Is Yeast Alive? Lab

This is the first lab I run every semester because it can be done with just yeast before fees have been collected to purchase organisms with. This semester, I bought two different kinds of water balloons at Wally World. The ones that come in the large plastic container (kind of like a tennis ball container) were TOO small to even fit over the mouth of the test tubes. The other ones didn't work very well either. I think last semester, I just bought small regular balloons and these seemed to work better.

One change I made to the procedures on Part 1 was to mix the yeast and water in a beaker instead of trying to mix it in a test tube. This cut down on prep time for the kids and worked just as well. They then just evenly distributed the yeast mixture into the four test tubes. I will do this again, so I need to change the procedures before printing them out next semester.

One student asked me how we knew that the production of the gas was an indication of life instead of just an indication of a chemical reaction. I told him that is one reason we are doing Part 2 of the lab also (which will show evidence of growth).

Infectious Disease Simulation

This activity was great fun and really drove home how rapidly infectious disease can spread throughout a community. I used the small plastic beakers instead of buying Dixie cups and they worked fine. After each round, I had students clean out their cups well and return them to the front demo desk where I refilled them with either water or .1M NaOH (only one cup). We actually had time to run through the lab enough times to where they did 5 interactions on the last round which infected all but about three people in the classroom, which was pretty powerful. Great Lab!

Differential and Selective Media Lab

This lab worked beautifully! We used Staphylococcus epidermidis, E. coli, Serratia marcescens, and Enterobacter aerogenes. All of them responded exactly as expected.

MacConkey and EMB agar

Next week's lab on Differential and Selective Media calls for MacConkey and EMB agar. I made 800 mL of both this morning.

MacConkey--40 g powder in 800 mL of distilled water

EMB agar--28.8 g powder in 800 mL of distilled water

Both required heating and stirring until dissolved.

I only had 100 g of each of these powders, so if I have two sections of Micro in the fall, I will need to order a bottle of each.

Protozoans Lab a HUGE Hit!

The kids had an absolute blast today looking at all the different types of protozoans, algae, etc. that I ordered in for today's lab. In fact they had so much fun that they want to continue this same lab on Monday. Sure hope the specimens can live until then!

Finding time for transfers

I am trying to find time to make transfers of my original cultures to try to keep viable, pure cultures on hand, but there is just NO time! I did start making them today, but did not get finished before someone grabbed me for yet another something I HAD to do for the Academy. Oh, well, maybe I'll get to finish them tomorrow. These slants are perfect though, so I need to be sure to use that recipe next semester or whenever else I need to make more slants.

Making Nutrient Agar Slants

The nutrient agar slants I made last time didn't set well, so I'm doing it a little differently this morning. Here's the recipe I found:

23 g. nutrient agar per 1000 mL
BOIL and STIR until thoroughly dissolved.
Aliquot 5-7 mL per screw-cap test tube.
Sterilize
Set out to cool on a slant.

Hopefully this will work better.

The first REAL flop in lab!

Nothing, absolutely NOTHING worked today in lab as I had expected! In fact, the only positive I can think of about our results today might be to let the kids see that in a real science lab, where you are doing real research, things OFTEN go WRONG!

To begin, in our antibiotic resistance lab, there were NO zones of inhibition in anyone's plates. I have no idea why that might be. The antibiotics were all new and had been stored in the refrigerator, so should have been very effective. I did ask the students to brainstorm possible things we could try to do differently. They hypothesized that if we had inoculated the plates on the same day that the antibiotics were placed on the plates, that might have made a difference. Someone else said that maybe we should have wet the antibiotic disks, a fact that was supported by several plates which had developed "cracks" in the agar emanating from the antibiotic disk which seemed to suggest that the disks had absorbed the water from the nearby agar. So, I had 20 agar plates left that we could use. Students who wanted to reinoculated those plates and either placed disks on them as before or wet them with some distilled water. I had a few drops of sterile water in the aluminum foil which held the inoculating loops I had sterilized, so one group dipped their disks in the sterile water instead of using distilled water. I guess we will see if any of this changes the outcome of this lab so I'll know how to proceed next year. If nothing changes, I probably will not do this lab next year.

On the switchgrass front, only two or three of the transfers showed any growth today and probably one or two are contaminated. We will continue to watch them for the next little while. So that all students will have a plate of fungus to look at when we study fungi in the next couple weeks, I let them all make transfers out of the fully developed plates I have of two of the fungi I brought with me from UT. I really want them to be able to see the fruiting bodies and make slides of the spores if at all possible. This switchgrass pathogen project may be too ambitious for a large group of high school students, especially without Rifampicin to inhibit bacterial growth.

