PCR happy ending

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2014-01-17 18.04.26

Me taking a picture of the envelope taped to the front door before leaving Friday evening. Just in case.

When I wrote the previous post about my anxiety regarding the PCR reaction of the soil cultures, I did not explain the main reason for it: this was to be part of an actual laboratory class. Those of us who teach lab classes know the feeling of slight embarrassment when things go awry. You expect a reaction that does not happen or the microbes don’t look the way they should. Reasons abound: from human mistakes and carelessness to expired reagents, bad batches, or plain biology. Sometimes it can be made a teachable moment, but not always.

So last Thursday I went to the lab hours earlier and did a mock run, which was a good idea, because I made a number of mistakes, one being walking away after pressing the Start button. Turns out the machine wanted me to press an additional OK, then asked about 2 more settings, and finally made me confirm that I was ready to run. Luckily after half an hour I had started wondering why the machine was so quiet. Duh.

By the time the students rolled in I was feeling much better, and after pipetting the reagents of the first tubes, I asked for volunteers. There were several, and as they were enthusiastically pipetting the 1 ul volumes I started wondering if that was a good idea. None of those students had ever used a micropipette before.

But they were delighted and enthusiastic, something that all participants of the Small World Initiative have reported. Ownership of a project comes hand in hand with motivation, and once the tubes were in the machine and the students returned to their plates it was a joy to watch them discussing their soil isolates.

When I ran the electrophoresis the next morning, I was a bit disappointed but not surprised. One lane had a clear band, some had faint bands, and the rest were smeary. As I expressed to one of the students the day before: we want the first experiment to work, but not perfectly so we can improve it for the next time. A first flawless experiment is almost always a fluke, and we cannot learn from it.

So I ran the samples again, this time in the blessed quiet of the lab. Things looked much better. I called the company that would do the sequencing, and scheduled a pickup around 2 pm. Put the samples in an envelope, left them at the front desk, and went on with my things. At 5 pm, the envelope was still at the front desk. Called the company, and they told me the courier was on its way. “LA traffic, you know.”

Our building closes at 6 pm, as the receptionist dutifully reminded us around 5.50 pm. The envelope had not been picked up. I was leaving town next morning, and started panicking. Three frantic phone calls later somebody picked up the phone at the company. I explained that the building would be locked in minutes, and was advised to tape the envelope to the front door. At 6.30 pm, I had to leave with a heavy heart.

Next morning I had not received any emails or calls concerning envelopes taped to the front door, so I assumed things were under control. It was in LAX 10 minutes before boarding the plane that the email rolled in: Please log in to see your sequences…

The next few minutes were almost rapturous. I logged in and discovered the majority of the sequences were “great” or “ok.” Few had issues. Knowing that students were using the long weekend to work on their poster presentations and they needed the data, I frantically copied and pasted the sequences into a document and sent them away before boarding the plane.

It was not until late in the evening that I was in internet range again. First I ran a sample I had sequenced before and used as a control. The hotel wifi was a bit sluggish, but Blast finally came back with the correct result: Pseudomonas. I almost hollered.

The sequences came out interesting- some expected genera, others less expected. It will be interesting to see how the genetic data compare with their traditional microbiology tests.

I am happy.

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The small details that make love great

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Image

Water…

There is a saying in Spanish about “los pequeños detalles que hacen grande el amor.” The small details that make love great.

I was thinking about it today.

Nuclease-free water.

We are doing PCR Thursday as part of the soil project. First PCR reaction in our brandy new PCR machine, and then second electrophoresis run ever in our brandy new electrophoresis set. Laugh if you want. I used to do research in Cuba, using stockings at the end of chromatography columns and candle jars instead of CO2 incubators. Life is good.

I had decided to play safe. No PCR from scratch, let’s get the ready-made beads. For the first trial, we can do with ready-made buffers. Let’s not risk the experiment.

But I forgot the small detail. High quality, nuclease-free water, per the protocol.

We’ll get it by Thursday, somehow. But it is funny, at the end.

We made all those considerations about PCR, ready-made beads and the sequencing place, the ethidium bromide disposal, and the filter to take pictures with the transilluminator. But we forgot the water.

And David Foster Wallace’s commencement speech comes to my mind:

There are these two young fish swimming along and they happen to meet an older fish swimming the other way, who nods at them and says “Morning, boys. How’s the water?” And the two young fish swim on for a bit, and then eventually one of them looks over at the other and goes “What the hell is water?”

