Martin Černý

• Master of Science
• PhD Student at Masaryk University

Dear colleagues,

I recently jumped in this field and I was trying to recreate a study, using absolute qPCR quantification for comparison between the abundance of certain bacterial specie in two groups of samples. After optimization of my protocol, I ran 2-step qPCR on 96-well plates with samples in triplicates and I ran the reaction three times in order to get the variance.

I am going for absolute quantification, with a standard curve with Ef ranging from 1,979 to 1,891 (98,95 to 94,5 %) with LunaMix from NEB. However, even though I used the same protocol, my values have a big variance between the plates. I would like to normalize them to use them in the same data set in order to have a more robust analysis. The ratio between the diseased and healthy group (16/8 at each 95well plate) is the same at each plate.

Is there a way to normalize the data in order to use them in one dataset? Also, if you are just looking, it is necessary to have more than 1 reaction apart from technical replicates?

I will much appreciate your help.

Sincerely, Martin

Dear fellow researchers,

I have stumbled upon a rather trivial thing during writing down my thesis. I have obtained several strains from DSMZ which provides a rather wide list of cultivation media, which fits most of the growth parameters of their provided strains. The DSMZ admits that cultivation in these media ensures growth and as such, the media recipes are freely accessible from the internet. During my studies, I have slightly adjusted the given media, but I want to properly cite them.

I know, this might be trivial, but I want to ensure that I credit everybody for their work.

All that is at their page, is the following " © 2017 DSMZ GmbH - All rights reserved " at the end of each medium.

My suggestion is that the medium should be cited as follows:

...the cultivation of the pure strain was undergone in the medium ABC derived from medium 823 (823. HALOBACULUM GOMORRENSE MEDIUM, © 2007 DSMZ GmbH)...

or perhaps...

...in the medium ABC derived from medium 823 (© 2007 DSMZ GmbH)...

I would be thankful for any replies, suggestions or experiences regarding this problem.

Since me and my supervisor weren´t able to determine what is going on with my media, I am seeking help among more experienced and skilled. I will be thankful for every kind of help, which may give me a bit of insight on where I was doing mistakes.

I will have various questions with photos which may help with description of my problems. I am really sorry for my grammar as my language skills are not as advanced as they should be.

I am having this problems for about the month and half, since the winter begun. We are maintaining 21 degrees of Celsius in laboratory, so this may not play role. Whenever I am working with my cultures, I am working in the laminar box or outside, always with the burner working nearby.

I´ve never witnessed this condition before, since I am preparing the medium, so I don´t know what may be going on.

Also, after inoculating the vials with my cultures, the medium turned white after 15 minutes, with precipitation containing what I believe are lysed cells, since I was unable to see any living cells on the native preparate, nor on the Gram. (I was working with DSMZ cultures of Methanobrevibacter smithii and Methanospirillum hungatei, inoculating 1 mL via pregassed syringes.)

This weekend I was working on the medium again, but we had different (smaller Volume) gas bomb containing the H2/CO2 (5:95). The number of failed media went bellow 20% but there are some with the whitish haze.

So my hypothesis is, that since I can´t afford the oxygen scrubber, it might be contaminated by the oxygen or something, since the problem introduced after the bomb was low and started running out. But I am not sure, because I´ve never had such problem before, working with anaerobic media.

I wanted to ask what are the most frequent anaerobic or facultative anaerobic contaminants during the work with anaerobes. Since I am unable to isolate and sequenate the samples at the moment due to the finances, the information would come in handy.

I was working with rather-pure cultures with possible minor contaminations. I processed the cultures with antibiotics (Vancomycin+Clindamycin) and ended with what u can see in the picture no. 3.

The picture is 1 mL filtrate stained with DAPI seem to contain the Methanospirillum itself with something representing round cells, sometimes forming pairs. I am unable to recognize the source, or the contaminant itself which may be some kind of methanogen as well.

Are there any handy and fast techniques to prevent it, or to identify the methanogens (I assume I may use the fluorescence microscopy by Doddema and Voegels 1978) which I may use in the laboratory.

Thank you very much for your help and time, I will be grateful for anything.

Martin Černý

Bc. student at the University of Masaryk

Good day,

Does anybody know, where can I get a peak into this publication? I found, that the Sydney library has those, but they are unable to share it via librarian sharing program.

Is there any other chance, where can I get this manual?

Anaerobe laboratory manual : by the staff of the Anaerobe Laboratory, Virginia Polytechnic Institute and State University ... /​ 4 th ed. by Lillian V. Holdeman and W.E.C. Moore. 1977

Thank you very much for your help.

Hello,

I am currently testing various methanogenic mediums for growth of methanogenic archaea, but the MM medium keeps failing to create a blackish sediment of Na2S in the solution after the preparations. Instead it creates milk-like haze in the solution with white precipitation on the base.

I am preparing medium in the flask under anoxic condition of CO2/H2 (g) atmosphere with the N2 (g) atmosphere of the other solution. 

I have checked the temperature of the solution prior to adition of Na2S if it was cool enough, I have tackled the pH with 0,1 M NaOH solution, or I left it unchanged and I´ve regulated it after completing it.

I´ve been working on the MM medium for some time and I´ve never been adjusting pH or watched the temperature of the solution. I also made a new solution of Na2S and managed to get a new, pure substance, but nothing has changed.

Dis somebody have this issue with MM medium preparation? If yes, please, help me with this step, thus I can continue in use of this medium for cultivation.