Microorganisms such as bacteria, fungus, mold, and yeast are present and common in almost every environment on earth. The normally microscopic organisms can easily be seen using differing types of agar, which creates an ideal environment for the organisms to form colonies, which are groups of hundreds of organisms that can be seen with the naked eye. In order to see individual microorganisms, it is necessary to use the magnification of a high-powered microscope.
These techniques of microbiology are used in the following five experiments. The first experiment used Trypticase Soy Nutrient Agar (TSA), which can grow a wide variety of organisms and contains casein and soybean meal and a minute NaCl, to study the effectiveness of alcohol as a skin antiseptic. The second experiment tested the effectiveness of different kinds of mouthwashes as antiseptics using TSA as well. Experiment number three explored the normal human flora existing on skin and in nasal cavities, and two types of agar were used, including TSA and Mannitol Salt Agar, which contains manitol sugar, phenol red, and 7.5% NaCl. Mannitol Salt Agar tests for the presence of staphylococci bacteria that can survive in the salt that inhibits the growth of most other bacteria. Some forms of staph bacteria ferment mannitol and produce a yellow color around the colonies, which can easily be seen against the red background. The fourth experiment studied the number of bacteria in a diluted sample of uncooked hamburger meat using nutrient agar and a Quebec counter to count the colonies. The final experiment involved the growth of yeast cells under aerobic and anaerobic conditions.
-Trypticase Soy Nutrient Agar
-Mannitol Salt Agar
-Sterile alcohol swab
-Tubes of nutrient agar
-Diluted hamburger meat
-Quebec colony counter
-Anaerobic yeast culture
Experiment 1. Effectiveness of alcohol as an antiseptic
1) All experiments must be done in a sterile environment; Lysol can be used to sterilize the area.
2)Divide a petri plate filled with Trypticase soy agar into 4 quadrants. Label them A, B, C, and D.
3)Press the pad of a dirty thumb onto quadrant A.
4)Swab the thumb with sterile water, allow to dry, and imprint it onto quadrant B. The sterile water acts as a control.
5)Press the other dirty thumb onto quadrant C.
6)Swab this thumb with the alcohol swab, allow to dry, and imprint on quadrant D.
Experiment 2. Effectiveness of mouthwash as an antiseptic
1) Divide a petri plate with Trypticase soy agar into 2 quadrants.
2) Swab the inside of your mouth with a sterile cotton swab, inoculate one half of the agar.
3) Rinse your mouth with Listerine brand mouthwash. Wait 5 minutes after rinsing.
4) Swab the inside of your mouth with a clean sterile swab, inoculate the second half of the agar.
Experiment 3. Normal human flora
1)Obtain two petri dishes containing one half Trypticase Soy Agar and one half Mannitol Salt Agar.
2)Moisten two sterile swabs in sterile water, remove excess water, and then use one of them to swab the inside of your nose, and the other to swab a patch of skin, like your neck.
3)Inoculate each dish of agar with either the nose or shin swab on both sections.
Note: For experiments one through three, each petri plate should be taped shut and stored upside-down for one week at room temperature to incubate the microorganisms. During the second week, the plates with microorganism colonies can be observed.
Experiment 4. Standard Plate Count of Meat
1)Liquefy two tubes of nutrient agar in a boiling water bath.
Experiment 5. Growth of Yeast
1)Obtain anaerobic yeast cultures, made from yeast cells inoculated into bottles of sterile apple juice and allowed to ferment the sugars without contact with air.
2)Degas the culture by allowing it to stand opened for one hour at room temperature.
3)Make 10ml dilutions of 10%, 20%, 50%, 70%, and 100% yeast solution with fresh apple juice.
4)Record the absorbance of each dilution in a spectrophotometer set at 650nm.
5)Prepare an iodine and yeast solution by adding about 4 drops of iodine to the 10% yeast dilution sample.
6)Using an eyedropper, touch a tiny drop of this iodine/yeast solution to the loading groove of a clean hemacytometer. Allow two minutes for the cells to settle before counting.
7)Count the number of cells and then the number of micro colonies in ten of the medium squares.
In the first experiment, our tests showed that the sterile alcohol wipe was extremely effective in killing any microorganisms that existed on a previously dirty thumb. There was over 95% fewer visible colonies on the agar where it had been inoculated by the sterile thumb. The water did nothing to decrease the number of colonies, in fact they appeared to be denser in the quadrant inoculated by the water-wiped thumb.
In the second experiment, the Listerine we used decreased the bacteria colonies by about 50%. The pre-washed side had a lot of large tan colonies and dense, small colonies. The Listerine side seemed to destroy all most all of these small colonies, but the larger colonies were actually more numerous. The other mouth washes used were salt water rinse and Scope. The salt water did almost no damage to the bacteria, and the Scope killed the most.
In experiment three, there were a lot of different kinds of organisms persent on the agar. On the plate that contained nasal micro-organisms, large yellow colonies appeared on the Mannitol Salt Agar, indicating Staphylococcus aureus. On the TSA side, small creamy white colonies were also present. These could possibly be yeast. On the plate that contained the skin swab, there was a large amount of large, fuzzy white and black growth, which is mold. There was a single light-orange colony on the TSA side, possibly a growth of Flavobacterium.
In the fourth experiment, there were a number of different kinds of colonies on the EMB plate with the meat swab. There were black dots, which indicate the presence of E-coli, pink dots, which indicate lactose-fermenters, and a few white and gray colonies.
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