Getting Bacteria Samples: May 2014

Friday, May 30, 2014

Day 10

Lab Day 10

We checked our results from the day before, our lab group samples.

Nasal Swab on the Mannitol Salt Plate:

There was growth on the plate however, there was no yellow coloration on the plate. Therefore there was no MRSA present in the sample.

Throat Swab on the Blood Agar Plate:

There was beta-hemolysis on the blood agar plate, meaning that there was Staphylococcus present. However, we placed a bacitracin tablet on the plate to check if it is Group A or Group B Staph.

Urine Sample on the EMB Plate:

There was no growth on the plate therefore there is no E. coli in the the sample that we collected.

Then we wanted to check to see if our bacteria was resistant or non resistant to specific antibiotics.

We placed 5 tablets into a plate of our specimen:

P: 7mm in diameter,

Neomycin: 15mm in diameter, intermediate

Cinnamon Oil: 25 mm

Tetracycline: 20mm very sensitive to Pro sysnthesis

Augmentin: 23mm very affective

Lemon Oil: didn't wok at all

Real Time PCR- sero conversion- present antibodies against antigens

We completed an Antibody-Antigen Reaction in Agar exercise, and we Tested for Food Purity.

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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Tuesday, May 27, 2014

Lab Day 9

Today we took a series of samples from members of our group.

Nasal Swab: For this test we took a swab sample, using aseptic technique, and smeared the sample on a Mannitol-Salt plate because we are testing for MRSA/staph. When taking the sample we needed to use a saline solution, because the area being tested is not moist, and then rotated the swab 2-5 times counterclockwise and clockwise.

Throat Swab: For this test we also swabbed the back of the throat using aseptic technique. In this test we are testing for strep, therefore we swabbed the sample on a Blood Agar plate.

Urine Swab: For this test, a member in our group preformed a urine test. We then placed some of the urine on a EMB plate, because in this type of test we are testing for E. coli we use either an EMB or MacConkey plate, and used a sterilized loop.

After we preformed these tests we placed these plates in the incubator and we shall observe our results tomorrow.

Next we started our Antibiotic Sensitivity Test. We spread our unknown bacteria onto a new nurtient agar plate. Then we added several antibiotics onto the plate. We sterilized tweezers using 95% ethanol.

Antibiotics

Neomycin
Tetracytine
Penicillin
Lemon Oil
Cinnamon Oil
Agumented

Tomorrow we shall see how our bacteria reacts to specific antibiotics.

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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Saturday, May 24, 2014

Lab Day 8

Today in lab, we observed our results from the tests we did the day before.
MacConkey Agar: the results of this test was that the strand of bacteria turned a bright pink color meaning it was a positive test. Our bacteria is a lactose fermenter.

Eosin Methylene Blue Agar (EMB): The strain of our bacteria was a green color meaning our bacteria was a strong lactose fermenter. If it was pink color, it would mean our bacteria was a weak lactose fermenter.

Mannitol Salt Agar: This test tests for staphylococcus which means only halophiles would appear positive and the color would turn yellow. Our bacteria was negative for halophiles therefore it is not staphylococcus and our bacteria does not ferment mannitol.

Phenylethyl Alcohol Agar (PEA): Our bacteria is gram negative therefore, it won’t grow on the PEA agar.

Blood Agar: The result of our test was that the bacteria turned green which means it was alpha-hemolitis. This means our bacteria was partial hemolysis and you can use this test to test for strep.

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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Thursday, May 22, 2014

Lab Day 7

May 22

Part 1 of today's lab: checking our experimental results from yesterday

1: Indole (Tryptophan Degradation) Test

Purpose: to determine the ability of some bacteria to split the amino acid trypotphan into indole and pyruvic acid.

Result: positive

We added 15 drops of Kovac's reagent. A red layer formed on the top of our sample and this indicates a positive test for the presence of indole. This means that Trypotphan was hydrolyzed.

2: Urea Hydrolysis Test

Purpose: to determine the ability of a bacterium to hydrolyze a urea.

Result: Negative

A few bacteria use the enzyme urease to rapidly degrade urea into CO2 and Ammonia. The color didn't change from from the original color and this signified a negative test. If ammonia was produced, the color would've been pink in alkaline ph due to the breaking down of urea with the urease enzyme.

3: Citrate Utilization Test

Purpose: to determine if bacterium can utilize citrate as its sole source of carbon and energy.

Result: negative

The ph indicator bromothymol blue turns blue in alkaline ph. A change in color of the medium from green to blue denotes a positive test. Our color did not change and therefore, our bacteria didn't have the enzyme to break down citric acid.

4: Nitrate Reduction Test

Purpose: to determine if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas.

