AUDREY-ANN & ALEXANDRE SIMARD
Ages 15 & 17 | St. Bruno Lac-Saint-Jean, Quebec
This project is an experiment that aims to discover the quantity of bacteria present in reusable bags. Nowadays, reusable bags are increasingly prevalent with the present ecological movement. It is believed that their hygiene is often neglected and that, due to their contact with food, they should be better maintained. With the collaboration Mr. Sabin Harvey, a natural sciences professor, samples were taken on the bags in a biology laboratory of the Collège d’Alma. Bacterial cultures, analyses, and statistics were carried out. The hypothesis was that there would be a strong presence of bacteria in the bags, and that this situation could have an important impact on the cleanliness of food.
METHODS
Preparation
Thirty-five (35) bags1 were collected, numbered and the following characteristics were noted: the last wash of the bag, its usage, the location where the bag is stored, its number of uses per week and its length of use.
Laboratory Procedures
Seventy (70) templates2 were disinfected with alcohol and placed in a radius of 30cm beneath a flame to create a sterile zone. Two templates were fixed in the bottom of a bag with adhesive tape. A culturette3 was activated by plunging the swab in Amies Agar gel4 so that it was moist to collect the microorganisms. The culturettes were labelled with the number of the bag, the index “a” or “b” and the date. Samples were collected by stratifying5 the 100 cm2 surface determined by the template and the culturette was then tightly closed. This process was performed for each bag. Next, Petri dishes6 containing TSA7 were identified with the sample number, date, and time. The Petri dishes were opened within a radius of 20cm under the flame and inoculated by striation with the cotton swab,8 which was done by rolling the swab between the thumb and the index finger. The Petri dishes were then closed and deposited outside the sterile field. This process was performed for each sample. The Petri dishes were then placed in an incubator9 at 37.1 degrees Celsius and removed after 48 hours. Lastly, the number of bacterial colonies10 present in each dish were counted with the help of a colony counter pen.11 The results were noted, and the samples photographed.
Gram Straining12
A number was attributed to six different colonies based on their colour, appearance, texture, and size, then the colonies were circled. Microscope slides13 were cleaned with 70% isopropyl alcohol and deposited in the sterile field. With the help of a wash bottle,14 one drop of distilled water was deposited onto each slide. An inoculation loop was sterilized with a flame15 until it became red. This inoculation loop was used to take a small part of a colony and rub it onto the drop of distilled water. After the surface was dry, the slides were quickly passed through the flame three times to fix the sample with the heat. A few drops of a solution of gentian violet16 were then placed on the slide and left to react for 45 seconds, then rinsed. Following this, a few drops of Lugol17 were deposited on the slide, left to react for 45 seconds, then rinsed. A bleaching solution was then placed on the slide for 6 seconds and rinsed immediately. Lastly, a few drops of safranin18 were placed on the slide and left to react for 45 seconds, then they were rinsed and left to dry. The slides were studied using a microscope with x100 immersion objective19 and strong lighting. The size, morphology, grouping and Gram of each colony were noted. These steps were performed for each chosen colony.
Figure 1: The Gram straining process.
MATERIALS
The following is the list of materials used during the experiments: questionnaires, pencils, culturettes, adhesive tape, 10 X 10 cm templates, plastic reusable bags (32 used and 3 controls), Petri dishes containing TSA, burner, camera, colony counter pen, latex gloves, rubbing alcohol, microscope, microscope slide, wash bottle filled with distilled water, safranin, Lugol, empty container, gentian violet, inoculation loop, incubator and bleaching solution.
RESULTS AND ANALYSIS
Below are the questions which were answered through this project. The data which was used to obtain these results are presented in Table 1.
Do the bags that contained meat products contain more bacteria?
Considering that the variable TNC (too numerous to count) is represented by the number 2500, an increase of 7.2% for bags having contained meat products was observed. Therefore, it was assumed that the bacteria would be those found in meat. There could thus be E. Coli and Salmonella present, for instance.
Does the usage frequency of the bags have an impact on the quantity of bacteria?
Yes, curiously, it was observed that the samples from bags used 1.5 to 2.5 times a week had 32.5% less bacteria than those used less than 1.5 times a week. It is believed that when they are minimally used, the bacteria have more time to multiply.
Does the age of the bag play a role in its cleanliness?
Yes, it was observed that bags 5 to 10 years old had 61.2% more bacteria than those less than 5 years old. Thus, one can deduce that the bacteria accumulate significantly over time.
Do the new bags already contain a lot of bacteria?
The samples from the 3 control bags had, on average, 24.67 colonies. This result is very little when it is taken into consideration that the average of all the bags analysed was 656.37 colonies on a surface of 200cm2.
The data having served to answer the previous questions is found in Table 1. It was determined that TNC=2500, as in certain cases, there were too many colonies to count, while other times, they were not as numerous, but their shapes made the exact number difficult to determine.
