Day 5: Taking the 'Un' out of 'Unknown'

Final Observations

For our last day of lab, after checking out of our dorms and dragging our luggage across campus, we took another look at our incubated inoculations from days 3 and 4 so we could determine the identity of our unknown bacteria (unknown F). The casein and triglyceride tests that we re-incubated yesterday still showed negative results. We can conclude that our bacteria does not use casein or triglycerides as energy sources.

Our re-incubated litmus milk test also looked negative at first glance. A true negative result would indicate that our bacteria doesn't use glucose, sucrose, or lactose. This didn't agree with the results of our earlier individual carbohydrate tests, which showed that our bacteria uses all three of these sugars. Since the purple litmus milk didn't turn white, litmus has not been reduced (it didn't accept hydrogen). The lack of a deep purple color at the top of the liquid indicates that the bacteria does not use casein, which is consistent with our other casein test. But the milk should have turned pink since we know that our bacteria converts lactose to lactic acid (from the positive lactose test). We compared our inoculated tube with a control and observed a slight difference in color, but we concluded that since the litmus milk contains such a small amount of the three sugars, we shouldn't be dissuaded by the lack of pink. It is still safe to say that our bacteria uses glucose, lactose, and sucrose for energy.

Our TSI (triple sugar iron) test came back positive today. It showed an acid slant in addition to an acid butt, showing that our bacteria did indeed use lactose and fructose. We knew our sample was acidic because the solid turned more yellow than it had been. Since the butt (the part below the slant) did not turn black, we can conclude that there was no hydrogen sulfide gas produced.

We re-examined our gelatin test and ended up with the same negative result we found yesterday. Our gelatin tube was a liquid when it first came out of the incubator, but became solid when we placed it in the refrigerator. We can conclude that our bacteria does not produce gelatinase.

Our citrate test was still negative as well - there was no color change in the tube, so we can conclude that citrate is not our bacteria's sole carbon source.

Selective and Differential Results

After double-checking our Day 3 inoculations, we moved on to those from Day 4. We started with the test for strep throat since our classmates were very curious about what it would say of their health. The blood agar plate we inoculated was equipped to provide information about the presence of strep bacteria in the throat as well as what group of bacteria they were. Group A (S. pyogenes) causes strep throat, but Group B (S. agalactiae) does not. We first looked for beta hemolysis of the blood agar to determine if either A or B was present, and then looked at the reaction to the bacitracin disc to differentiate between A and B. Group A is sensitive to bacitracin, while Group B is not.

Although both cultures showed sensitivity to bacitracin, they both resulted in alpha hemolysis rather than beta. We concluded from this that the bacteria present were not S. pyogenes (or S. agalactiae), so both 'patients' were negative for strep throat.

This was a particularly exciting experiment because it is definitely something we will do in a clinical setting. We learned the procedure for taking throat swabs and all of the science behind how the lab tests work, so now we are that much more prepared for nursing careers or diagnosing friends on campus.


The other groups who tested for MRSA (methicillin-resistant S. aureus) via nasal swabs taken yesterday also found negative results, much to everyone's relief. This is also a very important test for us to understand. MRSA colonizes in the noses of carriers, so testing the nasal swab checks for the presence of MRSA in a patient. If this test comes back positive, the patient needs to be rid of the colonization before undergoing anything like surgery, as that would allow it to spread to the rest of the body.


Next we returned to the selective and differential plates that we had inoculated with our unknown bacteria. Our blood agar plate showed gamma hemolysis (no lysis), a clue that we used later in the day to help determine our bacteria's identity.

Our mannitol salt test checked to see if our bacteria was S. aureus. If this was the case, the plate would have turned yellow after incubation as a result of the acid produced from the use of mannitol. Our sample had a slight yellow tinge, but not enough to conclude that mannitol was being used, especially since our mannitol sugar tube was negative. Therefore our bacteria is something other than S. aureus, and we have more results to view to find out what it really is.

Our MacConkey agar plate also showed a negative result. Only Gram-negative bacteria will grow on a MacConkey agar plate, so the lack of growth is an indication that our bacteria is Gram-positive. This is consistent with our earlier conclusions from the Gram stain. The MacConkey agar plate is important in clinical settings because it is used as a defining diagnosis for ETEC (E. coli) infections.

