SO...WHAT IS IT??? -Identifying our unknown bacteria!

After a semester with our unknown bacteria, we have studied its physical characteristics, how it grows, how it moves, how it reacts in different conditions, and what it uses for energy. All the results of these tests have given us clues as to our mysterious unknown. Using the flow chart provided for us, we were able to FINALLY identify it!

These were the determining characteristics when using the flow chart:

• Gram positive--narrowing it down to Corynebacterium spp., Bacillus spp., and Lactobacillus spp.
• Spore-forming--narrowing it down to Bacillus spp.
• Positive mannitol test--narrowing it down to the species B. megaterium and B. subtilis
• Positive Voges-Proskauer (VP) test-- Bacillus subtilis!!!!

Now that we know what our bacteria is, we decided to do a little research on Bacillus subtilis. Here are some interesting facts that we found:

• naturally-occuring
• widespread
• used for its natural fungicidal properties to prevent crop disease
• Used in commercial Japanese and Korean food production
• Can convert explosives into nitrogen, carbon dioxide, and water
• Used as an immunostimulatory agent in alternative medicine

References:

• http://epa.gov/biotech_rule/pubs/fra/fra009.htm
• http://en.wikipedia.org/wiki/Bacillus_subtilis

Cow Antigens & ELISA Test

Testing the purity of bovine extract and albumin. The Petri dish is dived into three sections and we begin by making four holes in the agar for each section with a pipette.  Then we fill the holes with the substance indicated in the work sheet and checked the results next lab. In section one we added green dye in one hole, red dye in the second, Barium Chloride in the third and Potassium Sulfate in the fourth. The second section got Bovine Albumin to the first hole, Goat Anti-horse Albumin to the second, Goat Anti-bovine albumin to the third and Goat Anti-swine Albumin to the fourth. The third section got Goat Anti-horse Albumin, Goat Anti-bovine Albumin and Goat Anti-swine Albumin and Hamburger extract. Our results were a little confusing because the agar did not change colors like the other groups, but I don’t think there was any contamination that we could see.

Cow Antigen Results

The ELISA was to determine if Patient 3 and/or 7 have HIV.  We marked a plus sign on the wells to make a positive control sample, and used a negative sign on the wells for negative control samples, “3” wells for Patient 3’s results, and “7” wells for Patient 7’s results.  The results were that Patient 3 does not have an HIV infection, but Patient 7 does.

ELISA Results

UV Light

The UV wavelength has the unique property of being able to kill microorganism. This really cool feature is being studied scientists and in some hospitals they use UV lights as a sterilization method to clean hospital rooms after a sick patient leaves. However, UV light is also known to cause mutations, and so in a hospital setting only the longer UV Light C wavelength us used just in case someone was to walk in the room by accident.

UV Light bulb and Petri dishes undergoing sterilization.

In Lab we decided to see if this was true and if UV light would kill or retard the growth or our bacterias. Everyone made a carpet spread of bacteria on a Petri dish. Since UV light is blocked by any physical barrier except air, to conduct the experiment we simply covered half the Petri dish with paper, and exposed each dish to one minute of UV light. Oh, and remember to take the glass lid of the Petri dish because that will block UV light too! Next lab we checked the dishes and found that there was still bacterial growth on the UV treated side of the plates, but it did seem to be a little less growth then the non-UV light treated side. We thought that maybe the experiment did not work very well because we set the wave length on too low a setting. No one checked the setting before we began.

Making Yogurt!!!

Today we get to apply what we learned about microbes to the real world - or rather the kitchen -- because we are going to make yogurt and Kefir!

The process was really simple. We start out with whole milk and boil it in the microwave to kill any pathogens. Then we filled two insulated cups and added a spoonful of yogurt to one and kefir to the other, mixed well, and let sit over night in a warm incubator. Another method would be to leave it in a stove with the light one. The light will provide just enough heat for the microbes to grow. Next the cups had turned from liquid to a solid cup of yogurt and a creamy cup of kefir! Most the students that tried them agreed it was good but a little sour. Just add a little sugar and berries and you have you're own homemade yogurt!

Sweet way to mix yogurt : )

Finished yogurt & kefire ready to eat!

Disclaimer: If you try this at home and get sick it's not my fault : )

Oxidase Test

Performing an oxidase tests lets us see if our bacteria have cytochrome oxidase, as participant in electron transport during respiration. To check, we simply take the old unknown bacterial plate we used to the antibiotic test and a vial of oxidase. It it turns blue right away the test is positive, but if it does not turn blue or turns blue after 30 seconds then the test is negative. Our plate only turned a little blue after a few minutes indicating that our unknown bacteria is NEGATIVE for Oxidase.

