Thats all folks!

Hello all,

My time in Uganda is quickly coming to an end. Tuesday I leave for Tanzania for a week and a half and then back to the good ‘ol USA-----it has been a while (lets hope the British Airways strike ends by then). Given that, yesterday was the last day that it was worth collecting samples, and even though it would have been nice to go out with a bang, there were no patients. Again, good for the kids; I can always take solace in the fact that if I’m not busy, well the kids are healthy and that is good.

Although I haven’t blogged in a while, it has been an eventful (as always) last week or so. Labwise, I got the –80 freezer up and running----it involved plugging it in for the most part. It has maintained between –83 and –78 for the past 5 days, so I think it is working great (even despite the periodic power outages). The liquid N2 transfer vessel arrived, which was the last item on the list of things that were shipped from the US. Thus, everything that was on our list, 3-gas incubator, biosafety cabinet, refrigerated centrifuge, -80 freezer, millipore water purifier, pH meter, pipet aids, liquid N2 storage tank and transfer vessel, are here and fully operational.

I spent most of this week just culturing, writing up protocols, and demonstrating and then observing Sam and Moses culture. I think some practice and mistakes, and then they will be proficient in culturing parasites. On that note, Sam doesn’t believe that they can afford to purchase the N2 gas when I leave, and thus he is thinking about finding some candle jars. This is not my domain, but despite the problems with the Oxy-gas people (see below, again), I think results are much better with the controlled incubator----that’s for the bosses to decide. Certainly, long-term, we should setup our own N2 generation system.

The only setback this week was, once again, the N2 gas. I called ahead of time to have two good tanks reserved (one shouldn’t have to fill two tanks at a time, that is why there is a backup, but the backup leaked three quarters before I realized it was leaking and needed some thread tape). Moving on, I called on Monday to have tanks delivered on Wednesday. I called again Tuesday to remind them---but oh no, now they can’t deliver on Wednesday because Wednesday was just deemed a national holiday to remember the 8 Ugandans that died along with Dr. John Garang, vice-president of Sudan, in an apparent helicopter crash (or hijacking or shot down by a missile or sabotage, all depending on which paper you read and who you believe). Anyways, so I asked for two tanks to be delivered early Thursday morning. Well, after waiting until noon, I called Oxy-gas and asked where the delivery was, they said the delivery truck had broken down, “can you come pick them up?” So I call Nuhu, one of the drivers here, and went over to pickup the tanks (again a 3hr ordeal). Bring them back and set them up-----great, they are at 9% O2. Better than the 14% before, but still not what we need. Anyways, it seems the 9% O2 hasn’t killed the parasites, and the two tanks lasted until today so all is well.

The other excitement this week was finding out that the Uganda customs police confiscated the jacket that my parents sent me to climb Mt. Kilimanjaro. They said it looks like a potential UPDF (the Ugandan Army) jacket. I guess it is likely green, but that, I’m sure, is the only possible resemblance. When I called the customs agent, he said, “well, if you come down here, maybe we can work something out.” Ahhh, yes the old hold it ransom trick. I already paid 80% import tax on the value of the shipment, and so I decided I will not go down there, especially when down there, involves a 45minute drive to Entebbe. After, I hung up the phone and stormed out to take a walk to calm down, Vicky (an intern here for the summer) kindly called the man back and figured out a plan where I can pick up the jacket before I leave the country-----thus the jacket never enters the country, I don’t pay more tax (officially) and I get my jacket. She is so kind!

I guess other than that, everything has gone well. The preliminary results of the culturing are as follows. We had 8 of 9 falciparum samples mature to schizont stage parasites, which is certainly a better percentage than expected. We encountered 1 episode of vivax malaria and 1 episode of malariae malaria (it was fun viewing the differences in the microscope, yes I’m bringing back the slides). Neither of these samples grew in culture. We were able to culture 3 new strains to the point of freezing them down, and one of those strains we froze down 2 times. Thus we have 4 different samples to attempt restarts from in SF. In fifteen days, we collected 11 out of 17 possible non-asymptomatic samples. We missed 6, one because we were too far away to come and get the sample, and 5 because the doctors periodically forgot to inform us---although, they were pretty good about this (4 of these were on this last Sunday when they just thought I wasn’t working). The one sad note is that one of these missed samples had a parasite density of 558,000, which is almost triple that of any other parasite density that we collected. When patients have a parasite density of 500,000 or above, the doctors immediately put them on Quinine. With this high density, I likely could have frozen the sample down immediately and retained some for culturing as well, too bad. Moving on, thus, out of 9 falciparum samples we were able to culture 3 isolates well (to the point of freezing down, ie >9% rings in 0.5ml RBCs). That is 33%, which I think is good. I think this percentage would have certainly increased if we didn’t have such fluctuations in controlling the %O2. Also, we started culturing just when the number of malaria cases decreased fairly dramatically---into the dry season. When we arrived, they were seeing 20 patients a week for malaria. As you can see we observed only 17 during the 15 day period we were up and culturing.