Switchgrass progress 2

On Thursday, most students were able to find tiny growth to make culture transfers of. There was not a lot of growth in only two days, but there was enough to get them interested. Heather from Junior League was VERY impressed with what she saw and encouraged me to apply for the grant again. I need to make lots more plates of PDA for them to make new transfers to next week. I really need to order the smaller petri dishes for this part since they can only put one transfer into a plate. I'll add that to my wish list for next semester.

Making PDA for switchgrass transferes

I decided to make 1000 mL of Potato Dextrose Agar today instead of 800 mL like I usually make when I make media. That way, there should be plenty of plates available for them to make transfers of any growth that may have come out of their plants since Tuesday. I used 39 g/1000 mL of distilled water to make the PDA.

I also decided to make more water agar and let the students surface sterilize some more plant material so they can each be sure to work with at least a couple different specimens after they are isolated.

Heather from Junior League is coming to visit us today to see what we are doing with the grant money they awarded us in the fall. I wanted her to be able to see several different steps which are involved in the research the kids are undertaking, so she will be able to see them taking cuttings of their live plants, looking for growth out of their samples we did on Tuesday, and hopefully making transfers of fungi growing out of their water agar cultures.

Working with Switchgrass

Finally, yesterday we began the lab work on the switchgrass we've been growing. It was so SLOW getting it started, but today we were ready to begin. Students evaluated their plants for disease and removed some of the diseased tissue (leaves or roots) using scissors they had sterilized in a flame using 70% ethanol. We had a near accident when one student started to go back into the alcohol with the scissors while they were still burning, and I had forgotten to put out glass petri dishes they could use to extinguish such a fire, so I have GOT to remember to put those out any time kids are sterilizing instruments with alcohol and flame.

After making their cuts, students followed the following sterilization protocol:

1. Soak one minute in 95% ethyl alcohol
2. Soak three minutes in 20% clorox
3. Soak one minute in 95% ethyl alcohol
4. Let dry on a piece of paper towel

After their samples dried, they placed the cuttings on water agar, sealed them with parafilm, and placed them in the incubator set at 37 degrees C.

Everything's ALIVE!!!

The switchgrass looks better than it's ever looked! Perhaps I should have been watering them all along from the bottom. My pathogens are doing well, except for two that weren't sealed with parafilm. They are getting a little dry. Guess I'll let the kids make some transfers out of those dishes today. I'm making water agar this morning--going to let the kids surface sterilize the plant material today and put it on plates of water agar. I sure wish I had some Rifampicin to put in with the agar, but I don't. I have autoclaved all the inoculating loops, to help prevent contamination, but I don't know if that will work. Maybe the lactic acid will be here to make PDA plates with when we do the transfers.

Back after Break!

I wonder how many of our switchgrass plants will still be alive tomorrow? I guess we'll see in the morning if you can water for 10 days from the bottom of the pan without drowning them!

Switchgrass progress

We have a good supply of young switchgrass now for our projects if I can get them to live over spring break with no attention. Right before we left school on Friday, I raised the lights as high as they will go above the plants to keep from burning them up, the kids watered them, and then we filled the trays they are sitting in up with at least 1 to 1.5 inches of water. I'd say that will either keep them alive or drown them, no real way to know which. But I felt there was no way to keep them alive with no water for 10 days, so I'm hoping watering them from the bottom will be enough.

Two of our pathogens look great. I had the kids who are going to be my lab techs next semester make clean cultures of the pathogens I had started on PDA. With no rifampicin or lactic acid, getting cultures that aren't contaminated with bacteria has been a challenge. I've got to have one or the other to do this project with students. Sarah is supposed to be ordering me some lactic acid after break, so maybe that will work. I have also autoclaved some inoculating loops to use only with the switchgrass project to help reduce contamination.

However we may not need my pathogens. The plants we are growing are showing signs of disease as well. When we get back, I need to have the kids start some new plants from seed and I need to let them surface sterilize the plant tissue we have and plate them on water agar to see what grows out of them. We may do that on the first day back in lab.

Thioglycollate broth is really cool!

As you can see, the thioglycollate broth, which has a color indicator identifying oxygen level within the broth, turned out quite nicely. Thioglycollate broth has a low oxidation- reduction potential and therefore contains litte free oxygen. The pinkish/blue region at the top has more oxygen in it and the clearish yellow region at the bottom is almost oxygen free. The students inoculated their tubes yesterday with E. coli, Lactococcus lactis, and Pseudomonas fluorescens. E. coli is facultatively anaerobic and will grow best at the bottom of the tube, but will have good growth throughout. P. fluorescens is aerobic and should only grow at the top of the tube (if their cultures were clean). Lactococcus lactis is fairly anaerobic and should grow best at the bottom of the tube, but may exhibit some growth farther up the tube.

