Results of Differential Media and Catalase Test!

Last class we set up a couple of tests on agar plates and now we have found the exciting results!

Mannitol Salt Agar: 

Above are the agar specimens in which our bacteria grew! Mannitol Salt Agar isolates bacteria based on salt tolerance, and differentiates it by its ability to ferment Mannitol. Fermentation is evident when the agar medium changes from red to yellow. Since the foundation on which our bacteria grew remained red our bacteria cannot ferment Mannitol. However, because it grew in this agar, it is proved that our bacteria must be of the genus Staphylococcus because this genus is the only one that is able to grow in a high salt concentration (also known as a Halophile).

MacConkey Agar: 

The pictures above are from the MacConkey Agar Test. As evident by the lack of visual growth, our bacteria did not thrive in this environment. This is because MacConkey agar differentiates between gram-negative enteric bacilli, based on their ability to grow and ferment glucose. It keeps our results consistent, since our bacteria is not gram-negative nor a bacilli. In the first picture, the right side of the agar plate had much growth and was able to ferment lactose, only because it was not our bacteria, but another specimen from the class. Lactose fermentation is evident by the pinkish color appearing in the agar.

Eosin Methylene Blue (EMB) Agar: 

The picture above depict the EMB Agar test that was carried out last class. As seen by the visual aids, there is no growth on our portion of the agar plate. This is also consistent with our data because the purpose of this media is to isolate and differentiate gram-negative enteric bacilli. Since, our bacteria is neither gram-negative nor enteric bacilli, it makes complete sense! The other sample (on the right side of the top picture), though hard to depict from our picture, has a green metallic color to it. This proves that our friend has bacteria that can ferment lactose. Ours, obviously, cannot.

Catalase Test: 

We added hydrogen peroxide to the sample that grew (Mannitol Salt Agar) our bacteria, to test if it had the enzyme catalase. Since bubbles formed on our bacteria the hydrogen peroxide was successfully broken down into water and oxygen. This proves that our bacteria is an aerobe. Previous tests have also shown that our bacteria fits this description as well, being a facultative aerobe.

We are getting so close to finding out the identity of "F"! Next class we should be able to completely identify him for sure, but for now we have a guess! We hypothesize that "F" is Staphylococcus epidermidis. 


Join us next time when we find out if we were right!

Litmus Results and Selective and Differential Tests!

We know you were waiting anxiously for the results of the Litmus Test, so we are going to go right into it! Since the suspense is so thrilling, we won't make you wait any longer! 

Litmus Results: 

The pictures above all depict the Litmus Broth specimen after 7 days in the incubator. The purple broth substance that occupied the test tube before has now vanished and turned into curds and whey. Our bacteria sufficiently ferments lactose into an acid and denatures the milk protein Casein, forming a firm curd. The results of the test are interpreted to be an acid curd (determined by the red color at the top) with litmus reduction. 

Exciting, I know right?! Can you handle what happens next...

Selective and Differential Tests: 

We next carried out a series of selective and differential tests in order to identify our bacteria further. One test was to isolate and differentiate gram negative bacilli (Eosin Methylene Blue Agar Test), another tested for salt tolerant gram positive cocci (Mannitol Salt Agar), and finally the last tested the ability to differentiate among gram negative bacilli and to ferment lactose (MacConkey Agar). Using the aseptic technique, we inoculated each of these agars with our unknown "F" bacteria and placed them in the respected incubator. 

Join us next time to see the results of these tests and the meaning behind differential and selective media! 

The Results! Gelatin and Fermentation

Welcome back!  The moment we have all been waiting for!  What happened with all those tests from earlier this week? Does our bacteria feast on Gelatin? Does it ferment glucose? Let's see!

Gelatin Hydrolysis Test:


Recall from last time, we used aseptic technique to inoculate our sample in a medium composed of gelatin and placed it in an incubator.  After 48 hours we immediately placed the sample into the refrigerator for 15 min.  After, retrieving the sample, we had to check for liquifaction by tilting the tube slightly.  If "F" has the enzymes to consume gelatin, the sample would remain liquified once cooled.  Let's see!

The Results:

Because the gelatin remains intact, "F" does not have the enzyme gelatinase; our Gelatin Hydrolysis test is negative.

Fermentation of Carbohydrates:


Recall, last time we inoculated three broth tubes: one containing sucrose, one containing lactose, and one with glucose.  After 24 hours we recorded the results!

