Download Microbiology lab TAs/BIO 351

Laboratory Exercise 18
Litmus Milk Test
Expectations of this lab:
 Understand the components of the litmus milk medium.
 Understand how litmus serves as a pH indicator as well as an
oxidation/reduction indicator.
 Understand the different reactions associated with the litmus
milk test results
 Develop the ability to interpret overlapping results.
Introduction
Milk is a nutritious medium that contains casein and lactose and provides
the growth requirements for many bacterial species. Litmus milk test is used
to differentiate members of the genus Clostridium and also the family
Enterobacteriaceae from the other Gram-negative rods like Pseudomonas
and Legionella because of the ability of the members of Enterobacteriaceae
to reduce litmus. Another group of bacteria that can easily grow in litmus
milk is the lactic-acid bacteria. A few examples of the common lactic-acid
bacteria are: Lactococcus, Lactobacillus, Enterococcus, and Streptococcus.
Litmus milk medium contains skim-milk and the litmus indicator. Litmus
serves two purposes. It is an oxidation/reduction (O/R) indicator as well
as a pH indicator. Litmus is also known as azolitmin. When oxygen is
removed from the medium the litmus becomes colorless and then when
oxygen is reabsorbed the blue color comes back. This medium is around pH
6.8 when it comes out of the autoclave. At an acidic pH the color is pink
(red), and blue at an alkaline pH.
There are four types of basic reactions that occur when bacteria are
introduced to the litmus milk medium. These basic reactions are summarized
below.
Lactose fermentation/Acid reaction- The disaccharide lactose in this
medium will be hydrolyzed to glucose and galactose by β galactosidase and
lactic-acid bacteria will ferment these sugars and form organic acids. As
more acids are produced casein will be denatured resulting in precipitated
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proteins leading to a firm clot (acid clot). The acidic pH turns the litmus
pink (entirely pink or pink band on top of a white clot). Whey is the clear,
watery fluid, on top of the curd. Some bacteria that form an acid curd will
also reduce litmus to a colorless compound. The reduced litmus will make
the acid curd appear white, starting at the bottom of the tube. A very tiny
amount of white at the very bottom of the tube may indicate precipitated
casein and not be indicative of litmus reduction. If there is gas production
there will be fissures in the clot. Extreme gas production may break up the
clot and show stormy fermentation. e.g.,Clostridium perfringens.
Reduction of Litmus- The lack of oxygen at the bottom of the tubes with
the formation of curd leads to litmus reduction, and therefore showing a
white color. e.g., Lactococcus lactis
Casein coagulation– The phosphoprotein casein gives milk its white color.
These large molecules dispersed in milk are denatured by the acid produced
by lactic acid bacteria from lactose fermentation. With sufficient acid
production casein will denature and its structure will be changed from the
tightly folded protein to an unfolded protein that is formless, semisolid, and
gel-like. This process is called casein coagulation.
Casein coagulation can also be a direct result of the initial steps of enzymatic
casein hydrolysis.
Casein hydrolysis - Casein hydrolysis can occur when bacteria produce
enzymes like rennin, trypsin, and chymotrypsin. These enzymes will digest
the protein to polypeptides. This will result in the formation of a soft curd
which will be “runny” when the tube is tilted. Unlike the acid clot, this soft
curd will not dissolve in alkaline solutions. Straw colored whey will also be
associated with the soft curds. Some proteolytic bacteria will further digest
the polypeptides to peptides and amino acids. This can happen with both
the acid clots and soft curds. The digestion of casein will increase the pH in
the medium initially by the release of NH3, giving the tube a blue color, and
the medium will be clear after the digestion is complete. Peptonization is
the conversion of protein into soluble peptones and digestion and
peptonization are both basic reactions.
casein proteases amino acids deaminases NH3(pH increases)Blue
(alkaline reaction)
Alkalinization – Partial digestion of casein leading to increased NH3 which
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subsequently raises the pH. A blue band forms at the top of the medium.
Summary
Litmus test can also be used to characterize Gram-positive bacteria (e.g.,
Lactococcus, Clostridium) as well as Gram-negative bacteria in the family
Enterobacteriaceae. It can also separate Gram-negative rods like
Pseudomonas and Legionella from the members of the family
Enterobacteriaceae. The following is an easy guide to interpreting the litmus
milk test.
 Pink color – Acid reaction due to lactose fermentation. The pH indicator
azolitmin (litmus) is pink at a pH around 5 and blue around 8.
 White color – Litmus reduction. Remember that azolitmin (litmus) is an
oxidation-reduction indicator too.
 Firm clot (curd) with a watery fluid (whey) – Due to the denaturation of
casein by acid production from lactose fermentation. This firm clot
dissolves in alkaline solutions.
 Soft clot – Due to enzymatic denaturation of casein. This clot will not
dissolve in alkaline solutions. Bluish color.
 Cracks in the clot – Stormy fermentation due to gas production by
fermentation
 Blue color – Increase in pH due to partial digestion of casein by products
such as ammonia and amines. Usually blue color precedes proteolysis.
 Proteolysis – Casein digested leaving a dissolved clot and a clear medium
 Peptonization – Conversion of casein into soluble peptones leaving a
dissolved clot and clear fluid.
These changes described above will occur in the inoculated litmus milk
tubes starting from day 1 and will continue until results are read in 7 days.
Materials:
Litmus milk test tube
Sharpie
Assigned Unknown
Microincinerator
Inoculating Loop
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Demos: Lactococcus lactis, Alcaligenes faecalis, Bacillus cereus,
Enterococcus faecalis, Escherichia coli
Procedure:
1. Obtain a test tube containing Litmus Milk Medium. Label it with the
necessary information (name, date, lab section, medium type, and unknown
number).
2. Using the stock culture of the given unknown, aseptically inoculate the
Litmus Milk Medium using the techniques listed in exercise 3: Common
Aseptic Transfers and Inoculation Methods section.
3. Incubate the tube at 35±2˚C for 7-14 days.
4. Analyze and record results. Compare results to the provided demos.
Data/Results:
Maintain detailed notes of your results in your lab notebook. Clearly labelled
drawings denoting the details and colors are recommended.
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Figure 18-1 Litmus milk test after 7 day incubation at 30oC.
1 Top left: Uninoculated litmus milk tube
2 Top middle: Alcaligenes faecalis
3 Top right: Lactococcus lactis (after a 7 day incubation period)
4 Bottom left: Escherichia coli
5 Bottom middle: Bacillus subtilis
6 Bottom right: Lactococcus lactis (after a 2 day incubation period)
Insert mpp3509
Common problems and tips:
 This test needs a long incubation period. The changes described above
will occur in the inoculated litmus milk tubes starting from day 1 and
will continue until we read results in 7 days.
 A shorter incubation period may not show the accurate results
associated with a given bacterium.
 Shaking, tilting, and mixing the contents of the tubes may make it
difficult to read results.
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References:
Claus GW. 1989. P297-300. In Understanding microbes: A laboratory textbook for
microbiology, W. H. Freeman and Company, New York.
Leboffe MJ, Pierce BE. 2015. P411-413. In Microbiology laboratory theory and
application, 4th ed, Morton publishing Company, Englewood, Colorado.
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