Oh, btw, Lactococcus did not grow at all on nutrient agar, so here is the email message I received from Dr. McPherson about a growth medium for it:

This medium is called Corynebacterium Agar, but it can be used for the
cultivation of L. lactis.

Agar                      15 g
Tryptic digest of casein  10 g
Glucose                    5 g
NaCl                       5 g
Yeast extract              5 g
dH2O                    1000 ml

pH 7.2-7.4 at 25 degrees Celsius

The formulation of Tryptic Soy Agar is

Agar                              15 g
Pancreatic digest of casein        5 g
Papaic digest of soybean           5 g
NaCl                               5 g
dH2O                            1000 ml

pH 7.1-7.5 at 25 degrees Celsius

The formulation of Nutrient Agar is

Agar          15 g
Beef extract   3 g
Peptone        5 g
dH2O        1000 ml

pH 6.6-7.0 at 25 degrees Celsius

So, TSA is closer in formulation to the Corynebacterium agar. 
If you don't have all the components to make the Corynebacterium agar,
you could try adding a little glucose to TSA. 

Getting ready for the Oxygen Requirement Lab

I decided today to let students keep the plates from the Effects of Temperature lab if they had good clean cultures of E.coli and Pseudomonas flurescens to use as seed cultures for next week's lab. I will have the stock tubes available if we need them. We also needed 6 cultures of Lactococcus lactis for the Oxygen lab. It came in yesterday but there were no real instructions for growing it out. It is currently in a milky liquid broth that I did store in the 37 degree incubator. I looked on the internet for growth requirements, but didn't find anything useful. I email Dr. McPherson and she emailed me back that she's never grown it but she thinks I could use tryptic soy with a little glucose added to it. The problem is that I need the cultures Monday and we don't have any glucose in stock. I'm ordering it soon though for a lab after Spring Break, so I'll have it next year.

I did let one of my students go ahead and make transfers to just a nutrient agar because I already had those ready. We'll see if they can grow just enough to do us some good for next week. There's a lot of "wait and see" and "trial and error" this semester, and the learning curve is pretty steep!

I made the thioglycollate broth today after students finished up their lab so they could see what that process is like. It was pretty cool because there was a pretty color change as the broth was mixed and brought to a boil and oxygen was added to the broth. I'm looking forward to seeing how this lab works out! I think the kids will like it if I can keep them from shaking their tubes when they make their transfers.

Effects of Temperature Lab complete

Overall this lab went quite well. The Bacillus stearothermophilus were not ALL dead. We did have a couple of plates with growth in the 42 degree incubator. However, I have decided NOT to carry this organism over until next semester. It is just too hard to maintain. We also had a couple of plates where Serratia had grown in with other organisms which served as an excellent reminder to my students that they need to really sterilize their inoculating loops before moving into another culture.

Antibiotic Resistance

Effects of Temperature Lab

Yesterday, we began the Effects of Temperature lab with the cultures I had plated last week. I think we were probably fine with the exception of B. stearothermophilus, the only thermophile I had. I plated that out and stored the plates in a 62 degree incubator as instructed, but over the weekend the plates dried out completely. I doubt seriously if there was anything left alive on them unless they might have produced endospores to survive the drought. I did have a few groups try scraping the plates to see if they could get anything off of them to do a culture transfer with, so we'll see if it works or not.

The cultures I used for this lab were Bacillus stearothermophilus, E. coli, Staphylococcus salivarius, Serratia marcascens, and Pseudomonos fluorescens. I believe all the other cultures were fine. There probably is some question about S. salivarius, but I think there were some living colonies there. Time will tell.

Each group had 3 cultures plates which they divided into four quadrants and inoculated a different organism in each quadrant. All three plates should have been inoculated the same way. Then one plate was put in the refrigerator, one on the counter at room temperature, and one in a 42 degree incubator.

Getting ready for the "effects of temperature" lab

Based on what appeared to be growing well right now, I decided to use the following four organisms next week: B. stearothermophilus, Serratia marcascens, Pseudomonas fluoroscens, and E. coli. That will give me one thermophile, two mesophiles, and one psychrophile (in order above) for the kids to experiment with. I had two viable plates of B. stearo., 2 tubes and 2 plates of Serratia, 2 tubes and one plate of P. fluoro., and 2 tubes of E. coli. I decided to make some more plates of everything except Serratia, so I made 2 more plates of each. I'm storing the P. fluoro in the fridge, Serratia and E. coli in the 37 degree incubator, and B. stearo in the 60 degree incubator, hoping for lots of colonies by Monday when this lab begins.

