Microbial and physical contamination –

potential souce of inaccurate result in

microbiology experiment

Paula Širola

Sunderland University, Faculty of Health

Sciences and Wellbeing, School of Pharmacy and
Pharmaceutical Sciences

Research And Practical Skills In

Biopharmaceutical Science
BPS114

28st February 2020

Word count:1077

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 ABSTRACT

This report explores if contamination has any effect in microbiology experiments.

Contaminating products in everyday practise can cause incorrect result or failure of
conducted experiment. What is more, it is quite possible to infect not only yourself but other
people in the aera as well. The main focus was on observing ever-present microorganisms in
the controled enviornment.
Research was divided in two sections. The method used in first section was based on
cultivating bacterias on several petri dishes. The often found sources of contamination were
used as samples that were compared with sterile agar plate. During the whole experiment
sterile tehnique was adapted. The second section was looking at physicaly contaminated
samples and identifying contaminant and its possible source.
The most contaminated sample was one with unwashed fingerprint, which proves that gloves
are mendatory in laboratory. One of the sample showed postive result on bacterias in the air
of laboratoy. With that in mind, the dishes used in experiments needs to be sterilised before
and after usage to achieve a sterile state. In other words, it is one of the procedures to curtail a
risk of contamination in lab. This report contains remedial action for possible sources of
contaminations provided in samples. Overall, it was shown that microorganisms are
everywhere around us and even on us. Therefore, it is certainly easy to contaminate
pharmaceutical products to various extents.

KEYWORDS
Microbiology experiments; contaminations; microorganisms; cutures; bacteria; fungi;
Tryptone Soya Agar plate; Sabouraud Agar plate

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INTRODUCTION

One of the most often reason for failed experiments is contamination. There are 3 types of
contamination which can appear in pharmaceuticals, such as chemical, physical and
biological. To begin with, biological contamination has its subtypes : bacterial, fungal and
viral (which is more rare). All the equipment used in a laboratory should be treated as it is
already contaminated. Therefore, when it comes to working with cells and tissues, it is
necessary to use sterile technique. Sterile technique intends to prevent contamination of
laboratory samples which can be easily contaminated by external sources along with
microbial contamination of co-workers. Multiple sterile procedures are used in everyday
practice. Heat treatment is used on a large scale with 3 ways to achieve: red-heat sterilisation
by using a Bunsen flame, dry heat sterilisation by using a hot-air oven or moist-heat
sterilisation by using laboratory autoclave. Furthermore, to ensure sterile conditions it is also
possible to use radiation, filtration and chemical agents. For culturing microorganisms, solid
media (agar plates) or liquid media can be used. Eilshemius (1850) was the first one who
suggested using agar to cultivate bacteria instead of gelatine, which was used at the time.
Agar is a complex polysaccharide from red algae that is partially immune to degradation by
most bacteria. although media for culturing microorganisms provide nutrients necessary for
their growth, some other conditions also need tio be accomplished, such as optimal
temperature, pH value, ionic ans osmotic conditions, as well as atmospheric requirements.
The way to prove presence of contamination is by using bacterias ability to be cultivated.

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METHODS

Tryptone Soya Agar (TSA) and Sabouraud Agar (SAB) were provided for this experiment.
The first section was about microbial contamination and how different causes of
contamination affect on growth and variety of colonies. To prepare the first samples, TSA
plate and SAB agar plate were left open in open space around the Faculty for 30min. The
second set of samples were prepared with 0,1mL of tap water spread with a sterile spreader
across plates, while the third set of samples contained 1mL of sterile deionised water. The
fourth sample was divided in half with a drawn line (A and B). A side was used for unwashed
fingerprint, while B side was used for washed and dried fingerprint. Next, hair with root was
placed on the fifth sample, in this case, the TSA plate. Afterwards, a coin was placed and
shortly after removed. For the sixth sample, the microbes from the surface of the chosen
object (jeans) were collected with a swab and then run across the TSA plate. Lastly, the
seventh sample was agar itself that was left unopened as a check if it is sterile. All of the
above samples were left 24 hours in the dark area to allow bacterial growth. After 24 hours
the samples in Petri dish were observed with the eye of the observer and results obtained.
The second section was about physical contamination. In that case, five contaminated bottles
were provided to identify by looking at them what is a contaminant and what their possible
source could be.

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RESULTS

Results obtained from section 1:

PLATE NUMBER OF DIFFERENT COLONIES OBSERVATIONS
TSA SAB
Open settle plate on 6 0 Very small colonies
the bench
Tap water 0 0 No colonies visible
Sterile deionised 0 0 No colonies visible
water
Unwashed fingers 34 35 Circular in shape
and bigger than with
washed fingerprint
Washed fingers 7 25 Small colonies,
punctiform in shape
Hair/coin 4 19 Punctiform in shape
Everyday item 23 11 Small white colonies
(Jeans)
Un-inoculated 0 0 No colonies seen
control
Figure 1: Growth of microorganisms on TSA and SAB plate in controled enviorment. Data
represents number of colonies visible on the plate.

Results obtained from section 2:

BOTTLE NUMBER CONTAMINANT
A Hair particles
B Glass particles
C Thin foil
D Rubber
E Oil mixed with
water
Figure 2: Psysical contaminants obvious in provided bottles.

