Inoculation Results
From the moment we made it into the lab today, we were kept plenty busy checking for and interpreting the results of yesterday's many inoculations. Some of these required a few more experimental steps to really see results, but soon we had a lot more information about our unknown bacteria.For the starch test, which had already been incubated, we flooded the starch agar plate with Gram's iodine. Since the iodine remains brownish-orange on top of areas of the plate that contain starch, a clear area around the squiggle of bacteria growth would indicated that the bacteria had catabolized the starch in that area. Our plate didn't have this clear area, so this was a negative starch hydrolysis test and we can conclude that our bacteria does not use starch as an energy source.
The casein test uses the same concept of a clear zone around the bacteria growth indicating a positive result - in this case, hydrolysis of casein. Our plate showed negative results, which would mean that our bacteria does not use casein, but we reincubated this plate and will check again tomorrow to be sure.
We checked for triglyceride hydrolysis in the same way. A clear zone would indicate that the bacteria produces lipase to break down lipids, which would be a positive test. Our plate had no clear zone, so we recorded it as a negative test and placed it back in the incubator for another day.
The test for gelatin hydrolysis was slightly more complicated than checking the color of an agar plate. When we removed our gelatin tube from the incubator, it was a liquid rather than a solid. This could either be because our bacteria produced gelatinase (and therefore uses gelatin as an energy source), or because it was at a high temperature and the liquidation was only a state change. To test this, we put our tube in the refrigerator for 15 minutes. If it was still a liquid after this, we could consider it a positive test and conclude that our bacteria produces gelatinase. However, our tube was full of solid gelatin after 15 minutes, so we recorded this as a negative test and returned it to the incubator for another day to be double-checked later.
The durham tubes for carbohydrates checked for two different results. Each one had a special tube inside that tested for the presence of gas bubbles, but none of our sugar tubes produced any gas bubbles. Each durham tube was also colored to act as a pH indicator - a change in color means that acid is produced as a result of the bacteria using the particular sugar that the tube is testing for. Our bacteria tested positive for sucrose, lactose, and glucose use and negative for mannitol.
The MR-VP tube that we inoculated yesterday was split into two separate tests today. We poured half of the liquid into a clean test tube so we could use one tube for the MR test and the other for the VP.
For the MR (methyl red) test, we added 6 drops of methyl red to our designated MR tube and swirled. The methyl red acts as a pH indicator for the broth - the presence of a red color is a positive test, which means that the bacteria is able to ferment glucose through mixed acid fermentation. This test is significant for healthcare because it recognizes the presence of E. coli. Our MR test was negative, so we can conclude that our bacteria does not use mixed acid fermentation and is not E. coli.
The VP (Voges-Proskauer) test had two major steps: we first added 15 drops of VP reagent A and then 5 drops of VP reagent B to our designated VP tube. We shook the tube to aerate it and then let it sit. If the top had turned pink or red, this would be a positive test and we could conclude that the bacteria used the butanediol fermentation pathway. Our test came back negative (no color change), so our bacteria does not use this pathway.
For the results of the citrate test, we looked for color changes from the slant's original green. Ours was negative (no color change), so we returned it to the incubator for another 24 hours. If it is still negative, we can conclude that citrate is not our bacteria's sole carbon source.
Our next job was to add 10 drops of Kovak's reagent (very carefully!) to our tryptophan tube. Since no red layer appeared after this, we can conclude that our bacteria does not convert tryptophan to indole.
Then came the nitrate test. We added 7 drops of reagent A (sulfanilic acid) and 7 drops of reagent B (dimethyl-alpha-naphthylamine) to our incubated nitrate broth tube. We shook the tube and waited a few minutes to see if a pink or red color would develop. Since no color appeared, this first stage of the test was negative for nitrate reduction (negative for presence of nitrite). This could be either because nitrate was not used or because nitrate was reduced to nitrite which was then reduced further. To test which was the case for our bacteria, we added a small amount of powdered zinc to the test tube. The broth eventually turned red, so this test was negative for nitrate reduction - our bacteria does not use nitrate.
The triple sugar iron test has significance in healthcare because it checks for stomach bacteria. There were no noticeable color differences in our slant after 24 hours of incubation, which suggests a negative test. However, since this test checks for the use of glucose, sucrose, and lactose, all of which the carbohydrate tests showed that our bacteria used, we returned the test tube to the incubator and will check back tomorrow to hopefully find a positive result.
Our urea test was decidedly positive, as evidenced by a beautiful deep pink color. This means that our bacteria uses urea, breaking it down to produce ammonia and carbon dioxide.