I'm still waiting on a purchase order to come through so I can buy Lactococcus lactis for the oxygen lab which is week after next. I need to check on that today.

Fascinating day in lab today!

Today we pulled out our first plates of Serratia marcescans which some of the students used to redo the antiseptics/disinfectants lab. The cultures were bright red and fascinated the students. We used them to make wet mount slides and some of the kids chose to do Gram stains. They expected the bacteria themselves to be red, but they were gray, rod-shaped bacteria which Gram-stained negative. The red is just a pigment produced by the bacteria. I wonder why they produce that pigment. I'm looking, but haven't gotten the answer to that question yet. They are motile, though and some of ours could be seen moving since we did not heat set them in the wet mount slide. The kids loved it!

The zones of inhibition part of the lab was also interesting today. We pooled all the data and the hydrogen peroxide was again very effective (shown in the bottom quadrant in the picture above) as was the iodine-based pre-surgical sterilizer someone had brought from a hospital. Hand sanitizer was not very effective at all, much to the dismay of the students.

Bacillus stearothermophilus

The nutrient broth tubes of B. stearothermophilus I made last week show no discernable growth in an incubator set at 37 degrees C. So, today I streaked some nutrient agar plates with broth from those tubes as well as from the original agar slant that it came on from Carolina last week. Those slants have also been stored at 37C. I'm hoping elevating the temperature to 60-65 degrees C will promote growth, so I've uped the temp in one of the incubators. We'll see if that does anything. The only other issue is that I noticed the slants are actually on tryptic soy agar, so I'm not even sure B. stearothermophilus will grow on nutrient agar. Time will tell, I guess.

Switchgrass pathogens problems

Ok, so right now the switchgrass pathogens project is in dire straits. So far, I've been unsuccessful in growing uncontaminated cultures. I'm getting lots of bacteria in my cultures which ruins them. I looked for Rifampicin in all my supplier catalogs and didn't find any. (That's the antibiotic we put in the media at UT last summer to prevent bacterial growth.) I emailed UT to see if I can buy some from them and they aren't really set up to do that, but she told me I can buy some from Fisher Scientific. They have 250mg for $34 but it is in powder form. It is only soluble in things I cannot have in a high school lab, like chloroform. It is slightly soluble in acetone and methanol, so I may have to try that, but Fisher can't tell me how I make it into a liquid, so I'm back to the drawing board. My contact at UT did tell me I can put one drop of lactic acid into each petri dish as I pour my agar, and swirl it around to reduce the pH to a level where the bacteria will be inhibited, so I may have to try that. I'm getting a little flustered though. I just knew I could make this work for my students, and now I'm not so sure.

Nature of Science At Its BEST!

The kids were so excited about the results of this lab when we put them on the board today, that they want to continue this Antiseptic/Disinfectant Lab more next week. They want to streak the plates from all four directions and then test some new items. They chose to retest hydrogen peroxide (which seemed to inhibit growth best) and then add Lysol, hand sanitizer, Febreeze, and a cleaner called Recal that is used in the vet office one of the students works in. We talked about how typical this is in science. The more you learn about a topic, the more questions you have!

I did have enough growth (after a week) in my pathogen plates on water agar to make transfers onto PDA which is what I think I should have put them on to begin with. I still haven't ordered Rifampicin. It's $33 for a small quantity, so I may see if I can do without it. Dr. Ownley emailed me that I can use a drop of lactic acid in the PDA plates to lower the pH enough where bacteria don't do well, but fungi can still do OK. So, I may try that.

Antiseptic/Disinfectant Lab Results

Yesterday, the students observed their plates from this lab. We did not get full lawn growth on any plate. I think next semester, we need to streak the plates in all four directions instead of just in two. This would assure better coverage of the plates with organisms and I will be sure that any results we get were a result of the work of the antiseptic/disinfectant, not just poor streaking techniques. However, we were able to see some definite zones of inhibition which they thought was really cool! We also combined all of our data in a large table on the front whiteboard in the lab, which made for some very interesting comparisons. Be sure to do that again next time. When we look at this data in class today, it will be a great time to discuss the difference between observation and inference to teach Nature of Science.

Getting Ready for the Temperature Lab

Yesterday I received a freeze-dried sample of Streptococcus salivarius and a slant of Bacillus stearothermophilus from Carolina Biological. The freeze-dried sample was easy to revitalize except I really needed a 5mL pipette that was sterile. The only ones we had that were sterile were 10mL and that wouldn't fit into the container the sample came in. In my opinion, they should have shipped one with it or at least let you know that you must have one.

