B028a - Methylene Blue Milk Experiments

Methylene Blue is blue when oxygen is present, but colorless in absence of Oxygen

In the old days dairies used methylene blue to judge the amount of bacteria present, however, the kind of bacteria present also affects the degree of blue lost. A few students have written me seeking info in mb, therefore I am starting this page.

Find Henerici book and put some of that info here on b028a

prior page is b028.htm It has more general info on using milk for media.

see page b028b for details of the chemstry of curdling, stormy fermentations, peptonization, etc

Info collected by not yet digested will be stored below.

ct: individual study Date: Mon, 3 Aug 1998 00:02:42 +0100 From: "Dipak Sodha" <dipak@netcomuk.co.uk> To: <indbio@disknet.com>

dear sir/madam, i am an 'a' level student doing my biology individual study, on bacterial populations in milks of different types, using methylene blue indicator. i would appreciate some information about similar studies or any related information. thank you shivani sodha

ps my email is shivani@netcomuk.co.uk

ubject: individual study Date: 5 Aug 98 05:45:02 -0500 From: dipak@netcomuk.co.uk To: Indbio

dear harold, thanx for taking an interest in my study. ok, well i am an A level student and thats what my study is part of, if u don't understand our system then this is just before i go to university, we do A levels in high school or some people decide to do them in college, after we obtain good A level grades we apply to university. Anyway, my study is as follows: investigating bacterial populations in milk of different types; 4 types of milk will be tested whole pasteurised, semi-skimmed, skimmed and sterilized. once M.B has been added the various milk samples will be incubated at different temperatures, these being at room temp, water bath at 37c, and in the fridge: this is to observe whether temp has any effect on bacterial populations. another factor to be tested is the age of the milk, milk will be bought fresh and tested, then after it is one day old and then after it is one week old.

i have put my hypothesis as milk of higher fat content will decolorize faster than other milk as there is more nutrient for the bacteria to live and mutiply from. and that milk at room temp will decolorize faster (as it is warm but unlike the water bath not too hot so that bacteria may die)

thats about the whole of it! any info on this or related studies and research would be welcomed thanx evers so much


ubject: Milk production Date: 7 Aug 98 12:49:24 -0500 From: dcarr@gsinet.net To: Indbio

Dear Sirs: I am an eighth grade science teacher in Derry, New Hampshire, USA. This year, for the first time, I am going to be doing a unit on the chemical reactions that happen in dairy production (specifically milk). I am also going to be discussing how a dairy farm works and what goes on at the farm. I grew up close to a city and I have NO idea of what goes on behind the sciences of a dairy farm. I would really appreciate any information you could give me so I could become more knowledgeable in this area. Thank you for your time. Dorina Carr 8th Grade Science Teacher

ubject: Re: milk science project Date: 30 Jan 99 17:46:12 -0500 From: indbio@disknet.com To: Indbio

BacteriaStudyList - http://www.disknet.com/indiana_biolab/b.htm

henry1@uscom.com wrote: > > hi, my name is Tim. I am a high school freshman. i'm doing a science > project on long milk stays good after the "sell by" date. I plan to use > methylene blue to measure the presence of bacteria and will do time tests > to see how quickly the color of the methylene blue disappears. I am > wondering if you believe this is workable. I emailed several dairies to > ask about acceptable bacterial levels. how will i know, without smelling > or tasting, when i've reached a high enough bacterial level to say the milk > is undrinkable by using the methylene blue? or won't I? > > I would appreciate any information you can give me. Thanks! I have not found much info. 10 ml of 1% aqueius methylene blue is added to 100 ml of milk in the identification of enterococci but I do not know how that is used. So you would add 1 gram of methylene blue to 100 ml of water and autoclave that and then add 10 ml of that using a sterlie pipet or sterile graduate to to 90 ml of milk that you want to test. After autoclaving, the cooled, shaken methylene blue will be blue. After addition to milk and mixing by vortexing, the milk will be blue. The blue color will fade away as the Oxidiation-reduction potential of the milk changes. However, kind of bacteria and number of bacteria affect the redox potential. As far as safe drinking goes, the disappearance of the blue is a very crude measurement and does not say much about the kind of bacteria or the number of bacteria. One needs to use diagnostic media anc pure culture methods to learn the precise number of various kinds of bacteria present. Since some of the bacteria found in milk may be beneficial to us and others can cause illness, the methylene blue test is pretty worthless and probably is not used much anymore. -- Harold Eddleman Ph.D. Microbiologist.

BacteriaStudyList - http://www.disknet.com/indiana_biolab/b.htm

Dear Tim In my e-mail I gave you the formula for methylene blue milk for assay of enterococcus which is 0.1% or 1 gram of methylene blue per liter. I do not know the amount of methylene blue used in the old days when methylene blue was used to estimate the number of bacteria in milk. Ulrich milk which is used to propagate bacteria and determine the action of bacteria on milk uses .005 grams of methylene blue per liter of skim milk. Which is 1/200 as much methylene blue. I have always used .005g of methylene per liter in my classroom. Since the amount of blue color seen after incubation with methylene blue depends upon the number of bacteria and the kind of bacteria, I would change the objective of the project. Since the amount of color does not tell you whether the milk is safe, I would forget about using methylene blue to tell you how long the milk is safe to drink. I suggest you just check how long it takes the milk to become colorless. I think the milk was incubated at 32 C in the old methylene blue tests in dairies. For more fun you might begin with one sample of milk, add methylene blue, and incubate at several temperatures and see how long it takes to lose the blue color. Use what ever temperatures are available to you. You could try refrigerator (about 4 C), room temperature, and whatever higher temperature have. If you have any identified bacteria available to you. Or if you have isolated some bacteria as pure cultures. test each one in methylene blue milk and check how they compare. Several students are isolating 10 different bacteria and running tests on them. I suggest they test all ten on methylene blue milk at any convenient temperature between 30 and 37C. -- Harold Eddleman Ph.D. Microbiologist. mailto:indbio@disknet.com Location: Palmyra IN USA; 36 kilometers west of Louisville, Kentucky http://www.disknet.com/indiana_biolab = Agriculture, science projects

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Ulrich Milk

100 g Skim milk powder
    5 mg Methylene blue
  15 mg Phenol red
    1 liter tap water

This medium works exactly like Litmus Milk but is more dependable and probably cheaper. Colors differ from Litmus Milk. Phenol red is red at alkaline pH. Phenol red is yellow at acid pH. Oxidized methylene blue is blue. Reduced methylene blue is colorless. Recall red plus blue is purple. Results are shown in the table:

Reaction Color
Uninoculated medium Medium bluish-gray
Slight acid Pale yellow-green
Acid with reduction Pale yellow-orange
Alkaline Purple clot or milky
Alkaline with reduction Red clot or milky
Alkaline with peptonization Clear, transparent red
or clear purplish

37oC ... SO4

First installed Feb 1998      Revision #2 1998 Feb 5       indbio@disknet.com
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
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