Test results for fermentation of our bacteria

Test Results for Fermentation of Our Bacteria

Class: Thursday, October 24th, 2013

Last week we prepared a bunch of tests (seven) and today all we did was observe those tests and determine what they meant (positive or negative for fermenting the media on the agar plate). If the bacteria fermented and grew, the result proved positive. And today our bacteria did not prove itself totally boring, IT WAS POSITIVE FOR SALT TOLERATION! IT LOVES SALT! I guess you could say its a HALOPHILE! Sorry, as you can see, we were pretty excited about having a positive test for once.. This rarely happens.

FIRST TEST: THIOGLYCOLLATE

With this we were testing our bacteria on whether it would grow in the presence of air, no air, or both. If it was able to grow in the presence of air, this would be a positive test for classifying the bacteria as obligate aerobe. If it grew and multiplied without the presence of air, this would classify the bacteria as obligate anaerobe. And lastly, if the bacteria can grow with and without the presence of oxygen it is classified as facultative aerobe.

Our Facultative aerobic bacteria

SECOND TEST: DNase DNA HYDROLYSIS TEST
Before we could determine the ability of our bacteria to hydrolyze DNA, there was a step that we had to take. We poured 1N HCL into the plate, flooding it. This allowed us to examine the plate for a clear area around our bacteria. If there were a clear area around the bacteria, this would be a positive test for our bacteria to posses a digestive  enzyme (DNase).

Negative for DNase :(

HCL used to flood the plate

What a positive test would look like

THIRD TEST: EMB: EOSIN METHYLENE BLUE
In my opinion, this test was a little pointless for our bacteria. This is because our bacteria is gram -  positive, which we already established in one of our earlier labs. This test was performed to distinguish between those bacteria that ferment lactose and /or sucrose and those that do not. The EMB agar uses dyes that will inhibit growth of gram - positive bacteria (our bacteria). So going into this test, we can assume that our bacteria will test negative and therefore will not ferment lactose and / or sucrose.

Negative for lactose and sucrose fermentation

FOURTH TEST: MANNITOL SALT AGAR
It was this test that I mentioned earlier that we all got very excited over! Our bacteria finally tested positively! In this test, we determined the salt tolerance our bacteria may have. --It may or may not love salt. Only bacteria that is salt tolerant will grow on this salt agar plate. And it just so happens a positive test is common with gram - positive bacteria (our bacteria!).

Our beautiful HALOPHILE!

FIFTH TEST: MACCONKEY AGAR
Another test that is in my opinion pointless as related to our bacteria. This test is used to differentiate among gram - negative (not our bacteria) based on their ability to grow on the medium and to ferment lactose. So again, gram - positive bacteria are inhibited by the media agar (crystal violet and bile salts). If our bacteria colony is colorless, it will be negative for fermenting lactose. However, if the test shows a pink to red in color our bacteria will be positive for lactose fermentation.

Negative for lactose fermentation

SIXTH TEST: PHENYLETHYL ALCOHOL (PEA)
Today has just been a bad day for our gram - positive bacteria. This is another test that isolates gram - positive bacteria from bacteria containing a mixture of gram - positive and gram - negative bacteria. If the bacteria grows in the presence of PEA than it is a positive test and if it does not grow than it is a negative test. --Really not much different than any other test that we've run today.

Another negative test for our bacteria :(

SEVENTH AND FINAL TEST TODAY: BLOOD AGAR PLATE
In this test we see if our bacteria has the ability to lyse (destroy) red blood cells (hemolysis). There are three classifications of hemolysis-- α-hemolysis, ß-hemolysis and γ-hemolysis. In α-hemolysis, partial disruption of the red blood cells by the bacteria results in a greenish color around a colony. In ß-hemolysis, the bacteria secrete enzymes that completely dismantle the red blood cells and a clear zone appears around the bacterial colony. And in γ-hemolysis, the bacteria is nonhemolytic where it does NOT damage the red blood cells.

Our bacteria is γ-hemolytic

PICTURE DAY! 

MY Bubbles & More Tests as Usual

MY Bubbles & More Tests as Usual 

Class: Tuesday, October 21, 2013

FINALLY.  Bacteria J had it's first reaction today!  Last week we made a streak plate of our bacteria (shared the plate with another group...theirs is yellow) so that this week we could test how it reacted to a bath of hydrogen peroxide.  A reaction indicates the presence of the enzyme catalase, which breaks down the H2O2.  Usually the reaction will occur in aerobic, aerotolerant, or facultative anaerobic bacteria .  