I also made nutrient broth cultures so the students can make culture transfers for me in class today after they finish the Antiseptic/Disinfectant lab. I mixed up 200 mL of nutrient broth to make the 12 tubes and probably had enough left over to do another 2-4 tubes. Each tube contains 10mL of broth.

I also did something different with the autoclave yesterday to speed up the process during 4th block. In the morning, I got the broth made, and aliquoted the broth into the tubes, and placed them into the incubator. I had already put water in the base. I got everything ready to go except putting the oil on the seal and placing the lid on the autoclave. About 1:30, I came into the lab and plugged in the autoclave, put oil on the seal, and put the lid on it so it could begin heating. I left the exhaust valve closed. At the very beginning of 4th block, I check on it and there was absolutely no danger. In fact, I could have probably turned it on 30 minutes earlier, but for sure another 15 minutes earlier wouldn't have hurt.

I need to make 800mL of nutrient agar today for the Temperature Lab for Monday. It takes 3 plates per group (one for each temperature). Probably 6 per group would be better, but I'm getting low on Petri dishes.

Antiseptic/Disinfectant Lab

We will do this lab today, but I don't have enough sterile filter paper circles for everyone to do their own petri dish. I have 50 that I purchased, but that would be enough for each group to do the lab together. I think I'm going to do one plate per person and use circles I cut out of coffee filters so everyone can have his/her own plate. I wish they had been sterilized, but it's too late for that. NEXT semester, I need to cut the circles and then autoclave them before the lab. I should have done that last week.

I am going to use Clorox, Quat 50, Iodine, 70% alcohol, 95% alcohol, and antibiotic ointment for them to choose their 4 antiseptics or disinfectants from.

Potato Dextrose Agar

This morning before school I made 800 mL of PDA (which made 40 plates)for plating out the fungal pathogens. I think this afternoon, I'll go ahead and transfer a sample of each to PDA because the transfers I made to water agar don't appear to be growing yet. Maybe I'm just impatient, but they might grow better on PDA.

Getting Ready for Antiseptic/Disinfectant Lab

After school this afternoon, I decided for sure what lab I would do next week. I am still waiting for more supplies from Carolina Biological, so I'm sort of stuck until they get here. However, the Antiseptic/Disinfectant Lab will be doable before those supplies arrive. So today, I made 6 nutrient broth tubes of E. coli and 6 of Bacillus megaterium to use in that lab. That will mean that every two groups will have a tube of each bacteria to use in the upcoming lab. That should be plenty. They have from Thursday until Monday to grow and I put them in the incubator so they can grow quickly. Actually, the nutrient broth tubes I used were those nutrient agar tubes that didn't have enough agar in them, so they hadn't set up. I just couldn't bring myself to throw them away as tight as funds are. I don't see why they won't work.

Growing Plant Pathogens

This morning I finally got around to making transfers of the plant pathogens I brought with me from my summer research in the Plant Path Lab onto water agar plates. I didn't have my lab notebook with me when I did it, so I think I remembered that we transferred them onto water agar, but I'm not sure. I'll look in my lab notebook from this summer to find out. Anyway, I put them in the incubator at 37 degrees C to grow for a couple of days. I did not transfer the Bipolaris because of the risk of disease transmission. The next time I run the autoclave, I need to kill that and get rid of those cultures.

Water Agar for Switchgrass

In preparation for growing out my pathogens for the switchgrass research project, today I made water agar. I didn't have my lab notebook from the UT research project this summer, so I just Googled water agar recipes to find out how much powder to put in the water. I made 400 mL of agar, so I put 8 g of agar into the 400 mL of distilled water. I labeled the plates with a purple stripe and will use black ink to mark the nutrient agar plates in the future.

I re-sterilized the nutrient agar I had left from yesterday's lab and poured up about another 20 plates for future use.

REALLY need a deeper water bath!

So, today I improvised for the deep water bath we don't have and took my crock pot to school. I set up a thermometer on a ring stand in it to monitor the temperature of the water, but the only problem was, there was no way to adjust the temp of the crock pot other than High and Low settings! So, the crock pot DID get the agar to melt, but it was either too hot, or by the time I got ready to pour the plates, almost ready to set up again. But, this took the problem of the agar being so hot that it might kill the organisms out of the picture. Anyway, we made it! Other than that, the lab we did today (Can Your Hands and Lab Table Be Sterilized?--Flinn #9) went well. It did not take the whole class period, so I had time for some notes as well. We will do the observations and comparisons on Thursday.