I have samples of every day, for every culture to genotype when I get back to SF. It will be interesting to see whether, and if so, how the genotype changes during adaptation to in vitro culture. Do we start with a multiple infection and select for one strain, etc?

As for the samples, I am trying to arrange shipment in our new dry-shipper to San Francisco, instead of bringing them with me on the plane. I have a contact, Jimmy at World Couriers that hopefully will take care of everything on Monday (this is who Walter Reed uses). They allow net-30 billing in the US, so I thankfully won’t have to front the money here, or figure out how to get it. I’ve put Joe and Felix as the people to contact about billing.

Given the above, I certainly think we have accomplished our goals, which is great given the numerous obstacles that one ends up facing here. The lab is a lab, with benches and air conditioning and operating equipment. And, although we did not isolate as many samples as we would have liked, we certainly have demonstrated proof of concept. More importantly, it has been a wonderful, yes trying at times, but wonderful experience nonetheless. I certainly have gained a lot from learning how things get done in the non-western world. I probably still haven’t learned enough patience, but I think I’m making progress, albeit slowly. I certainly think there are wonderful opportunities for further research here in Uganda, and now we have the facilities. It is just a matter of sitting down and figuring out what are the most feasible and interesting questions.

I would like to thank the higher-ups, both Joe and Phil for providing the support for this little endeavor---brain support and financial support. Also, Sam and Moses for helping us setup the lab, and teaching me some new microscopy tricks. As well as everyone else here and in SF, working on the MU-UCSF Malaria Research Collaboration, particularly Kenneth and Catherine for being so helpful with everything, especially all the shipping issues.

Again, I leave Tuesday, the 16th, for Tanzania and I’ll be back in SF on August 30. In Tanzania, I’m setup to climb Mt. Kilimanjaro and then I’m likely to head to Zanzibar, but these plans are flexible. I am leaving again on September 2 to visit my family in WI---so I truly won’t be back working in the lab until the second week in September or so.

Jenny, Terry, and Dustin got back from their Ugandan safari all intact and healthy Thursday afternoon. They have some wonderful stories and pictures. They headed out today to go rafting on the Nile, and then they fly back on Monday. I assume they’ll be back in SF on Tuesday, SF time.

With that, I sign off, and leave this blog up to the next crew of Derisilab people that head to Uganda (although I may update the blog about my travels in Tanzania, depends on internet access).

See you all soon,

Adios,

Nate

As for the lab

Once again, on the work side of things...Things are going fairly well, despite a drastic slowdown in the cases of malaria (again good for the kids, bad for us). I have not been able to freeze down any more cultures yet, but I hope to freeze down two tomorrow, assuming they reinvade as expected. If so (big if), that would mean we've been 33% effective at culturing the parasites for at least one invasion cycle after being drawn from the patient. Furthermore, one invasion cycle seems to be all we need to get enough parasites to freeze down, at least for samples that start with high parasite density. The best indicator thus far of whether a sample will grow decently in vitro, has been the starting parasite density of the patient. Additionally, we have had 9 out of 10 samples mature to schizont stage parasites in vitro, which is better than expected----now, if more would reinvade...

Anyways, other pertinent information is that the -80 freezer arrived. Getting it into the lab was quite an ordeal-----Ugandans do not use dollies for anything, not the 200 pound gas tanks that I carry myself into the lab, nor the gigantic and very heavy freezer. Thus, 15 guys carrying this upright freezer into the lab was fairly interesting (and disconcerting at times as well). Anyways, the freezer is here, now I'm just waiting to get another voltage regulator to plug it in (not going to make that mistake twice). Also, the UV-light bulb arrived, and now we have a sterile hood-----although, we haven't had any trouble with contamination yet--------Must be a testament to Jenny and My uncanny abilities' at sterile technique. I guess the only thing left, that was ordered in the US, is the liquid N2 transfer vessel.