Bacteria streak before reaction. Bacteria J is
the orange one on the left

Jess adding the hydrogen peroxide 

Woohoo! We have a positive test result.  It may
look like the bubbles are coming from the other
bacteria, but they really are from Bacteria J! Also note how
Bacteria J seems to be dissipating as the reaction occurs....interesting

MY bubbles.  

Test 1:

      Culturing Bacteria J in an anaerobic environment.  The culture medium is broth that has thioglycollate added to it.  The thioglycollate is a reducing agent which dissolves some of the oxygen in the tube so that there are oxygenated and deoxygenated areas for the bacteria to possibly grow in.  The location of growth indicates the bacteria's oxygen requirements.  

Here, we can see the differentiation in oxygen distribution that the thioglycollate produced.  There is a small section of pink broth at the top while the remainder is clear.  Oxygen is only present in the pink section and absent in the rest of the culture medium.  The results of this test will indicate Bacteria J's true oxygen requirements.  

The rest of the tests we did today are both selective  and differential.  Selective mediums favor the growth of one type of bacteria over another.  Differential mediums distinguish between a few different types of bacteria.  The results will look different depending on the bacteria present 

Test 2:

      Eosin Methylene Blue (EMB) Agar.  This growth test is used to isolate gram-negative bacteria that ferment lactose or sucrose.  EMB is a selective medium because the eosin and methylene blue dyes inhibit the growth of gram-postive bacteria and it is a differential medium because the lactose and sucrose sugars it contains definitively distinguishes between bacteria that do ferment sucrose/lactose and those that don't.  There are a few different outcomes here that we could see in growth and color:

1. No bacterial growth = gram-positive bacteria
2. Dark blue colony w/ green metallic sheen = significant fermentation of sucrose and/or lactose
3. Pink colonies= some fermentation
4. Colorless (but still present) colonies = no fermentation

Sierra inoculated the EMB agar plate in the pattern F U S.  Because we're cool like that, ya know? 

Sierra, expert inoculator.  

Test 3:

      Blood Agar Plate.  Another differential and selective media.  Blood agar (from sheep's blood) is highly enriched with nutrients and thus helps isolate fastidious bacteria that have trouble growing else.  I bet Bacteria J is like this, the fussy little thing.  Because blood agar contains well, blood cells, it differentiates bacteria that completely lyse RBCs (beta-hemolytic), partially lyse RBCs (alpha-hemolytic), and do not lyse RBCs (non-hemolytic).

Test 4:
      Phenylethyl Alcohol (PEA).  PEA selectively inhibits gram-negative bacterial growth because it interferes with DNA synthesis.  Preeety simple.

Look at that focus! 

Test 5:
      Mannitol Salt Agar.
      Selectivity: salt tolerance; bacteria must be able to tolerate a 7.5% NaCl concentration
      Differentiation: mannitol fermentation; acid from mannitol fermentation changes the pH indicator in the medium (phenol red) from red to yellow.  If the bacteria can ferment mannitol, a yellow zone will appear around the colony.  Pathogenic bacteria have this mannitol fermentation ability, so let's hope we see some yellow! (yes, we actually want a pathogenic bacteria....it's exciting).

      

Test 6:
      MacConkey Agar. MacConkey Agar selects for gram-negative bacteria.  The crystal violet and bile salts in the medium prohibit the growth of gram-positive bacteria.  The presence of lactose in the medium differentiates between lactose fermenting and non-lactose fermenting bacteria.  This is indicated by the color of the colonies; lactose-users turn pink-red.  non-lactose fermenters remain colorless.

Can I get an F U S??

Test 7 (last one, I promise!) 

      DNase hydrolysis test.  To determine bacteria's ability to digest DNA, which they could do if they secrete DNase enzyme.  Pathogenic bacteria use DNase to clog up infected tissues so they can move more easily from cell to cell.  We will be adding HCl to the plate in the next lab period to truly investigate the DNase production of Bacteria J.  

Does it eat DNA? Only time shall tell....
Also, we shared a plate again with Mary and Jess
They're pretty neat.  We like 'em.  

Now time for some lab group lovin'. 

Matt, you know you love us.  

More Tests on Mystery Bacteria J

More Tests on Mystery Bacteria J

Class: Thursday, October 17

In class today, we looked at the results of a few tests on Bacteria J as well as complete a few more tests.