The litmus milk test checked to see if our bacteria uses lactose and/or produces gas. The liquid started out purple, but it would turn white if the bacteria oxidized lactose. We didn't observe any color change, so this tube is also incubating until tomorrow. This extended incubation should also tell us if there is any curd formation, which could be due to either acid or renin.
The next group of tests checked for growth in aerobic and anaerobic conditions. We already knew that our bacteria is capable of growing in aerobic conditions, so we wanted to find out if it is facultative (if it can grow under both aerobic and anaerobic conditions), or if it is an obligate aerobe. Bacterial growth in the GasPak (anaerobic conditions, shown on left) showed that unknown bacteria F is facultative.
Results from our thioglycollate test (right) confirmed this conclusion. We inoculated this tube yesterday by stabbing the broth from top to bottom with an inoculating needle. After incubation, we could see where the bacteria was able to grow. Since ours grew both at the top (where there is oxygen) and at the bottom of the tube (where there is no oxygen), we can further conclude that it is facultative.
To see the results of the oxidase test, we added a few drops of oxidase reagent to a small piece of filter paper. We transferred a visible amount of bacteria from an agar plate to the reagent-infused filter paper using a sterile plastic tip and watched to see if the paper/reagent would change color. Since there was no color change within 10 seconds, the test was negative. We can conclude that our bacteria does not have cytochrome oxidase (which can be used for nitrate reduction when oxygen is not available). It's still safe to say that our bacteria is facultative.
For the catalase test, we added hydrogen peroxide to the inoculated and incubated catalase plate. The quick appearance of bubbles indicated a positive test. We can conclude that our bacteria uses the enzyme catalase to degrade the hydrogen peroxide that results from oxygen reduction.
Our motility stab tube showed that our bacteria is non-motile. We saw bacterial growth along the line of our inoculation stab, but no movement away from the line of inoculation. This is a negative result, but we'll incubate it longer to be sure.
While day-to-day nursing routines aren't likely to involve motility stabs and Kovak's reagent, it will be important to understand that different types of bacteria can be identified by their different, testable properties. Effective treatment will require the recognition that not all bacteria act in the same way.
Differential and Selective Plates
Once we finished going through all the inoculation results from yesterday, we moved on to differential and selective plates. We inoculated blood agar, phenylethyl alcohol agar, MacConkey agar, and mannitol salt plates. These select for salt-loving bacteria and differentiate one from another. We inoculated all of these using aseptic technique and our unknown bacteria and placed them in the incubator until tomorrow.
Antibiotics
Another blood agar plate was used to culture throat swabs. This was an exciting experiment because it's something we will do in healthcare to check for strep throat, and we can even perform this test in the lab now instead of waiting for expensive results from another doctor's office. We used sterile cotton swabs to take a sample from the back of each of our volunteers' throats and applied them to the blood agar plate after marking whose bacteria would be on each side. We added an antibiotic disc to each side of the plate as well. If strep bacteria is present, it will kill the red blood cells in the plate but won't go near the antibiotic disc. This is incubating until tomorrow.
The other lab group did a nasal swab instead of a throat swab. This tested for MRSA, which colonizes in the nasal cavities. Since MRSA is methicillin-resistant, it won't grow around the methicillin antibiotic disc.
Next, we tested a variety of antibiotics against our unknown bacteria sample. We smeared our bacteria all over the surface of two agar plates and divided each plate into four quadrants, labelled 1 through 8, with a china marker. We used sterilized tweezers to place an antibiotic disc in each quadrant:
- penicillin
- novobicin
- neomycin
- streptomycin
- tetracycline
- erythromycin
- amoxicillin
- oregano
Since the amoxicillin and oregano were not in disc form, we placed plain paper discs in their quadrants and added 5M of each to their respective disc by pipette. Both plates are incubating until tomorrow, when we will be able to see our bacteria's response to the various antibiotics.
Making Yogurt
Our final adventure for Day 4 was to try our hand at making our own yogurt. Dr. P swore that he knew the most delicious way to do this, so we had to test it out in spite of a little skepticism. To do this, we heated milk and then cooled it to 37 degrees Celsius before adding a small amount of pre-made Greek yogurt and putting the concoction in an incubator overnight (this time it went in a specified yogurt incubator to stay away from all our bacteria). Our sample of the Greek yogurt was quite sour, so hopes aren't too high, but we'll know more tomorrow...
While we probably won't spend our time as nurses personally making yogurt for our patients, it will be important to know and to tell patients that eating yogurt has many health benefits. The probiotics found in yogurt are important to consume while taking antibiotics so the gastrointestinal tract doesn't get overly irritated. These probiotics also work to prevent yeast infections because they create an acidic environment, and yeast grows well in alkaline conditions.
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