Tomorrow will be another day.

Out of the frying pan…

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One of the student pick-and-patch masterplates

One of the student pick-and-patch masterplates

It has been months since I wrote a blog post, party because of busy professional times (applications, abstract submissions, deadlines), plus holidays, a nasty cold, and family. Nothing out of the ordinary.

I started a microbiology course last week, one that is greatly relevant for me: it is the adaptation of Yale’s Small World Initiative to our university’s accelerated schedule. Even more, it is actually an attempted merger of our previous lab curriculum with Yale’s research-based approach. One week into the course I am cautiously optimistic that we will pull it out.

During the pilot partners workshop last summer, it was obvious that the accelerated pace would be one of our main issues. The original Small World Initiative (SWI) was developed with a traditional semester course in mind (14-15 weeks). On the other hand, it also assumes shorter lab sessions and students being engaged in a number of other classes and activities. National University‘s model is based on students being focused on one course at a time, and lecture and lab are considered one course each running concurrently. This means long (2-3 hour) labtime chunks 2-3 times per week, for 8 weeks.

So developing the schedule has not been too bad. Of course, the main issue will come once the soil cultures are tested for antibiotic producers. We would like students to have something with activity against the tester strains so they can move forward. If this does not happen, in a semester there is time to go back and swab new soil samples. In 8 weeks, that may not be possible. I have some cultures to provide as positive controls and if it comes to that, something to work on if there are no producers.

The other issue has been also the student population and their majors. Most of our microbiology students are heading to nursing or other health-related careers, not research. It is part of the curriculum and also our obligation as instructors to introduce students to the importance of aseptic technique, the role of disinfectants, antibiotics and hand-washing, plus let them practice some of the most common medical microbiology procedures (throat and urine cultures, bacterial typing, some serology etc.)

Yale has developed a very nice laboratory manual for the SWI, which is basically open source. Obviously, it does not contain any of the medical microbiology kind of experiments. My original idea was to use that manual with handouts for the other exercises. However, things got soon complicated. The beginning of the SWI process is quite mellow: swab soil samples, observe, pick and patch. Even including all kind of discussions about soil microbes, culture media, and colony characteristics, the lab procedures are rather quick. It made sense to use the extra time to get students going with smears and stainings, and to practice aseptic technique and microscopy.

So at some point during the holidays I realized that it was easier for me to start assembling a new manual that mixing and matching two.

Yeah, I know.

I am writing the manual as we go, basically. I use a live Google document that my students have access to, but cannot edit. I also make copies of the procedures as most of them, tech savvy as they are, like paper.

In a way I am glad. For years we, the microbiology instructors, have dreamed with writing our own lab manual. While we go from the manual for most of the basic exercises, we have our own tweaks to many others plus the added little exercises developed over the years.

But…it is quite a chore, especially as it was not really planned.

What do they say? Out of the frying pan…into the FIRE (cue Meat Loaf)!

And that is all for today. I’ll try to post updates about the logistics of the course and how the project is going. As of today, students have colonies and they have made their master plates. With MLK weekend coming, we will play with some other techniques this week (streaking, differential media) and get started with antibiotic production testing next week.

It will be fun 🙂

Microbial updates

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This has been a pretty hectic week, and with lots of food for thought. The situation in the world concerns me, and also other issues of the bigger picture. But today I will focus on updates on bugs. Taking care of microbes growing has  a zen-like quality to it. Call me old-fashioned, but waiting for a hot loop to cool is relaxing.

One of the bugs I brought from Yale, a perky Pseudomonas, has proved to be a funky one. Last week I strode into the lab and decided to try everything available in the fridge, and inoculated all kinds of media to get more information. Results came out along the expected ways (negative fermentation and MR-VP among others), but the nitrate was negative. The sequence had been pointing at a weird fish-killing Pseudomonas, but  P.plecoglossicida is nitrate positive. My bug did not seem to be fluorescent under UV light, so P. fluorescens seemed to be out of question, but it is nitrate negative, so I inoculated a couple of gelatin deeps and am waiting for the results, gelatinase production being one of the distinguishing features. I have been around labs enough to not get too excited about weird stuff, and I just cannot believe that the soil at Yale University is harboring some kind of special bug. But at the end that is not so important…it is the antibiotic production that matters. So I am also repeating the spread/patch plate against four bugs, including E. coli & P. aeruginosa. After that, if things repeat, then I will have to make some decisions about what to do next.

