Eureka?

 Since my last write up in January 2020, my 13 flasks have been mostly growing very well. Biofilms have been forming and have been shaken off and then new biofilms forming. I have taken regular photos to track progress, though due to Covid lock downs my visits to the lab and work with the flasks has been less regular than I wished.

I was delighted to find that the cultures survived this lack of attention. It occurred to me that not only was the process of shaking off of biofilms important to the process but also everytime it was done it allowed a change of air around the filter cap flasks and that could be important too. I don't know how quickly the air is depleted in the box holding pretty tightly packed in flasks, but the sense of a vaccum when opening the box made me think that the air had been depleted somewhat.

Over the last few months it has been clear that the biofilms were growing more slowly and starting to disappear, so the big question was, had tuberculomucin (Tbm) developed? 

The test fot Tbm is a couple of drops of 20% acetic acid. In previous tests of some old cultures which had been left in an incubator without attention we found that there was a reaction of cloudiness/white bits whereas just growing medium had no visible reaction. However Friedrich had clearly stated that the cloudiness reaction was not what we were looking for and likely indicated the presence of albumins and was not therapeutic and should be disposed of. What we were looking for was a coherent clumping after shaking.

At the beginning of December we were at last able to do the acetic acid test on the growing medium in the flasks. Imagine my excitment when we got a clear clumping reaction in those flasks which had been regularly shaken and not in those which either hadn't grown well or hadn't been shaken!

Take a look at the shape in the liquid. This is a few minutes after adding 2 drops of acetic acid. The vial has not been shaken.

This vial has been shaken and the form is more definitive

11 days later we took photos again

 Vial W3 is Weleminsky broth with H37rv strain of TB - this grew well, though took a longer time than in Oxoid nutrient broth to get going, and has been regularly shaken. Lots of clumping - hopefully this is Tbm. W4 has a clinical TB strain which never grew very well and has not been shaken. No indication of Tbm.

So I'm pretty excited. I really think we have Tbm!

Judy Weleminsky

Recreating Tuberculomucin - a citizen science project

When this blog was set up in 2012, we recognised the ambitious nature of the tuberculomucin project. Over the next five years, nearly all of Friedrich Weleminsky's German papers and archive from the Charles University, Prague, were sourced and translated. The translators were Rita Drobner, Stephanie Eichberg and Krystin Unverzagt. Professor Paul Weindling offered further information about medical refugees from Czechoslovakia to the UK; and UCL donors, John and Ann-Margaret Walton, generously funded the early laboratory and historical research. Jennifer Willis, a Human Sciences student at UCL, wrote her BSc dissertation on Tuberculomucin (2013), and Carole Reeves published the first paper in Transactions of the Medical Society of London (2015).

Then, in 2017, Judy Weleminsky, working in Professor Tim McHugh's Centre for Clinical Microbiology at UCL, became the project's first 'citizen scientist'.

Can people without a specialist background do 'serious science'? Citizen science, according to a report in Nature (2018) 'is growing bigger, more ambitious and more networked.' Many citizen science projects involve data collection by large groups of volunteers - over 100,000 in the case of Galaxy Zoo - which analyses images of distant galaxies. Individual citizen scientists like Judy are a rarity. She has shown remarkable tenacity and ingenuity in pursuing her goal of recreating tuberculomucin as it was first made by her grandfather over a hundred years ago. Judy's blog postings give a good indication of the difficulties posed by this work in a twenty-first century laboratory but it might be argued that only a citizen scientist, unfettered by the rigid protocols of modern biomedical research, could attempt such a project.

Historical research has determined the context in which tuberculomucin was successfully produced and tested. The increasing prevalence of multi-drug and extremely drug resistant tuberculosis has established an imperative to explore all routes to new treatments for this major global health problem.

Shaking off

From Judy Weleminsky - blog 5

 Friedrich's instructions are to shake off the biofilms every 3 or 4 weeks, leaving a small amount on the surface from which a new biofilm can grow. As mentioned in the previous blog, other scientific accounts of growing TB biofilms seem to stop at 5 weeks. We initially started 8 flasks to grow H37rv biofilms and at 4 weeks growth we tried the 'shaking off' with 5 of the flasks leaving three flasks unshaken.

