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【Contamination Prevention】What is contamination? Measures that need to be taken to prevent it.


Hello again!

In this article, I will write about contamination in cultures.

This is a problem that causes ‘cultures’, such as cell cultures and organ cultures to mainly fail.


What springs to mind when you think of the word ‘contamination’?


People who conduct biological research typically associated it with other words, such as:


"Dirty", "Infected", "Redo", “Repeat”

It is a wholly negative image, isn't it?


When I was doing my own research in this field, as a postgrad, I sowed the cells and came to exchange the medium the next day. However, when I did so, I noticed that the medium had turned completely yellow. Moreover, some of the medium was cloudy and it smelled strange. . .


The experiment I had done the day before was for nothing, and I had to start over from scratch. I think that the image (or rather, the real experience it was formed from) is the main thing that comes to mind.

The professor jumped into the laboratory of the medical university to help me (in my role as engineer and research student), and supported the actual research, but I was troubled by this contamination.


That time, it was the content that was able to solve the problem.



Table of contents

  1. Conclusion

  2. What is contamination in the first place?

  3. In order not to increase bacteria, use an ‘antibacterial agent’

  4. Follow the "aseptic technique" to prevent bacteria from entering in the first place

  5. Ex vivo perfusion is regularly affected by contamination

  6. Summary




Conclusion

Even if your contamination measures are correct, wash, wash, wash, anyway.



What is contamination in the first place?


‘Contamination’ in English means ‘pollution’ (Weblio https://ejje.weblio.jp/content/contamination).


In addition to simply getting dirty, such ‘pollution’ also involves getting dirty with material different to the type being used for the one original purpose. Especially in the field of culture, contamination is called contamination if something other than the target is cultured.


Therefore, if yeast grows when bacteria are cultured, it is contamination and conversely, if bacteria grow when culturing yeast, it is also contamination.


In the example at the beginning, when culturing mammalian cells, the medium turned yellow overnight, cloudy, and smelled strange, so it is believed that bacteria had contaminated the culture of the mammalian cells.


Since ancient times, measures against contamination have been considered.


The main concept of the countermeasures are:

・ Do not increase bacteria

・ Do not put bacteria in the culture in the first place

I believe these are the only two that used to be mentioned.



In order not to increase bacteria, use an ‘antibacterial agent’


Antibacterial agent is generally added to the medium in cell culture, specifically to suppress the growth of bacteria. Types of effective bacteria and mould may vary depending on the manufacturer and may be referred to by them as ‘antibacterial spectra’.

Gibco's Penicillin-Streptomycin (10,000 U/mL) (https://www.thermofisher.com/order/catalog/product/15140148#/15140148) was at a concentration of 100X, so as instructed, 1% of the medium amount was added.


However, I was one of the researchers who was troubled by contamination that still happens.



Follow the ‘aseptic technique’, to prevent bacteria from entering in the first place


When opening the lid of the culture dish, do so on a clean bench to prevent the contamination of falling bacteria.


This is called the ‘aseptic operation’, but according to Wikipedia, the ‘aseptic technique’ is a general term for unique procedures that prevent microbes contained in a sample from mixing with other substances, when microbiologists conduct experiments. (Wikipedia https://ja.wikipedia.org/wiki/%E7%84%A1%E8%8F%8C%E6%93%8D%E4%BD%9C accessed June 17, 2020).


Work among clean benches, wear gloves, spray 70% ethanol, and prevent objects and hands from passing the medium with the lid open are lessons that many researchers learned from a lecture as a senior, immediately after being assigned to the laboratory.

These measures can be said to be measures of the idea of ‘not putting bacteria in the medium’.


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Ex vivo perfusion is regularly affected by contamination


Until now, we have mainly talked about cells, but communication problems also occur while performing perfusion culture of individual organs in Ex vivo. From my personal experience, contamination occurs more often than a successful cell culture.

Of course, contamination does not occur in the organs of a living body, so whenever contamination occurred, I was impressed at the natural defence of the immune system (although you are probably not as impressed!).

Now, why does contamination occur in the perfusion culture of the organ alone?


