Our Unknown REVEALED!

After all that testing in 5 short (long) days, we were able to conclude what our unknown "K" bacteria was! One of the first tests done at the beginning of the semester was to perform the gram stain.  Our gram stain revealed that our bacteria was gram negative.  Under the microscope, our bacteria were rod-shaped tiny bacteria.  These two little observations allowed us to narrow what our unknown bacteria was.  After all the tests were done and analyzed as discussed in previous blogs, we were able to conclude what the unknown bacteria was.

We followed the flow chart and using our results, we followed these steps:

1.) Gram- Negative (that eliminated many bacteria)
2.) Lactose: positive (what could it be?!)
3.) Indole: positive (Oh no!)
4.) Citrate: negative.... (WAIT FOR IT...)


Escherichia coli!!!!

The chart we used to identify

 

That's all, folks!

Day 5: Antibiotics testing

During our last day, we analyzed the antibiotic sensitivity test to determine if the unknown bacteria was resistant or sensitive towards certain antibiotics.  If the bacteria grew around the antibiotic area, it was resistant. If there was a ring around the antibiotic area, the bacteria was sensitive to it.  We spread the bacteria on the agar culture plate and placed four different antibiotic areas in four labeled locations on the plate.  We did the same thing for the second plate.

         

Antibiotic tests 1-4

Antibiotic tests 5-8

      After incubation, the best antibiotic for the bacteria was Amoxicillin. 
The measured diameters of the bacteria surrounding the antibiotics are as follows:
             Penicillin: 8 mm (resistant)
             Novobiocin: 0 mm (resistant)
             Neomycin: 14 mm (intermediate)
             Streptomycin: 19 mm (sensitive)
             Tetracycline: 16 mm (intermediate)
             Erythromycin: 0 mm (resistant)
             Amoxicillin: 22 mm (sensitive)
             Oregano: 20 mm (sensitive)

Measuring the diameter

Oregano was almost the same distance as our best antibiotic, Amoxicillin, so we concluded that it is sensitive.  Compared to Novobiocin, oregano is a much better antibiotic for the unknown bacteria.

We also analyzed the selective and differential media plates.  We used mannitol salt agar plates to see if Molly had the bacteria Staphylcoccus aureus (MRSA). Using a sterile cotton swab, we dipped the swab into saline solution and swiped it around the inside of Molly's nostrils.  We then spread the swab around on the mannitol salt agar plate to cover the entire plate.  We incubated the plate at 37°C (the temperature of Molly's body).  After 24 hours, we did not find the Staphylococcus aureus bacteria in Molly's nostrils (Thank goodness!)

Negative!!

The next experiment was throat swabs! This test s used for detecting the bacteria streptococcus (strep throat).  Lindsey and I both had our throats swab because I had a sore throat and Lindsey wasn't sick so we wanted to compare the two.  Using a sterile cotton swab, we got bacteria from the throat and spread it around the blood agar plate, covering the entire plate.  We incubated the plate at 37°C (the temperature of the human body). Both plates turned out to be α lysis which is a particle sensitivity to hemolysis.  However my half of the plate showed a spot that could be the streptococcus bacteria!

Our throat swabs!

The final four selective and differential plates were the blood agar plate, MacConkey agar plate, Phenyethyl alcohol agar plate, and EMO plate.  On each of the different plates, using the aseptic technique, we drew a squiggle line and incubated the plate at 37°C overnight.  

The blood agar plate showed α partial hemolysis. 

Our results are on the left

Our alpha results on the right

The MacConkey Agar was positive because there was no color change. Gram-negative bacteria grows.
Phenyethyl alcohol agar plate was negative because only gram-positive bacteria grows and our bacteria is gram-negative. 

Our bacteria on the right- no growth

 Our EMO plate was positive as there was clearly growth!

Look at that thing!