The Results:

Above: Sucrose

Above: Lactose

Above: Glucose

From Left to Right: Sucrose, Glucose, Lactose

Remember from before all the tubes were a red color.  Fermentation of the sugar resulted in a color change due to a pH indicator added to the broth.  It is hard to notice, but there is also a Durham tube located in each tube.  We had no bubbles in these tubes; therefore, our bacteria does not produce gas as an end product to fermentation.  However, "F" ferments glucose and lactose, producing acid as an end product.  This is indicated by the yellow color found in glucose and the orange color found in lactose.  Our bacteria does not ferment sucrose.

Triple Sugar Iron Agar Test (TSIA):


Recall, we used the aseptic technique to inoculate an agar slant containing triple sugar iron agar (consisting of  1% lactose, 1% sucrose, 0.1% glucose).  After incubating for 24 hours, we examined our slant!

The Results:

The slant was determined to have an acid slant and alkaline butt.  Our bacteria consumed the glucose, producing an acid as an end product to fermentation.  Once again, our bacteria didn't produce a gas as a by-product because there was no break in the agar.  This is a good result because it is consistent with the previous carbohydrate test. However these results are not typical.  There may have been some systematic error in our experiment.

Methyl Red/Voges-Proskauer Test:


After incubating our bacteria in a MR-VP broth for 48 hours, we separated its contents into two tubes.  To one, we added 5 drops of Methyl Red dye, and to the other, we added 15 drops of VP-A and 5 drops of VP-B.

The Results:
Methyl Red Test

After adding the drops, if our bacteria was positive for acid production, which it was, it would retain the red color of the dye.  However, if an acid was not the by-product of the experiment, the color would have faded to a distinct yellow.  Thus, our bacteria tested positive for acid fermentation.  A bacteria that commonly has an acid by-product is E. coli.  However, if you were following previous blogs, this could not be the case because our bacteria is a small cocci bacteria.  E. coli is bacilli.

Voges-Proskauer Test:

In a positive test, which this is not, there would be a reddish tint nearing the top of the tube.  Our bacteria did not show any change in color; therefore, it is negative for the fermentation of glucose via butanediol fermentation.  This means our bacteria does not degrade acids into non-acids.

Join us next week for more Microbiology Lab Adventures!  The Litmus Test will be ready!

The Preparation of Tests, Tests, and more Tests!

Today, we prepared more tests to identify "F" even further. While, results were not analyzed today, the preparation was a very interesting procedure!

Test #1: Gelatin Hydrolysis Test


For this test, we first used the aseptic technique to isolate a colony from our pure culture into a gelatin agar slant. We used an inoculating needle in order to transfer the colony and proceeded to gently stab the gelatin 3/4 of the way through with the needle. After, we placed the specimen into the 37 degree Celsius incubator to grow.

This procedure was completed in order to determine whether our bacteria has the enzyme gelatinase and can therefore digest gelatin. After the agar is cooled to 4 degrees Celsius, if there is liquid present in the tube, our test will be positive for the presence of gelatinase.

Test #2: Fermentation of Carbohydrates


Using the aseptic technique, we inoculated our bacteria into phenol red-sugar broth tubes with a Durham Tube (used to trap gas bubbles) inside, each containing one of three sugars: sucrose, lactose, and glucose. After the inoculation we placed each of the tubes into the incubator (37 degrees Celsius) until next time.

This test was completed in order to determine "F"'s ability to ferment particular carbohydrates. We will be able to see this depending on the color of our broths. If the broth tube changes from red to yellow or gas bubbles are trapped in the Durham tube, fermentation of the particular carbohydrate had occurred.

Test #3: Methyl Red/ Voges-Proskauer Test


For this test we used an MR-VP broth and inoculated our bacteria into the broth using the aseptic technique. Later, we placed the sample into the incubator at 37 degrees Celsius.

This test determines the bacteria's ability to ferment glucose by means of butanediol fermentation. Voges-Proskauer in the broth reacts with the acetoin produced by the butanediol fermentation changing the color of the broth from yellow to red.

Test #4: Triple Sugar Iron Agar Test (TSIA) 


This test involves the usage of Triple Sugar Iron Agar slant and the aseptic technique (no surprises there;)). After inoculating the inoculating needle we injected the needle into the agar 3/4 of the way through, smearing the needle on the top of the agar at the injection site. This was then placed into the incubator at 37 degrees Celsius.

This test will determine our bacteria's ability to ferment glucose, sucrose, and lactose and to produce an acid as a result of fermentation. Also, it more clearly differentiates between the Gram Negative Enteric Bacilli.