We were pleased to find that even with quite vigorous shaking that while most of the biofilm swirled around in the liquid and then dropped to the bottom of the flask, some returned to the surface. And three weeks later we were even more pleased to find that the biofilm had regrown in those which had been shaken and we were able to shake them off again.

Close ups of the biofilms in flask 3

For contrast here is the picture of the unshaken biofilm which is forming a great mass sinking under its own weight.

I would love to know more about the changes that are happening in the biofilm but there are no resources in the lab for me to test these easily and we are not yet at a stage where the lab is willing to commit significant other resources to this work.

Growing TB biofilms

From Judy Weleminsky - blog no 4

Eventually, after proving myself to be a somewhat competent microbiologist and able to follow the lab safety rules, it was agreed that we could start trying to grow TB according to Friedrich's instructions. My work with BCG had persauded me that the freshly made growing medium according to Friedrich's instructions was the best, but also I had quite a lot of problems with contamination. This  problem followed me into the CL3 (containment level 3 - extra security) lab and a number of our initial attempts to grow TB had to be discarded.

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Oxoid Nutrient Broth

While waiting to access one of the additional ingredients I required to make the growing medium - Peptone - I had used some ready made medium called Nutrient Broth and made by Oxoid. This had proved to be very good in growing BCG so we agreed to give it a try for the TB and at the same time reduce the likelihood of contamination by changing our methods of adding the additional ingredients.

Despite the TB growing well it failed at first to form biofilms - films of TB colonies on the surface of the liquid which is what we needed. With a little experimentation we found that one issue seemed to be the amount of TB we added to our growing medium. 7ml of recently flagged (using a MGIT) in 100 ml of medium proved to be successful and working with the most studied strain of TB - H37rv - we were rewarded with a nice biofilm after three weeks of growth in the incubator.


This flask shows the growth of the early biofilm after four weeks growth. and a close up pic of the biofilm

Close up of the biofilm

It is interesting to note that most reports of TB biofilm growth only cover a period of 5 weeks growth as for example this study by Ojha et al in 2012.  A big issue in TB treatment and other diseases is the antibiotic tolerance which is often associated with the formation of biofilms. Biofilms provide an important reservoir of cells that can repopulate colonized sites upon removal of drug treatment. The biofilms are also linked to drug tolerance as in this study so it is clearly important to study the underlying mechanisms.

As the biofilms in vivo are long lasting, it is curious that the work with biofilms is over such a short period. The issue appears to be that in addition to the fact that biofilms have not in the past been extensively studied, when the biofilms are grown in vitro in the lab after a couple of months the biofilm appears to sink under its own weight to the bottom and the biofilm is lost. As I explain in the next blog, Friedrich's methodology has circumvented this problem.

What on earth would substitute for a Swan Necked Flask?

From Judy Weleminsky - blog no 3

Friedrich specified the use of a swan necked flask for growing the bacteria and although we have no picture of his flasks, I imagine them to be similar to the glass flasks in the picture below of Louis Pasteur  in 1862 disproving ‘spontaneous generation’

Pasteur with his swan neck flask,

Well there was no way that I would be allowed to use such easily breakable and bulky glass flasks in a modern lab, but I nonetheless needed to find a suitable substitute. Key requirements were that they be of sufficient size to hold at least 100ml of growing medium (that’s pretty large in microbiology terms) and allow air or oxygen to get in but not allow water vapour or airborne bacteria out! A bit of hunting and I found a plastic flask with a filter cap which allowed some air in but didn’t allow the bacteria out. Unfortunately, I found it allowed a considerable amount of evaporation and needed to find a way around this problem. 

I trialled keeping standing it in water - but that was problematic for sterility and also did not help very much. Eventually when we were growing the TB in the CL3 lab we filled big plastic box with the flasks and that seemed to do well both to reduce evaporation and to keep the flasks well contained and easy to move out of the incubator and into the cabinet for observation and shaking.

