Thanksgiving SOP musings

So, what has Thanksgiving and SOP’s have to do with each other? PLENTY! Every recipe you use, is an SOP. Same with every instruction manual, etc. Just as with SOP’s, there are good ones and bad ones. The good ones, may be annoying and pedantic, but they explain every step in the process. Many recipes look like this (a Famous Sidney Harris cartoon)….

A recipe that is like the above image, is one I tried after Thanksgiving. In that recipe, one was using leftover stuffing (like there is any stuffing leftover!) for dumplings. The way the recipe stated how to make the dumpling one wan not sure what the mixture was to make the dumplings besides the stuffing. It took me several readings and sitting down with a beer to really figure out what they wanted.

It was not a well written recipe and not what your recipe (SOP) should look like. Every step needs to be explained and that is so anyone, in your organization (or for recipes anyone in a kitchen), can reproduce what you are doing. This is the key, with SOP’s, BE PEDANTIC but, also use plain language. So many owner’s manuals, for TV’s, etc., are written in engineering language and that does not help the average user.

So, if you need help rewriting or making your SOP’s understandable, GeoMetrick Enterprises is there to help.

David Slomczynski, Ph.D; GeoMetrick Enterprises

Fermentation for Everyone

Fermentation – what a broad topic. People think that most fermentation types are different from each other and they really are not. Fermentation is the production of a product in a tank by usually a mono culture (but not always – Belgium ales, kombucha, etc) be it, fungi, bacteria, algae or yeast. Some have unique aspects, like totally anaerobic fermentation, which is really susceptible to oxygen. But, if you know the particulars of the organism, the basic process is not that different.

The big trick, with most fermentation processes are understanding the what the product is and what parameters (micro-organism biochemistry) are needed to produce it. These are considered the parameters of the process. For example, there are many bacterial enzymes, such as alpha-amylase, which are produced by starving the culture for carbon (discussed in papers such as Regulatory factors affecting alpha-amylase production in bacillus licheniformis. ). Knowing the fermentation parameters is important as is what was discussed in 3 previous posts, cleanliness and contamination – B. cereus about contamination – part 1.

An example of dealing with process parameters:  long fermentation times can have issues with contamination.  The longer the process time, the more susceptible the process is. This is where understanding the process and the micro-organism’s characteristics can help define the proper parameters. For example, I performed a 7 day fermentation to produce an enzyme I was interested in studying. The fungus I was studying was grown at a low pH (~pH4.0) to help prevent contamination. Since I understood the growth and  biochemical characteristics of the organism in question, it was easy to determine the best way to alleviate possible contamination and this was by lowering the pH of the initial fermentation broth. This helped to relieve any possible contamination issues. This is just one example of how understanding the parameters can bring you to a successful fermentation.

If you need help with fermentation design, scale-up, or contamination issues, GeoMetrick Enterprises can help.

David Slomczynski, Ph.D; GeoMetrick Enterprises

Calibrations: The More the Merrier?

Actually, more isn’t necessarily better but none is not an option either.

Calibration curves are used to actually determine concentrations of compounds of interest in your samples.This idea being that the lowest and highest unknown concentrations are bracketed by at least 2 standard concentrations from your standard curve. https://terpconnect.umd.edu/~toh/models/CalibrationCurve.html  If a unknown is outside you calibration range, then you need to redo, to bracket that concentration in your curve, as long as the curve is still linear. If the curve cannot be increased, then dilutions are needed to bring the unknown concentration in the calibration range.

One can talk about the time interval between calibrations  (weekly, monthly, etc.). This interval depends on how complex or dirty the samples are. If degradation of the separation is occurring, then a calibration is warranted. EPA test methods, in particular, say what the recommended calibration interval is. Also, if the column is removed for any reason, then a calibration is warranted.

Also, there is the use of duplicate samples, calibration check samples, and spiked samples that can be used during a run. These samples are to access the viability of the calibration curve.  All of these should be used to some extent.

There is quite a bit of information in this post. The point is, one should not ignore these points because they are hard or make analysis more complicated. These should be used to better the analysis.

David Slomczynski, Ph.D; GeoMetrick Enterprises

 

A Reflection on Antoni van Leewenhoek’s Birthday

Today marks the 384th Birthday for the Father of Modern Microbiology. Many things have changed but many things still remain. We have DNA sequencing, genetic engineering, and other sort of marvelous technology yet, there are estimates that we only know less than 1 thousandth of  1% of all micro-organisms out there – http://www.newsweek.com/new-estimate-there-are-1-trillion-species-microbes-earth-454714   .  This fact is lost on most lay people. For all our advances, there is a whole universe out there that we know nothing about.