I've been trying to take Sam and Moses through some of the culturing steps, hopefully I will get them more interested and culturing their own flask this week.

That is all for now. Hope all is well,

Nate

Hotel, Laundry, and Tinidazole

I guess it has been a few days since my last blog, so here is an update on the craziness of the past few days. To start with I woke up thursday morning and friday morning absolutely covered in bites, Thursday I thought it was mosquitos, but then again friday morning I realized it was BED BUGS. Oh well, they don't carry any nasty diseases like the mosquitos, but they itch like crazy, and are extremely difficult to get rid of. Thus, I decided that I can't sleep in that apartment anymore, and so I booked a room at the Hotel Catherine, ohhh it is so nice to have my own bathroom again-----and I arranged to have all of my clothes laundered (to make sure I don't bring the little bastards with me). Then I was sitting pretty in my new self-contained single room when my intestines started cramping up again (they have on and off pretty much since I got here). Thus, I finally decided to take some Tinidazole that I had been recommended to take (by the many infectious disease docs working here) and despite a magnetic taste in my mouth, I feel much better-----ie, I had a bit of Giardia tying to survive inside of me that the Tinidazole nocked out.

So, as for life outside of the lab, a new room, clean and to myself, all clean clothes, and giardia free (for now)--------Hotel, Laundry, and Tinidazole has got me feeling much better than a few days ago.

Nate

On a more serious note

Just to update you on our progress, yes we've recovered some ground from our latest setbacks. The new gas tanks did arrive this afternoon, and they are working great! I called the manager at Oxy-gas to thank him for being cooperative and let him know these tanks are working well-------he finally informed me that the last tanks I ordered were filled during the initial operation of the N2 generator, and he said they didn't really know how to work it. But now, everything is good, and the gas should be okay from now on out, I'm assured.

Anyways, the major success we've had is one of our samples proliferated to the point where we could freeze it down. Sample U057, and I remember the little person it came from------I suppose that is inappropriate in a double-blinded study, but for me it is pretty neat if we actually end up studying this sample-------observing the poor child that the parasite was bothering, it kind of brings it home. Thus, our primary goals coming into this project were to setup the lab, get all the equipment here and working, and obtain some new malaria isolates. And, despite the seeming slow progress, and numerous frustrations, I have to say we have succeeded. Stepping back, it is pretty amazing to see this building move from remaining physical renovations like electricity, water, ceiling, shelves, benches, and chairs----all the way to a relatively fully functional little lab in Uganda, both successfully In vitro culturing parasites and conducting PCR to genotype samples at the same time. Pretty neat.

Out of la la land, despite the arrival of good gas today, our parasites certainly took a dive during the 36 hours of high O2. After looking at the slides today, most of the cultures look a bit unhealthy, and certainly did not proliferate as expected. This may be due to multiple reasons, but I suspect the gas. I hope some of them recover and we can turn our isolate into isolates by the time we go home.

That is the update,

Nate

Spoon Saves the Night

So tonight I was working on some genotyping for one of Grant's studies---the first study in Uganda assessing the efficacy of the drug CoArtem, so pretty interesting. (For the malariaologists in the readership, the genotyping is done by looking at the size and pattern of repeats in the MSP2 gene by PCR, and they/we do this to determine recrudescence vs. new infection). Anyways, I setup the first set of reactions, and took them over to the PCR room which appropriately houses the PCR machine. Set the reactions in the PCR machine and started the appropriate cycle. I then turned around to leave, but to my dismay the door wouldn't open. I had the keys in my pocket, and thus I checked to see if it was locked---nope. "How the hell did I get locked inside a room that isn't locked?" I realized that the door handle on the inside was broken and wasn't turning the bolt. Now what? After about 10 minutes of contemplating whether I should break the door down, call and wake Jenny up to come free me, or spend the night on the floor in the PCR room, I finally found what I was looking for. A shiny spoon was lying on the ground in the corner of the room. It was just thin enough to half-fit into the screws that were holding the door handle in place. Thus, I quickly undid the screws with the spoon and opened up the door handle/lock--which then allowed me to turn the knob from the inside manually and I was free. Given the scarcity of objects in the PCR room (new lab and all), I have no idea what a spoon was doing in there, except to save me from a sleepless night staring at the blinking lights of the PCR machine.

