Bacteria Description

Our unknown bacteria, L-25, is of the genus Serratia, and we believe of the genus marcesens. Here are descriptive qualities we have obtained through testing in the lab-

Morphological Characteristics
Cell shape: Bacilli
Size: Small
Spores: none
Gram's Stain: Negative
Motility: Motile
Capsules: none
Special stains: Acid-fast

Cultural Characteristics
Colonies: red
      Nutrient agar: red
      Blood agar:  white, partial lysis
Agar slant: red
Nutrient broth: pink
Gelatin slab: turbid
Oxygen requirements: facultative anaerobe
Optimum temperature: 25 degrees Celsius (but varies, also grows well at 37 degrees Celsius)

Physiological Characteristics
Fermentation

• Glucose: positive
• Lactose: partial
• Sucrose: positive
• Mannitol: negative

Hydrolysis

• Gelatin liquefaction: positive
• Starch: negative
• Casein: partial
• Fat: positive

iMViC

• Indole: positive
• Methyl Red: negative
• Voges-Proskauer (acetylmethylcarbinol): positive
• Citrate Utilization: positive
• Nitrate Utilization: positive
• Urease: negative
• Oxidase: positive

Litmus Milk

• Acid: no pH change
• Alkaline: no pH change
• Coagulation: soft curd formed

All of these characteristics indicate that our unknown bacteria is Serratia marcesens
 

Identifying our Unknown... FINALLY!

Today, we identified our unknown bacteria, L-25! The genus is serratia, we're still working on the genus. We also started our research project in which we're testing different essential oils their bacteriocidal effects. We made a bunch of paper discs to be autoclaved, after determining we would use 10 microliters of water and 10 microliters of each oil to saturate the discs to place on the streak plate of streptococcus pneumoniae. 

Streptococcus pneumoniae

We also checked our plates from last week, finding that the anti-bovine solution reacted and formed a precipitate with the hamburger solution and the bovine solution.

Antibodies and Antigens!

Today, We learned about the ELISA tests and Secondary Antibody tests. We performed a test to determine the purity of a meat sample by poking little holes into agar and injecting bovine and hamburger solutions with Anti-Bovine horse, goat, and pig solutions, to see which ones would react. We also did a control with red and green dye. Then we performed the ELISA test used to detect hepatitis and HIV. First, you coat the bottom with an antigen, rinse with a buffer wash, then add a positive antibody solution, negative antibody solution, and other samples. After washing, add a secondary antibody solution, let sit, rinse, then add a substrate that will change color to indicate a positive test. It was really cool to see tests that are used to detect such serious viruses. We also smelled essential oils that we will be utilizing in our research projects. We also saw that our bacteria that was stored in the freezer had grown when inoculated on an agar plate! 

Yum Yum Yogurt!

Today, we ate the yogurt we made on Tuesday! It was interesting, with the probiotic tablets making a yeasty tasting yogurt and the kefir making a sour cream like yogurt. Then we checked our UV plates, finding that our unknown bacteria, L 25, was not affected by 30 seconds under UV-C light. We made a little fieldtrip to the greenhouse, then got our environmental samples out of the -80 degree freezer. We prepared a streak plate from the environmental samples and put the little tubes back in the freezer. Then we watched a movie on biological terrorism and how scientists in the U.S. traced the anthrax poisonings in 2001.

Yogurt-Makin' :D

Today, we prepared yogurt as a class using Vitamin D milk, Kefir, and Mrs. Pathakamuri's homemade probiotic tablet yogurt! First, we microwaved the milk and let it cool to 37 degrees Celsius. Then we poured the milk into three different labeled cups, Kefir, Yogurt, and Control. We added about a teaspoon of Kefir and yogurt to their respective cups, then covered and put the cups in the incubator at 37 degrees. Tomorrow, the cups will be moved to the fridge and Thursday, WE EAT! We also prepared smear plates of our unknown bacteria and subjected half of the plate to long-wave UV-C light for 30 seconds. After that, we inoculated a beaker of water with unknown bacteria and sterilized it with Dr. Pathakamuri's UV SteriPEN! Today's lab had really practical applications to the real world, I'm so excited to learn more about microbiology and pathophysiology next semester. 

More Results!

