Re: Methylene Blue as a bacterial assay.

Hi Linda,

It's true, methylene blue can be used to detect the presence of oxygen. There is actually a page about the principle used, it's at texas university.

However, there are several things that might go wrong in your experiment. I don't know the exact protocol one should follow, cause I never did this experiment. Below I'll summarize some bottle-necks..

The most common bacteria to grow in milk are lacto-bacteria. These bacteria produce yoghurt, cheese and other diary products, but also make milk turn 'bad'.
These bacteria however don't need to use oxygen to grow, they produce lactic acid. Nevertheless, they can survive in an oxygen containing liquid like milk.

Also, other bacteria do consume oxygen, but in milk fresh from the carton, there should be no bacteria, or when they are present, not in amounts to be detected with methylene blue. There should be quite a lot of bacteria to consume the oxygen and care should be taken to prevent oxygen from re-entering the liquid (so, no stirring, shaking, mixing, but use a bottle which can be capped and fill it completely before capping).

Of course the concentration of methylene blue you use is improtant, if a small amount is used, changes in color can be detected faster. However, I don't know which concentrations to use ....

Something that does change in milk when lactobacteria grow is the acidity (pH). In a lot of fermentation processes the pH is a measure for the microbiological activity. pH can be detected with indicator paper, but there are also several pH indicator-solutions available. Usually sterile milk will have a pH of about 6-8, but when lactobacteria start to grow it will drop significantly. If you find a nice indicator on your 'lab shelf' that will change colors somewhere in this range, it might work.

If you really want to have quick growth, you could see if you could find yoghurt of which the lactobacteria aren't killed (not pasteurized or sterilized) and add a small amount to fresh milk with the indicator, store the 'culture' for one or a few days at a place at room-temperature of a bit hotter (30C) and mix once in a while. Bacteria should start growing... (WARNING: DON'T EAT THE CULTURE, OTHER HARMFULL BACTERIA COULD BE GROWING IN HERE TOO!)

One of the easiest ways to detect bacteria is using a 'rich' (ie containing enough nutrients) clear solution and see the bacteria make the solution more and more turbid...

Good luck with the experiments..

Rolf Marteijn Wageningen Agricultural University dept. Foodtechnology and Nutrional Sciences division of Bioprocesengineering Animal cell culture workinggroup