Next, we checked our inoculated phenylethyl alcohol agar plate. This one is selective for Gram-positive bacteria (Gram-negative bacteria won't grow on the plate). Our bacteria did grow, so this is further confirms our conclusion that it is Gram-positive.

We also inoculated one more plate - an EMB (eosin methylene blue) plate. We used a sterile plastic tip to transfer our bacteria from our working stock to the EMB plate, and then placed it in the incubator for Dr. P to check later. This plate contains multiple dyes that act as pH indicators, and is frequently used to isolate fecal coliforms in samples. Ours came back negative.

Antibiotic Susceptibility

After finishing with our selective and differential plates, we moved on to checking the results of the various antibiotic discs that we placed in our inoculated agar plates yesterday. To check our bacteria's susceptibility to the drugs, we measured the diameter of the sensitive circles - the areas around the antibiotic discs where there was no bacterial growth. The diameters of the sensitive circles for each drug are as follows:

1. Penicillin - 25 mm
2. Novobicin - 20 mm
3. Neomycin - 10 mm
4. Streptomycin - 15 mm
5. Tetracycline - 17 mm
6. Erythromycin - 0 mm
7. Amoxicillin - 25 mm
8. Oregano - 13 mm

We then compared our measurements with a data table that showed the measurement ranges for resistance, sensitivity, and an intermediate for each drug so we could draw empiric conclusions about our bacteria's reaction to all the drugs. These conclusions, expressed as "Drug - conclusion (range from data table)," are:

1. Penicillin - resistant (<28 mm)
2. Novobicin - intermediate (18-21 mm)
3. Neomycin - resistant (<12 mm)
4. Streptomycin - sensitive (>15 mm)
5. Tetracycline - intermediate (15-18 mm)
6. Erythromycin - resistant (<13 mm)
7. Amoxicillin - sensitive (>20 mm)
8. Oregano - somewhat effective*

*Since the oregano oil used is not a clinical drug, we evaluated its effectiveness by comparing the size of its sensitive circle to the other drugs we tested. It was less effective than Amoxicillin, which was the most effective drug against our bacteria, but it still inhibited bacterial growth to a certain extent.

Based on these data, we can conclude that Amoxicillin is the most effective drug (of those tested) against our bacteria, but Streptomycin would also work. Our bacteria is resistant to Penicillin, Neomycin, and Erythromycin, so these would not work to treat a bacterial infection caused by our bacteria (unknown F). 

Antibiotic sensitivity and resistance is an important concept for nursing. It is crucial to recognize that not all antibiotics work against all bacteria, and knowing the difference between drugs that will help clear an infection and drugs that won't (or knowing how to find this information) can be a matter of life and death in some cases.

ELISA Antibody Test

After getting through the last of the Petri dishes, we learned about the antibody test used to detect HIV. We listened to a short lecture on the basics of how the test works, and then tried it for ourselves. 

We were given different wells that contained different substances for different parts of the experiment. Some of these substances were different patient samples, the HIV antigens, and unknown liquids for testing.  

Then we received a microplate strip, which consisted of 12 connected wells. We labeled 3 wells with a "+" and 3 with a "-" to indicate the control samples that were positive and negative for the HIV antigen. The last 2 sets of 3 wells were labeled with the initials of the 'patients' that the unknown samples belonged to. Our goal was to test those for the antigen to determine whether they were HIV-positive or -negative. These wells had already been treated with the unbound antigen serum by Dr. P and washed out with a buffer.  

Next we transferred 50 microliters of the positive control sample (in the violet tube) into the wells with "+" on them, and 50 microliters of the negative control (from the blue tube) into the wells labeled with "-".  We then transferred 50 microliters of each of the patients' samples into the wells with their initials. We waited 5 minutes to allow the serum samples to bind to the antigen on the walls of the wells and then rinsed them out with buffer like we watched Dr. P do earlier. After the wells had dried properly, we added 50 microliters of the secondary antibody (SA) from the orange tube into all 12 of the wells. After waiting another 5 minutes to allow for bonding, we washed and dried as previously stated - twice - to ensure that no secondary antibody was left in the wells.

