UNKNOWN #16
SUMMER 2013
INTRODUCTION
Purpose
The purpose of the lab is to separate characterize and identify microbes from unknown mixed culture using the morphological and biochemical methods.
Theory
The process of identifying bacteria is a systematic and careful process which uses various techniques to narrow down the bacteria types which are present in an unknown culture of bacteria. This process produces advantages for many factors of research of microorganisms and assists in the proper treatment of patients (Aneja 10). Several tests were performed to give the fermentation abilities, availability of specific enzymes and specific biochemical reactions.
MATERIALS AND METHODS
The experiment started off by the streaking of the unknown bacterial culture: unknown #16 using the three sector technique streak isolation with the materials; TSA plate and the unknown bacterial culture. The procedure for the three streak isolation is as follows:
- Begin by sterilizing the wire loop.
- The loop is cooled by touching the sterile TSA plate with it
- The loop is then dipped into the unknown culture which contains the bacteria mixture. the mixture of bacteria.
- The lid is lift a little to allow the loop to be inserted, then the loop is dragged over the top 1/3 of the plate in a zigzag manner. With this, thousands of bacteria have been picked by the loop and are spread over the surface of the agar.
- The loop is then sterilized by heating.
- The plate is then rotated at angle of 90 degrees, while dragging the loop through the area which has been streaked two to three times and carrying on with the dragging of the loop in a zigzag manner in the remaining half of the plate without touching it again.
- The loop is then sterilized by heating
- Rotate the plate at an angle of 90 degrees, repeating the procedure.
- The plate is then incubated for 24 hours. If streaked correctly, isolated colonies in the third sector can be seen. The first sector will have the heaviest growth, the second sector with slightly less growth and some isolated colonies and the third with the least growth with isolated colonies will be in the first sector.
This was done labeling the each plate as TYPE A and TYPE B. After the streaking, a gram stain test was done to identify the type of bacteria.
Gram staining
Materials:
- Crystal Violet
- Gram's Iodine solution
- Alcohol solution
- Safranin
- Microscope
- Oil immersion
- Bunsen Burner
- 24 hr. TSA slant of TYPE A
- 24 hr. TSA slant of TYPE B
Procedure
Smearing procedure
The smear was prepared using an aseptic technique. A loop-ful of distilled water was placed on each slide. The bacteria were then obtained from the slants using an aseptic technique. After air drying and heating the gram stain procedure followed.
Gram stain procedure
- Cover the TYPE A slide with crystal violet for 20 seconds.
- Gently rinse off the stain with water and shake off the excess.
- Cover with iodine for a minute.
- Run the alcohol solution down the slide until the solvent runs clear
- Rinse with water to stop the action of the alcohol.
- Cover with Safranin for 20 seconds.
- Gently rinse off the stain with water. Blot with bibulous paper and clean off the bottom of the slide with 95% alcohol.
After that the slide is observed with a microscope using oil immersion. Following the gram stain, the flow chart of preparing a MSA plate was undertaken, taking the TYPE A streak and incubating it at 37 degrees C. a fermentation testing procedure was also undertaken to further identify the unknown bacteria.
The same procedure was applied to TYPE B slide and a blood agar test undertaken, incubating the TYPE B bacteria at 37 degrees C.
RESULTS
TYPE A
Gram Stain
For the other tests that is the Mannitol Salt Agar and fermentation testing, a yellow color was obtained which also indicated the abilities of fermentation.
TYPE B
Gram Stain
An additional spore blood agar test on TYPE B due to the presence of spores, indicated no some growth but there was no change in the medium surrounding the colony.
DISCUSSION
In performing the gram stain on TYPE A, it was observed that the color turned to purple. Gram -positive cell walls stain purple with the Gram stain. It was also noted that the shape of the bacteria was round. This indicated that TYPE A was a gram positive Cocci. Further tests using the MSA obtained a yellow color which indicated that the bacteria type being dealt with was a Staphylococcus Aureus. This is because Staphylococcus Aureus produces acid on a Mannitol Salt Agar resulting in a yellow colony; which is the reason for the yellow coloring. The fermentation test conducted had a change of color from red to yellow recorded. It is only microbes under the genus Staphylococcus which yield this positive result for fermentation. Therefore the TYPE A bacteria is a Staphylococcus Aureus (Schlegel and Zaborosch 522).
On the other hand, the test conducted on TYPE B, starting with the gram stain obtained the purple coloring which is observed with Gram positive bacteria. However, the observed shape which was rod-like indicates that the TYPE B is gram positive Bacillus. In the process of observing the slide, it was noted that the endospores were present or visible, this narrowed to the spores blood agar test to clearly identify the type of bacteria. The blood agar test resulted in the observation that there was some growth though there was no change in the medium. This indicates the presence of cellular amalyse which is found with Bacillus Megatarium. Therefore, TYPE B is a Bacillus Megatarium (Schlegel and Zaborosch 522).
CONCLUSION
This report summarizes the results of the tests which determine the morphological feature, physical necessities and biochemical reactions which are mandatory in the identity isolation of an unknown microorganism. These tests could be thought of as a sequence of questions in the classification; Dichotomous key which utilizes the elimination process to identify an unknown microbe. For the case of this experiment, the unknown microbes fit the test results as criteria for identity were Staphylococcus Aureus (TYPE A) and Bacillus Megatarium (TYPE B).
Works Cited
Aneja, K R. Experiments in Microbiology, Plant Pathology and Biotechnology. New Delhi: New Age International, 2005. Print
Schlegel, Hans G, and C Zaborosch. General Microbiology: []. Cambridge [u.a.: Cambridge Univ. Press, 1995. Print.