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West Coast & East Coast kombucha. Just refilled the East Coast one, that’s why it is in the bottom.

As for kombucha, as they say, no news is good news. It has been growing placidly in the corner, by now in its 3rd of 4th tapping. The first ones had 1/2 cup of sugar per liter, and were very sweet in the beginning and very tart at the end. Then I halved the sugar amount, and the resulting drink was way mellower but also bland. I had started experimenting with diluting the strong concoction with fruit juices, which seems to produce a pleasant flavor. Another option is to add chia seeds, which give a nice chewy quality to the drink not to mention the added protein. I finally relented and bought a set of big jars for the cultures. The West Coast one has been steadily growing, and I can see it becoming equal to the Yale one, especially after today as I started to give away babies (tearing away a layer). Next stage will be experimenting with different teas and sources of sugars.

My next post will be about scaffolding assignments for a General Bio class. I had a kind of epiphany about grading rubrics the other day while banging my head in frustration because of students not following instructions. Stay tuned!

Small World Initiative: update on the babies

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The last day of the Small World Initiative at Yale my roommate and I walked down to the UPS store and shipped a few streak plates home. They were to arrive on a Thursday, the day I had to drive down to San Diego for a couple of meetings, and then I was leaving town on Friday. I asked the labtech to put the plates in the fridge.

I completely forgot that the little box was not labeled anything special, and as normal mail it was lovingly placed on my chair in the office. When I returned to campus the Tuesday after, I started opening the box with apprehension. “It will smell bad,” announced to the labtech as I started opening ziplock bags. “One of them seems to be a Pseudomonas.” Indeed, when the last bag was opened, the musky smell of earthiness surrounded us. However, the babies looked ok. The colonies were big and fat, but the plates were not overgrown.

That same day the 16s sequences had arrived via email, and after a quick Blast, the predictions based on Gram staining and colony morphology were confirmed. One was a Pseudomonas, the other a Bacillus.

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Inhibition zone on the E.coli spread plate around one of the colonies. Broken agar, my bad. Ugly lawn too.

With a microbiology course happening on campus, I looked for bad bugs to test, and found E.coli and Staph. aureus. I did a quick spread/patch plate with the 2 candidates. Today the Pseudomonas was showing a clear inhibition zone with E.coli, and a possible one with the Staph.

So this is exciting, although hardly earth-shattering. As an anthropocentric cell biologist (mea culpa, mea culpa) I knew a lot about antibiotic resistant nasty bugs such as P.aeruginosa, but I just realized there are a lots of other Pseudomonas in the soil that have to do with protection of plants from diseases. And those antibiotics are of the phenazine type. I just got started in the literature research, so I do not know much more, apologies.

That said, I do want to finish the SWI sequence, so next time it will be testing against the whole battery of ESKAPEs, and starting some traditional microbiology. Where is a Bergey’s when you need it?

I know it is not P. aeruginosa, as even when spread on a P agar it was not green. And I don’t think it is P. fluorescens because it does not fluoresce under UV light. Grateful for your advice as to how to proceed.

The other baby, the Bacillus, did not do squat to E.coli.

Still love them both. What do microbiologists say? “You get attached to your bugs.”

Amen to that!

Small World Initiative: what’s next?

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picture showing heads sticking out from a building

Heads watching from one of Yale’s older buildings

Last day of SWI was a tad more relaxed than the previous. After breakfast the four working groups presented their ideas about course implementation as either non majors or  majors general biology, cellular/molecular biology, or microbiology. I was in the majors biology group, one of the largest and most diverse regarding student characteristics and type of course. For some students, this would be their first lab course; for others it would be the second or third. Some courses were planned as pure lab, others had lecture components. The non majors group was also very diverse and with the added burden or beauty of teaching students not heading to sciences. Our presentations reflected that, mainly concentrated on process skills and providing examples of approaches we thought useful, such as scaffolded graphing assignments, or a stepwise progression in poster presenting skills from critique to design, with plenty of feedback opportunities. Our group’s most popular contributions were related to quantitative analysis, with emphasis on introducing graphing and statistics applied to colony numbers and diversity. That science is indeed a collaborative process was evidenced by the fact that these ideas came from the ecologists in the group. On the other hand, the cellular/molecular bio and microbio groups presented more detailed and structured proposals, delving into rather specific areas such as materials and stock cultures, ASM guidelines, and even tentative schedules.
After that, it was Jo Handelsman’s turn to give a kind of final words and to outline strategies. She used the word “historic” and expressed hope that this workshop would indeed start a major expansion of research based  science courses in the field of biology, joining others such as SEA-phages and of course GEP. We participants of this first cohort will be known from now as SWIPPs (from SWI Pilot Partners), which provoked some chuckles.
As we go home, many of us plan to pilot the course or parts of it soon, while others well implement it next Spring. Working groups of volunteers were formed to tackle tasks such as standardization of test cultures, materials, methods, sample and data collection, web page design, and social media presence.
Picture taking and lunch followed, then we returned to the lab and the pungent odor of soil microbes emanating from the incubator. My two samples, picked out as afterthoughts, looked pretty after being streaked, and I put parafilm around their edges to ship home.