The last step of this experiment was adding 50 microliters of enzyme substrate (SUB) from the brown tube to all 12 of the wells. This substrate acts as the indicator for the test results because it binds with the secondary antibody present in HIV-positive samples, turning the colorless solution blue. After 5 minutes we observed that the wells with the positive control (labeled "+") turned blue, along with the patient samples labeled "FN". We concluded that they were positive for HIV. The wells of the negative control (labeled "-") and the samples from patient "AF" remained clear, indicating that no HIV antigens were present and that they were negative for HIV.

Understanding the basics of this HIV diagnosis is going to be useful in our nursing profession because we will need to know how to recognize and treat patients infected with this virus. Because these immunocompromised patients are so extraordinarily susceptible to disease, it is important to treat them with extra precautions and often different drugs, doing everything in our power to prevent them from contracting a disease.

Identifying the Unknown Bacteria

It was the end of our last day of lab, and finally time to solve the mystery of our unknown bacteria (unknown F). To do this, we began by compiling the results from all of the tests we performed this week. The summary is as follows:

• Morphological Characteristics
• Cell shape: round (cocci)
• Arrangement: mostly clusters
• Size: small
• Spores: no
• Gram stain: positive
• Motility: non-motile
• Capsules: yes
• Special stains: not acid-fast
• Cultural Characteristics
• Colonies
• Nutrient Agar: convex, raised, white/rose
• Blood Agar: gamma lysis
• Agar Slant: raised, white
• Nutrient Broth: turbid
• Gelatin Stab: negative
• Oxygen Requirements: facultative
• Optimum Temperature: 37 degrees Celsius
• Physiological Characteristics
• Fermentation
• Glucose: positive
• Lactose: positive
• Sucrose: positive
• Mannitol: negative
• Hydrolysis
• Gelatin Liquefaction: negative
• Starch: negative
• Casein: negative
• Fat: negative
• Others
• Indole: negative
• Methyl Red: negative
• Voges-Proskauer: negative
• Citrate Utilization: negative
• Nitrate Reduction: negative
• Hydrogen Sulfide Production: negative
• Urease: positive
• Catalase: positive
• Oxidase: negative
• Litmus Milk (reaction: time)
• Acid: N/A
• Alkaline: N/A
• Coagulation: N/A
• Reduction: N/A
• Peptonization: N/A
• No Change: 48 hours

With all this information handy, we were able to use various flowcharts to determine and double-check our bacteria's identity.

We used the above flowchart for our initial identification. Since we started off with the knowledge that our bacteria is Gram-positive and round, the next question was whether it is positive or negative for catalase. We determined that it is catalase-positive, so the chart told us that we were dealing with either Micrococcus spp. or Staphylococcus spp.. The next differentiation was the fermentation of mannitol, which our bacteria was negative for. This meant we had either S. saprophyticus, S. epidermidis, M. luteus, or M. varians. Telling the chart that the bacteria is pigment-negative and positive for fructose fermentation led to a conclusion: our unknown bacteria is Staphylococcus epidermidis.

To double-check, we used a second flowchart. This one also started with Gram-positive cocci, which includes Micrococcus, Staphylococcus, Streptococcus, and Enterococcus spp, and then asked whether catalase is produced. Our bacteria is positive for catalase, which narrows it down to Micrococcus or Staphylococcus spp. Next, we knew that our bacteria was negative for mannitol fermentation, so we could rule Staphylococcus aureus out. The chart's next question was whether our bacteria's colonies had a yellow pigment. Since ours did not, the possibilities were narrowed down to the Staphylococcus spp. After this, the determining factor was the bacteria's sensitivity to novobicin. Our measurements showed that our bacteria is sensitive to novobicin, which confirms our initial finding that unknown F is Staphylococcus epidermidis.