Test #5: Litmus Milk Test 


Using a Litmus Milk based broth we inoculated our bacteria (DON'T FORGET THE USE OF THE ASEPTIC TECHNIQUE) and placed it into the incubator at 37 degrees Celsius.

This test will determine the bacteria's ability to utilize lactose, protein, and litmus that is found in milk. Results will be determined by the color and curd formation.

Tune in next time to see these exciting results!!

ps. Check out the New Disclaimer from Franciscan University of Steubenville.

"F." Is he Anaerobic and what does he eat?

Lab today revealed much about the treacherous life of "F" and how likely he is to survive!  In which conditions does "F" thrive? Let's find out!

The Oxygen Requirements of "F"

Recall, from the last blog we placed our bacteria in a GasPak in order to test if they could thrive in an environment without Oxygen. The chemical bags inside the GasPak eliminated the Oxygen. Also recall, that we made a Thioglycollate Broth culture of our unknown bacteria.

Results:

 The bacteria that was stored inside the GasPak is shown above. Note, that there is some growth on the plate, thus our bacteria is able to grow in a deoxygenated environment. Remember from previous labs that we ALWAYS grew our bacteria in an oxygenated environment as well. Therefore, because it grows in both conditions our bacteria is considered "Facultative".

To further prove that "F" is facultative, we analyzed the results of a Thioglycollate Broth. This broth also depicted large amounts of our bacteria growing throughout it. Bacteria was present in the reddish area depicting Oxygen, towards the top of the broth. Also, it was noticed reaching throughout the yellow broth towards the bottom of the tube, depicting its ability to grow without Oxygen. Growth was equal in both areas.

Starch, Casein, and Triglyceride Testing: 


Next, we proceeded to do tests to see what kind of nutrients our bacteria feasts on! To do this we acquired three agar plates: one containing starch, one containing a milk protein (known as Casein), and the other containing a lipid (known as a Triglyceride). Using the aseptic technique we inoculated the surfaces of each of these plates and placed them in the 37 degree Celsius incubator for 24 hours.

In the above photo the nutrient plates are as follows: Starch, Triglyceride, Casein.

Later... with the results...

The nutrient plates are as follows: Casein, Starch, Triglyceride.

Starch: 


In order to check to see where Starch was present in the agar, we covered the plate in Gram's Iodine for 30 seconds - 1 minute, waiting for the purple color to appear.

Notice the top half of the plate, this is where good old "F" is found (the bottom is a sample from a different set of bacteria for comparison in our lab). The Starch plate contains Amylopectin and Amylose. If there were a halo around our bacteria streaks (located on the top) then our bacteria would contain the enzyme Amylase, and thus be able to digest Starch. However, since our bacteria lacked this halo and had a blue black precipitate under the streak, it was unable to feed on the Starch present.

Casein: 

This Casein plate revealed no mysteries, other than the simple fact that neither of the bacteria feeds on the nutrient Casein. This is revealed through the opaque white surface that covers the entire plate, even after incubation. If Casein eating bacteria were present there would be a halo around the streak of bacteria, thus our bacteria does not produce the enzyme Casein Protease.

Triglyceride: 

The photo at the top depicts both of the bacterias -- ours is to the right with the blue color (also pictured separately above). The dark blue precipitate appearing in our sample with the bacteria proves that  the triglycerides in the agar underwent partial hydrolysis. This means that our bacteria was positive for the enzyme Lipase, and thus gets its nutrients from fat. The other sample pictured tested negative for the enzyme, take time to note the aesthetic difference.

Join us next time to discover more mysteries about "F". Now, we are going to go enjoy our lunches, though I can assure you it won't be the same lunch as "F" is eating right about now.

Timeless Storing of Bacteria and Culturing an Anaerobic Bacteria

This class we took a break from staining and made various cultures of our bacteria. In past blogs, we described storage of the bacteria in a refrigerator; however, this class we discovered a technique in which we could save our bacteria FOREVER! We know our children's children are going to come back wanting to see this sample! Using this method we are able to cryoprotect the bacteria. Bacterial membranes are delicate already, but when placed in a refrigerator these membranes may break from rapid cooling. Using the chemical Glycerol halts this process.

In order to begin our cryprotection, we first measured 300microLiters of 100% Glycerol using a pipet. Then we proceeded to empty the contents of the pipet tube into a small storing vesicle.

Second, we discarded the plastic pipet tube and attached a new sterilized tube to the tip. This prevented contamination of the bacteria and chemicals. After, we measured 700microLiters of the bacteria broth, making sure to mix the broth thoroughly before drawing the bacteria into the pipet tube. We added this sample to the Glycerol, mixing it by drawing and emptying the contents of the vesicle.