A Gram staining was also performed in order to differentiate between different categories of bacteria by their cell membrane. In fact, the Gram-negative bacteria would tend to be more resistant than Gram positive bacteria. The results of this test are presented in Table 2.
Figure 2: Differences between Gram+ and Gram–
Table 2: Results of Gram test.
A Gram staining was also performed in order to differentiate between different categories of bacteria by their cell membrane. In fact, the Gram-negative bacteria would tend to be more resistant than Gram positive bacteria. The results of this test are presented in Table 2.
NOTES
135 bags (selected sample size): 32 bags to evaluate and 3 control bags.
2Template: model that serves as a reference for a dimension. (LeParisien, 2016)
3Culturette: a swab (absorbent tampon serving to take samples) with a transport medium. (Oxford, 2020)
4Amies Agar gel: sterile and isotonic gel, a solution that reproduces good conditions for the survival of microorganisms, due to its osmotic pressure being similar to the liquids of organisms.
5Stratify: make parallel lines by rolling the cotton swab between the thumb and index finger. (CNRTL, 2012)
6Petri dish: plastic dish used for bacterial cultures in a solid medium. (Larousse, n.d.)
7TSA (tryptic soy agar): popular culture medium for developing bacterial cultures.
8Cotton Swab Striation: technique consisting of making lines with a cotton swab.
9Incubator: machine furnishing heat for the growth of microorganisms. (>atg, 2017)
10Bacterial Colony: group of bacteria cultivated from a single bacterium. (Wikipedia, 2020)
11Colony Counter Pen: pencil whose exercised pressure on a Petri dish triggers a count. (Dutscher, n.d.)
12Gram Staining: colouration that serves to highlight the properties of the bacterial wall and to use these properties to distinguish and classify the bacteria. (Scienscope and l’Université de Genève, 2017)
13Microscope Slide: small glass plate containing a sample during microscope observation. (Wikipedia, 2014)
14Wash Bottle: container producing a jet of liquid used during washing in a laboratory. (Larousse, n.d.)
15Inoculation Loop: tool consisting of a shaft, stem, rigid metal thread with a closed loop of 2mm. (Glossaire de bactériologie)
16Gentian Violet: violet colouring for biological examination, also known as “Gram staining” or “cristal violet”. (Pilou, 2014)
17Lugol: iodine compound that allows for the fixing of the violet in the bacteria.
18Safranin: pink colouring used during Gram staining. (Éducalingo, 2020)
19Immersion: with a drop of special oil. (Lavallée, 2008)
REFERENCES
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>atg start. (2017). Faire pousser des organismes: l’incubateur. http://www.atgstart.be/index.php/these/11-le-chimiotactisme--le-moyen
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Differences between Gram – and Gram +. Docplayer.fr. https://docplayer.fr/docs-images/59/42863136/images/228-0.png
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Lavallée, J. (2008). Microscope et objectif à immersion. Le monde en images. http://monde.ccdmd.qc.ca/ressource/?id=60781
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Pilou, J-F. (2014). Violet de gentiane – définition. Le journal des femmes santé. https://sante-medecine.journaldesfemmes.fr/faq/36585-violet-de-gentiane-definition
Université de Genève BIOutils. (2017). La coloration de Gram. https://www.bioutils.ch/protocoles/5-la-coloration-de-gram
ACKNOWLEDGEMENTS
Acknowledgements
We would like to thank:
Mathieu Simard. November 15, 2019. He oriented us in our project.
Sabin Harvey. January 23, 2020, February 10, 2020, February 11, 2020, February 13, 2020. He is our scientific supervisor, accompanying us during the laboratory trials and helping us collect the reusable bags.
Mathieu Bilodeau. February 13, 2020. We benefitted from his collaboration during the inoculation of the Petri dishes.
Marilou Laprise. During the project, she helped us in completing the forms and gave us information concerning the science fair.
Guylaine Simard. February 17, 2020. She explained the Gram test.
ABOUT THE AUTHORS
Audrey-Ann Simard
My name is Audrey-Ann and I am 15 years old. I have been passionate about science for quite some time. I do theatre, improvisation, create stained glass, and I am also part of my school’s student council as Minister of the Environment. I love to write stories and read novels, despite the fact that I am very picky in this area. In August of 2020, I will be entering my last year of high school. In the future, I would like to study medicine or mathematics. Others say that I am considerate, talented, and that I am very empathetic.
Alexandre Simard
My name is Alexandre Simard and I am 17 years old. I have just finished high school at Curé-Hébert school. In the 2020 school year, I will be at the Collège d’Alma in their natural sciences program. I am a hard-working, generous, and punctual person. I am a very good public speaker, in addition to being gifted in French. I am very interested in biology, which is why I hope to go into general medicine in university. One of the values close to my heart is family.