I did this lab differently from the way it called for in the lab manual because I didn't have enough empty screw-capped test tubes available. I made about 1300 mL of nutrient agar last week (in two flasks). That's why I needed to melt them today. I just poured the agar into the plates when the kids were ready. It worked just fine and was a lot less prep work. Next semester, if I have the Lab Techs, we may do the tubes, but if not, this works fine.

Switchgrass is growing, but very slowly. I have GOT to get the pathogens growing, so I can inoculate the plants with them soon. Just not enough TIME!

Technical difficulties!

This was a tough lab! Only 3 of the 4 cultures I started on Tuesday were ready by today. The fourth one (B. catahallis) didn't do anything. I don't know if I went in while the inoculating loop was too hot or what. I also forgot that the Endospore stain required boiling water, so at the beginning of the lab, I had to go to the chem lab to borrow equipment to boil water with. The kids were great though! They were very patient and helpful. They definitely realized today why I need Lab Techs for next year. Also with only one set of stains (remember this was from the kit that came from Carolina), the kids had to share today. They did well, but I need more of these stains before next semester. However, the stained slides they made looked great! Micrococcus luteus reacted VERY well to the capsule stain! It was very interesting--probably my favorite.

I made some nutrient agar slants this morning and only 8 of the 20 set up. Maybe I can use the other 12 as nutrient broth tubes next week. I'll need some soon anyway. I need to make transfers of the original cultures that were given to me. I think next time I'll pipette them out while they are on the stir plate. I know the agar powder settled to the bottom while I was aliquoting it out. This was definitely a day of trial and LOTS of error. Even trying to melt the agar for Monday's lab went awry. I need to purchase a deeper water bath because only the agar in the bottom of the flask melted, but I am FAST running out of money. I think on Monday I'll have to take a crock pot to set the 1000 mL flasks in while the water heats enough to melt the agar.

Getting ready for endospore and capsule stain

Last week I received the High School Microbiology Kit from Carolina Biological which contained 5 organisms to be used in various staining techniques. I will do a lab with them on Thursday, so today I transfered the culture from the agar slants on which they were growing when I got them to nutrient agar plates. These cultures must grow from 24-48 hours before using them. Anyway, I did the streak-plate method to transfer them to petri dishes instead of going first into nutrient broth as I would have done with a little more time. I hope they grow sufficiently for 12 groups to get colonies out of these dishes. I probably should have done more than one petri dish for each organism. I need to remember to do that next semester.

I'm needing to do transfers of all the cultures I was given by UT to keep them fresh, but all of my screw cap culture tubes still had E. coli and B. megaterium in them, so today I autoclaved all of those tubes, washed them out, and disposed of the contents in a biohazard bag. I'm a little nervous about doing these transfers outside of a laminar hood, but I really don't have much choice. That is a little out of our school budget price range. :(

Lab Technicians for next school year

I have recruited some Microbiology Lab Technicians for next school year from my Microbiology students. They will be helping me in the lab as their Senior Portfolio placement. I will be able to show them how to do all the things I do to get ready for my labs, but I also want them to embark on some sort of research project of their own. I hope they will do research that enables them to participate in the Southern Appalachian Science and Engineering Fair at UT in March/April of 2011. Ryan and Penny will be working with me during the Fall Semester and Jordan and Jun will be my lab techs during Spring Semester. I am very excited to have their help.

Autoclave issues resolved!

The leakage problems with the autoclave have been resolved. After speaking with the technician about them, I have made a few key changes to the way I get it started. I now apply olive oil to the entire beveled edge of the unit where the lid makes contact with the base unit. That needs to be done every time the unit is used instead of every 5-8 times. Also, when I put the lid on and tighten the bolts, I make sure the lid is perfectly level, using two opposite bolts as before. If I will make sure the lid is level, the unit does not leak. That has significantly decreased the amount of time I have to sit with the unit.

I also tried another time-saving technique he told me about. Instead of exhausting at the beginning of the heat-up, I let the unit come to temperature and pressure, release the valve and let it exhaust for 5 minutes, then close the valve and let it build to pressure again for sterilization. That worked well too.

Gram Stain Round 2

The Gram stains went well again today (Thursday), but we did have some mixed results. Some of the students got results that were just as nice as the first time (cultures are now 72h old instead of 24h old). However, there were a couple groups that got results I couldn't explain. Overall, I feel our results were better the first time. I did have some students who got good results the second time that were totally unsuccessful the first time, so I guess practice makes perfect.

