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
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
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 starts in tube bottom
3. casein protein coagulation
|| solid (clot or curd, in lower tube)
4. casein protein hydrolysis
(or nearly complete
(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
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
- Obtain a tube. (If you do not have a lab partner,
it is recommended that you repeat this test with a second tube.)
- Label each tube.
- 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.
- 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
- 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.
- 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:
- First, determine if there is white in the tube. This is from
reduction of litmus and it can make determining the pH difficult.
- Second, determine the color of the media, use your
observations on day 2 or 3 if needed. Then follow the steps below.
- Third, after determining the color, determine if the media has
- Fourth, look for specific characteristics of each reaction, such as gas
production in a clot, etc. See the steps below.
- First, determine if there is white in the tube, especially at the bottom
(check that is not sedimented milk).
- If there is no white in the tube, there is no reduction of
litmus. (Result so far is No Reduction which is usually not
- If there is white in the tube, you have reduction of litmus. If the uninocculated controls have the same amount of white in the bottom,
assume it is sedimented milk. If in doubt, use a marker to mark the area
of white and reincubate the tube. If the area of white expands with time,
you have litmus reduction. If the litmus has been totally reduced and you
have no color other than white, and you did not make an observation on day 2 or
3, then it will be difficult to proceed with this test. (Result so far is
Reduction which may occur with any of the below results.)
- Second, determine if the tube is (or in the case of reduction was pink).
- If the tube is pink, it means that lactose fermentation has occurred
resulting in the production of acid end products (result, an Acid
- If the tube is pink, check for bubbles or check for fissures or breaks in any
clotted milk. The bubbles usually rise to the surface and pop at the first
disturbance, so they are difficult to see. If any of these are
present, there is fermentation with the
production of gas (result, Acid and Gas).
- If the tube is pink, check to see if any of the tube contents are solid,
especially in the tube bottom. The acids that accumulate from fermentation
may cause casein to clot. Usually, one can tip the tube and immediately
tell if the tube is solid or not. If needed, you can flame a loop and
gently stick it into the media to test if any of it is solid. Keep the
tube sterile in case it needs to incubated further, but be careful
not to damage the loop! If there is a solid pink clot, coagulation has
occurred (result, is an Acid Clot, with or without gas).
(Note, an acid clot always occurs with an acid reaction which is
probably why it is not called "an acid and an acid clot.")
- If there is an acid clot, it is easier to observe gas production from
fermentation due to the presence fissures in the clot. If enough
gas is produced it can break up the clot and some call it stormy
fermentation, but we will keep things simple and just call it gas
- Third, determine if the tube is or was blue.
- If the tube is blue (especially at the top), the media has become basic from
the partial hydrolysis and degradation of proteins, especially casein, which
tends to release ammonia. (Result is a Basic
- If the tube is blue, look to see if there is any solid media especially in the
bottom of the tube (see the discussion above for an Acid Clot). If there is a blue solid,
it may only be in the bottom with a fluid
whey on top, then there is coagulation of casein (result, a Basic
reaction and a Curd).
If the tube is or was blue, check to see if there is is
any clarification of the media, where the milk has become thin, as if watered
down. The top may appear as a transparent fluid that is dark, or brownish
(or grey if there is reduction). The curd appears to be
partially or fully dissolved. If so, this is digestion of protein that is
nearing complete hydrolysis and peptonization. (Result is Digestion
or Peptonization which always occurs with a Basic reaction, with or
Fourth, determine if the tube is (or in the case of reduction, was purple).
If the tube is purple, then the pH of the media is
If the tube is purple, check to see if there is a solid, especially in the
bottom of the tube (see the discussion above for an Acid Clot). If
there is a solid, then there is coagulation of casein by enzymes released by
your unknown without an acid or a basic reaction (result is a Curd).
- If the tube is purple and if none of the above have occurred, then there is no change in the
tube. If there is no change, sterilize a loop, cool it, stick it the
litmus milk, and streak it across a general purpose agar plate to ensure
that there is bacterial growth in the tube.
- If few colonies grow on the
plate, then the strain of bacteria
did not grow in the litmus milk (result is Little or No Growth).
- If there are many colonies, then the results of the experiment
are valid (result is No Change).
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.