The Methylene Blue Test

A few months ago I wrote a cheese-focused article on the safety and organoleptic qualities of raw milk. During that post I mentioned the methylene blue test, which is a quick and easy test that can be used to get an idea of the bacterial load present in milk. This generated some interest among my readers here, and on a cheesemaking group I am a part of. As promised back then, here is a post on how to perform this test at home.

How the methylene blue test works

The concept behind this test is fairly simple. Methylene blue is, as the name suggests, a blue dye. Bacteria (and other contaminants like yeast and fungi) tend to reduce (add electrons) to the materials around them. This includes the methylene blue, which when reduced becomes colourless. Oxygen in the atmosphere counters this, stealing electrons (oxidizing) the decolourized methylene blue, rendering it blue again. Thus, if a known amount of methylene blue is added to milk, the speed in which it becomes decolourized is dependent on the number of bacteria (and other microbes) present in the milk.

When done correctly, the time it takes for the methylene blue to reduce to its colourless form is a good guide for the cleanliness of your milk:

• Under 30 minutes: Milk is of very poor quality and should be disposed of immediately.
• Under 2 hours: Milk quality is poor and it should not be used unless cooked.
• Between 2 and 6 hours: Milk is of medium quality. Raw milk, collected using best practices, will typically turn around the 6 hour mark. The risk of a food prepared from this milk developing contamination is modest*.
• Between 6 and 8 hours: Milk is of high quality. The risk of a food prepared from this milk developing contamination is low*.
• Over 8 hours: Milk is of excellent quality. The risk of a food prepared from this milk developing contamination is very low*.

*assuming contamination isn’t introduced from elsewhere

A caution

This test only informs you of the net microbial load of the milk. It does not tell you whether those microbes are pathogens, food spoilers, or something innocuous. If you were to add a cheese or yogurt culture to some sterilized milk and run this test, you’d get a “poor” or “medium” rating. Likewise, minute amounts of pathogens could lead to a “high quality” rating, while still representing a risk to you.

This means that this test is of limited use. A few places where it is useful to the cheese maker (or maker of other milk-based products):

1. If you raise your own dairy animals, this is a good way to test whether your animal husbandry and milking procedures are allowing you to collect milk that is relatively free of contamination. As mentioned above, freshly collected raw milk should rate close to the boundary between medium and high quality (or better). Lower grading than this can be a sign of an unclean barn, milking practices which allow for contamination, or an issue such as mastitis.
2. If you pasteurize milk yourself, this is a good test to confirm that your pasteurization procedures are working properly. Freshly pasteurized milk should score as being of “excellent quality”.
3. You can use this method to confirm that your milk provider is providing quality milk. If you test their milk and it comes out as medium or low quality, you know they are not collecting it properly, not storing it properly, or that they are providing you with old milk.
4. As a test of raw milk before using it to make a raw-milk cheese or other uncooked milk product. This can give you some peace of mind when working with raw milk.

Performing the test

To perform the test you will need (links are to Amazon Canada, but similar items are available through many different sources):

• 1% solution of methylene blue. This is sometimes available cheaply as a treatment for aquarium fish, but be careful as some of these include other antimicrobials or may be 3% methylene blue.
• Sterile 15 mL centrifuge tubes. Sterile tubes that can hold 11 mL of liquid. 15 mL centrifuge tubes are typically the most common and affordable form that are readily available. Sadly, these are single-use. If you can find glass test tubes, they can be cleaned, sanitized, and reused.
• Sterile single-use (individually wrapped) transfer pipettes. These too are single-use, which is why you want ones that are individually wrapped. Bulk-wrapped pipettes can become contaminated once the bag is opened.
• Instapot (or other electric pressure cooker) with a yogurt setting, or a yogurt maker with selectable temperature control.

To perform the test:

1. Using a sterile transfer pipette, transfer 10 mL of milk into a sterile 15 mL centrifuge tube.
2. Using a second sterile transfer pipette, transfer 1 mL of methylene blue into the same tube.
3. Cap the tube and invert 4 or 5 times to mix.
4. Loosen the cap (so air-exchange can occur) and place in the instapot/yogurt maker. A good way to get the tube to stand up is to place it in a small cup filled with water. Fill the instapot or yogurt maker with as much water as you can without the tube flipping over or becoming submerged.
5. Set the instapot/yogurt maker to hold the sample at 37C (98F). Check every hour for a colour change. A tube is considered “changed” once the bottom half of the tube has gone from blue to white (see the “organic” sample in the image below for an example of a tube that has just “changed”).

It’s really that simple! If you are doing multiple tests at the same time you can cut back on the number of transfer pipettes you use by measuring out the milk with separate pipettes, and using a single pipette for the methylene blue.

Too prevent contamination of your test materials, never open tubes or pipettes until the last possible moment. Contamination of a tube or pipette may invalidate your results. Likewise, the container of methylene blue shouldn’t be left open for any longer than needed to perform the test.

A bit of fun with commercial milk

I teach this method in a microbiology lab course I run. In the lab we test a variety of store-bought milk, as well as home-pasteurized and raw goats milk from my farm. We also have a positive control, which is milk that we inoculate with E. coli, and a negative control where we filter-sterilize the milk. It is interesting to see how commercial brands stack up. To the left you can see a photo of this year’s lot after 7 hours of incubation.

The lactose-free is a UHT-pasteurized milk, and the filtered is a filtered milk. The organic and regular milk samples are both conventionally-pasteurized products. The organic milk is at the 50% converted stage, meaning it would have a “high quality” rating – this also shows what a tube looks like at the time-point where you’d consider the tube to have “changed” (decolourized). The regular milk converted at 6 hours – essentially the legal minimum – and actually converted faster than the positive control (+ control)!

That latter finding is not unprecedented. In fact, we saw the exact same result with the same brand last year (Neilson brand skim milk, for those in Canada who are interested). This is a “budget brand” of milk, and I suspect they are pasteurizing to the legal limit and not one bit farther. This would minimize the shelf-life, meaning more people would not finish the milk before its best-before date than if they had bough better-pasteurized products. This is also reflected in the “best before” date on these milks. The filtered and UHT milk has best-before dates nearly 2 months away from the date of purchase, the organic 1 month, and the normal a mere two weeks. This is just a guess on my part as to why the “normal” milk barely passes the test, but clearly this is more than a chance occurrence.