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.