Nate

3 days of false hope

After being overly excited/relieved and confident that things are going great--------and for good reason, all four of our samples thus far matured from ring stage parasites into beautiful schizonts, we have once again hit a wall. As alluded to in the last blog, the Nitrogen was running out and the first thing I was going to do on Monday was get some new tanks. Thus, I spent 4 hours at the gas company (Oxy-gas ltd) trying to exchange our bad tank of N2 (our incubator sensor said it was 13.5% Oxygen) and purchase a replacement for our one good tank of gas. The guy insisted on testing our bad tank, and told me he'll let me know tomorrow when he gets done "just come back again tomorrow to pickup the replacement". I said that was not acceptable, and I need to leave here today (NOW) with two tanks of the purest N2 that they've got. He finally agreed to test the tank, and wow, it took about 5 minutes and we had our answer. Their testing method, which was absolutely incredible to watch, did eventually agree with our O2 sensor, said that the tank had between 12 and 15% O2. The method involved about six tubes of Ammonia and Copper Sulphate, an absorption matrix of which I couldn't get the answer to what it was made of, 4 lengths of glass pipes, and a guy holding a rubber tube connected to a bottle containing more ammonia/copper sulphate---of which was leaking and spraying ammonia into the air-------it was a fairly amazing sight, and smell. Evidently, he puts some gas in the system, and then pumps it a bit (that is about all I can describe it as), and where the buble ends up on one of the glass pipes, tells you how much O2 is present-------there are just some man-made hash marks (unlabeled)on the glass pipe. Anyways, he said that the bubble stopped at about 13% O2 and thus his method agreed with our Incubator. Thus, I asked him to test the available tanks of N2 for the most pure, and give me those two. Thus, everything looked great, and after another 2hrs and 8 signatures, on 8 receipts (seriously, people are crazy about paper work and receipts here), I was leaving with two tanks of N2, that I was assured were 98% N2 or better.

I should have known this was coming, but I was still quite dismayed when I hooked up the new tanks to the incubator (of which was at 10% O2), the oxygen level immediately began to climb----and then stabilized at about 14%. $#%%^$#@. I called the manager at Oxy-gas immediately, and he said his apologies, but also said that those were the last two tanks of "N2" they have, but he'll have a better product for me on wednesday. I guess the guy either didn't test them, or tested them incorrectly, or what he claimed was an agreement between there testing method and our incubator before, was just a bit of a stretch.

The way I see it, there are five options, of various probability of occurring. 1. He delivers new tanks early wednesday and it is good gas (least likely, I give it 5%). 2. He delivers new tanks wednesday afternoon and it is good gas (15%). 3. He delivers new tanks early wednesday and it is bad gas (20%). 4. He delivers new tanks wednesday afternoon and it is bad gas (25%). 5. No delivery occurs at all, and I will have to drive there again (35%). Yes that adds up to 100.

On a different note, after maturing nicely, our parasites have taken a bit of a dive, poor reinvasion, but do seem to still be alive----great. This may be normal, or it may be due to the high Oxygen (14%) in the incubator. We did try some other tricks to decrease the O2-----------of which you may or may not want to know, we tried burning candles in the incubator, but they went out at about 16-17% O2. Thus, perhaps 14% is already below what the actual O2% is in candle jars??? Thought about blowing gas coming from liq. N2 into the incubator (decided against that one). Anyways, these weren't working, so we decided to wait.

I guess that is all. Hopefully we'll have better gas tomorrow, and we (well I by then, Jenny's heading out), can continue without worrying about O2. I do think that a Nitrogen generator would work fine here, and it certainly would have been a better option than trying to buy gas from the crazy ammonia/copper sulfate guys at Oxy-gas.

Adios,

Nate

We've Matured...