Are falling bacteria attached to cells?


Is there less antimicrobial agent?

I have faced this problem many times, both as a researcher and as an engineer, and tried solving it in every way I could think of at the time. In particular, the main target when I was initially conducting my research was the small intestine of rats, so it was a situation where contamination was even more likely.

Countermeasure 1. Prevention of falling bacteria

To prevent falling bacteria, work on a clean bench or use clean gloves and clothing during removal procedures.

Will you ultimately undertake the procedure with sterile gloves, sterile gowns, masks, and disposable clean caps, like you are going to perform surgery? I dressed as if I was going to, from the start of the perfusion procedure.


The frequency of contamination will decrease, but contamination can and may still occur.

Countermeasure 2: Increase the concentration and types of antimicrobial agents

The concentration of antimicrobial agents added to the medium during perfusion was increased from 1% to 2%, and antifungal agents were also added as an additional measure.


Although the frequency will decrease, contamination can still occur.

Countermeasure 3: Thorough sterilisation of organ chambers and tubes

This measure is quite effective in suppressing the frequency of contamination. The organ chamber is autoclave-enabled, and the tube is autoclaved after passing 70% of ethanol or peracetic acid solution.


In particular, we are able to reduce the frequency of contamination that occurs when re-experimenting with a set-in which contamination had occurred during previous attempts at the procedure.

However, even still contamination can and does occur at times.


Countermeasure 4: Put sodium hypochlorite in a cultured solution

It's getting worse and worse! During perfusion culture, the outside of the organ is often contaminated, so we poured a little sodium hypochlorous acid into it and continued the culture.


No contamination until the end of culture! Oh, I did it! It was bleached splendidly but, by the end of it, the cell was dead. I didn't have contamination, but my essential organs had died.


Countermeasure 5: Wash with a large amount of saline

We obtained important information from our clinical professors on this measure. They advised us to wash with a large amount of saline during the operation, such as peritonitis, clinically. The amount was about 1L.


Indeed, I tentatively thought whether or not to do it, and then washed the outside of the organ with saline after it had been set in the organ chamber. I washed it a number of times (roughly 50 times with a 10 ml syringe), to use up the 500 mL drip bag, and then cultured it.


Then, I did not combine it the next day, or the day after that.


Conversely, it created an atmosphere that was likely contaminated. However, I was able to culture for up to 14 days without a contamination event. Moreover, even when repeated twice or three times, contamination still did not occur!!!


In addition to the basic measures for contamination prevention, above ("Do not increase bacteria" and "prevent bacteria from entering"), we discovered a third countermeasure: "wash away the bacteria itself" or "supply a clean medium before the bacteria increases".



In conclusion: Contamination measures in organ culture (wash, wash, wash!)

We optimised the procedure and settled on the protocol of washing the outside with saline containing antimicrobial agents. Today, we have been able to stably culture a rat’s small intestine, consistently, following this protocol.

Related article: [Ex-vivo organ perfusion] Application example to gastrointestinal research (rat small intestine perfusion example)


Related article: [Ex-vivo organ perfusion] Application examples to muscle and skeletal research (14 days long-term perfusion)


It is speculated that in the process of removing organs, there are many bacteria that have contamination sources around them (especially from animals), and if you culture them together, contamination will occur. It is thought that thorough washing means washing away bacteria from the original animal that has found itself attached to the organ.


Paradoxically, this method can be adopted in all perfusion cultures, including cell perfusion, and I think that the idea of "supplying clean medium before the bacteria increases" can be applied to cell perfusion.




Summary

Contamination is only contamination when objects that aren’t meant to be there appear.

  • In cell cultures, which are often contaminated by bacteria and mould.

  • In cell cultures, contamination can be prevented, to some extent, by antibacterial agents and aseptic techniques

  • In organ culture, it is solved by washing, washing again, and thoroughly washing a third time (at least!).


How about that?


We have told you about suitable measures that can be adopted, based on our experience of contamination that everyone performing similar procedures can expect to face, at some point at least.


I hope that it will be helpful to you, in your own research.


Thank you, once again, for reading to the end.