On Tuesday, we completed the Citrate Utilization Test to determine if our mystery bacteria possessed the membrane-associated transporter citrate permease. Our test came back negative.

Mystery bacteria negative Citrate test

Dr. Pathakamuri enjoying Max & Sam's positive Citrate test

Next, we checked our results for the Urea Hydrolysis test.  The reason for this test is to see if the bacteria secretes an enzyme called urease.

Mystery bacteria negative Urea test

For our next test, called the Indole test, we wanted to determine if our bacteria would split an amino acid called tryptophan into indole and pyruvic acid.  To do this, we added ten drops of Kovac's reagent into a broth culture of our mystery bacteria.

Adding Kovak's reagent to our mystery bacteria

If our mystery bacteria produced the byproduct of indole, then it would be positive for undergoing the reaction for splitting the amino acid tryptophan.
If positive, a red layer at the top of the broth tube would quickly appear.  If negative, there would be no presence of a red layer, meaning that tryptophan was not hydrolyzed.

Mystery bacteria negative Indole test

As the picture indicates, our mystery bacteria tested negative for Indole.

Another test we completed is a respiration test, specifically called a Nitrate Reduction test.  For this test, its purpose is to determine whether our bacteria is able to reduce nitrate ions to either nitrite ions or ammonia (nitrogen gas).  To test this, last class on Tuesday we inoculated a nitrate broth tube with our mystery bacteria.  Today, we added five drops of sulfanilic acid and five drops of dimethyl-a-naphthylamine, and gently mixed the broth.

Adding sulfanilic acid to inoculated nitrate broth

Adding dimethyl-a-naphthylamineto inoculated nitrate broth

If there is a presence of nitrate, the broth turns a red color. However, if there is no color change this does not mean that the nitrate test is negative.  It is possible that the nitrate ions were not reduced to nitrite ions.  In this case, a small amount of zinc would be added to reduce the nitrate ions to nitrite ions.  We did not need to do this last step, because our broth turned red after adding the two reagents.

Negative nitrate test

Matt checking out our initial nitrate test

The next test is another respiration test, called Oxidase test.  This test is to determine whether our bacteria has the cytochrome oxidase, which plays a role in electron transport during respiration.

To do this test, we swabbed our mystery bacteria and dripped oxidase reagent on the swab.  Within ten seconds, if the test is positive, the bacteria should turn blue/purple, indicating the reaction is positive for oxidase.  If nothing happens, then the bacteria is negative for oxidase.

Adding oxidase reagent to mystery bacteria

Negative oxidase test

For our next test called Methyl Red Mixed Fermentation test, we tested the pH of our bacteria after adding methyl red pH indicator.  This would indicate products of mixed-acid fermentation of glucose.  To do this, we added 5 drops of methyl red to our mystery bacteria-inoculated broth tube.

Negative mixed fermentation test

Another test we completed is called the Voges-Proskauer test or Butanediol Fermentation, which would determine the ability of our mystery bacteria to ferment glucose through butanediol fermentation.

To test this, on Tuesday, we inoculated a methly red Voges-Proskauer broth tube with our mystery bacteria.  Today, we added 15 drops of Barritt's reagent A and 5 drops of Barritt's reagent B to the tube.

Adding reagent A

Adding reagent B

Negative test for Butanediol fermentation

We are a bit disappointed in our mystery bacteria - all our test results have come back  negative! What is to come?

Test our bacteria's ability to ferment, utilize, degrade,reduce,andhydrolyze

TEST OUR BACTERIA'S ABILITY TO FERMENT, UTILIZE, DEGRADE, REDUCE, AND HYDROLYZE

Class: Tuesday, October 15th, 2013

We put our bacteria through five different tests today! Too bad we won't be able to see the results until next class because they'll be too busy incubating. So our job today was pretty easy, we were only preparing the tests. There was not much more to do than inoculate the test tubes with our mystery bacteria J and place them in the incubator until next lab.

4 of the tests ready to be inoculated!

Indole test was our first test that we prepared. The purpose of this test is to determine whether our mystery bacteria J can degrade tryptophan into indole and pyruvic acid. To result as positive, our bacteria will have to use tryptophan as energy to degrade the amino acids to make pyruvate. Indole and pyruvic acid would be the left overs that aren't used by our bacteria.

