The Litmus Milk Test, Atlas p. 64.

The Litmus Milk test is a rather complex test containing milk, litmus (a pH indicator), lactose, and casein (a milk protein) all of which can be metabolized by some bacteria.  Litmus milk also contains sulfur and many nutrients that are found in milk.  It is an undefined and differential media that is used to help differentiate and identify Enterobacteria, Clostridium, and Lactic acid bacteria. 

A number of results may be obtained in the Litmus Milk test, but there are four main reactions: lactose fermentation, litmus reduction, casein coagulation, and protein hydrolysis.  Some or all of these reactions may occur at the same time, and many of them have multiple or similar sub-reactions (for example, casein coagulation can form a solid clot (as in an acid clot) or casein coagulation can occur as the casein is converted into a solid curd in the bottom and a liquid whey on the top (as in curd formation). 

Four reactions can occur in Litmus Milk: Litmus Color pH Notes

    1.  lactose fermentation (without gas)
         (or fermentation with gas production)

4.5 and below
(4.5 and below) 
(acid with bubbles or fissures)

    2.  reduction of litmus

white any white starts in tube bottom

    3.  casein protein coagulation

any any solid (clot or curd, in lower tube)

    4.  casein protein hydrolysis (partial) 
         (or nearly complete hydrolysis)

(clear & dark)
8.3 & above
(8.3 & above)
basic (especially at tube top)
(basic and media is thin)
    There can also be no reaction and
    any combination of the above reactions
    (see Table 6-2, Atlas p. 64)
purple shades 4.6 to 8.2 near neutral pH
other pH uncertain  

As you might have noticed, it is rather complex, for further details, see Atlas, p64-65.  It is required that you be able to use and interpret the data in Table 6-2 and Fig 6-50 (Atlas, p. 64) when provided with the table and/or figures.


  1. Obtain a tube.  (If you do not have a lab partner, it is recommended that you repeat this test with a second tube.)
  2. Label each tube.  
  3. Use your wire loop to aseptically inoculate the Litmus Milk tube with your unknown bacteria.  Add a lot of bacteria as some species do not grow well in this medium.
  4. Remind your instructor to set up the control uninoculated tubes (they will be useful for comparing with your tube).  Place your tubes in the rack for this test, they will be incubated for one week at 35-37 degrees C.
  5. The tubes should be observed in 2-3 days to check for the color of the media (or the pH).  Continue incubating the tubes for a total of one week.  The media color should be checked because at this early date, the litmus will not be totally reduced.  When the litmus is totally reduced, it is difficult determining the pH.
  6. Record your results of media color after day 2-3, and all your results including color after 1 week.


Obtain your tubes after one week of incubation, checking for gas bubbles before you handle the tubes as these disappear after handling.  Obtaining results for such a complex test can be tricky if several reactions occur together.  Follow this guide for assistance:

Note to Instructors:

Students often struggle with the results of this experiment.  As the pH of the tube must be known to determine the result and instructors will not likely have observed the tube after about two days of growth, it is handy to use some pH paper when attempting to assist students in interpreting the results, especially when multiple reactions are occurring at the same time.  To keep from contaminating the tubes in case further incubation is required, one can remove a small sample of solution from the tube using a sterile pipette or loop and transferring it to the pH paper.  Once the pH is known and the reduction of litmus has been determined, most of the above results can easily be distinguished, except for maybe digestion.  In determining if digestion has occurred, first realize that a basic reaction must come first, where protein has been partially hydrolyzed.  One can determine if there is a basic reaction by referring to the pH.  If there is a basic reaction, next determine if there is a basic reaction and a curd by looking for solids.  Lastly, look for signs of digestion where the proteins are nearing complete hydrolysis.