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.