The next experiment was the ELISA test. The ELISA test demonstrates the use of antigens and antibodies.  Antigens produce antibodies to kill substances foreign to the body.   The test was used to see if the HIV virus was present in different samples.  First, we labeled each of the 12 wells properly.  Then we added 50 microliters of the purified agent into each well and let that sit for 5 minutes so the antigen can bind to the well.  After 5 minutes, we poured the solution onto a paper towel and washed the wells with a wash buffer.  Now, it was time to add the antibody. We then added 50 microliters of the positive control to the first 3 wells, 50 microliters of the negative control to the next 3 wells. We then added 50 microliters of the first sample, 20, into the wells 7-9 and the 50 microliters of the second sample, 43, to wells 10-12.  After 5 minutes, we washed the wells with the wash buffer.  We then added 50 microliters of the second antibody to all 12 wells. After 5 minutes, we washed the wells with the wash buffer twice.  Finally, was added 50 microliters of the enzyme substrate to the 12 wells.  After 5 minutes, we observed the sample 43 was HIV positive. 

ELISA test

Our materials

Positive patient 43!

Day 4: The Results!

Day 4 was mainly a day for analyzing all our our results from the tests done in Day 3.  So after all the test were finished in day 3 and inoculated, we analyzed the bacteria and came up with these results. These results are important for the health science field when treating a patient.  The caretaker must properly understand and identify the bacteria or illness before they can treat it. 

The starch test was negative meaning our bacteria did not use starch, a carbohydrate made from glucose. The use of starch is by alpha-amylase enzyme. 

Our negative starch hydrolysis plate

The casein test was negative meaning our bacteria did not use casein.  The gelatin test showed some liquid but after it was placed in the refrigerator, there was no liquid, meaning the test was negative and our bacteria did not use gelatin.  Our lipid triglyceride test was negative meaning our bacteria did not use lipid.  There an no enzymes that can break the lipid.


Next, we analyzed our bacteria's ability to ferment carbohydrates.  Our results showed:

Lactose: Bubbles were present (presence of gas); the color changed to yellow. There was bacterial  growth in the tube.

Glucose: Bubbles were present (presence of gas); the color changed to yellow. There was bacterial  growth in the tube.

Sucrose: There was on tiny bubble at the top (small presence of gas); the color remained orange-red (no color change). There was bacterial growth in the tube. 

Maltose: Bubbles were present (presence of gas); the color changed to yellow. There was bacterial  growth in the tube.

All positive but sucrose

Our next test was the methyl-red and Voges-Proskaus tests. We poured half of the liquid into a clean test tube and put 8 drops of methyl red dye into the tube. This test was to determine if we have E. Coli or not. To follow to test, we used 5 drops of B reagent and 15 drops of A reagent.  The test was positive, allowing us to highly consider E. coli as our unknown bacteria!!


The Voges-Proskaus was next. This test was to determine that if there was butanediol, there would be a color produced. The bacteria would be E. aregenous if a rose color was produced. There was no color produced so the test was negative.  

Our negative VP and positive MR tests

For the indole tryptphan test, we added 10 drops of Kovac's reagent into the tube. This reagent is dangerous so we had to wear gloves! There was a red line at the top of the liquid. This was a positive result telling us that our bacteria used tryptophan. 

THE RED LINE!

For the nitrate test, the first step was to add 10 drops of A-sulfanilic acid and 10 drops of B- dimethyl- α-naphthylamine to the tube.  Our liquid turned pink so we didn't need to add the zinc powder.  Our results were positive and our bacteria used nitrate to produce nitrite.  

Positive nitrite test because of that bright color change!

For the TSAI test which used 0.1% glucose and 1.0% lactose and sucrose, our slant turned yellow meaning acid was produced and our acid butt was positive also  However, our results were negative for H2S.

The urea test was negative so our bacteria did not contain urease.

The litmus test allowed us to find out whether our bacteria uses lactose, protein, and litmus.The litmus test showed a color change from purple to a pinkish purple.  This test was negative for gas because no fissures were produced. The test was positive for using lactose to produce lactic acid.  We had a white acid curd at the bottom of the tube meaning litmus was reduced.

Our acid curd

The motility test showed that our bacteria is non-motile. 

The GasPac test was positive for growth, allowing use to know our bacteria is falcultated (grows under aerobic and anaerobic conditions).  The thyogylcolate test also proved our bacteria is falcultated because there was bacterial growth in the middle and top of the tube.

