PCR happy ending


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.

Nudging my Dad

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Dad and I, in the very beginning.

As I write these lines, my dad is already up in Havana. He stays up late at night and gets up early, sometimes in the middle of the night if inspiration hits him. After breakfast he will sit and fire up the oldish PC and check his email through a dial-up connection. As most people in Cuba, he does not have access to internet at home, but he can send and receive emails, the main way of communication between us. Through the years I have subscribed him to newsletters, and copied and pasted big chunks of information and articles into bodies of email for him to read. In his 80s, my dad is still very curious about the world.

That is one of the reasons I subscribed him to my blog. Other reasons include an added motivation for me to write regularly. But my main hope is that he will be eventually writing his own blog and that way widen his circle.

ana papi sepia

Dad and I, last year.

My dad has many stories to tell. He wrote an autobiography some years ago where he tells about his “lives in succession” from his native Spain to Cuba. After the first book, the writer’s bug got him, and he has several books in the making related to his experience in criminal justice and history. But we kids are currently enjoying his newest work, coming in email installments, about the adventures of a globetrotting character born in the island of Lampedusa.

In the book The End of Big, Nicco Mele tells the story how his elderly father wrote a book by transcribing his letters from WW2 and publishing it as an ebook through the internet (with his kids help, of course). Besides being a fascinating read for the family, it turned out a way for the father to connect with a number of people who had similar stories and experiences, enriching his social life.

My dad taught me many of the important skills in life, from swimming to driving, from how to give IM injections to how to avoid getting drunk. He introduced me to chess, the love of philosophy, and in general the joys of intellectual pursuits. Over the years we have been close or far, depending on which corner of the globe I happened to reside. And communications have ebbed and flowed depending on the demands of life.

But as of today, I am enjoying this common interest in writing, and I do hope we will become blogging buddies in the near future. Looking forward it 🙂


The small details that make love great

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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.

Lecture cleanup

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Becoming superhuman?

I happened upon Tim Ferriss’ book The 4-hour body a year ago. I was desperately looking for a system that could keep me more or less fit in spite of the regular academic crunch times. I confess I do not have the discipline of finding some time for exercise no matter what. My tendency is to work on a task basis, which means taking no breaks until done. That meant hours and hours sitting in front of the computer, and it was taking a toll.

The book was fun to read, and the diet and exercise advice worked for me. I do not follow it as rigorously as before, but it is still the basis of how I eat and how I exercise. (Just to be clear: I am not endorsing it or anything, just sharing that it worked for me).

If you are not familiar with Tim Ferriss, he is a sorts of accelerated learning expert as explained in his bestsellers (the latest being The 4-hour chef). In a self-deprecating and humorous manner, he shares his ways of mastering new skills, from new languages to gourmet cooking.

I was thinking about it as I started reviewing my lecture powerpoints for my current microbiology class.

One of Tim’s main points is that most activities can be mastered in an accelerated optimized way. As my teaching takes place in an accelerated environment, most of us teaching at NU explore learning strategies supportive of this approach. Traditional lecturing is not really feasible- many of us combine short lectures with group activities, audiovisuals, or more active approaches to chunk the content and keep students’ interest.

However, as I went over my lecture slides, I started noticing things. Apologies in advance that I am going to talk now about metabolism…that was the chapter I lectured on.

For starters, there were slides that did not make sense where they were except for me. For example, after an introduction to metabolism and metabolic pathways, there was a slide about oxidation-reduction. I know that this is an opening to what is coming next, which is the topic of carbohydrate catabolism. But why talk about oxidation-reduction in abstract first, when I will have to explain it later anyway when referring to what happens during cellular respiration?

The second thing I noticed was lots of material irrelevant for the particular student audience (this case pre-allied health students). Yes, it is pretty to show the names of the components of the electron transport chain, but in all honesty, I do not remember many of them (and I was a biochem major).

Tortora chapter 5

Oxidative phosphorylation, summarized.

I started cleaning up my slides. I took away all information that seemed superfluous, and arranged the slides so there was a clear narrative from beginning to end.

Lecture was yesterday. I followed the narrative of the slides, repeating several times the basic messages. Once students got the relationship between oxidation, reduction, electrons, and hydrogens, I made them look at the metabolic pathway diagrams and count carbons, NADHs, and ATPs. I was stoked seeing their faces, deep in thought as they followed the path of energy. I asked them to turn to their neighbors and explain things with their own words. They asked questions. They were engaged. This chapter is one that I usually dread to teach, but last night was an exception.

We often say “less is more,” but it is not always easy to let go of material or approaches that seemed necessary to achieve something. How much of it is really necessary, and how much of it is tradition and inertia?

Dear educators, how do you achieve a balance between the content that one is supposed to teach and what is really relevant to teach?

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 🙂

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