Negative Oxidase Results

We also added hydrogen peroxide to the plate to see if it would react and our bacteria fuzzed a lot! This means our bacteria can use aerobic respiration.

Results of Protien-Indole, Citrate, Nitrate, and Urea.

Protien-Indole Results: Negative. When we added the Kovak's solution after 48 hours, the broth did not turn red. Therefore our bacteria does not have the ability to use tryptophan as an engergy source.

Protien-Indole Restults: Negative

Citrate Results: Negative. Our slant had a tinge of blue on top, but the rest of the tube remained green. Therefore our bacteria does not use citrate.


Protien-Indole Results: Negative


Nitrate Results: Positive. When we added the A&B solution nothing happened. Then we added the zinc dust nothing happened either. If no change occures when the zinc dust is added after ten minutes, then the test is positive for nitrate reduction and nitrite ions are present.

Nitrate Results: Positive

Urea Restults: Negative. Our bacteria does not have the enzyme that breaks down urea because the test tube turned yellow. If the tube remained red it would indicate a positive result.

Urea Restults: Negative

Reading the Results of our Antibiotics Test

Upon retrieving our antibiotic test from the incubator, we saw the following...

We measured the diameter of the clearing around each of the antibiotics used to determine the sensitivity of our bacteria to each antibiotic. We found...

• Penicillin: 27mm--sensitive
• Vancomycin: 22 mm--sensitive
• tetracyclin: 25 mm--sensitive
• Erythrocyclin: 31 mm--sensitive
• Chloromphenicol: 30 mm--sensitive
• Neomycin: 20 mm--sensitive

In conclusion, our bacteria is VERY sensitive to a lot of antibiotics...yet another distinguishing characteristic!



Protien-Indole (tryptophan), Citrate, Nitrate, and Urea Tests.

Uninoculated tubes:

Above (left to right) citrate tube, indole tube, nitrite tube, and urea tube

The Citrate test determines whether a bacterium can utilize citrate as its sole source of carbon and energy. If our bacteria does, it has the enzyme, citrate permease and will turn the green agar blue. Our bacteria turned slightly turned the agar blue, therefore, our bacteria does utilize citrate.

The indole test determines the ability of some bacteria to split the amino acid tryptophan into indole and pyruvic acid. If the bacteria produces indole, a red layer would form at the top of the test tube. Our bacteria did not produce a red layer, therefore, this test is negative. Our bacteria does not use the amino acid, tryptophan.

The nitrite test determines if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas. Our sample did not turn red upon adding the zinc, therefore our bacteria is positive and is capable of reducing nitrate ions.

Finally, the urea test allows us to determine whether a bacteria has the ability to hydrolyze urea. We inoculated our bacteria into the urea-containing broth and if the tube turned bright pink, the bacteria would test positive, showing that it has the enzyme urease. Our bacteria did not turn pink but remained the same color. So, our bacteria does not have urease and therefore it cannot hydrolyze bacteria.

During the second part of lab, we prepared a petri dish of our bacteria and then placed seven different antibiotic tablets in the plate: Penicillin, vancomycin, novabiocin, tetracydine, erythromycan, chloramphenicol, and neomycin. We will find out next lab whether our bacteria are sensitive to the antibiotics...

Gelatin Test, the Carbohydrates (sucrose, lactose, and glucose) Fermentation Test, TSI Agar Test, VP, Litmus Milk Test Results and Methyl Red Test

The results for our VP, gelatin, carbohydrate fermentation, TSIA, and the litmus tests are in! The following images show what we found... 

Above is the result of our VP test. The VP test determines the ability of our bacteria to use butanediol fermentation to ferment glucose. If the test is positive, the presence of oxygen will turn the liquid red upon addition of the VP reagents. As you can tell, our product has turned a reddish color (it was originally yellow) and therefore our test is positive, which means that our bacteria uses butanediol fermentation to ferment glucose.

The fermentation of carbohydrates tests (above) allow us to determine our bacteria's ability to ferment a particular carbohydrate. There are three carbohydrates that we used: sucrose, glucose, and lactose. If our bacteria uses one of these sugars for energy it will produce organic acids as waste products. If this is the case, the phenol red will turn from red to yellow in the presence of acid. As you can see from the picture, the sucrose and glucose tests were positive, meaning they use those sugars. Our bacteria does not use lactose because the medium is still red.

