Lab Day 7

Thursday, Day 7

In lab today, we looked at the results of yesterday’s inoculations.  Yesterday, we inoculated tubes of tryptophan, urea, citrate, nitrate, and MR-VP, to see which amino acids our bacteria is capable of breaking down.

Starting with the tryptophan degradation test, we added 15 drops of Kovac’s reagent.  This test was to see if our bacteria had the ability to split tryptophan into indole and pyruvic acid.  If the Kovac’s reagent causes it to rapidly turn red, then it’s a positive test.  Ours showed no red, so it was a negative test.  Therefore, we know that our bacteria does not break down tryptophan.

Next we looked to our Urea Hydrolysis test, to see if our bacteria had urease, which breaks down urea.  If the urea was broken down, it would have turned bright pink, because the urea would have broken down into carbon dioxide and ammonia, causing it to turn alkaline.  This pH change is detected with phenol red, which turns bight pink in alkaline medium.  Ours was a negative test, with no color change.  Therefore, we know that our bacteria does not break down urea.

The results of the Citric Acid test were easy to observe.  If our bacteria had the enzyme citrate permease, which breaks down citrate, then the byproduct of the breakdown reaction would be CO2, which combines with NA+ in the medium to form NaCO3, an alkaline compound.  The pH indicator, bromothymol blue, turns blue in alkaline pH.  Therefore, all we need to look for is a deep blue in our Citric Acid tube.  We observed plenty of blue, so ours was positive.  Therefore, we know that our bacteria breaks down Citrate.

Next, we looked at our Nitrate Reduction test, to determine if our bacterium is able to reduce nitrate ions to either nitrite or nitrogen gas.  This reaction is anaerobic respiration, because Nitrate is accepting the electrons instead of Oxygen.  It needs Nitrate reductase enzyme to convert Nitrate to Nitrite.  So, essentially, if we’re looking to see if our bacteria has Nitrate reductase, then we’re looking for the end product, which is Nitrite.  To to this, we use two reagents: Sulfanilic acid (reagent A), and dimethyl-alpha-naphthylamine (reagent B).  If Nitrite is present, the medium will turn pink or red.  Ours was immediately positive, because it turned red.  Therefore, we know that our bacteria breaks down Nitrate.

Finally, we did our MR-VP test to see if our bacteria is able to ferment glucose via mixed acid fermentation.  The products of this fermentation are acidic, so the addition of methyl red (pH indicator) will turn the solution red.  Our methyl red test was negative, indicating that our bacteria did not ferment glucose.  

However, the results of our Voges-Proskauer (VP) test were positive, indicating that it did ferment glucose.  We added 15 drops of Barritt’s reagent A (alpha-naphthol) and 5 drops of Barritt’s reagent B (KOH).  After about five minutes, it began to turn red.  This means that our bacteria fermented the glucose and produced acid, but the acid was further converted to 2,3-butanediol.  The methyl red was initially misleading because it didn’t find any acid, because the acid had all been converted to 2,3-butanediol.  The VP, however, found the 2,3-butanediol and turned the                                                                                  medium red.  Therefore,        

                                                                           we know that our bacteria          

                          MR                                            ferments glucose.                                                   VP

Also, because we had forgotten to do it yesterday, we took a look at our now complete litmus test.  There was definite curd formation, and there was no color change.  The color didn’t change because the pH didn’t change, and the soft curd formed when cadein was altered by the bacterial enzyme renin.