Methylene Blue

[planetmars]

Hi,

 

I'm investigating using methylene blue (redox indicator) and I am aware that in the precense of bacteria it decolorises as it is reduced. However, what specifically causes the indicator to reduce? Is it related to bacteria cell structure?

 

Regards

[YT2095]

IMO, (as stated in our chat room) it will be (in the case of Live bacteria) that they are consuming the Oxygen and excreting reductive agents, or in the case of dead/dying then certain organelles in each cell contain Enzymes that are responsible for cell destruction, I know one sort gives off Hydrogen Peroxide, so it won`t be that, maybe something in the Lysosomes?

[planetmars]

I was thinking about Lysosomes but bacteria are prokaryotic cells and therefore i thought that their cell structure does not consist of lysosomes.

 

Why do the bacteria consume the oxygen?

 

Thanks for your reply

[YT2095]

aerobes and anaerobes, the sort that would consume the O2 would be the Former.

[planetmars]

aerobic respiration involves consuming oxygen and just to clarify, sorry - what do you mean by 'Former'?

Bacteria tend to respire anaerobically, without oxygen

[iNow]

aerobes and anaerobes, the sort that would consume the O2 would be the Former.

aerobic respiration involves consuming oxygen and just to clarify, sorry - what do you mean by 'Former'?

YT <translated> --> "the sort that would consume the O2 would be the Former aerobes."

[planetmars]

But bacteria are anaerobes not aerobes

[YT2095]

no, there are 2 sorts

 

Examples of Obligate Aerobic Bacteria: Nocardia (Gram-positive), Pseudomonas aeruginosa (Gram-negative), Mycobacterium tuberculosis (acid-fast), and Bacillus (Gram-positive).

[planetmars]

ah, I see - so the bacteria consume the oxygen causing the methylene blue to become reduced and therefore decolorise. Does the bacteria consume the oxygen for aerobic respiration? I am looking at bacteria formed from milk in particular. I know that the oxygen actually destroys the bacteria.

[YT2095]

I assume so, but it`s probably best to wait until someone better qualified answers, this is Not my area.

[planetmars]

Sure, thanks for being helpful YT2095

[CharonY]

Basically it is correct. Bacterial reduction of methylen blue is usually done by oxygen consumption and secretion of reducing metabolites. Bacteria do not have organelles as lysosomes. Also, the reduction of oxygen does not reduce methylene blue per se, it only prevents the re-oxydation of reduced methylen blue.

Finally, there are different kind of bacterial strategies with regards to oxygen consumption. The obligate aerobes require oxygen as terminal electron acceptors, that is, they essentially respire oxygen as we do. The facultative anaerobes can do the same, but they are able to switch over to anaerobic metabolism, which does not require oxygen to generate energy. This can be facilitated by respiration of different substances with a sufficiently low redox-potential like e.g. Fe(III) to Fe(II), or they can do fermentation.

Then there are obligate anaerobes that not only cannot use oxygen as terminal electron acceptors but are often sensistive against oxygen. These are the ones that can actually killed by oxygen exposure.

The so-called microaerophiles can use oxygen, but only in low amounts and then there are some forms in-between.

 

The composition of milk bacteria is somewhat complex. Two often found genuses, lactobacilli and propionibacteria for instance, are facultative anaerobes. Exposure to oxygen won't kill them. However if methylen blue is reacts with oxyge, reactive oxygen species are formed which in turn can harm bacteria.

[planetmars]

Firstly, thanks Charony - that was very helpful

 

Is methylene blue affected only by dead bacteria or does harmed bacteria affect it too?

How do the reactive oxygen species harm the bacteria? Is it by destroying DNA and interferring with respiration?

Finally, does the methylene blue react with oxygen from the air or is the oxygen produced by the bacteria? Also, if the number of bacteria decrease, will the methylene blue indicator darken?

 

I would appreciate any help

Thanks

[zule]

Is methylene blue affected only by dead bacteria or does harmed bacteria affect it too?

In fact, it’s mainly affected by healthy living bacteria, although colour also changes due to dead and damaged bacteria.

 

 

How do the reactive oxygen species harm the bacteria? Is it by destroying DNA and interferring with respiration?

Reactive Oxygen Species (ROS), not only harm DNA, but also oxidize other molecules as polydesaturated fatty acids and amino acids.

 

 

Finally, does the methylene blue react with oxygen from the air or is the oxygen produced by the bacteria?

The oxygen comes fro the air.

 

 

Also, if the number of bacteria decrease, will the methylene blue indicator darken?

I think that you not only would have to reduce the number of bacteria, but you also would have to eliminate thepreviously produced metabolites.

[planetmars]

Does this make sense? --> With an antibacterial the number of bacteria decrease so the methylene blue darkens. The greater the concentration of the antibacterial substance, the faster the no of bacteria decrease and so the methylene blue becomes darker faster. Hence, the rate of reaction increases since the color change occurs faster.

[CharonY]

Well partially. The problem is that oxidation of methylene blue (causing the blue colour) is not directly connected to bacterial presence per se. Of course one could infer that the absence or reductive power is an indirect proof of absence of bacteria, on the other hand, mixing the solution (and thus add oxygen influx) will also easily result in a blue stain.

Moreover, there are numerous studies that show that especially in complex liquids like milk, ethylene blue stain (or rather destain) does not directly correlate with bacterial numbers (as there are other reductive processes not conducted by bacteria).