Today is our second day of having samples hopefully growing in the incubator. Yesterday there were two cases of malaria---Jenny came in and took care of the case in the morning, and I took care of one in the afternoon. There has been one case of malaria today thus far, and I've got it in the incubator. Briefly, our current protocol consists of:

1. When a child has a fever, and their thick smear is positive for parasites the doctors in the clinic text message us to let us know they have a positive diagnosis. At this point, the doctors draw blood from the child. They try and draw a total of 10mLs, 5mLs into an anticoagulant tube (purple top), and 5mLs into a tube with nothing in it (the red top). On a big side note, the doctors draw blood from children on a regular basis as well, even if they do not have a fever. For example, blood is drawn every day 14 and day 28 after treatment was begun. Also, every child gets a routine blood draw every 90 days. Thus, some patients are asymptomatic; have parasites in their blood, but do not have fever. I asked the doctors if we could get this blood as well-----the problem is, if the child does not have a fever they do not read the smear right away. Most of the time those smears are not read until the end of the day or even the next day, and thus the blood has already been taking over to be frozen. Thus, if we want to get some asymptomatic blood, we need to come in and read the slides ourselves. I am still thinking about this, if they would let me (although I have a feeling I'll just be in the way), I could just quickly blow through the routine slides looking for parasites, just before they take the blood away. We'll see-----it also may be of no need if we start getting a few more cases of malaria a day. We have been averaging 2 cases a day, but sometimes there is none, and sometimes there are 6----------having 6 a few days this week would be great (for us).

2. Anyways, after the text message from the doctors, Jenny or I (who ever is on call at that time) come running to the lab with an eppendorf tube. We use a small 5mL syringe to poke into the purple top, then turn it upside down and draw out approximately 500microliters---------definitely 500microliters if the purple top is full as it should be, but if there is only a small amount of blood we accordingly decrease the amount that we take out. We then carefully (remember several of these children are likely to HIV positive) take off then needle and transfer the blood into the eppendorf tube. Then, run back up to the lab.

3. Next, we spin the parasites down, 2 minutes at 1400rpm in our fancy new Eppendorf 5415-R microcentrifuge (it is awesome).

4. Aspirate the serum, and discard, then wash with 1mL of hopefully prewarmed RPMI wash. This whole prewarming media would be much nicer if we had a water bath, which was supposed to be ordered ages ago. As it stands, we just put aliquots of RPMI and RPMI complete in the incubator at the start of the day (hopefully).

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-----------------WOOOOO, POWER OUTAGE--------------------I'll let you know in a few minutes whether everything starts back up all right, biggest worry obviously the incubator.-------------------------Well everything looks fine, the power was out for only about a minute, but the main thing is that the incubator came back on fine, that is no blown fuses, and the sensors seem to be correct, great----the voltage regulator must be doing its job.
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5. Continuing... Next we aspirate the RPMI wash and transfer the parasites into a 10mL tissue culture flask. Add 200microliters of 50%washed blood, and then add 10mL of culture media.

6. Here I take a 300microliter aliquot out, and use 50microliters of that to make a thin smear. The rest of this, 250microliters, gets frozen so that later on we can genotype the parasites. We plan to take a small aliquot daily for genotyping, to watch and see whether the genotype changes during adaptation to culture, and whether in a multiple infection, one parasite population is selected for. For those who don't know, the genotyping protocol that the Rosenthal/Dorsey papers use is genotyping MSP2 by PCR. We, well I since Jenny is heading out soon, may do some of this genotyping here, it remains to be seen.

7. Lastly pop the 10mL TC flask containing the new isolate in the incubator and wait. As of now, we take care of the parasites as we do in SF, change the media once a day, and split when above 10% parasitemia----or so that is the plan, we've only changed media once.

RESULTS THUS FAR:

Today I did smears on our two isolates from yesterday, and they look wonderful. The parasites have MATURED (and so has the lab). High "parasitaemia" actually, the morning isolate has progressed to schizonts, the afternoon isolate has progressed to nice mid-trophs. There are lots of parasites, but I can't get an accurate count----visualizing the RBCs is hard in this microscope----it could be the microscope, I just need to get used to it, or the oil is different (I think we use type A at home, don't we?, here we have type B). Also, since we don't have EDTA or Tris, we haven't made any TE, and thus we have been mixing the giemsa in PBS-----I think I will go to the Walter Reed lab and ask if we can steal some TE (shouldn't be a problem). We'll keep you informed of the parasites progress (and ours).

