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Title: Sterile / Aseptic Techniques Objective: To learn how to execute aseptic techniques when working microorganisms To understand the

the principles of streaking agar in order to obtain pure culture To prepare a culture medium of Escherichia coli

Abstract: Introduction: The purpose of the streak plate is to obtain isolated colonies from an inoculum by creating areas of increasing dilution on a single plate. The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria. Isolated colonies represent a clone of cells, being derived from a single precursor cell. . Quadrant streaking involves distributing the inoculum into 4 quadrants. Aseptic techniques are procedures which are practiced under sterile conditions, e.g. flame sterilization. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Materials and Methods. A flamed inoculating loop was used to spread an inoculum of E.coli on nutrient agar medium using quadrant streaking. The inoculating loop was flamed each time a new quadrant was streaked. The culture was then inverted and incubated at 37 degrees Celsius. A prepared agar plate was obtained and divided into halves. One half of the plate was inoculated with unwashed hands, and the other half with washed hands. The plate was inverted and stored at room temperature. Another nutrient agar plate was divided into 4 quadrants. The quadrants were inoculated by a gloved hand that had touch the palms of 3 lab students. Conclusion: Aseptic techniques, such as applying bleach and flame sterilization, greatly reduce the chance of contaminating the agar while preparing the agar culture. Microorganisms are everywhere, even on the human body.

Methods/ Materials The table which facilitated the working area was disinfected by using 10% bleach to thoroughly wipe the table. The follow materials and apparatus were provided: metal inoculating loops, an inoculum of E.coli, pre-prepared nutrient agar medium, gloves, and Bunsen burner. The inoculating loop was first sterilized by holing the loop into the Bunsen burner flame until the metal portion became red-orange. The tube containing the culture inoculum was open by gently lifting the cap, and the tip of the culture tube was briefly flamed. The culture tube was then oriented at an angle, and the inoculating loop was inserted inside to remove a loop full of the inoculum. The lid of the agar Petri dish was slightly opened, just enough for the inoculating loop to enter. The loop was used to create a pool on the agar surface, as well the first quadrant of the streaking pattern. The lid was removed, and the loop was incinerated yet again, and inserted into the inoculum to obtain another loop full of the bacteria. The lid of the agar was slightly opened and the loop was used to make the second quadrant on the agar. The steps were repeated until 4 quadrants were made; care was taken not let the last quadrant touch the first quadrant. The culture tube and inoculating loops was re-sterilized by flaming and then put way for storage. The culture was inverted, and the name experimenter was written on the agar containing side plate. The plates were then incubated at 34o- 37oC. An agar plate was obtained, and a wax pen was used to draw a line on the bottom side of the plate, dividing the agar in halves. One half was labeled clean and the other half dirty. The lid was removed and the experimenters finger tips touched the dirty half. The plate was then closed. The experimenter washed hands with sanitizer and soap, removed the lid from the same agar plate and touched the clean half,. The plate was then closed, inverted and stored. Another agar plate was obtained and divided into quadrants 1, 2, 3, 4. Gloves were put on, and the instructor swabbed the left gloved and hand with cotton swab. The agar plate lid was removed, and the first two right fingers was used to touch the agar in quadrant 1. The lid was closed. Then the first two fingers of the experimenters right hand touched the left palm of another student in the group. The same right fingers were then used to touch the agar in quadrant 2. The same was done twice with two other students until quadrants 3 and 4 were touched. The agar plate was inverted and stored. The gloves were carefully removed and disposed of in the biohazard bag.

Results: The E.coli colony appeared circular, opaque and dull. Table Showing Result of The First Plate Inoculated with Unwashed and Washed Hands Side of Plate Dirty Clean Degree of Microbial Growth More than of the area was covered Small isolated areas of growth seen

Table Showing Results of the Second Plate Inoculated With Gloves On QUADRANT 1 2 3 4 Results Least microbial growth Some microbial growth Some microbial growth Most microbial growth

Discussion Aseptic techniques must be practiced at all times when working in the laboratory, especially when handling microorganisms. The purpose of practicing aseptic techniques is to safeguard the experimenters from infection and disases, as well others who will be using the lab afterwards, and to prevent the contamination of the samples by other microbes. The practice of aseptic techniques is based on the ubiquitous nature of microorganisms they inhabit almost all realms of the biosphere, especially where there is human activity. The purpose of the Bunsen burner was not only to incinerate the inoculating loop, but to also heat the surrounding air so as to reduce the number of airborne microbes present around the work area. The inoculating loop was repeatedly flamed (a form of sterilization) to prevent cross-

contamination. It is sterilized before so that you do not have any bacteria on it before you obtain your culture. It is sterilized after each quadrant so you do not leave bacteria on it for someone else to pick up. Care should be taken not expose the medium too much; therefore it is advised that during streaking, one should just slightly displace the Petri dish lid just enough for the loop to enter and streak agar. This is to prevent digging too deep into the agar with loop, as well as to limit the entrance of airborne microbes. Microbial growth was observed on the agar plates because the human body is inhabited by normal micrbiota microorganisms which have established a more or less residence in/on the body that do not cause harm under normal conditions. On the human skin, Staphylococcus aureus, and Corynbacterium are common microbiota. The dirty side of the dish was almost covered with microbes, while the clean side had a few areas of growth. The clean side may have experienced areas of microbial growth due to contamination from the dirty side, and from the subjects hands. The fact is that soap cannot remove all the bacterial population on the hands. Furthermore, if the subject dried his/her hand before inoculating the agar, the hands could have been contaminated in the process. The first quadrant had the largest amount of colony, but decreased in size and amount as the quadrants progressed; as such the fourth quadrant as the smallest number of microbes. Conclusion Aseptic techniques, such as flaming and applying bleach solution, are instrumental the occurrence microbes in the working environment. The purpose of practicing aseptic techniques is to safeguard the experimenters from infection, as well others who will be using the lab afterwards, and to prevent the contamination of the samples by other microbes. The purpose of the streak plate is to obtain isolated colonies from an inoculum by creating areas of increasing dilution on a single plate. The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop that is dipped into the inoculum which spread over agar in a pattern. Microorganisms are present everywhere, and aseptic techniques cannot fully eradicate microbial population. Post Lab Questions 1. Describe the growth on the plate labeled Open.

The colony morphology was circular. The colour was pale green, and the diameter of the colony covered almost all the agar plate. 2. Are organisms found in the air? What results support your conclusions? Yes organisms are found in the air. This has been proven by the fact that the open agar plate showed signs of microbial growth after some time. 3. What effect does hand washing have on microorganisms? Should you ever touch a sterile surface? Hand washing reduces the population of microorganisms that contaminate the hands, as well as the normal flora of the skin. There are chemicals, like alcohol and chlorine, in the soap that also destroys some of the microbes. 4. One person in your group had microorganisms swabbed on their glove. The others did not. From you results can you determine who had the contaminated glove? Yes, the quadrant with most microbial growth infers that it was inoculated by the person who was not wearing any gloves. 5. What conclusions can you draw from your data concerning where microbes are found in the environment? In concluding, microorganisms are found everywhere on our bodies, in the air, and on items we use.