile
field.
Works Cited:
“Bacteria and Viruses.” American Lung Association. http://www.lung.org/healthy-air/home/resources/bacteria-and-viruses.html. 2013. Web. 12 Apr. 2014.
2
Time of exposure to air (minutes)
1 minute 15 minutes
30 minutes
Gould, S.E. “How Bacteria Get into Your Home.” Scientific American. July 15, 2013. Web. 12 Apr. 2014.
Grice EA, Kong HH, Conlan S. (2009). Topographical and Temporal Diversity of the Human Skin Microbiome, Science, 324: 1190 - 1192.
Krotz, Dan. “Study Finds the Air Rich with Bacteria.” Berkeley Lab. 2006. Web. 12 Apr. 2014.
April 14, 2014