DISCUSSION

Sample one, plate settled on a bench with TSA showed a positive result on bacteria due to the
very small bacterial colonies that were shown on the TSA plate, 6 to be precise. On the other
hand, on SAB no colonies were formed which suggested that funghi were not present in the

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air, only bacterial. No bacteria observed in the samples of tap water nor sterile deionised
water. Even though results were negative, the experiment did not prove that water is sterile,
because the amount of water sample for analysis was not enough to complete the parameters
of World Health Organization (WHO) for verification of the microbial quality of water.
Parameter states that 100 ml of a water sample can not contain any E.coli nor other coliform
bacterias but it can contain other bacteries. Since the amount of sample was less than 100 ml,
there were fewer chances to cultivate bacteria on a plate.
The samples of both washed and unwashed fingerprints showed positive results on SAB and
TSA plates. Unlike sample with washed fingerprint, a sample of an unwashed fingerprint had
colonies in a circular shape and almost 5 times higher representation. It was no surprise when
both of the samples showed high positive results because out of whole human body the hands
are the most exposed to bacteries. With this in mind, the results were valid. Surprisingly, low
amount of colonies has grown from the fifth sample. On TSA plate 4 colonies in punctional
shape were observed, while sample on SAB plate developed 19 cultures in punctional shape.
According to Schlegel (1924) heavy metals, (in this case, silver coin) can slow the growth of
microorganisms or inhibit it. The effect is called bacteriostatic. Thus, coin stopped the growth
of all bacteria and fungi on both coin and hair root. It is assumed that coin destroyed the
enzymes responsible for microorganisms growth. The cause of high results on SAB plate is
the hair root, and on TSA plate is coin, since coin has more bacteria than hair root and
opposite. The sixth sample contained everyday item (in this case, jeans). On TSA plate 23
small white colonies were developed from sample obatined from jeans surface, while on SAB
plate there was only 11 colonies. Therefore, jeans were proved to have more bacteria than
fungus. There were no colonies observed in the last sample, so we can conclude that agar is
sterile. Last sample was un-inoculated negative control.

In the second section, by looking in the bottles, the contaminants and their possible sources
were identified. Bottle A had a hair particle, which possibly was human hair. To prevent
contamination like this from happening, the hair net should be used. Bottle B contained glass
particles, possibly from dish breakage, which is why it is important to clean thoroughly after
breakage. Bottle C had thin foil, which probably came from an unadvisedly done experiment.
Therefore, it is important for people that work in a lab to be conscious and educated about
sterile techniques. Bottle D had pieces of rubber inside of it, that maybe came from using a
rubber on a bench. To stop this kind of contamination from happening, the pen should be
used. Bottle E contained oil mixed with water, possibly from unwell washed dishes. To
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prevent it, dishes should be washed thoroughly and sanitised properly, because that is an
important section of every experiment.

REFERENCES

 Cooper, C., (2019), Microbial Growth, lecture slides, Introduction to cell biology and
biochemistry, BPS111, University of Sunderland, delivered 31 January 2020

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  Gottschalk, G., (2012), Discover of World, Bacteria, Archeae, Viruses, Weinheim,
Germany: Wiley-Blackwell,

 Guidelines for drinking-water quality: fourth edition incorporating the first addendum.
Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO

 Palmqvist, C., Samuelsson, A., Froeding, I., Giske, G.C. (2019). Surface
Contamination of CT and MRI Equipment- A Potential Source for Transmission of
Hospital-Acquired Infections, Journal of Radiology Nursing, 38, 254-260

http://eds.b.ebscohost.com/eds/detail/detail?vid=2&sid=c6f815d0-1ec2-4db4-af64-
cc1e1638e5ad%40pdc-v-
sessmgr06&bdata=JnNpdGU9ZWRzLWxpdmUmc2NvcGU9c2l0ZQ%3d
%3d#AN=140423318&db=edo Accessed:27/02/2020

 Reed, R., Holmes, D., Weyers, J., Jones, A.,(2016), Practical Skills in Biomolecular
Sciences, Harlow: Pearson Education Limited,
 Schlegel, G. H., (1924), General Microbiology, (seventh edition), Cambridge:
Cambridge University Press
 Sigward, E., Fourgeaud, M., Vazquez, R., Guerrault-Moro, M.N., Brossard, D., &
Crauste-Manciet, S. (2012). Aseptic stimulation test challenged with microorganisms
for validation of pharmacy operators. American Society of Health-System
Pharmacists, 69, 1218-1223. http://eds.a.ebscohost.com/eds/pdfviewer/pdfviewer?
vid=3&sid=3330f98c-f33c-48dc-a215-2a0b75b50e68%40sessionmgr4007 Accessed:
27/02/2020
 Zani, F., Minutello, A., Maggi, L., Santi, P., & Mazza, P. (1997). Evaluation of
preservative effectiveness in pharmaceutical products: the use of a wild strain of
Pseudomonas cepacia. Journal Of Applied Microbiology, 83(3), 322-326.
https://sfamjournals.onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-
2672.1997.00231.x Accessed on: 27/02/2020