Well, that is that. The next immediate objectives include: 1. getting another tank of N2. It seems that one tank is going to last only 4-6 days depending on the amount we are culturing. This is dissappointing, but I think it is because the N2 we are getting (from the only distributor in Kampala) is of poor purity. In fact we did get two tanks of N2, but, after thinking again that the incubator was busted, we figured out that one of the tanks of N2 is about 12% oxygen, which is a pretty pathetic tank of N2. Also, it takes more than we are used to, to decrease the Oxygen in the incubator----I think that also is a product of the poor N2, but may just be this incubator also. Hopefully, I can talk to Oxy-gas, the distributor, and increase the purity of the N2 somehow------or at least quality control check their tanks.
Other than that we need: 2. to get -70 freezer here and up and running (it should be here tomorrow), 3. get the UV-light bulb for the TC hood. 4. Write up manuals/protocols on how to use the new equipment and take Sam and Moses through culturing. 5. Get the Cryo-dry shipper charged with liquid N2 and find the FEDEX office to ship it to you guys back home. 6. Continue culturing, hoping we can get some long-term cultures to freeze down in order to have something in the Cryo-dry shipper.

Outside of the lab, I think Terry and Dustin get here on Wednesday this week, and then Jenny and they will head out for their Ugandan Safari (all kinds of fun stuff I'll let her tell you about). I then will stay on here until August 16, of which then I'm, yes, going to go climb Mt. Kilimanjaro, hopefully summit, and then spend a few days in Zanzibar-----I begin the trek back to SF on August 28, arriving back in SF on the 30th. See you then.

Hope all is well,

Nate

WOW

Well, after what can only be considered an extraordinary set of events, we are now up and running again. It started when Jenny and I were all happy and excited to go to work Thursday morning. It was a nice cool morning, it had just rained, and no cars were on the road---thus the air was cleaner than I had ever seen/breathed it in Kampala. There were no cars on the road because Thursday was deemed a National Holiday so that everyone could go out and vote in the national referendum about allowing/introducing political parties (turnout was a paltry 5-10%, and that is probably exaggerating, but as most Ugandans will say, this referendum is meaningless anyway, the country wide referendum is basically for show, its clear that political parties have already formed, etc.). Anyways, it was a pleasant morning and we were excited to start our first full day of just lab work (not building contractoring). BUT, the incubator had other plans. When we arrived the incubator was off, and we couldn't get it turned on. After taking off the cover, a quick glance inside indicated that we had blown a fuse on one of the circuit boards----quite dissappointing given the two hours of work I spent the night before, just to find and restart the incubator on a fancy surge protector. Evidently the surge protector did not live up to its name. Just a side note, the whole lab is supposed to have a giant Voltage Regulator, yet it has not arrived, hmmm, and I've been trying for weeks to get the electrician that has the contract for the building to put in a bunch of extra mini-voltage regulators for our expensive equipment, to no avail, hmmm, hmmm. Anyways, we thought, well a fuse is easy enough to fix, and we'll just purchase the voltage regulator ourselves. After about a 2 hour search for the proper fuse, we came back, popped them in the incubator and turned it on...Nothing. Now what, did something worse happen to the incubator than originally thought? After a bit of worry we tried some other fuses----and got it to work briefly---thus we decided that the blown fuse was the problem, but that our new fuses were just not up to par. Anyways, being a national holiday, we couldn't get an electrician there to help us out with the fuse issue, thus we waited 'till today. Today an electrician came, left, and came back with some new fuses, and well, the incubator worked like a charm, great! Thus, we asked the electrician to set up the rest of the equipment on mini-voltage regulators and ground some items, etc. He left and didn't come back for quite some time, so we went out to lunch. We came back and to our happiness both the TC hood and incubator were up and running on a fancy new voltage regulator. ALTHOUGH, upon further inspection, the incubator was dented in the back, one of the nozzles was bent, and the power plug was hanging out. Also, one of our gas tanks had a different nozzle on it. Furthermore, there were scrapes in an arc-like fashion on the wall. Well, it didn't take too long to figure out that the tank with the different nozzle had fallen over and bounced off the incubator and onto the floor. We asked around if anybody heard anything, and oh yes, they heard a big crash but couldn't find what had happened. Well, the nozzle that evidently broke off the incubator took me two days to find, and having a 150 pound steel tank land on our one and only very expensive incubator, somewhat frustrated me. Nonetheless we have a spare gas tank, and thus we hooked it up, put some new fuses in the incubator, again, and started it up---it is still working fine. The next question was, what happened to the nozzle that I searched two days for, and where had the electrician run off to----his tools were still here. After an hour or two, the electrician came back, and well----wouldn't admit to doing anything, or anything happening. I was quite flabbergasted at this. I know this tank fell over, the wall marks are there, there is a dent in the incubator-----and I know somebody put the tank upright and put a new nozzle on the tank (albeit the wrong kind). Finally, after walking the electrician through the evidence, the electrician admitted that he had accidently knocked the gas tank over. I felt like a father who found an empty bottle of licquor in the room of my child, and the child tried to deny drinking it---------wow, I wonder where that analogy came from.