Inoculating the tryptone broth tube

Nitrate Reduction Test is performed in order for us to determine if our mystery bacteria is able to reduce the nitrate ions to produce nitrite ions or nitrogen gas. How does this happen? This test is performed anaerobically so no oxygen will be present. If this will be positive, our bacteria successfully reduced nitrate ions to nitrite ions by adding electrons through the electron transport chain to the nitrate ion. These electrons facilitate the reduction of nitrate ions. If a gas is present in the results, our bacteria will be concluded as positive for reducing nitrate completely to molecular nitrogen. This forms a nitrogen gas.

Inoculating the nitrate broth tube

Urea Hydrolysis Test is used to determine our bacteria J's ability to hydrolyze urea. If our bacteria results as positive, then it had to degrade urea into two byproducts: carbon dioxide and ammonia. We're testing to observe if our bacteria has the enzyme capable to hydrolyze the urea.

Inoculating the urea medium tube

The Methyl Red Test is prepared so we can determine if our bacteria has the ability to ferment glucose through mixed-acid fermentation. Because the products of the mixed-acid fermentation include organic acids, the test observes the pH levels after methyl red is added. If the test results as positive, the methyl red remains red in color indicating the pH is 4.5 and below. A negative test will be yellow or orange.

Inoculating the MR-VP tube

Citrate Utilization Test is performed to determine if our bacteria can utilize citrate as its sole source of carbon and energy. If our bacteria results as positive, then we can conclude that our bacteria has membrane-associated transporter citrate permease. Once our enzymes gets inside of the cell's cytoplasm, they convert citrate into pyruvate (energy) and CO2. If positive, the citrate agar slant tube will transform from green to blue.

Inoculating the citrate agar slant tube

Test Results, Boring Bacteria, and a Class Pet

Test Results, Boring Bacteria, and a Class Pet

Class: Tuesday October 8, 2013

Today we learned what our little mystery bacteria J is really made of.  And folks, it ain't much.  Last week we inoculated several different culture mediums to test the enzyme activity of Bacteria J.  

The first test was on a Starch Agar plate, with culture medium

that is made up of, well, starch.  This test was to determine if the bacteria metabolizes carbohydrates.  Our bacteria grew very well on the plate, but we want to know what it actually used for its metabolic processes.  

 

In order to determine if the starch was in fact used, we flooded the plate with iodine solution...

and then looked for a distinct halo around the bacterial colony.  Ours has a very slight halo, but not enough to be significant.  

Okay, so maybe we'll have better luck with the next test....this time we are looking to see if the bacteria metabolizes casein protein found in milk.   The results? Well, if you look really closely, you may be able to see a teeny little spot of orange. Yes, that would be our bacteria, trying to grow but failing miserably.  Poor guy.  Clearly milk protein is not his food of choice.  Let's try something else!

Casein is not the only protein available in milk; it has lactose too! Maybe Bacteria J would grow better in there! If there is any metabolic reaction with the lactose, the culture would have coagulated and formed lumpy curds throughout or even curdled into a solid.  The color also would have changed from the original lavender purple color to a pink or reddish substance.  The color did not change at all, but just to be sure no reaction took place, we inverted the tube to check the culture's consistency...

...and again got no results.  That tiny little bit of sediment at the bottom of the tube is merely from the culture components settling over the course of the week.  C'mon Bacteria J, will nothing satisfy you?!  We want to see a reaction!!  

The next culture medium we grew Bacteria J in was a triglyceride medium, to asses how the Bacteria metabolizes fats.  If it has significant metabolism of fats, then the majority of the blueness from the plate would have been 'absorbed' into the bacterial colony on the Petri dish, leaving the rest of it clear and the colony appearing dark blue/purplish.  Do we have either of these results? Take a guess.  Nope, of course not! Most of the plate is still blue and the colony is now a gross orangish-purple color.  Not enough of a change unfortunately.  

Okay, Bacteria J, you have one more chance to prove yourself! Ready?! Go eat some gelatin!! In this test we inoculated a test tube with a gelatin culture medium to asses how the bacteria metabolizes, well, gelatin.  If it does, then the once-semi-solid tube of culture should be mostly liquified, even after refrigeration.  But no, our culture stayed nice and semi-solid like, meaning no metabolism of gelatin here.  Of course.  

Well, despite the abysmal performance by Bacteria J in the realm of metabolic functions, we did have one highlight to the class.  World, meet Striker, our little class pet.  

And you thought stink bugs were gross!