Above is the result of our gelatin hydrolysis test. The gelatin hydrolysis test determines if our bacteria has the enzyme, gelatinase, necessary to digest gelatin. If our bacteria does have gelatinase, it will cause the peptide bonds to cleave in the gelatin, causing the semi-solid to liquefy. If the gelatinous medium is still liquid at 4 degrees Celsius, the bacteria has gelatinase. It's hard to see in the picture, but our gelatin medium has liquefied, and so this test is positive.

The two above images are the result of the TSI (triple sugar iron) agar test. The agar in the TSI test contains lactose, sucrose, and glucose. The agar is originally red. If the bacteria uses any of these sugars, acid is a byproduct, thus causing the agar to turn from red to yellow. As you can see from the image, our slant is red and the butt is yellow. This means that our bacteria only ferments glucose and not lactose and/or sucrose. 

The purple medium in the test tube second to the left is the litmus milk test. The normally-lavender litmus milk medium allows us to differentiate the bacteria's ability to utilize lactose, protien, and litmus in litmus milk. The litmus acts as the pH indicator, turning pink in the presence of acid and blue in alkaline conditions. As you can see from the picture, our medium remained lavender in color. What you can't really see from this picture is that the medium produced a runny curd towards the surface of the medium. This means that our bacteria possesses the enzyme, rennin, to break down the casein. Our bacteria did not turn pink, meaning that it lactose fermentation did not occur.



Methyl Red Test Results: Negative



The above Methel Red test (Mixed Fermentation) shows us if the bacteria has the ability to ferment glucose via mixed-acid fermentation. 5 drops of methyl red are added to the test tube and if it turns red then the test is possitive. If there is no color change are the tube remains yellow, then the test is negative. Our test tube remained yellow so our results are negative.

Starch, Lipid, Casein, and Motility Results

At the beginning of today's lab, we retrieved our starch, lipid, and casein plates from the incubator along with our motility test slant.

Results:

Above is the result of our starch agar plate test. As you can see, not a whole lot happened here. Our test result is negative, meaning our bacteria cannot produce amylase and therefore, it does not use starch.

This is our skim milk plate that tests whether or not our bacteria uses casein. As you can see, there is a halo around where we streaked the plate. This indicates that our bacteria is able to produce protease and has used up the casein where the halo (clear spot) is. This test is positive!

This is our blue agar dish that tests whether our unknown bacteria can use lipids. Our bacteria has produced a halo around the streak, which indicates complete hydrolysis of triglycerides, positive for lipase production. The bacteria uses lipids, therefore, this test is also positive!

Although it is hard to tell from this picture, the motility slant to the right shows that our bacteria is motile because the bacteria has traveled from the original insertion line from the inoculation needle, causing the agar to appear cloudy.

Today, we also started the gelatin test, the carbohydrates (sucrose, lactose, and glucose) fermentation test, TSIA test, and the litmus milk test. We will let our bacteria grow in the incubator until next lab, when we get to determine the results.

Oxygen Requirements Lab Results and Starch, Lipid, and Casein Test

Today, we retrieved our thioglycolate broth tube from the incubator to see the oxygen requirement for our unknown sample. The result is as follows:

As you can see from the image above, our unknown bacteria thrived towards the surface of the broth and did not grow at all at the bottom. There are trace amounts of bacteria towards the center, meaning our bacteria is facultative anaerobic. We were unsure whether the bacteria in the middle is there because it grew there or whether we weren't careful and accidentally shook it when handling it. If that is the case, then our bacteria would be aerobic.

When we retrieved our petri dish from the GasPak, we discovered that our bacteria did not grow in the absence of oxygen. Therefore, our bacteria is aerobic/micro-aerophilic. This means that our bacteria needs oxygen to grow! 

Before we left lab, we inoculated three different petri plates: one was a starch agar plate, which tests whether the bacteria produces amylase to hydrolyze amylopectin and amylose into maltose, glucose, and dextrans. Another petri dish had skim milk and it discriminates bacteria that produce protease to hydrolyze the casein into clear amino acids. The last is the blue agar plate, which tests whether the bacteria produces lipase to break down lipids.

We also inoculated a solid motility medium to determine whether our bacteria has to means to move through the medium.

...the results will be determined next lab...

Oxygen Requirement Labs

Today, we got to find out whether our bacteria (environmental were aerobic or anaerobic. We performed two tests to determine if the bacteria needs oxygen and to what extent: one test we used was with thioglycollate broth and the other was with the GasPak.

