Identification of Unknown Bacteria Lab Aim: The objective of this lab to determine an unknown bacterium using different techniques in streaking and staining from previous labs. This lab will be done throughout a 3-week period, during these three weeks students will be introduced to new biochemical tests that will help in identifying their unknown bacteria. This experiment combines old techniques like gram staining, various streak methods combined with new tests like the catalase tests, rapid membrane analysis and the use of different, selective medias (NA plate, MSA plate, MAC plate, etc.) to see the different criteria needed to identify a unknown culture.
Materials: - 24-hour unknown bacteria culture broth (Sample #7) - Glass microscope slides - Bunsen - Inoculating loop - Permanent marker - Gloves - Nutrient agar (NA) - Mannitol Salt Agar (MSA) - Catalase - Esculin ID Membrane
Methods: Week 1- 1) Select a labeled unknown broth culture 2) Label the nutrient agar plate with sample number, name or initials, and date lab was performed. 3) Streak the unknown organism onto a nutrient agar plate, after sterilizing and cooling an inoculating loop. 4) Incubate the sample at 37 °C for 24-48 hours Week 2- 1) Once isolated colonies are seen and recorded from the first procedure, confirm with instructor, then continue with a gram stain a. Prepare smear and heat fix before staining b. Place the slide with smear (side up) over sink and then cover with crystal violet c. After letting it sit for 1-2 minutes, wash with distilled water to remove stain d. Then cover slide with Gram’s iodine and let sit for another minute or two e. Rinse again with water, then decolorize by washing the slide with ethyl alcohol for 2-3 seconds f. Then immediately rinse slide with water to stop decolorizing g. Drain off excess water into container, then cover the stain with safranin counterstain for another 1-2 minutes h. Rinse slide with water then blot with bibulous paper to dry
b. Incubate growth at 37 °C overnight and then observe growth and fermentation to determine which bacteria was the unknown
Discussion: Bacteria is ubiquitous, meaning it is present everywhere and once it’s found various methods and tests are performed to slowly identify which specific organisms it is. Identifying bacteria is important for various reasons, including testing for bacteria in samples of food, soil, water, blood, etc., successful identification of a bacteria ensures proper treatment and control (Arulanandu, 2022). There are various methods used to identify a bacteria’s genus and specific by isolation, as each test result allows for a closer determination of its identity. A big part of identifying these bacteria is the use of selective and differential medias. Selective media allows for select/isolate groups of specific bacteria while preventing other specific bacteria, and
tolerant staphylococci, while differential: contains carbohydrate mannitol and phenol red which essentially detects acid produced by mannitol fermentation (Arulanandu, 2022). When bacteria is placed onto a MSA plate and it does ferment mannitol it changes the coloration of the agar, while bacteria that doesn’t ferment mannitol won’t cause any changes to the agar itself. My sample did not ferment mannitol and did not cause any color changes to the MSA plate, eliminating Staphylococcus aureus. After all tests were completed, following the flow chart and the pigmentation on the nutrient agar plate resulting in yellow colonies it was determined that my unknown sample #7 is Micrococcus Luteus. Each test essentially gave another piece of the puzzle, as without the results of prior tests further experiments couldn’t be completed. A key variable to identifying the unknown bacteria was the use of various agar plates. According to Arulanandu (2022), the nutrient agar plate demonstrates bacteria in the environment, MacConkey (MAC) selective allows only for gram-negative bacteria to grow while differential allows for lactose fermenting bacteria to appear rink or red, mannitol salt agar (MSA) selective inhibits growth of most bacteria except for salt tolerant staphylococci and differential is essentially a pH indicator for detecting acid produced by mannitol fermentation. These were the main agar plates that played a crucial role in identification of sample #7. The NA plate especially, due to the one of the final factors in identification was viewing the bacteria’s pigmentation which was shown on the NA plate. The MSA and MAC plate also played a role as it helped show that my bacteria did not produce any growth or fermentation. Throughout the lab multiples errors could have occurs and caused repetition of tests, or the wrong results. Throughout the 3 weeks, many tests were performed, and each test affects the next. One test that almost did cause an error in my result was the gram staining done in week 2, I
almost added the wrong stain at the beginning of the experiment which would have not let me see the morphology or arrangement on my own sample. Other possible errors could be improper techniques in streaking which could have affected the bacterial growth in each step, not incubating for the certain amount of time or temperature also would have affected the growth, using the wrong plates or wrong tests at the wrong time could have been a significant error too. Despite any possibility of error, the process of identifying bacteria shows important in the regular world and especially in the medical world. It is tests like these that can help identify certain bacteria which can cause disease or infection.
Conclusion: My unknown bacteria sample was #7, it was with that unknown broth culture that various tests were done and completed to identify its genus and species. After various testing it was determined that my unknown sample was Micrococcus Luteus, which is a gram-positive, coccus bacteria. Each test helped in identifying the result of identifying sample #7, such as selective and differential media, gram staining, the catalase, ID membrane tests or simple pigmentation. This lab truly showed the important of each step-in identification and the important of identifying itself as it gives us answers in the medical world from helping determine a prognosis, which medication will be effective or safe, and even more. Identifying bacteria can help identify which specific bacteria is present in fighting diseases, causing diseases, causing resistance to certain medication, even helping to know which medication to use and not use, or just simple knowledge to better help the patient.
