Is it possible to make anaerobic media too reducing?

Question

I've been struggling to grow an anaerobe (P. gingivalis), I take every precaution to keep the culture conditions free of oxygen, I was wondering if the conditions can be so reducing that they inhibit microbial growth of an obligate anaerobe.

To make the liquid media I do the following:

• TSB supplemented with yeast extract
• plus 0.5g/L L-cysteine HCl as a reducing agent
• plus resazurin as a redox indicator
• heat-sparge with nitrogen until the resazurin goes completely colourless
• add hemin (5 mg/L) and vitamin K1 (1 mg/L) from stock solutions (1mL/L hemin in water, 200uL/L vit K1 in ethanol)
• filter-inject this media into glass tubes which have had the gas in them removed (this takes place in an anaerobic hood)
• all materials (syringes, needles, filters, tubes) had been left in the anaerobic hood before use so that no residual oxygen is left in/on them
• swap the headspace for a gas mix of 80:10:10 N₂:CO₂:H₂

I don't get any growth after over 72 hours @ 37^oC.

Other protocols I have found are much more vague/lax about removing the oxygen, many just say that the cysteine should be added, and the media should be incubated in an anaerobic hood for 24 hours with the lid loose before being used. This got me thinking that maybe I'm removing too much of the oxygen.

What do you think?

Answer 1

I notice that you don't specify pH, which might be important.

I don't know if you can put in too much reducing agent (I suspect not), but a large excess is often required as the equilibrium constants for disulfide exchange reactions are typically close to unity (see Cleland, 1963).

I also don't know if any of following has any relevant to your problem (again, I suspect not), but I want to briefly comment on two aspects of using thiols to as reducing agents in general.

Firstly, the oxidized (disulfide) form of thiols may react with a protein sulfhydryl. Using β-mercaptoethanol as an example (Scopes, 1993, p 320), something like the following may occur, where the reducing agent becomes (reversibly) bound to protein. If the reactive sulfhydryl is at an enzyme active site, this may lead to inactivation.

$$\ce{2 HSCH2-CH2OH + O2 -> HOCH2CH2-S-S-CH2CH2OH + H2O2\tag{1}}$$

$$\ce{HOCH2CH2-S-S-CH2CH2OH + HS-{Protein} <=>HSCH2-CH2OH + HOCH2CH2-S-S-{Protein} \tag{2}}$$

As stated, equilibrium constants for thiol exchange reactions are often close to unity (Cleland, 1963)., $$\ce{R1-S-S-R2 + R3-SH <=> R1-S-S-R3 + R2-SH \tag{3}}$$ For this reason, many protein chemists prefer to use dithiol analogs of threitol (dithiothreitol, DTT, or Cleland's reagent) or erythritol as reducing agent, as the oxidized form of these compounds are cyclic disulfides, where the equilibrium constant is displaced to the right (see Cleland, 1963).

There is also the question of stability.

The half-life of thiols in solution are markedly pH and temperature dependent, as the table below shows. The half-life of β-mercaptoethanol at pH 6.5 and 20℃ is greater than 100 hours, but at pH 8.5 and 20℃ it is only 4 hours.

 

The above table was taken from Stevens, R, Stevens, L & Price, N.C (1983) The stabilities of various thiol compounds used in protein purifications, published in (the now-defunct) Biochemical Education. The full article is freely available as a pdf from the Wiley website