Anyways, the short story of this very long blog is that everything is working again, and the electrician is scared shit-less of me. Bring on the malaria.

Hope all is well,

Nate

Good News

Well, just excited to have ample internet access so I decided to blog again. We have both fixed the intermittent internet issues and trouble shot the inordinately slow incubator problem (wasn't decreasing the oxygen level below 12.8%). As for the internet, the ISP provider (InfoCom) changed some of our permissions and now we can actual "surf" the web----yet now the wireless router that Heidi brought with her just got fried in an electrical surge, and thus wireless is gone (for now). As for the oxygen, after fiddling for a couple hours and switching tanks, we figured out that one of the nitrogen tanks is, well, about 12.8% oxygen, thereby preventing us from getting below that level of O2. Now all I have to do is move through the beaurocracy to get the tank exchanged--perhaps a whole days worth of work.

Also, it is pouring rain out, so its likely that we will have more cases of malaria than we can deal with in about 7-10 days------the mosquitos will be out tonight.

On the other hand, our first culture, of which we quasi-incubated all night seems to not have taken (to be expected)------There are a couple of rings, but mostly those beautiful black dots observed in dying cultures---we'll see...

Adios for now.

Nate

WE'RE UP AND RUNNING!

Hello all,

Well 4 weeks on the dot since we arrived, and today, we are all up and running. The incubator arrived late Monday. We spent yesterday (Tuesday) putting the finishing touches on the gas connection to the incubator and filling the incubator with water etc. Then, after a 12 hour stabilization period overnight and a restart this morning to get the CO2 sensor working, we are all set (although, it seems it is taking an inordinate amount of time to decrease the O2, hopefully nothing is wrong). Thus, everything has arrived and is "working" except for the -70 freezer (presumably next week), and the UV light bulb for the TC hood (Alissa and Peter in the Rosenthal lab are working on getting that to us). "Working" is in quotes, because many things work intermittently. Despite putting in a separate water line to the millipore machine, we are still having pressure problems. Depending on how full the water tank is, determines whether the millipore machine works. Also, the internet which finally got installed last week, is extremely finicky---for reasons we don't know yet, some websites work and some don't, and nothing is consistent. We need to contact the ISP. Oh yeah, we also don't have a water bath yet, so we are storing aliquots of media in the incubator to keep it ready.

There was one case of malaria yesterday at the clinic, and we went through the motions on how we are going to transfer the parasites up to the lab. Briefly, the clinic staff text messages us when they have a positive diagnosis and we coming running to the lab with an eppendorf tube. Then, the doctors draw blood and we use a tiny syringe to remove approximately 200-500 microliters of blood from the anticoagulant purple top vacuum tube. Then pop the needle off and transfer the blood to the eppendorf tube. Hike up the hill to the lab, spin the blood, wash it once with warm RPMI, then put it in media in a 6 well plate/petri dish/small TC flask.

Now, we're just waiting for a sick child (as most of these clinical studies go, bad for the person/child, but good for us). Thus, we'll start culturing whenever the next positive diagnosis comes.

This last weekend I went whitewater rafting down the river Nile, it was great, and then on Sunday I went with Jenny and others to the Entebbe Zoo and Lake Victoria-----we all had freshly caught and deep fried Tilapia.

Hope all is well.

Nate