CHAPTER III
METHODOLOGY
This chapter includes all the materials and procedures that were properly followed to gather the needed information and necessary data for the study.
A. Sources of Test Organisms
The bacterial isolates (Staphylococcus aureus, Escherichia coli and Streptococcus) used in the experiment were obtained from Institute of Biological Science, University of the Philippines Los Baños, Laguna. B. Maintenance of Microbial Cultures
Bacterial isolated were subculture on slants of Nutrient Agar (NA). Incubation of the subcultured specimens for bacteria was done for 7-14 days in Isolation Room. C. Media Preparation for Assay
Three tubes with nutrient agar were prepared. The cultures were inoculated onto
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G. Testing the Effectiveness of the Formulated Hand Sanitizer
Using two sites of the arm, the effectiveness of the hand sanitizer was tested. Swab head was moisten with sterile water and rubbed three times on the surface in rotating motion. This swab head was put in the tube. The tube was then shaken making 50 complete cycles of 15 cm in 10 seconds. A 1:10 dilution of the rinse solution was prepared. On Tripticase soy agar, the plate 1.0 and 0.1mL portions of the resulting suspension was poured. The plates were incubated at for 370C 1-2 days. The number of colonies per mL of the rinsing solution was calculated. Using a specific formula, the microbial load of the 4cm2 surface of the skin was determined.
The second area which was marked was wet and rubbed with sanitizer for 15 seconds. This area was lathered for 60 seconds without soap and rinsed for 15 seconds with sterile water. Some steps are repeated to determine the CFU/cm2 of the second washed area. H. Data Analyses
Extracts from leaves of Ashitaba were applied on bacterial cultures. Detection of antibacterial activity was done after 7 days. The plates were compares in terms of the diameter of zone of inhibition. Hand sanitizer was then formulated using the extract. This was tested on a specific part of the skin. The abundance and type of microbial load of treated and untreated surface of the skin were compared.
This is just a part of my science investigatory project. I will upload the rest of the
Dirty hands is the common source of spreading infection. It is very important to keep hands clean to avoid getting infected and spreading infection in the community. It is important to wash hands to keep hands clean. There are two ways to keep hand clean, one way is wash hands with soap and warm water while rubbing hands together for minimum 15 to 30 seconds. Indication of washing hands with soap and water is when hands are visibly dirty, before and after eating, feeding, using the toilet, after coughing or sneezing, after using gloves, taking care of patients. There is also second way to clean hands, but it is advisable to wash hands with soap and water all the time, but it can ignore when soap and water is not available so it is okay to use hand gel or foam in the form of sanitizer. This helps to clean hands or kill germs when hands are not visibly dirty.
A mixed culture of two unknown bacteria was provided by the instructor. The methods used for
This experiment illustrates the importance of handwashing and proves that hand washing is worth it. Since our hands are constantly coming into contact with ourselves and others, touching surfaces, grabbing objects, being sneezed into, etc., keeping our hands clean is one of the most effective, yet simple way we can take to avoid getting sick and spreading germs to others. Many diseases and conditions are spread by not washing hands with soap and clean, running warm water. “The human skin is a host to anywhere between 10,000-10,000,000 bacteria per square centimeter and since health care providers come into contact with pathogenic bacteria by being engaged in patient care, hand washing can reduce the risk of spreading diseases (page 3).” The objective of the experiment is to test the effectiveness of hand washing and demonstrate normal flora. This report presents the procedures and materials for the experiment, the experiment's results, and an analysis of those results.
The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is
The purpose of this study project was to carefully isolate and identify two unknown bacteria from a mixed culture. The ability to properly evaluate biochemical test results is also necessary for the identification to be successful. The goal was to apply all of the methods and techniques that have been learned in the microbiology laboratory course for the proper identification of unknown bacteria. A certain amount of bacteria that were used throughout the course were possible bacteria that could be found in a mixed culture. The bacteria that were identified in the mixed culture were Staphylococcus Aureus and Kocuria Rhizophila.
In a laboratory setting, it often becomes necessary to identify an unknown organism. In this experiment, researchers classified an unidentified bacterium based on its physical structure, colony morphology, optimal conditions and metabolic properties. A Gram stain using crystal violet, iodine, and safranin and a simple stain using methylene blue characterized the organism’s cell wall. Cultural behavior was classified by inoculating the organism onto nutrient agar and incubating it at 37° C for 48 hours, and observing its behavior, as well as using SIM medium to test for motility. Optimal growth temperature was
The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible
After all results have been recorded, pour all of the solutions down the drain, rinse equipment and clean up the
The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were oxidase, catalase, lactose and sucrose fermentation, Kugler/iron agar, nitrate reduction, gelatin hydrolysis, starch hydrolysis, manitol salt, MR-VP, citrate, bile esculin,
Please complete the entire experiment as instructed in the lab manual except for any modifications noted below. Fill out the
Unknown lab report# 24 Introduction Microbiology is a study of organisms that surrounds us every day. It requires an educational understanding to identify organisms, and the ability to distinguish different bacteria’s. In applying the learning process of the different bacteria’s, unknown bacteria samples are given to be studied and identified.
In order to obtain well-isolated discrete colonies, the quadrant streak and spread technique was used. This allowed dilution of the original microbial material over the entire surface of the plate. As the original sample was diluted by streaking and spreading it, over successive quadrants the number of organism decreases. Usually by the third or fourth quadrant only a few organism were transferred on the by the inoculating loop and theses produce a few isolated
2. Introduction: Each student was given unknown bacteria and was instructed to perform a variety of experimental tests that would help to identify their bacteria. During the process of identification, the unknown bacteria was added to many different testing medias using aseptic technique. They are as follows: lactose fermentation on eosin methylene blue (EMB), TSI (Triple Sugar Iron agar), Phenol red sucrose, the SIM test, H2S by SIM, IMViC (indole, motility, voges-proskauer, and citrate), Urease (urea broth), PDase (Phenylalanine Deaminase), Lysine Decarboxylase, and Ornithine Decarboxylase. Colonial morphology on EMB was used to
Nutrient Broth and Nutrient Agar were used to inoculate bacteria taken from different surfaces. Nutrient agar plate was inoculated with a sample taken from skin surface. A sterile cotton swab was first immersed on sterile water, then, rubbed against the skin with swirling motion and transferred to an agar plate by rubbing
Infectious diseases that are commonly spread through hand to hand contact include the common cold, and several gastrointestinal disorders such as diarrhoea (WaterAid, 2006). Human hands usually harbour microorganisms both as part of a person’snormal microbial flora as well as transient microbes acquired from the environment (Lindberg et al, 2004).