Plate showing a petri dish with beautifully glowing microbial colonies

“You get attached to them, the little bugs.” Unknown microbiologist, any century after the microscope.

Another treasure that I am bringing home is a Falcon tube with a brownish mass: a kombucha starter. Besides being a fermented and supposedly healthy probiotic drink, it is also a great model of a microbial ecosystem, as described in Ashley Shade‘s research. Once I knew she was in the same building, we communicated via email and then chatted in person about fermented foods and drinks. Can’t wait to plop the stuff in sugary tea and see it grow. As one of the microbiologists said to me: “you get attached to your bugs.” She was the one who found out the rules to mail biological samples, and in the afternoon we walked together to the UPS store to send our plates with the babies home. The kombucha  however crossed the TSA inspection in the ziplock bag together with my contact lens fluid. I guess the tube looked like a funky container and nothing else.
I write this on the Atlanta-Orange county leg of my flight home. The four last participants in town who had to wait for a crack of dawn flight shared a great tapas dinner at a place named Barcelona Friday evening, and today we are heading back to campuses scattered all over the country. Whatever happens in one year, rest assured that something will be presented at the ASM meeting, or ASM-CUE. And materials well emerge soon: from a Yale newspaper to professional channels, we expect the word to spread.
Dear readers, would you consider implementing SWI, or other research-based science course?

The unbearable fun of science

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Image

Some of our extracts under UV light.

The Small World Initiative workshop has been galloping rapidly for the past 4 days. The group (24 biology educators from a variety of institutions) recreate the activities that students are to do in the prospective research course. The soil sample spread on the plates produced a number of colonies, pale bacterial ones that later became yellow or even fluorescent, intermingled with a haphazardly growing mucous bacillus whose name I have forgotten. We picked and patched, making master plates; and then spread and patched, confronting our colonies with harmless relatives of ESKAPE organisms (Enterococcus, Klebsiella, Pseudomonas, Acinetobacter, Staph, Enterobacter). We got all excited when discovering zones of inhibition, we retested, Gram-stained, did colony PCR to make a 16s sequencing, extracted with organic solvents, and as of today, we are eagerly awaiting the results of a spot assay of the aqueous phase against one of the bad guys.

Most of our time, however, is spent either in small groups developing our learning goals and objectives, as well as assessments; or sitting in a very cold big room to learn more about backward design, diversity, or IRB forms. The rest of the time we eat, and drink copious amounts of coffee or tea.

To make things more interesting, Jo Handelsman just became nominated for the position of Associate Director for Science, Office of Science and Technology Policy of the Administration. It is to the team’s credit that in spite of the news and all possible changes this may cause, things keep running smoothly. As smoothly as possible with 20 something scientists packed in a lab playing with microbes while learning new techniques. The course methodology incorporates such a variety of techniques, that most of us have something to learn.

The blog site I referred before is closed now for participants only, as we are uploading our working documents. There will be an official website with all the bells and whistles coming out in the near future.

Besides the workshop curriculum, we are learning lots from each other. We exchange ideas and tips, show each other techniques, software, apps, and tricks we use in research and teaching. I installed MEGA yesterday thanks to my roommate, and have been happily making little phylogenetic trees. Can’t wait for the sequence of my two finalists to come out.

And that is all for today. In a small scale (our workshop) we are having fun doing science and are worrying about how to make it happen home. In the large scale it is fun to think that somebody who knows science and science education well will be in such a high and responsible position. Personally, I hope that we are reaching a tipping point, where the powers to be realize that science education HAS to change and adapt to the realities of our century.

Is it time for dinner so soon?

2013-07-31 11.24.10

I can see some inhibition zones! Spread and patch plate.

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