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.