Result: positive

Nitrate accepts electrons instead of oxygen. We were looking for a nitrate reduction enzyme.

This test can be two part:

Nitrate ----> nitrate

Nitrite ---> ammonia

However, because our test was positive for nitrites upon adding Sulfanilic Acid and Dimethyl-alpha-naphthylamine, we did not have to test further in case our bacterial nitrate broth had undergone both reactions and turned into ammonia. It turned a cherry red which signifies that it produced nitrites and is therefore positive.

5: MR-VP

Purpose: to determine the ability of some bacteria to ferment glucose via mixed-acid fermentation.

MR result: positive – VP result: negative

If our MR-VP broth used glucose and fermented it for acid, upon adding methyl red (a ph indicator), you should see a color change to red. Ours reacted this way when it was added. VP test tests butanediol for non-acidic end product but if MR test is positive, VP is negative.

6: Litmus Milk Reactions

Purpose: to differentiate among bacteria as to their ability to utilize lactose, protein, and litmus in litmus milk.

Results: The curds that were present yesterday, were even more of a pink color today. The pink color signifies that our bacteria can ferment lactose. The cracks in the curds signify the presence of gas.

When testing for acidic and non-acidic curds, we turned the tube upside down. If they were to come down, it would be non-acid curd but being that our curds remained immotile when turned, we can tell that our bacteria form an acid curd in litmus milk.

Part 2 of todays lab: Selective and Differential Plates

The procedures for each of these tests were the same. Practicing aseptic technique throughout, we inoculated each of the following plates.

1: MacConkey Agar

Purpose: to detect and differentiate among gram-negative enteric bacilli, based on their ability to grow on the medium and to ferment lactose.

2: Eosin Methylene Blue Agar

Purpose: To isolate and differentiate gram-negative enteric bacilli.

3: Phenylethyl Alcohol Agar

Purpose: To isolate gram-positive bacteria from a specimen containing a mixture of gram-positive and -negative bacteria

4: Blood Agar Plate

Purpose: 1. To isolate and support the growth of fastidious bacteria. 2. To differentiate among bacteria based on their ability to lyse red blood cells.

5: Mannitol Salt Agar

Purpose: to isolate bacteria based on their salt tolerance and differentiate among these isolates for mannitol fermentation.

After inoculating each of these plates, we placed them in an incubator set at 37 degrees Celsius.

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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Lab Day 6

Part 1-Observations

Today in our lab we observed the results from the food tests that we did the day before. Our results are going to help us figure out what our unknown sample of bacteria is.

We also looked at our virus test that we did yesterday. Our "Europe" did not turn out very well. The other group's "Awkward", however, turned out very well.

The first test we checked is the starch plate. A positive test would show a halo on the plate around the bacteria. This halo did not show up on our plate, therefore our bacteria had a negative starch test. We used Gram's Iodine to check to see the halo.

The next test was the skim milk test, which tested caesin. This was a negative result for us. Dr. P told us to incubate the plate longer. We shall see more results tomorrow.

The lipid, fat, plate was the next plate we observed. There is clear zones due to break down of the lipids. This test was negative for us.

A positive DNA plate when loaded with N. HCL will show a dark halo. Our plate did not show a dark halo when the solution was added, therefore our DNAase test was negative.

The next set of tests we observed were found in the tubes.

Sucrose Test: negative

Litmus Milk: lactic acid curds milk, pink hue, cracks in the curd meaning that there was gas produced. We had a positive test result, however, Dr. P told us to re-incubate.

Maltose: positive

Glucose: positive. There was color change

Lactose: positive. Change in the color

Triple Sugar IA: positive. Both the slant and the butt showed a yellow color which meant that glucose, lactose, and/or sucrose were fermented. Meaning that the slant and the butt were acidic.

Part 2-Tests

The next part of the lab that we did was complete more tests on our bacteria.

Oxidase Test: to determine if our bacteria has cytochrome oxidase a participate in electron transport chain during respiration.

We took the lid of our bacteria plate placed filter paper on it. Then we squeezed two drops of oxidase reagent and then we added the bacteria. The bacteria turned yellow not purple on contact with the reagent, therefore, the results were negative.

The next set of test that we did are going to set overnight to incubate.

We inoculated 5 different tests.

Urea

Citrate

Nitrate

MR-UP

Protein Indole

Today during lab we noticed that Dr. P had a cut on his hand. He showed us a cool way to decrease the bleeding of his cut. He took a piece of chalk and crushed it and the placed the chalk on the cut. This because there is calcium in the chalk the coagulation increased.