Just to be sure and to broaden our understanding, we looked up S. epidermidis in Bergey's Manual to compare the characteristics listed with what we found in our experiments. The information was consistent with our results, so we concluded that our identification was correct. Being able to use clues like this to determine the identity of a microorganism or the cause of a disease is an important healthcare skill, so this was good practice for our deductive reasoning skills even if we won't be going through these exact procedures as nurses.

More specifically for our bacteria, it is also clinically important to differentiate between S. epidermidis and S. aureus. The former is generally harmless and part of the indigenous microflora, but the latter can cause harmful infections and skin lesions. Knowing the difference between the two is crucial for treatment.

Discovering our bacteria's identity drew our week to a close. Unfortunately, we were unable to celebrate with a yogurt feast as the yogurt wasn't done forming in the incubator, which hadn't been at quite the right temperature. We got over that disappointment pretty quickly when Dr. P invited us outside for a game of frisbee in the sun until it was time to leave. All that's left is to go home and study until all of our experiments make sense.

Day 4: Results and Antibiotics

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:

1. penicillin
2. novobicin
3. neomycin
4. streptomycin
5. tetracycline
6. erythromycin
7. amoxicillin
8. 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.

Day 3: Inoculations!

Results from Day 2

By the time we made it into the lab for day three, yesterday's samples were ready to view. Results varied - our streak plate worked well, and we could see several different colonies (pure cultures), one in each quadrant. The spread plate, though, was less successful. There was too much of the bacteria sample on everyone's plates, so no one could see any individual growth other than a solid layer of bacteria on the agar plate.

We looked at our streak plate under a dissection microscope to determine the colony's characteristics. This investigation showed us that our bacteria are cream and rose-colored in terms of pigmentation and circular in terms of colony form. The colonies were also raised and convex. These characteristics will help us identify our unknown bacteria later in the week.

Our second environmental sample was about as successful as our first. There was no visible bacteria growth, which could indicate a problem with either the swabbing or incubation, since this time we made sure to choose an environment that we knew would have bacteria. We returned the sample to its incubator and will check for growth tomorrow.

Next we looked at the stocks we had prepared, which were more successful overall. Our working and reserve stocks grew bacteria, and we were able to use our working stock throughout the rest of the day for our inoculations. Our broth culture did not appear to yield any bacteria growth, so we prepared another one that is now incubating so we can use it to test our sample's motility tomorrow.

Since our unknown bacteria grew in the slant tubes and agar plates, which are environments with oxygen, we can conclude that our bacteria is either an obligate aerobe or facultative. Further tests will determine if it is also anaerobic. Differentiating between aerobic, anaerobic, and facultative bacteria is important

More Stains

After a short coffee break, we started preparing various stains to further examine our bacteria samples. Materials: working stock with unknown bacteria, inoculating loop, clean slides, bunsen burner and lighter, slide tongs, bibulous paper, wash bottle with distilled water, staining rack, sink, stains (nigrosin, safranin, malachite green, Ziehl-Neelsen carbolfuchsin, methylene blue), droppers, china marker, microscope and immersion oil, hot plate, large beaker, filter paper, acid-alcohol

Procedure: the first step in most of the stain preparations was to make a slide with a fixed smear of bacteria. To do this, we drew a large circle on a clean slide to delineate the smearing area and placed a small drop of distilled water inside the circle. We then touched the inoculating loop to the bacteria in our working stock (using the aseptic technique, of course) and then smeared this around with the water on the slide. After allowing the smear to dry, we ran it quickly over the bunsen burner flame a few times to fix the smear, being careful not to get the slide so hot that the bacteria were damaged.

The first stain we prepared was a negative stain. The beginning procedure was a bit different for this one - we started by placing a small drop of nigrosin on one end of a clean slide and smearing our bacteria directly into that, being sure to keep it on only a small portion of the slide. Next, the edge of a second clean slide was used to spread the nigrosin/bacteria along the length of the first slide so that we ended up with a thin film that trailed off at the end of the slide (we tried this a few times to get it right). After this dried completely, we were able to view it under the microscope with the oil immersion lens.

After viewing the negative stain, we used it as a starting point for preparing a capsule stain. We placed the smeared slide on a staining rack over the sink and covered it with safranin. This was then gently rinsed off with distilled water and blotted with bibulous paper before being viewed with the oil immersion lens.