We labeled it and then stored it within a -80 degree Celsius storage unit. Keep tuning into the blog in order to find out if our bacteria survives until the end of the semester!

Culturing an Anaerobic Bacteria: 


To determine if our Unknown "F" Bacteria is Anaerobic (thrives better without Oxygen) we inoculated it into a Thioglycollate Broth. This chemical reacts with Oxygen to form water; where Oxygen is present in this broth the broth appears red.

Notice the red color at the top of each of the test tubes; this is where the Oxygen is present within the broth.

Using the simple aseptic technique, we isolated a sample of "F" into the broth and placed it into a 37 degree Celsius incubator. We are going to leave it to culture until next class where we will determine whether our bacteria is aerobic or anaerobic. This will be identified as to where the bacteria grows: in the red or yellow portion of the broth (Oxygenated or Deoxygenated respectively).

We also conducted a GasPak Anaerobic System to check the requirements of Oxygen in our bacteria. To do this we prepared a nutrient agar plate of our sample using the aseptic technique.

The GasPak (due to its catalyst Palladium) reduces Oxygen and produces Carbon Dioxide in the container. If our bacteria thrives in this container by next class, then we will know that it does not need Oxygen in order to grow and reproduce.

See you next time when we unravel the mystery of Oxygen intake of our Bacteria!

Creating an Endospore and an Acid-fast Stain

Welcome Back! This week, though it started off slow, really heated up when we started our stains! To understand even more about our unknown bacteria, we created Endospore and Acid-fast stains.

The Endospore Stain: 

To start the stain we followed the same beginning procedure as the simple stain (smearing our bacteria, letting it air dry, and heat-fixing). This procedure is found in the previous blog pertaining to simple stains. We then placed the slide over a beaker full of boiling water (suspended with a slide drying rack), covering the smear with Bibulous paper. We saturated the paper with drops of Malachite Green dye.

We continuously added dye to the paper to keep it saturated for 5-6 minutes. It was important not to let all of the stain evaporate, or the slide would not properly set. After the time was over, we removed the slide with the forceps, removing the paper from the slide as well.

We let the slide cool, and then rinsed the slide (for about 30 seconds) with deoxidized water to remove excess dye from the slide. It is important not to rinse too thoroughly or all of the dye will be removed from your slide.

After the slide was rinsed, we suspended the slide on a drying rack over the sink and added Safranin dye directly onto the slide, letting it sit for 60-90 seconds. This dye is a very deep red color. When the allotted time was through, we rinsed the slide carefully and blotted with Bibulous paper.

The results: 

Notice the colonies of our small cocci bacteria. Also, note the color of our bacteria in this sample. The Green Malachite dye remains visible when endospores are present. Since all of the green dye was rinsed away and the bacteria was only visibly stained with the Safranin, there are no endospores present in our bacteria.

This picture was taken from http://archive.microbelibrary.org. This is a website that shows many more examples of stains, if interested. Notice how the endospores in this picture are stained green. This obviously contrasts with our sample, not having any visible green endospores.

The Acid-fast Stain:


To start this procedure, we once again began with the steps for making a simple stain. After, we placed the slide over a beaker of boiling water suspended by a slide drying rack. Then we covered the slide with Bibulous paper and saturated the paper with Ziehl-Neelsen Carbolfuchsin.

Like the last procedure, we had to keep the slide saturated with the dye while it steamed, but this time for 3-5 minutes. After the allotted time we removed the slide carefully from the rack and removed the Bibulous paper, throwing the used paper away in the designated receptacle.

After cooling, we rinsed the slide with deoxidized water to remove the excess stain. Also to remove more stain, we used Acid-alcohol. To do this we held the slide at an angle over the sink, and added Acid-alcohol to it until the magenta color stopped running.

Immediately we rinsed the slide free of decolorizing agent so that the rest of our process was not inhibited. We then covered the slide directly with Methylene Blue for 2 minutes. After this time frame, we rinsed the slide clean of excess dye and blotted the slide dry.

The results: 

Once again the small cocci colonies are present within our slide, but this time with a different stain color and meaning. The blue appearance reveals that our bacteria is Non-acid-fast. If the magenta stain remained after using the Acid-alcohol, then our bacteria would be considered Acid-fast. In an Acid-fast bacteria the first dye (red) is trapped by waxes in the cell membrane.

Join us next week for another exciting installment of Microbiology Lab! See you then!