I received a new kit from Carolina Biological (part of the grant from DonorsChoose.org) that I plan on using on Thursday of this coming week. We will be doing some capsule and endospore staining using the stains and organisms they supplied with the kit. (The name of the kit was High School Microbiology Kit.) I'll need to make some transfers of organisms Monday or Tuesday from the culture tubes they sent so they will be ready for Thursday.

Gram Stain Round 1

When I got to school today and looked in on our petri dishes, they were PERFECT for doing a Gram stain today--lots of isolated colonies. So instead of waiting until Thursday as planned, I checked to see if the lab was going to be used during 3rd block, and it was free. Also, I had emailed Dr. McPherson about whether or not our samples would still be good come Thursday. She emailed me back to say that when she has her college students do Gram stain, they do use 24h cultures, but that we should still be OK with 72h cultures. Anyway, I took advantage of the empty lab and we did Gram stains today.

After only about 3 broken slides because either they heat set for too long or because they were impatient and didn't let the slides air dry first, we had excellent results from our Gram stains. I was so proud of these kids! They were able to accomplish what many students in college struggle with doing properly. We will do them again on Thursday for more practice. This will also let me know if my 72h cultures act like they did after only 24h. I wish someone besides me could understand what a great job these kids are doing!!!

From nutrient broth to agar plates

Today, the kids took a few minutes to analyze their nutrient broth transfers by comparing them to the originals. I believe all of them were successful in making the transfers and the microbes seemed to have cooperated by forming either turbid or sediment samples in the new tubes. Kids were pleased!

After that, they learned how to make a transfer from their broth tubes to the agar plates I had ready for them. Some of them are still having trouble with their Bunsen burners (getting them adjusted properly). They were learning the streak plate method today. I did not have them flame their inoculating loop after changing quadrants on their plates. I guess I'll see if that was a mistake when we get back in lab on Thursday.

I also finished creating their Gram stain trays today. I bought inexpensive metal bread pans and some hardware cloth (metal screening with larger holes than door or window screening). I cut the hardware cloth and fit it into the trays to support their slides when they are doing the staining and washing. Gram staining will be on Thursday. I am somewhat disappointed that all of the cultures I have on hand are rod-shaped bacteria. Most are Gram negative. I have only one Gram positive organism for them to look at on Thursday. I'll have to adjust that before next semester.

Aseptic Technique Lab

I made some changes to the Aseptic Technique Lab from the Flinn Lab Book (#4, I think). I took some of the pressure off of the kids by letting them practice more than one time into the nutrient broth tubes. I also noticed that the excellent diagram provided showed transferring from one tube into another, but the directions in the lab had students transferring from a petri dish into a broth tube. So I rewrote the lab slightly to just allow the kids to transfer from tube to tube. I will not have a control to check their aseptic technique, but I decided comfort level was more important to me right now. They will still be able to see if they contaminated one sample with their lab partner's sample because the E. coli samples should be turbid, while the B. megaterium should have a sediment. The kids really seemed to enjoy this lab. After they finished with their transfers, I let them use the microscopes again to practice their technique on some prepared slides. The slides with all three types of bacteria on them that I bought from Carolina Biological were terrific! The kids are really getting good at using the oil immersion objective. I was very pleased!

Autoclave Update

I tried again on Thursday morning to run the autoclave. It continued to have leaks which prevented it from reaching the green zone (17-21). It stopped at 15 pounds of pressure and just sat there. Frustrated, I called for help and finally got in contact with a very helpful technician. He told me a couple of things that will help me. I am going to buy some olive oil, on his recommendation, to improve the seal (instead of using petroleum jelly as directed in the directions). Also, he said to cover the entire slanted surface, not just the pointed edge or bevel. Thirdly, when tightening the screws on the lid, doing them opposite screws at a time, I need to check to see that the lid/seal remains level at all times. He said to tighten and check, tighten and check, etc. until the lid is seated and level on the unit. When I checked, I did see that the lid was slgithly crooked so I guess that explains the inability to reach pressure. However, he did tell me that sterilization will take place at 15 psi, so at least I didn't waste my whole morning.

Autoclave issues

Today, I was unable to use the autoclave because after 1.5 hours, it still was nowhere close to temperature and pressure for sterilization. I did have some leakage going on in one place on the lid, so maybe that was the problem. I'm hoping that I just had gotten some lubricant on the seat instead of just on the bevel. I'll try it again tomorrow.

Nutrient broths looked OK!

I was quite pleased to check on my nutrient broth transfers this morning. They looked fine. Both the E. coli were turbid as they should have been and the Bacillus megaterium both had sediment at the bottom of the tubes. So, I think they will be fine.

During fourth block today, I made the transfers into student nutrient broth tubes--16 of E. coli and 16 of B. megaterium.

