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Introduction:
The goal of this project is to learn how to perform and master laboratory techniques used in microbiology to identify an unknown bacterium. Practicing these techniques gives students experience with scientific investigation and report. This is a valuable exercise for many reasons: it helps students understand the procedures of each test, learn standard bacteriological techniques, characterize and identify the species level of the bacterium, and practice scientific method and thinking. This project enables the student to understand the bacteria’s characteristics and also experience hands-on learning in laboratory settings. One of the main reasons for identifying bacteria is associated with issues of pathogenic bacteria. Understanding bacteria helps to find out how these tiny lifeforms behave, deduce what diseases they can cause, and find solutions to prevent, reduce, or solve these issues. Meanwhile, accurate and definitive microorganism identification, including identification and detection of pathogenicity, is essential for correct disease diagnosis, treatment of infection and tracing back of disease outbreaks associated with microbial infections (1). Materials and Methods

List the materials:
1 tube sample of original unknown bacteria #57.
Tube agar slant (for reserve stock #57)
Incubator for 25 & 37 degree celcius and refrigerator
Agar streak plates
KOH solution
Sterile pipettes and sterile water
Gas Pak kit
Inculating loop and inoculating stab
Glass slide
Microscope
Crystal violet, safarin, iodine, alcohol, decolorizer, water(gram stain) IMVIC= tryptone broth tube, MR and VP phenol red tube, citrate slant Fermentation tube (phenol red
Lysine decarboxylase medium, mineral oil
Tube of urea agar

Unknown bacteria # 57 was randomly selected from our instructor. The aseptic technique was performed throughout the testing process. The unknown bacteria, including the working and reserve stocks, were maintained in 25 degree C incubators or the refrigerator, respectively. Results were read after for 48 hours incubation unless otherwise specified. The Cultivation Technique was first performed to find the optimal growth temperature for the bacteria and make a reserve stock. The first procedure was to streak the unknown on two Trypticase soy agar medium plates using the streak method to test the purity of the unknown. The plates were dated, labeled and then incubated. Incubation after 48 hours in 25 and 37 degree celcius, morphology was observed and recorded. The Gram Stain distinguished gram positive from gram negative bacteria and rods shape to cocci shape. A sample of the unknown was smeared on a glass slide and heat fixed then flooded with primary stain, crystal violet which rendered the bacteria uniformly violet for 50 seconds, the slide was flushed with water and the smear was treated with a few drops of gram’s iodine for a few seconds which serves as a mordant. The slide was flushed with water and decolorized with acetone for 20 seconds and flushed with water. Adding a few drops of Safarin and waiting for 30 seconds, flushed with water and dried with a drop of oil it was observed under a light microscope. Observations were recorded. The Gas Pak System determined whether the unknown grew either anaerobically or in a reduced oxygen environment. Using the inoculationg loop, a sample of the unknown was streaked on two TSA plates and labeled aerobic and anaerobic. The anaerobic plate was placed in a gas poch with a generating packet sealed and incubated at 37 degrees Celcius with the aerobic plated for five days. Results were observed under a light microscope and recorded. Two t-streaked TSA plates were placed in a Gas Pak pouch, sealed in a Ziploc bag along with a wet paper towel to supply moisture, and incubated at 37 degrees. Excess air was pushed out and the Gas Pak’s oxygen levels were monitored by the oxygen-reacting pellet included in the pouch, results were observed under a light...
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