With the first test using the thioglycollate broth, we used the ascetic to add our unknown bacteria to the liquid broth. We then placed the inoculated broth tube in the incubator (set at 30 degrees). There, it will grow so that next lab we can determine whether our unknown sample is aerobic or anaerobic. The results can appear as follows:

1. Obligate Aerobe

2. Facultative Anaerobe

3. Aerotolerant Anaerobe

4. Obligate Anaerobe

For the second test, we used the GasPak (shown below)

To use the GasPak, first we used the aseptic technique to transfer bacteria onto a new Petri dish. Then we placed the dishes into the air-tight chamber so that no oxygen could be present. Before we placed the lid on, we inserted a methyl-blue pad inside that would act as an indicator, turning from blue to colorless in the absence of oxygen. 

     

...And now we wait until next time to see the results...

Capsule Stain

Some bacteria produce a capsule or a slime layer for protection. Bacteria that have capules or slime layers make themselves very difficult to attack and kill, or scrape off of surfaces. After preparing a capsule stain, if the bacteria have a capusule, there would be a clear area that does not pick up the stain around the bacteria. If the bacteria does not have a capsule, a clear area will not be present.

Results: No Capsule




Endospore Stain

Ever wish in stressful moments you could just curl up in a shell and weather it out? Well if you're a cell with an endospore you can! An endospore is a hard capsule that the cell shrivels up into when there is stress in the environment that makes it's habitat unlivable, such as a lack of nutrients, extreme temperatures, or lack of water. A cell can remain in this state for an indefinite time, and when favorable conditions return...POP! out comes the cell from its vegetative state and is ready to start living like nothing ever happened.  Our bacteri does have endospores and this is fairly characteristic of gram positive microbes.  The way we can tell is that there are little green spots inside which are the endospores.

Bacteria with Endospores

We lost our picture of bacteria with endospores. The above picture is a picture of Bacillis subtilis with endospores from the internet.

Acid Fast Stain

Acid-fast bacteria are gram positive and have large amounts of glycolipids, especially mycolic acids. This makes their membranes difficult to penetrate and also resistant to chemicals.

We found our Unknown E. bacteria was acid-fast positive because it turned red at the end of the staining procedure.




Acid-Fast Stain Results

 

Motility Test

Motility in a bacteria depends on the presence of flagella. We used two different techniques to check for motility in our bacteria. One was instering a straight inoculating wire into an agar tube and checking for cloudiness the next day. If the tube is cloud it means the bacteria have spread around the tube and are therefore motile. If the tube is clear, then the bacteria where not able to leave the original insertion streak and are thus not motile.

Results:
Unknown: Motile
Environmental #1 (Lauren) Motile
Encironmental #2 (Kelly): Not Motile

The second mothod involed viewing the bateria under a slide immersed in water to physically see if they were swimming or not. This is done by placing a drop of liquid agar inoculated with bateria a few days before on a cover slip. Dot the corners of the cover slip with vasaline and place a welled slide on top of the cover slip. Then slide is flipped over and this leaves us with a drop of water suspended from the cover slip and not touching anything else so the bateria are free floating in the drop of water and can be observed.

Results:
Only Environmental #1 was motile and can be seen swiming around in this video link.

These little guys are fast!

Gram Stain

Making a Gram Stain shows us if the bacteria cell wall is negative or positive which indicates the structure of the cell wall. Gram positive cell walls are thicker and have a bigger layer of peptidoglycan and teichoic acid and lopoteichoic acid molecules while gram negative cell walls are thinner due to a smaller layer of peptidoglycans, but have a complex layer of lipd macromoecules. Medically it is important to know if the cell wall of a bacteria is possitive or negative because gram negative bacteria are usually more resistant to antibiotics due to thier cell wall structure.

Two dies are used in the gram stains, Crystal Violet and Gram's Iodine. The Crystal Violet is added first, then rinsed with ethanol and then stained again with Gram's Iodine. The concept behind this is that because a gram positive cell wall is larger it will pick up the crystal violet stain while gram negative's cell wall is too small to hold the die. When we rinse the slides with ethanol, if the stain did not absorb into the cell wall it will be rinsed off allowing the next stain, gram's iodine to stain it. If the bacteria is gram positive it will already be fixed with the red crystal violet and will not have room to pick up the purple gram's iodine and will remain red. The gram negative bacterial will stain purple because they did not have room in their cell wall to pick up the crystal violet stain and it gets washed off.

Results:

Unknown bateria is Gram Positive.

Environmental #1(Lauren) is Gram Negative.