Today during our lab period, the students finished their observations of the cultures they had taken last Monday from various places around the school. It was odd, two of the cultures taken from the restroom door handles actually dissolved the nutrient agar, turning it into liquid. The rest of the samples looked good and the students seemed to enjoy comparing their growth with what they observed on Thursday.

After they finished with the cultures, we planted switchgrass seed in soiless potting mix and put them under the grow lights. I almost used Miracle Gro, but instead, I bought organic potting mix. I need to pick up some litle watering cans for the kids to use to keep them moist while they germinate.

Making transfers to nutriet broth

This was a little scary. I haven't done this in 30 years. I sure hope I didn't contaminate the original sample while doing it. I made the transfer from the agar slant that was given to me as a pure culture into the nutrient broth tubes I had made yesterday morning. I actually did the inoculations yesterday afternoon, but didn't have time to blog about it until this morning. I inoculated two tubes of Escherichia coli and two of Bacillus megaterium and placed the tubes in the incubator at 37 degrees C. I was planning on making transfers into the students tubes today or going to school on Saturday to make the transfers if there didn't appear to be enough growth, but schools are closed today due to an impending snow storm. When I do inoculate the tubes for the students though I need to do one for each student. That's a lot of inoculation of tubes when you have 32 students in lab! Maybe I'll go to school in a little while to see if I can get in the building before the bad weather gets here.

Making Nutrient Agar Plates

400 mL distilled water, 9.2 g Nutrient Agar powder

autoclave 15 min. at 121 degrees

Sterilizing the nutrient broth

The directions on my autoclave say always run at least 35 minutes after reaching sterilizing pressure, but the directions on the bottle of nutrient broth powder say to autoclave at 121 degrees for only 15 minutes. I decided to follow the directions on the bottle. When I removed the lid from the autoclave, the tape did show that sterilization had occurred, so maybe I made the right choice. It was difficult to hold my autoclave at that temperature, so there was a time or two when the temp got several degrees higher. I hope it didn't damage the nutrient broth. Guess we'll see.

Nutrient broth

At 7:00 a.m. I mixed up 4 g nutrient broth powder with 500 mL distilled water, poured 5 mL per test tube (wishing I had pipettors, but I don't), put on lids (not tightly), and put in the autoclave (about 7:30). I made two per student and about 8 extra for me to use to grow cultures or in case of accidents. I didn't have quite enough screw-top test tubes, so I stoppered the rest with cotton balls and lightly covered them with aluminum foil. I wonder if I should have put the cotton balls in after the autoclave process, but I guess I'll find out soon enough. I started off the semester with a 100g bottle of nutrient broth. I'll be interested to see how long it lasts.

Fundraising

Realizing how expensive this course was going to be to teach, I soon began searching for grants that might support my cause. I was sent an email through the school mail about an organization called DonorsChoose.org. I quickly put together a couple of proposals for basic supplies for the class, posted them and waited. To date, two grants in support of this class have been fulfilled by DonorsChoose.org and I received another grant from the Junior League for supplies as well. In all, approximately $1500 have been awarded to help me fund this class.

Another way I have received support for Microbiology is from our local STEM professionals organization. I let them know that I needed lab coats for my students and several different companies donated either lab coats or money to purchase them. Some of my students wanted their own and so purchased them. The local university referenced in the last post even granted my students a 20% discount on the lab coats sold in their bookstore, making them extremely affordable. I have discovered that if the community knows of a need, they do come through in many ways. I cannot tell you how invaluable their support has been in this endeavor.

The adventure begins...

About a year ago, as we at Hardin Valley Academy were putting together course offerings for our science department, I ran an idea by our principals of offering a high school level microbiology course. This course would be ideal for our Health Science Academy and our STEM Academy students, I believed. Our awesome administration agreed and the project began!

Considering the fact that no other high school in our area offers Microbiology, I began seeking out folks to discuss the course with. I met with teachers from our local community college who taught Microbiology and I also met with teachers from our city's major, research-based university. Both were helpful, but the teachers from our university are unbelievably cooperative, allowing me to attend the Introductory Microbiology Lab to refresh my lab skills and be current on lab techniques and safety procedures. I owe my sanity to them!

My county was also extremely cooperative in this endeavor. They allowed me to spend my textbook money allocated for this new course on oil immersion microscopes capable of magnifying up to 1000 times, something I felt was critical if I wanted to train my students to move seamlessly into a college level Microbiology course. I am truly lucky to teach in a system that is that supportive. The county science department also purchased an autoclave for me so I didn't have to raise funds for that.

However, I did need to raise funds, for I quickly determined that this would be a terribly expensive course to teach!