Growth on the top and middle

The backside of the growth

Our bacteria grew under anaerobic conditions too!

The catalase test was postivie because bubbles were produced meaning H2O2-> H2 +O2. We poured hydrogen peroxide into an agar plate with our unknown bacteria.  Because our bacteria started to bubble, there was gas produced, showing the presence of the enzyme catalase.

Bubbles!

The oxidase test was negative because there was no color change.  We added the reagent onto filter paper and applied the bacteria on the color change. The results were negative.

The citrate test was negative. We inoculated the tube with our unknown bacteria and inoculated it at 37°C overnight. If the tube turned blue, the test would have been positive. However, our tube remained green. 

 Our negative citrate test

 We also tried to make yogurt but that turned out to be not as successful as we had hoped.

Our yogurt samples

Day 3: More staining

We began the day by making a variety of stains. The first stain we made was a Capsule stain to see if our unknown bacteria had any capsules.  We used Nigrosin to stain and added the bacteria, using the aseptic technique, from the working stock to the stain drop.  The slide was held at a 45 degree angle to spread the dye and bacteria by dragging another slide across it in one, smooth movement. After drying, safranin was added to cover the slide for 60 seconds.  The slide was rinsed with water and blotted dry. The nigrosin stains the background of the cell and the saphrinin stains the cell wall so that the capsules surrounding them can be clearly seen. Because there was no white and both the bacteria cell wall and background were dyed, there was no capsule. By using this stain method, health science majors can determine the bacteria and properly treat the bacteria.

Bacteria with no capsule

The next stain was did was endospore stain to see if our bacteria was producing endospores.  We drew a circle of the slide with a china marker to know where to place the bacteria.  We added a droplet of water and bacteria from the working stock. Once it was air-dried, the slide was placed over boiling water and covered with bibulous paper.  The slide was saturated with malachite green dye for 5 minutes. After the malachite green began to steam, adding additional stain to the slide to prevent it from drying. After 5 minutes, the paper was removed from the slide and placed in the biohazard bag. The slide was left to cool. It was rinsed with water then covered with safrinin fro 60 seconds and rinsed with water again to removed excess safranin. The slide was blotted with bibulous paper. After examining the slide under the microscope, there was no endospores. 

Saturating with malachite green

Rinsing the slide

                                  

Bacteria under the microscope

No Endospores!

         Our next stain was the acid-fast stain. After preparing a slide with our bacteria using the aseptic technique, the slide was placed over a beaker with boiling water.  Bibulous paper was placed over the slide and saturated with Ziehl-Neelson carbolfuchsin.  The stain was covered for 4 minutes. While the stain was evaporating, more stain was added to prevent it from drying.  After 4 minutes, the slide was removed from the heat to cool and the paper was placed in the biohazard bag. To remove excess stain, the slide was rinsed with water.  The slide was held at a 45 degree angle and decolorized with acid alcohol. The slide was rinsed with water and then covered with methylene blue for 2 minutes. The slide was rinsed with water and blotted dry with bibulous paper.

Saturating the slide with Ziehl-Neelson carbolfuchsin

Bacteria under the microscope

The next part of the day consisted of doing a multitude of test to determine our unknown bacteria.
        Our first test was the starch hydrolysis test. This test would determine if our bacteria contain amylase, which digest starch. Using a starch agar plate, we inoculated it by streaking a squiggle like on the surface using the working stock culture. We incubated the plate at 37°C overnight.
        We preformed the casein hydrolysis test to see if our unknown bacteria contained caseianse, a protease that digests casein (how clever!). We inoculated a skim milk agar plate  by streaking a squiggle line on the surface of the agar with our working stock culture. The plate was incubated at 37°C overnight.
       Next, we did the urea hydrolysis test, the lipid test, to see if our bacteria contained urease.  We used urea-containing broth. Using the aseptic technique, we inoculated the urea with our working stock culture.  The tube was incubated at 37°C overnight.
       Next was the gelatin hydrolysis test. Using a nutrient gelatin test tube culture, we stabbed the nutrient gelatin with our bacteria, inoculating it.  We incubated the tube was incubated at 37°C overnight.