Our next trick was to prepare an endospore stain. We started with a slide that we had fixed a bacteria smear to and placed it on a staining rack over a beaker of boiling water. We covered the smear with filter paper and saturated the paper with the malachite green stain. This stayed on for 5 minutes as we added more stain to keep the paper from drying out as the original evaporated. Once time was up, we removed the slide from the heat, disposed of the paper, and let the slide cool before rinsing it for 30 seconds. The smear was then covered with safranin for 90 seconds, then rinsed and blotted so it could be viewed with the oil immersion lens.

Finally, we prepared an acid-fast stain. This also started with a fixed smear of our bacteria sample placed over a beaker of boiling water. This time, the filter paper cover was saturated with Ziehl-Neelsen carbolfuchsin for 4 minutes. The slide was then removed from the heat, cooled, and rinsed. After rinsing the slide with distilled water, we decolorized it with acid-alcohol (drop by drop until the color stopped running). This also had to be rinsed off before the slide could be covered with methylene blue, which was rinsed off after 2 minutes. After blotting with bibulous paper, we were able to study our slide under the microscope.

Results and conclusions: by observing both our negative stain and capsule stain, we were able to conclude that our bacteria does have a capsule. The bacteria looked smaller after the capsule stain than it did with only the negative stain, which suggests that there is a capsule present in the extra space colored by the capsule stain.

The other stains showed that our bacteria has no endospores and is not acid-fast (because only the blue stain was visible).

Significance: while we probably won't use these particular techniques in a nursing setting, it will be important to recognize that different types of bacteria have different properties like capsules and endospores that necessitate different treatment plans if the bacteria are to be destroyed.

Inoculations

To wrap up our third day in the lab, we prepared a multitude of inoculations to test various properties of our unknown bacteria.

Matierials: bunsen burner and lighter, inoculating loop, inoculating needle, working stock with unknown bacteria, starch agar plate, skim milk plate, spirit blue plate, nutrient gelatin deep tube, sugar tubes with gas and pH indicators (lactose, glucose, sucrose, mannitol), methyl red/Voges-Proskauer (MR-VP) broth tube, citrate agar slant, tryptone broth tube, nitrate broth tube, plain agar plates, triple sugar iron agar slant tube, urea broth tube, litmus milk tube, tube with gelatinous motility test medium, thioglycollate broth tube, GasPak anaerobic system, incubator (35 degrees Celsius), test tube rack

Procedure: this whole process involved little more than inoculating plate after plate and tube after tube with bacteria from our working stock:

The starch hydrolysis, casein hydrolysis, triglyceride hydrolysis, and gelatin hydrolysis tests will show us what digestive enzymes our bacteria uses. The first three involved inoculating plates while the gelatin test used an inoculating needle to stab a gelatin tube. These are all incubating and will be checked for results tomorrow.

The sugar (lactose, glucose, sucrose, and mannitol) tubes, MR-VP test, and citrate test will tell us what carbohydrates are used by our bacteria. We inoculated the sugar and MR-VP tubes with the sterilized loop and the citrate agar slant with the inoculating needle. These are also incubating overnight.

The tryptophan (indole) test in the inoculated tryptone broth tube will provide information about our bacteria's degredation of amino acids.

The nitrate reduction and catalase tests, as well as the GasPak, will tell us about our bacteria's respiration. We inoculated the nitrate broth with the sterilized loop to test nitrate reduction. For the catalase test, we . The GasPak test started with everyone placing their inoculated agar plates in an anaerobic jar (agar side up). The GasPak envelope was shaken and placed inside the jar, and the lid was attached to the jar by screw clamp. All these tests are incubating as well.

The other miscellaneous tests (triple sugar iron, urea, litmus milk, and motility) will also help us to identify our unknown bacteria. The urea and litmus milk tubes were inoculated with the sterilized loop and the triple sugar iron and motility tubes were stabbed (the slant portion of the TSI tube was then inoculated by drawing a wavy line with the bacteria-covered needle). These are also incubating until tomorrow.