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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Wednesday, May 21, 2014

Lab Day 5

Upon entering lab, we checked yesterday’s experiments. In our thioglycolate slants and we found growth on the top and the bottom, therefore signifying that our bacteria is a facilitative aerobe. Our stab was not motile because it remained in the line of inoculation and was not hazy throughout the tube. Our plate had growth and this further confirmed that it was a facilitative aerobe.

The second part of our lab consisted of seeing if our unknown bacteria had catalase. Any oxygen breathing organism can produce H2O2 because they contain the catalyse enzyme which breaks down H2O2 into Hydrogen and Oxygen. By pouring Hydrogen Peroxide onto the plate with our unknown bacteria, we could determine if our bacteria did in fact have cataylse. There were bubbles around the reaction which means our bacteria tested positive for cataylse. We are O2 breathing beings, therefore, also have catalyse and can produce H2O2.

During the last part of our lab, we prepared tests that would help us discover what foods our bacteria prefer to eat such as lipids, proteins, carbohydrates, or nucleic acids. To do this, we used the aseptic technique to inoculate many test tubes with different foods such as glucose, sucrose, maltose, lactose, and litmus milk, with our unknown bacteria. We took a test tube containing TSIA and stabbed our bacteria into the slant and then used the snaking technique over the surface of the slant. We also inoculated a starch agar with our bacteria. After 24 hours you stain the plate with Gram's Iodine. We inoculated a skim milk agar using the snaking method. The last two agar plates we inoculated were the DNA and Lipid plates.

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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Tuesday, May 20, 2014

Sick Days and Bacteria Tests

Day 4
May 19, 2014

Today was the day we all found out about our acceptance into the nursing program!! Lab began with Dr. P teasing Elizabeth about her acceptance. She was afraid she didn't get in and didn't want to get her letter from Dr. Miller but in the end, we got her to smile with the good news.

Saturday night, Pete, Dr. P, Mary Rose, Ben, and Adrianna went to the lab to take a culture of Mary Rose's throat, thinking that she had strep. The plate with the swab had incubated over two nights and we were ready to see the results.

The plate had obvious bacterial growth and as it turns out, Mary Rose tested positive for strep. Pete had experimented with the strep by adding Penicillin, cinnamon oil, and clove oil to the plate. We were able to link lecture to lab by observing the changes in the bacteria. The cinnamon oil killed RBC but did the best job of killing the strep. It did a better job at this than Penicillin! Clove oil also did an incredible job at killing the bacteria as did Penicillin. We were able to deduce the bacteria's death due to clear rings around each of the three substances on the plate.

Now we know! If you have strep or a throat infection, take cinnamon and clove!!

Part 1: Thioglycolate

Practicing Ascetic technique throughout the experiment, we took a loop and, taking a culture of our unknown bacteria, we dipped it into a test tube filled with thioglycolate. This substance was pink towards the top of the tube and a yellowish color on the bottom. This was due to the thioglycolate being oxygenated on the top and not the bottom. By stabbing our bacteria into this tube, we will be able to see if our bacteria is aerobic or anaerobic due to how it will grow in the incubator overnight.

Upon receiving Dr. P's permission, we repeated this procedure with Mary Rose's strep culture. This too we put into the incubator.

Part 2: Stab to test Motility

This we did by taking our loop with our unknown bacteria and stabbing it into an agar deep provided by Dr. Pathakamuri. We put it into the incubator and we'll see what happens tomorrow!

Part 3: Inoculation of a Slant Agar

Because our first slant samples melted we had to redo the two agar slant samples we did earlier last week. Taking our bacteria with a loop, and practicing ascetic technique throughout, we used the snake method to spread the bacteria on the surface of an agar slant. We'll see what happens to the bacteria tomorrow!

Part 4: Testing the effects of a virus on bacteria

We began this procedure by spreading 50ml of group 1's, our group, broth onto a plate. This we did by using a dispenser set to gather 50ml of the broth. Dr. Pathakamuri gave each of the students a small amount of an unknown virus and, taking a big tip, we each wrote something on the broth with it. Our group chose to write Europe! We're excited to be going officially now that we're in the program!

After that, we put the plates the incubator for viral growth.

Part 5: Culturing Anaerobic Bacteria

Taking a small sample of our bacteria, we spread this on a plate. We then put the plate into an anaerobic jar. This jar is used to test whether or not our bacteria is anaerobic. This jar expels all traces of oxygen by the use of hot gas packs in the air tight container. We also put an indicator strip into the box. This will indicate the presence or lack of oxygen. Pale white = no oxygen and blue = presence of oxygen.

We'll get all of our results tomorrow!!

Joanna Dawyot, Cassie Livingston, Mary Rose Capara

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