Our next experiments involved a variety of sugars. We inoculated three sugar test tubes  to determine our unknown bacteria’s ability to ferment certain carbohydrates. The sugars used were lactose, glucose, sucrose, and mannitol.  Using the aseptic technique, the bacteria from our working stock cultures were inoculated into each tube, respectively.  We did a mixed acid test of methyl-red and Voges-Proskaus.  Using the aspetic technique, we used the working stock culture to inoculate the liquid culture.  We used the same technique for the indole (tryptophan) and nitrate cultures.  The next culture was the citrate slant.  Using the aspetic technique, a needle of bacteria form our working stock culture was inoculated into the tube. 

Our sugar tubes

       Next, we tested our bacteria using the “Triple Sugar Iron Agar” or “TSIA” using the same technique as above. 
       Another test we performed was the Litmus milk test. The test allowed us to find out whether our bacterium utilizes proteins, lactose and litmus.

The next part of the day consisted of determining if our unknown was facultature, meaning both aerobic and anaerobic.  We known that our culture is not obigate anaerobic because our unknown bacteria has grown in oxygen (aerobic). In order to test if our unknown is facultature, we did a multitude of tests. 

To test for motility, we used the aseptic technique by dipping our inoculating needle into the working stock culture.  We stabbed the needle into the middle of the motility medium.  The tube was incubated at 37°C overnight.

       For the catalase test, after the aspetic technique, we drew a squiggle like of a nutrient agar culture plate. The plate was incubated at 37°C overnight.
        Finally, we wanted to determine if our unknown was facultature, meaning both aerobic and anaerobic.  We known that our culture is not obigate anaerobic because our unknown bacteria has grown in oxygen (aerobic). In order to test if our unknown is facultature, we did gaspac and thyoglycolate tests.  For the gaspac test, after the aspetic technique, we drew a squiggle like of a nutrient agar culture plate. For the thyoglycolate test, after using the aspetic technique, we obtained bacteria from the working stock culture and inoculated the thyoglycolate tube. The tube was incubated at 37°C overnight.

Placing our cultures under anaerobic conditions

The pictures of these tests are seen in the Day 4 post!

Day 2: All About the Stain

We brought out our bacteria samples from the fridge and observed them for any new growth. Turns out, all of our environment samples did not really grow overnight so we decided to collect new samples at the end of the lab. However, the bacteria from the fingerprint samples grew enough overnight for us to see different kinds of colony shapes. Our plate had bacteria with puntiform and filamentous colonies.

Our fingerprint bacteria with colonies

After viewing our samples, we observed a few on the 3D screen and even got to wear some pretty fly looking glasses.

Lab is a good time

Following this, we got some practice using the microscopes with slides provided for us. We were able to get comfortable using the microscope and then reviewed microscope information like resolutions and magnifications. We learned about the use of oil increases the resolution because it captures all of the light to the lens.

We then made a working stock and a reserve stock of our unknown bacteria to be able to use for future experiments.  We also made a streak plate and spread plate using our unknown bacteria.  The streak plate was used to get a pure culture by spreading out the bacteria to grow separately. For the streak plate, using the aseptic technique, we touched the unknown bacteria and spread the bacteria on the top region of the plate.  After doing the aseptic technique, we streaked the bacteria from the first region to the second region.  Then, after the aseptic technique was done again, we streaked the bacteria from the second region to the third region.  Finally, the aseptic technique was done again and the bacteria was streaked from the third region to the fourth region.  In the center of the plate, a squiggle line was drawn.  The plate was placed in the incubator at 37°C overnight.
        The spread plate was next and we used a spreader with 70% ethanol and a micropipet using 200 mL of our bacteria sample.  We placed 200 mL of our sample onto the agar culture plate and after doing the aseptic technique with the spreader, we spread the bacteria to cover the entire plate.   The plate was placed in the incubator at 37°C overnight.