Last week, we observed the results of the test tubes we inoculated. Our unknown, L 25, tested positive for Citrate utilization, meaning it converts citrate to citrite to ammonia! L 25 also tested positive for the Indole test, meaning to produces indole as a byproduct by utilizing tryptophan. L 25 tested positive for oxidase test, changing the swab instantly purple like Sarah Bartnicki's hair! L 25, an amazing unknown, was positive for nitrate reduction, because it is a facultative anaerobe, it can reduce nitrate completely into molecular nitrogen!  L 25 tested negative for urea hydrolysis, meaning it does not hydrolyze urea, a molecule used to excrete nitrogen in urine.

Enjoy the pictures, L 25 is almost identified!

Lots of Tests and Antibiotics

Today, we inoculated lots of different test tubes with our unknown sample to determine what enzymes it has and what it utilizes. These tests included the Citrate utilization test, Indole test, Nitrate reduction test, and the Urea hydrolysis test. We also streaked a plate and put 7 different antibiotic chips on to determine which antibiotics are effective. Results Thursday, enjoy the pictures!

EMB, Mannitol, and MacConkey Agar

We streaked EMB, Mannitol, and MacConkey agar plates with our unknown bacteria L 25, and determined that since our bacteria was pinkish on the EMB plate,  it is a gram-negative enteric bacteria, with a lower acid production because it only partially ferments lactose. The mannitol plate was negative, our bacteria does not ferment mannitol. Our MacConkey agar plate confirmed that our gram-negative enteric bacteria partially ferments lactose. It is a facultative anaerobe which oxideses H202, which means it has catalase. Hopefully next week, we will identify it!

Test Results!

Today, we checked the results from all the tests we performed on Tuesday. Our unknown bacteria was positive for gelatin hydrolysis. It also fermented glucose and sucrose, but not lactose, using the Durham tube tests. Our bacteria was negative for the Methyl red test, but positive for the Voges-Proskauer test. Our bacteria had a red slant and a yellow butt with a gas bubble for the TSIA test, which means only glucose was fermented. We will check the litmus milk test next week.

P.S. Sorry all of the pictures are sideways. 

Lots of Inoculation!

Today, we inoculated a LOT of test tubes to test for a variety of things. We are testing for gelatin hydrolysis, fermentation of the carbohydrates glucose, sucrose and lactose with a Durham tube, a Methyl Red/Voges-Proskauer test for fermentation of mixed acids, a Triple Sugar Iron (TSI) Agar test to see if the bacteria produced hydrogen peroxide, and a Litmus milk test to determine if the bacteria will utilize lactose, protein or litmus.

Oxygen Utilization and the Digestive Enzyme Test

Yesterday, we checked the results of our oxygen utilization tests of our unknown bacteria. Both the thioglycollate broth and the GasPak tests indicated our unknown bacteria L is a facultative anaerobe, which means it can utilize oxygen when present, but that it is also capable of thriving without oxygen present.

Next, we made streak plates to test the digestive enzymes of our unknown bacteria. We streaked a starch agar plate, a lipid plate, and a casein plate (skim milk). After 24 hours, we came back to check the results. After adding Gram's iodine to the starch plate, we determined unknown L 25 was negative for starch hydrolysis. The casein plate also indicated our bacteria was negative for casein hydrolysis. The triglyceride plate was different, our bacteria indicated positive for lipid hydrolysis! We're getting so close to identifying it!

Enjoy the pictures!

Long Term Storage of Bacteria and Determining Oxygen Requirements

Today, we made a tiny sample of our bacteria to store long term in the -80 degree freezer until the end of the semester. Using a pipettor, we combined 700 microliters of our environmental bacterial broth sample from last week  and 300 microliters of the cryoprotectant glycerol. Then to determine the oxygen requirements of our unknown bacteria, we performed two different tests. First, we inoculated our sample in a thioglycollate broth, which turns pink when oxygen is present. We will determine the oxygen requirements next Thursday after examination. Then we made a streak plate of the unknown sample to use in the GasPak Anaerobic system. The GasPak eliminates oxygen using a chemical reaction and produces carbon dioxide. An indicator strip inside the GasPak turns blue when oxygen is present, and stays colorless when oxygen is absent. We will check those results Thursday as well. Enjoy the pictures!

Acid Fast and Endospore Staining!

Last week, our group split the tasks of acid fast and endospore staining our unknown samples. I completed the acid fast stain by using Ziehl-Neelson carbolfuchsin on a slide that was over a beaker of boiling water. After decolorizing that stain with acid-alcohol and covering it with methylene blue for two minutes, I used the microscope's oil immersion lens to determine that our unknown sample  is non-acid fast, because the sample was purple. Annie also determined our unknown sample (L 25) had no endospores.