Yeast Lab day 2

Today was the day the students took their petri dishes that they had sprinkled yeast onto the agar in and made a wet mount slide from the yeast growth.  There was a great deal of bacterial growth as well, but I had them take their samples from the fungal growth and use aseptic technique in their handling.  The slides they made were great!  I did not have them heat set this year.  We did use a cover slip, and then we added the immersion oil to the top of the cover slip.  Each group was successful with this method.  I had to make up some 70% ethanol at the last minute because I had forgotten to check the bottles left in the lab over the summer.  We had used almost all of it last spring semester, so I mixed some up quickly this morning and refilled the bottles.

Jobs for the lab tech today: Finish refilling all of the bottles of 70% ethanol, fill up the distilled water bottles in the lab for the new semester, cleaned the 40X and 100X objectives on all scopes, got one round of old culture tubes ready to sterilize when I run the autoclaves on Wednesday, and cleaned a round of used slides that had soaked in clorox water.

On Wednesday, I need for her to make at least another 1600 mL of nutrient agar to have all the kids will want on Thursday for the Collection of Microbes lab.  She can also help me pour those plates and begin to clean out the test tubes after they have been autoclaved.

Yeast Lab Followup

I need to order some 7" balloons before I do this lab again.  Since Party City went out of business, I haven't been able to find that size.  I purchased 9" balloons, and they are acceptable, but we did not have the excitement of small explosions occasionally around the room like I have had in years past.  The kids love it when that happens!

Initial Setup

I also told second period to fill their tubes up to within about 1" of the top which will allow for more dramatic results.

Different Lab Group, but results after 25 minutes

Is Yeast Alive? First Lab of the New Semester

I almost messed up today!  Tomorrow is the first lab of the new fall semester--a lab titled Is Yeast Alive?  I got this lab on the internet a couple of years ago and have used it as the first lab every semester since, but I almost had an epic "fail" on my hands tomorrow which would not have been a great way to start a semester! There are two parts to this lab, one a test for metabolism (cellular respiration producing carbon dioxide gas that will fill up balloons) and the second a test for growth.  I remembered to buy balloons yesterday, although I only found 9" balloons, not 7" which I think is what I have used in previous semesters.  I hope those will be OK.  Today when I went to make copies of the lab for my students, I remembered that I also needed Petri dishes of nutrient agar for the second part.  I had not planned on making agar today, but frantically got everything mixed up and into the autoclave in time to get it processed and hopefully poured without staying too late.

My autoclave is a highly manual (as opposed to automated) version that is really just an overgrown electric pressure cooker and the entire process takes approximately 2 1/2 hours to complete.  There is no thermostat which regulates itself--someone must stay with the autoclave at all times once it comes up to pressure to be sure that it doesn't overheat.  If you are lucky, you can get the gauge set to where it will toggle on and off within the appropriate range, but I still will not leave it unattended.

Anyway, I mixed up 1600 mL of nutrient agar in two 1000 mL Erlenmeyer flasks (18.4 g of nutrient agar powder to 800 mL water in each to avoid boiling over), let the mixture stir and then placed both flasks with lids loosely screwed on into the autoclave to sterilize.  I had a new lab tech student helping me today, so even with teaching her how to pour plates, I was able to pour 74 plates from the agar I made.  

I did  finally have to buy new yeast this time.  I had my lab tech check the viability of the yeast that had an expiration date of October 2011 on it.  And, as expected, the foaming action was not what I had hoped.  New yeast this semester!  I'll be going to Publix after school today to try to buy another container of yeast.  I don't want to use the packets, but I may have to.  I need 4 grams per lab station, so I will need at least 56 grams.  Well at least that's an excuse to go visit our new Publix grocery that just opened up here in Knoxville.  LOVE that store!  I also had the lab tech place four balloons in each of the white baskets that hold student supplies for lab.  I'll just have to replenish them between 1st and 2nd block.

I am going to try to revisit and update this blog with each lab this semester.  I hope it will be beneficial to others and especially to someone here at my school if I end up somewhere else sometime in the future.  At that point, someone else would have to step in and teach Microbiology.  This blog should be a big help to them if that should happen.

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.