Real beef for the growing medium

From Judy Weleminsky - blog no 2

My initial training was with BCG – the live vaccine, which is used to inoculate mostly young people. It can be grown in a containment level 2 lab which has some biosecurity and is suitable for moderate hazards but not too high as it is not dangerous to humans. BCG is an attenuated version of Bovine TB.

pippettes

 While I was trying to get my head around sterile techniques, using pipettes, calculating and measuring small amounts, using petri dishes, making the growing mediums used in the lab, and awaiting the time when my skills would be considered sufficient to work with live mycobacteria tuberculosis, I also wanted to make a start on the first steps in my research. So, I persuaded the lab to let me trial making growing medium according to Friedrich’s recipe.

Powder used for most culture medium

The growing media 

usually used in the lab is made from dry chemicals. However, Friedrich was clear that this would not work for growing mycobacteria tuberculosis over many years in the hope that it would produce the biproduct – Tuberculomucin – which is the therapeutic substance. I found that the idea of growing TB for long periods was completely against the grain of current microbiological work. Most of the work in the lab involved growing the bacteria for weeks not years! 

However, I got permission to trial making growing medium, starting with fresh minced beef. My professor thought about it and said – I’m ok with a little cooking in the department! I bought some lean mince from the local shop, assuming that this wasn’t something I could order through the provisions ordering system in the lab!

Mince beef before autoclaving

I soon found that working with real mince in a lab had its own logistical difficulties. Despite the comment about cooking by the professor, there are no cooking facilities in the lab. But there is an autoclave which uses heat and steam to sterilise liquids and equipment and therefore seemed a reasonable substitute for the instruction to boil the beef for two hours. I got training in how to use it, but I found the glass bottles which they use to contain the substance to be sterilised were very awkward containers for putting mince beef in and ever more difficult for getting it out! 

Autoclaved beef before and after straining

And filtering the liquid to get a clear bouillon was also a challenge given the large quantities Iwas attempting to produce and the tiny paper filters which took an interminable time to use. Eventually, I worked out ways of doing things but it was quite a learning curve. 

Filtered beef broth

I needed to add a number of chemicals to my bouillon and then adjust the pH to get it to 7.6 as specified by Friedrich and then autoclave it again to make sure it was sterile. Nonetheless I still had problems with bugs which weren’t supposed to be there.

But the positive thing was that the BCG was clearly happier growing longer term in my growing medium than it was in the usual lab formula – so a first tick for Friedrich’s directions and methods. However the BCG did not form a biofilm on the surface of the liquid and this was an important stage in recreating the Tbm as a biproduct.

Recreating Tuberculomucin – getting started

From Judy Weleminsky - blog no 1

Dr Friedrich Weleminsky left an excellent unpublished paper on how to make and use Tuberculomucin. His granddaughter, Dr Charlotte Jones, updated it and attempted to get medical researchers interested. Thankfully University College London, where she had studied as a doctor, expressed interest. 

They arranged for Carole Reeves to do some historical research and her paper Tuberculomucin: A forgotten Treatment for Tuberculosis had a great reception when it was presented to the Medical Society of London on 13 April 2015.

 UCL also agreed to do some fundamental microbiological research, but the initial research they carried out failed and they discontinued it.  

Charlotte was finding it increasingly difficult to pursue progress on the research due to being in her late 80s and living in Wales, so I took over the mantle. I am Judy Weleminsky, another granddaughter of Friedrich’s but 23 years younger and I live in London. The lab explained that they didn’t have the resources of money or people to carry out further research.

Judy Weleminsky

Reading Friedrich’s paper it seemed to me that the process for recreating Tuberculomucin was clearly laid out though it was also obvious that it would take many years to achieve anything. As I am retired, I offered to do it as a volunteer and much to my surprise they agreed! I offered to commit to working for a number of years as the process involved culturing the bacteria over two or more years, so I suggested 3-5 years at 1 to 2 days per week and that seemed acceptable.  

Unlike Charlotte who was a GP during her working life, I have never studied medicine and the last science I learnt was as a teenager doing A-levels. But I was happy to give it a go and in October 2017, I started getting training as a microbiologist.