Before starting this, we checked out our anaerobic tests. These tests for some reason did not work out. The methylene blue strip was still blue, indicating the presence of oxygen. In order to still use these plates we prepared (that in the end had grown in the aerobic conditions), we tested this with hydrogen peroxide. If it formed bubbles, then it produced carbon dioxide as a result of the catalase enzyme breaking down the hydrogen peroxide. If there was no bubbling, then it produced no carbon dioxide. Ours produced a large amount of bubbles, while our partner's group did not produce any.
 |
| Our sample, upper right, large amounts of bubbles. |
Today began a very fast-paced day of inoculating tubes and spreading bacteria on plates. We prepared the tubes and plates for 14 different tests.
1.) Starch Hydrolysis Test
In this test, we are looking to see if our bacteria hydrolyzes/digests starch by the extracellular enzyme called amylase. This enxyme breaks the bonds that link the glucose subunits. If a bacteria has this enzyme, it will have glucose and maltose as biproducts, creating a large growth of colonies on this plate. Using aseptic technique, our pink bacteria was inoculated onto the slide and will incubate until tomorrow.
2.) Casein Hydrolysis Test
Casein is the predominate protein in milk, and can be used as a substrate to assess the production for proteinases. In this experiment, we will determine wheter our bacteria has the ability to digest casein, by inoculating some of our bacteria onto a skim milk agar plate, streaking a short line on the agar surface, and will incubate until tomorrow. The products for a postive test are amino acids and peptides.
3.) Gelatin Hydrolysis Test
Gelatin is a processed form of collagen, an important protein in connective tissue. When boiling the bones of cows and pigs, the product is gelatin, a semi-solid gel. This test distinguishes bacteria that have the enzyme gelatinase, which can break the peptide bonds in gelatin, causing the gel to liquify it into amino acides and short peptides. This procedure is slightly different than the other two above, in that this one calls for an inoculated needle to be stabbed into a gelatin test tube, to once again be incubated until later.
4.) Carbohydrates Fermentation
The four carbohydrate tests below will determine whether our bacteria will ferment a particular carbohydrate. This test looks for two results: a possible color change of the phenol red added to the test tube (yellow means acidic, red means basic) and possible gas formation from the fermentation of the following carbohydrates. We inoculated the test tubes, and incubated them.
a.) Glucose
b.) Sucrose
c.) Maltose
d.) Lactose
5.) Methyl Red Test
This test looks for products of mixed-acid fermentation of glucose into significant amounts of organic acids (acetic, succinic, and formic acid as well as gas). The test tubes have a pH indicator in them called methyl red (hence the name of the test) that remains red in a positive test (acidic) and turns orange/yellow in a negative test (alkaline). We simply used aseptic technique again to inoculate the broth inside the test tube, and wait until tomorrow for results in the incubator.
6.) Voges-Proskauer Test
Some bacteria have a 'butanediol fermentation pathway' utilized to ferment glucose. This test looks for the protein acetoin, a vital intermediate in this pathway. Organic acids are products of this pathway, and a red color denotes a positive test becuase the presence of oxygen gas and acetoin reacts with the VP reagent. No color change (yellow) is a negative test. We carefully inoculated the bacteria into the corresponding test tube, and put it into the incubator.
7.) Citrate Utilization Test
This test looks for the membrane protein citrate permease; an enzyme that breaks citrate into pyruvate and carbon dioxide. This test determines if our bacteria can utilize citrate as the sole source of carbon. If the bacteria produces carbon dioxide, it will bind with the sodium and form sodium carbonate, an alkaline compound that changes the color of the pH indicator (bromothymol blue) blue from the initial green color. After inoculating the agar slant with our bacteria, we wait until tomorrow.
8.) Indole - Tryptophan Degradation - Test
This test determines if a bacteria splits the amino acid tryptophan into indole and and pyretic acid. Though indole is a byproduct not used by the bacteria, it is the marker this test uses. If positive for indole, the bacteria will have broken tryptophan and oxygen into pyruvate, ammonia, and indole by the protein tryptophanase. We will know tomorrow if by inoculating our test tube, our bacteria uses trptophan as a source of endergy by degrading this amino acid. A red strip on the top of the broth tomorrow after added procedures will indicate a positive test for tryptophan use, and a negative test will show a yellow strip.
9.) Litmus Milk Test
This test, differentiates between bacteria as to their ability to utilize lactose, protein, and litmus in litmus milk. Litmus here serves as a pH indicator, and turns pink in an acidic environment (lactose fermenting into acids), and blue in an alkaline environment. If the solution becomes too acidic, the casein in the milk may become denatured and from a firm curd; some bacteria may even turn the litmus clear. If casein is altered by another protein called rennin, a soft curd is formed. Proteolytic bacteria digest milk proteins into peptides and amino acids; all the above observations are important in this test and if casein is converted to amino acids by proteases, and to ammonia by deaminases, the whole solution will turn blue. A lot will go on in the test tube overnight, but this is again just a simple inoculation- using aseptic technique of course... and we wait for tomorrow.
10.) TSIA Test
This test differentiates among gram-negative enteric bacilli, Enterobacteriaceae, (which means that gram positive bacteria will not grow), as to their ability to ferment glucose, lactose, and sucrose, and to produce hydrogen disulfide. The triple sugar agar contains lactose, sucrose, and glucose and if our bacteria ferments any of them, the pH of the agar will drop considerably because of the organic acid byproducts. If the bacteria starts to ferment the protein in the medium instead of (or after) fermenting the sugars, the pH becomes alkaline and liberages ammonia from the amino acids. If acidic the medium will turn yellow; if alkaline, the medium will turn red. Black precipitates indicate hydrogen sulfide production, and gas indicates the production of ammonium gas. We inoculate the agar plate and place it in the incubator.
11.) Triglyceride (Fat) Hydrolysis Test
This test differentiates bacteria that can break down lipids, which are primarily water-insoluble molecules. This test looks for bacteria that use the enzyme called lipase to break down the fat into fatty acids and glycerol resulting in a clear zone around the bacterial growth after incubation. Today we inoculated the triglyceride agar plate, and wait until tomorrow to determine the results.
*App of the Day - The reason why the dish bubbled when hydrogen peroxide was put on it was the same reason why wounds will bubble with the addition of Hydrogen peroxide. In either case, the H2O2 is being broken down by an enzyme (which in the body is catalase) to become H20 and O2. The bubble are actually bubbles of oxygen gas.
No comments:
Post a Comment