We also think we are the masters of this planet and that is probably far from the truth. Micro-organisms multiply and adapt much more quickly than we can. If you want proof, look the anti-biotic Staphlylococcus strains out there. They are getting increasingly more resistant to all the powerful antibiotics we can produce. Maybe this is a sign we need to co-exist in this world rather than trying to bend it to our will.  After all, there are more of them, then are are of us.  🙂

David Slomczynski, Ph.D; GeoMetrick Enterprises

Literature Searches – Where Computers have Failed Us

This topic really is tough for people to understand.  It is so easy just to go into various search engines and type in a topic and get out information but,  that is only PART of the information out there. So much information is located in physical media (books, journals, etc) that people have ignored by it not being ‘convenient’.  In the science fields, many researchers do not go back far enough in the literature and this is to their detriment. There have been some amazing science problems worked out and discussed in older papers. Just using only the last 20 years of research or so, means people are using an arbitrary point of reference. Yes, older literature means you have to look through actual physical media and not on a computer screen. I think researchers should not be intimidated by it, after all, the earlier researchers had to do it that way also.

I will give an example. In my MS and PhD theses, I referenced a paper from 1883. This was the first scientific paper describing the actual enzyme I was working on. Why was that important? The researcher, Yoshida, described the enzymatic reaction in a very succinct and beautiful way. Remember, this paper was published before the term ‘Enzyme’ was coined, so Yoshida used the term ‘diastase’. Beyond being historical, using this reference also showed how much interest there was in this research topic.

In another example, I found work that was done in the 1940’s by the USDA that superseded some of the research I was involved with which proved it was not unique or novel. Going back to the original literature, showed how people have misinterpreted the original work and did not read the original close enough to actually understand the research.

Literature research is important for both research and for patent purposes. This has to be one of the most important of all the work the researcher has to do. “Due Diligence” in literature search can save researchers time from re-inventing the wheel.  This is why it is important for researchers to do as much literature research as possible.

David Slomczynski, Ph.D; Geometrick Enterprises

HPLC Method Development around the World: Why it is so hard

Taking a method that was developed and published from another country and trying to use it in another country – why should that be so hard? Well, once, I was trying to reproduce an HPLC method from a Japanese group published in an Analytical Chemistry Journal (Honda et al 1991 Anal Biochem 199: 256). It turns out the columns used, in the paper, were not available NOR for sale in the US. I tried many columns, with similar characteristics (packing material, coating, etc.) and none worked (even with help from companies selling columns based on their knowledge). I basically had to start from scratch and develop my own system, which I did and it was totally different and more complex.

Another time, I was using an older method, which used high pressure solvent mixing, versus what I had, which was low pressure mixing and could not reproduce the separation. Again with modifications, I was able to get the right results. As it turns out, high pressure mixing, involves solvent compressibility calculations in its mixing algorithm and if not calculated properly can actually throw off the solvent mix the column sees.

So the take home points:

  1. Make sure all the instruments are the same (avoids void volume issues, etc.)
  2. Make sure Solvent and water quality are the same (this is easier now).
  3. Make sure the columns are the same and available.
  4. Use set standards and if possible the same standards

David Slomczynski, Ph.D; Geometrick Enterprises

B. cereus about contamination – Part 3

In part 2  (  http://wp.me/p7I0V6-1l  ) I covered problems with equipment. This post will cover cleaning. As was once told me by my graduate advisor, ‘Cleanliness is next to Godliness’. So, protocols for cleaning are important. Too many times people get accustomed to CIP (Clean In Place) but do not check whether or not the procedure actually works.

There are many tools to check for issues, one is from Hygienia and there are many others. This is a basic test to determine if there are intact bacteria, mold, etc. Then further tests, like culturing, etc. will determine what the microorganism is and what to do about it.

At one particular job, I was working on a project where sterility was important, since we were doing a continuous fermentation to a slow growing culture. It was important that the initial sterility and cleanliness were paramount. All the issues with dead spot, etc. were in place but how to clean was not discussed. We started with a CIP (Clean In Place) system but determined that the system caused issues, namely some Bacillus sp. that was found in the CIP (Clean In Place) material. We had to switch and tried several different vendors until one solution was found of a different make up.

In another instance, a simple brush was found to be the culprit in scoring the inside of a fermentor. It was these scratches, that allowed contamination to build up. We had dismantle the fermentor and polish the inside to remove these scratches before continuing with our process.

Cleaning protocols are not infallible and need to be tested to make sure they work properly.

All 3 posts show how important being observant and diligent in dealing with contamination can be.

David Slomczynski, Ph.D; Geometrick Enterprises