Day 7: 5/22

Follow Up on Many Tests:

Today we checked the results from the Tryptone, Urea, Citric, Nitrate and MR-VP test from the day before and checked the tests for litmus milk, Casein, Lipid, and Gelatin a second time.

First the Trypton test was complete by adding 5 drops of Kovac's reagent to the SIM agar deep tube with culture. This was to see if our bacteria uses trypton as a source of energy by degrading the amino acid to get pyruvate and indole is the by product. After adding the drops and using the shaking technique our sample had a yellow layer on top that turned to green. Therefore the result is negative.

Then we examined the urea broth tube for a color change. If our bacteria uses the enzyme urease to break down urea and ammonia would accumulate.  The tube would detect a pH change from the ammonia and turn a bright pink in alkaline pH. Our tube was a slight yellow orange and therefore a negative test-- so far, we will incubate it longer for further testing.

Onward to the Citric acid test with the Simmons citrate agar slant tube.  If our bacteria uses citrate as a sole source of carbon and energy it has a transporter called citrate permease. Once citrate is inside the cell it is converted to pyruvate and carbon dioxide.  The carbon dioxide will combine with sodium and forms an alkaline compound and the medium will change from green to a Prussian blue.  Our tube had the deep blue on the top and bottom was a dark green indicating a positive test. So our bacteria doe shave citrate permease.

Moving on we finished the testing for the nitrate.  In anaerobic respiration we are testing to see if our bacteria reduces ions to nitrate or nitrogen gas. such as this:

NO3 + 2H + 2e  (nitrate ion)        ------->    NO2 + H2O   (nitrite ion)

The respiration cycle goes on as usual (Glycolysis, Krebs Cycle, and Electron Transport Chain) But as a result instead of oxygen receiving the electrons it is nitrogen.  Some organisms continue this phase and convert the NO2 (nitrate) to ammonium in a process called denitrification.  This completely reduces nitrate to nitrogen.  To test which process our bacteria uses we added reagent A - sulfanic acid and reagent B – dimethyl –alpha-naphthlyamine.  If nitrate is still present the broth will turn a bright red.  Our broth tube did turn a bright red then a deep red meaning a positive test for nitrate reduction (meaning our bacteria does not continue on to denitrification).

But alas our encouragement from appositive test was short lived as we moved onto the MR-VP test to look for acid end products.  Unfortunately, we added the methyl-red too prematurely.  The reason the test did not working by just adding the methyl red is because the tube has been incubated for less than 48 hours.  An insufficient level of acidic acid may have accumulated on the broth and thus our test would result in a false negative.  Also, we were not supposed to use the same tube as the test broth is in because the 2 different reagents used will interfere with another. Fortunately for the procrastinators Pete and Ben they waited to add their reagent and were able to successfully add the two different reagents in 2 separate tubes.  But the rest of us inoculated a second tube to try the testing again tomorrow. (Second time’s a charm!)

The Second Follow –Up: Litmus Casein, Lipid and Gelatin

The litmus milk tube was a lighter lavender after 2 days and contained a small amount of curding.  The curd was mobile and therefore non acidic.  We still could not fully conclude about the bacteria’s ability to utilize lactose, protein and litmus milk so we but the tube back in the incubator for further testing.

We examined the Casein and Lipid agar plates and found that the results were still negative due to no clear zone around the bacteria in the casein and lack of clouding around the bacteria in the lipid that had grown and thus both results were definitely negative.  This means our bacteria does not secrete caseinase to break down Casein.  Also the bacteria does not secrete any lipases to break down triglycerides and therefore does not use fatty acids or glycerol for energy.

At finally we followed up on the gelatin test after placing it in the fridge overnight.  The medium in the tube remained solid, a negative test result again.  We put it back in the incubator to further test for gelatin hydrolysis tomorrow.

So in conclusion our unknown bacteria uses glucose, sucrose, lactose and maltose for energy.

Inoculation of Selective Differential Plate Agars:

Today we shared 5 agar plate with 2 other groups. Our materials we used to inoculate were: Bunsen Burner, inoculating loop, and a slant agar with the unknown bacteria culture “N”.  The five plates we inoculated were for the following tests: 

Blood agar plate: to isolate and support the growth of fastidious bacteria and to differentiate among bacteria based on their ability to lyse red blood cells.

The Eosin Methylene Blue (EMB) Agar plate: to isolate and differentiate gram-negative enteric bacilli. This inhibits the growth of gram-positive bacteria which is our unknown, minimal if any, growth is expected.

The Mannitol Salt Agar: to isolate bacteria based on their salt tolerance and differentiate among these isolates for mannitol fermentation.  This relates to salt tolerance of a gram-positive cocci, so we will see if our bacteria is salt-tolerant.

A MacConkey Agar: to detect and differentiate among gram-negative enteric bacilli, based on their ability to grow on the medium and to ferment lactose. (Which our bacteria should be able to do because it tested positive in the lactose test) But our bacteria is gram-positive and the crystal violet and bile salts will inhibit gram-positive bacteria.

And lastly the Phenylethyl Alcohol (PEA) Agar: to isolate gram-positive bacteria from a specimen containing a mixture of gram-positive and –negative bacteria.  Because this medium inhibits the growth of gram-negative bacteria our bacteria will grow in the presence of PEA.