Lauren's Environmental Gram - Stain

Environmental Sample #2: Gram positive

Simple Stain

Now that we have a pure culture, we want to see what this bacteria looks like up close. To do this we need to stain the bacteria so they can be viewed under a microscope to see their structure and shape. A drop of water is placed on a slide, bacteria added and swirled in the water, fixed by letting the water air dry and running it through a flame. Then Gram’s Safranin (Red) stain and/or Crystal Violet (Purple) stain is applied, let dry, and rinsed.

Crystal Violet and Gram's Safranin Stains

Environmental Samples under low magnification:

Lauren's Env. Sample stained with Crystal Violet

                                           Kelley's Env. Sample. (Upper image under low power and lower image under oil immersion)

In order to manage a close up of the bacteria, we need to look at them using immersion oil. This oil has the same refractory index as the glass slide, and when it makes contact with the microscope lens, it prevents the light rays from bouncing or refracting out of the lens, giving a much sharper image.

Applying imersion oil to slide

Environmental Samples under high magnification using imersion oil:

Lauren's Env. Sample stained with Gram's Safrinin under high magnification using imersion oil

Taking a Pure Culture from the Environmental Samples

The object of this lab was to issolate just one type of microorganism from our environmental sample. Most of the students found many different types of bacteria or mold growing in the Petri dish. Env. Sample #1 was one of the few that had only one or two types of bacteria growing, while Env. Sample #2 had many different microbes growing including mold.  Using aseptic technique we transferred only one strain of the bacteria from our first culture to a second dish to make a pure culture.

A pure culture is made by selecting a single colony that is not touching any other colonies with an inoculating loop and using aseptic technique, transfer the bacteria to a new sterilized Petri dish.  The idea is that it only takes one bacteria to make a culture, and if the colony (the yellowish dots seen in the picture) are not touching any of the other dots, it was made from only one bacteria, and thus it contains only one type of bacteria, or a “pure culture”.

Enviromental Sample #1:
From Lauren's Env.Sample #1 there were a few larger yellow colonies of bacteria that were chosen to become the pure sample bacteria that can be seen in the picture bellow.

Environmental Sample #1 with Large Yellow colony that was selected for the Pure Culture

The colony Lauren chose was very close to all the other bacterial colonies, and therefore very difficult to obtain without being contaminated with the whitish bacteria. As a result I did not end up with a pure culture and had to redo the procedure. This is normal and it often takes many attempts before a pure culture can be obtained. If the culture is given too much time to grow the colonies can grow into one another, making it impossible to obtain a pure culture. In order to ensure a single colony of my yellow bacteria I only touched the inoculating loop to the colony to only pick up a few bacteria, then spread them in wide streaks across a new Petri dish, and only allowed the bacteria to grow in the incubator for a day. The result was much smaller colonies that were not touching. From this I was able to make a pure culture. Bellow is the final yellow Environmental Pure Culture Bacteria sample.

Env. Sample #1 Pure Culture

Env. Sample #1 Pure Culture. In this view you can see how this species forms raised colonies and has a shiny appearance.

Environmental Sample #2:
Kelley's Environmental Sample:

The colony I chose was one of the smallest colonies on my petri dish. It was the only colony of its kind growing on the agar plate and I chose it because of its unique rose color.

The above picture of is of my original environmental swab culture. As you can see, there are many different kinds of bacteria and molds that grew.

Above is my original petri dish (on left) next to my pure culture that I grew from the original (on right)

Above is a closer look at my pure culture. Notice the rose color. It is hard to tell from the picture, but the bacteria is shiny and the colonies are circular. The colony margins are entire and the elevation is convex.

Collecting an Environmental Sample of Microbes

Our first assignment was to collect a microbe from anywhere in our school environment. The purpose of this is to see that microbes live everywhere all around us in our daily lives. Lauren collected Environmental Sample #1 from the woman's room soap dispenser handle, and Kelly collected Environmental Sample #2 from a bicycle seat outside of Cosmos & Damian Hall.  Gathering a sample of microbes from any given surface is done by soaking a sterilized swab in liquid agar broth, swabing the area to be tested, and then streak or wipe the swab all over an agar plate in every direction. The plates are then put in a room temperature incubator for 24 hours to see if anything will grow.

The agar plates went from looking like this...

To an explosion of microbial growth...

Enviromental Sample #1:

Environmental Sample #1

Enviromental Sample #1 Close-up

Enviromental Sample #1 Close-up

Enviromental Sample #2:

Environmental Sample #2

Environmental Sample #2 Close-up