Desulfitobacterium: growth medium, add salt to taste! ;)

Preparing a growth medium is as esay as following a recipe! :)
This is the colture medium I'm going to use with Desulfitobacteria hafniense DCB-2, D. hafniense TCE-1, D. chlororespirans, D. dehalogenans, D. PCE1.

Pay attention to respect the right concentration and follow the protocol i've posted to prepare anaerobic solutions.

Starting solutions:

• M1= K2HPO4 (0.2 M)  
• M2= NaH2PO4 * 2H2O (0.2 M) 
• M3= Resazurine (0.5 g/L) 
• M4= EDTA 500 (mg/L) 
• M5=

• Vitamin H (50 mg/L) 
• p-aminobenzoate (250 mg/L) 
• Pantothenate (50 mg/L) 
• Folic acid (20 mg/L) 
• Lipoic acid (50 mg/L) 
• Vitamin B6 (100 mg/L) 
• Nicotinamide (550 mg/L) 
• M6= Vitamin B1 (100 mg/L)
• M7= Vitamin B2 (50 mg/L) 
• M8= Vitamin B12 (50 mg/L)
• M9= NH4HCO3 (114 mM) + NaHCO3 (906 mM)
• M10= Na2S * 9H2O (1M)
• M11= CaCl2 * H2O (30 mM) + MgCl2 * 6 H2O (20 mM)
• M12= Yeast extract (4%)
• M13= Na2SeO3 (6 mg/L)

All solutions were made in anaerobic water and flushed with N2 (except for M9 and M10 which contain volatile compounds).

Basic medium

• 21 ml M1 
• 7 ml M2 
• 1 ml M3
• 971 ml “sub-boiled” water

Cold solutions

• Cold A: in anaerobic, sterile bottle with 20 ml sterile demi-water added were: 1 ml M4, 1 ml M5, 1 ml M6, 1 ml M7, 1 ml M8, 0,5 ml M13. 
• Cold B: 1 ml M10, 49 ml M9 filter-sterilised. 
• Cold C: ~30 ml M11.

Completing the growing medium

To 18 ml basic medium (M1+M2+M3) add: 

• 1 ml Cold B 
• 0.5 ml Cold A
• 0.5 ml Cold C
• 1 ml 4% yeast extract
• Inoculum (10%)

Desulfitobacteria hafiense DCB-2 (University of California)

So calculators in hand and good luck!
Soon to your screen the Best Pasta Ever recipe! :D

Standard Addition, the escape route! ;)

Tired of Internal and External Standard?
There is always a third solution... Standard Addition! :)


Quantification with Standard Addition is often used when the matrix of the sample can significantly modify the analytical sensitivity of the measurements. 
I've performed this procedure with dust samples(dirty enough!) and LC-UV analysis.


I'll try to explain this procedure in the simplest way. :)
Let's imagine to have four samples, same volume and unknown concentration of analyte.

David L. Zellmer (1998)

A series of increasing volumes of stock solution (solution with isotopically marked carbons) are added.

According to the literature: "The concentration and volume of the stock solution added should be chosen to increase the concentration of the unknown by about 30% in each succeeding flask".

My tip is to increase it more than 30%. I'll explain later why.

It's not necessary filling up the vials with solvents as shown in figure (in our case Vflask in the formulas is referred to our volume of sample+standard).

Just remember that this step has to be performed before the extraction, since standard solution and sample have to follow the same steps (extraction and cleaning up).


The concentration of analyte is given by:

The instrumental response of the analyte will be R = K x (concentration), so:





Now set Csa = CstdVstd/Vflask.





To obtain our CSA we should extrapolate for y=0, of course we need a positive value.

It's necessary, as for Internal and External Standard protocol, building a calibration line.

We should prepare at least six levels.

Levels are made by standard solution with different concentrations (the 1st level has standard concentration equal to zero).
They are useful to understand the real response of the machine to the different concentration.
Of course these levels go directly to the analys without any other treatment.
Plotting the pick areas, obtained from the measurement, with the concentration we should obtain a linear function.

The theoretical concentrations of standard solutions have to be divided by the calibration line's slope. In this way we'll obtain a new set of concentrations. Plotting these new real concentrations with the instrumental response gives us the right trend line and x-intercept, so the real analyte concentration.

As you can understand, drawing a right trend line is important to obtain the right x-intercept.

In my example the systematic error is quite big! :(
To avoid this kind of error we could: 

• add another measurement to the experiment, so that we could draw a best trend line with five points;
• choose bigger gaps between the increasing amount of standard solution, in this way we could move our points in the graph, and obtain a trend line more definite.

I think that's all folk, i hope i've been clear enough!

Good luck with Standard Addition!!! :)

Glove box! Such a cool invention! :)

Supsup?

Since I'm working with anaerobic solutions i had the opportunity to meet this elegant lady that occupes a big part of my small lab.

Let me introduce you the Glove Box! :)

According to me the glove box, it's really a great invention, and as it often happens behind a big invention there is a simple idea.
The mechanism in fact is quite easy to understand.
As I've already explained, I'm preparing solutions without oxygen.
Of course it's not easy forbidding that oxygen spreads into our fluids, considering also that air contains 21% of this gas. 
For this reason once we obtain an anaerobic solution it's always better closing the bottle with a rubber stop that has a best adhesion and does not permit to any gasses to enter.


Problems begin when is necessary working with our solutions, and maybe keeping our bottles opened for hours.
The glove box in this case results very useful. It consist in a hermetically sealed box, in which we can set the experimental conditions we need, in my case the experimental condition is "no oxygen".


Let's give an easy example.
I have to work with 24 serum bottle, with anaerobic solutions.
I'll need pipettes, tips, stoppers and of course of my bottles.
How could I put these materials into the glow box paying attention that air doesn't go inside???
Easy! :)

As you can see from the picture, it has an external smaller box. That's used to exchange material with the environment.

So I'll put everything into the exchanging box, I'll close it and I'll push the button connected to the vacuum pump, pressures depend by the dimension of the box, for the glow box shown in figure, 15 atm is enough.  

In this way all the air contained in the little box will be carried away. Generally another system is connected to the glow box to filter and dehumidify the air that is released outside.

At this point I'll fill the exchanging box with another gas different from oxygen, nitrogen for example that presents inert characteristics.

I'll repeat this procedure at least twice, so that I'll be sure that there will be no air inside.

Now I can open the door between the exchanging box and the glove box and carry the instruments inside.

Working with the glove box, on the other hand, isn't so easy because of the huge gloves that aren't so confortable specially if you have to work with little dimensions!

Anyway we just need practice! :)

P.s. Glove box is also useful if we are working with toxic gasses. In this case we want that what is inside stays inside, so all the procedure described should be performed before exchanging materials from the glove box to the environment. Same thing but in the opposite way! :)