Heating our spreader to kill off bacteria

 Next, it was time for the staining to begin!
Our first stain was the gram stain, which would tell us if our unknown bacteria was gram positive or gram negative.  After doing the aseptic technique, we placed our slide on the slide holder over the sink and covered the bacteria with crystal violet dye.  After 30 seconds, we rinsed off the excess crystal violet with water.  We added iodine to the slide and allowed it to sit for 1 minute. After the minute was up, was rinsed the excess iodine off with water and then added a decolorizing reagent (95% ethanol) until the color on the slide stopped running. Finally, was covered the slide in safranin dye, and let it sit for 1 minute.  Then, we rinsed the slide with water and blotted the slide dry with bibulous paper.
Gram-positive bacteria do not decolorize in the decolorizing reagent, meaning the crystal violet color is retained.  However, gram-negative bacteria decolorize in the decolorizing reagent allowing them to accept safranin's reddish color. Because our bacteria was reddish, our unknown bacteria was gram-negative!

Dr. P explaining gram-positive cell wall

Our gram-negative bacteria (RED!)

 Our staining days weren't over today! Day 3 continues with many more stains...

Day 1: The Beginning

           After final exams were done, 8 students decided the semester wasn't long enough and decided to take on one more class.  We entered the laboratory and Dr. Pathakamuri gave a small introduction to the class. He told us how this class is important to nursing majors and the health science relations, in general.  He said that patients will have germs and illnesses and we, as professionals, need to be able to aid in the treatment of the illnesses quickly.  We need to be able to properly identify the bacteria to be able to treat it efficiently.  Also in our introduction, Dr. Pathakamuri gave a brief introduction about the laboratory safety.  Once we enter the lab, we need to put on our lab coats, wash our hands thoroughly, and clean our bench area with disinfectant to get rid of any bacteria.  When we left the lab, we need to clean our bench area, wash our hands, and hang up our lab coats. 
                                                                                                 

 
 To really see and understand how clean our hands were after they were washed, we did an experiment.  Lindsey and I received a Petri dish with a culture medium. We divided our dish into four sections using a China marker. We pressed our thumbs into the medium and quickly covered the dish to prevent any contamination.  Then we washed our hands, we pressed our thumbs again into the medium.  We covered the dish and put them in a 37°C incubator.  This allowed the bacteria to grow overnight. 

Our Petri Dish with our thumb prints before and after washing

          Next, we made our own media culture from Nutrient Agar or Nutrient Broth.  Our group was assigned Nutrient Agar.  We measured 4.6 grams of Nutrient Agar and added it to a beaker of 200 mL of distilled water and covered the beaker with foil to prevent water loss.  The mixture was placed in the autoclave for 20 minutes under 15 lbs of pressure at 121°C.  The autoclave uses moist heat for sterilization.  Once the mixture cooled to approximately 30°C, the mixture was poured into Petri dishes to cover the bottom. The dish was covered to prevent contamination and the liquid had turned to gel within minutes. The dishes were placed in a 24°C incubator. 
                                                   

Weighing our Nutrient Agar

Placing our mixture in the autoclave

Removing our mixture from the autoclave

Gel media cultures

         
                       We learned about Aseptic techniques to transport bacteria from one tube to another under sterile conditions.   We lit the Bunsen burner and were given two test tubes of liquid.  The inoculation loop was sterilized over the Bunsen burner, the test tube was slightly heated to remove bacteria, the inoculation loop was placed in the test tube to get a thin film covering the loop, and the tube was heated and closed.  The second test tube was heated slightly and the bacteria was placed in the second tube.  The second tube was heated and closed.  The inoculation loop was heated a final time for sterilization. 

Sterilizing the inoculation loop

         Our final experiment of the day was to realize how many colonies of bacteria surround you. For this experiment, we decided to swab the silverware water container in the cafeteria.  We figured this would have tons of bacteria since this is where all the dirty silverware is placed.  We swiped the swab in the dirty water and spread it across the Petri dish.  We put the dish in the 24°C incubator (room temperature). We left the bacteria grow over two nights.
         We examined the colony morphology of the bacteria.  The whole colony form had three types of bacteria: puntiform, circular, and rhizoid.  The colony pigmentation was white/colorless and the colony margin was entire. The elevation of the puntiform colony was raised. The elevation of the circular colony was raised. The elevation of the rhizoid colony was undulate. 

Silverware Bin Bacteria