Casein, Lipid, Starch and Gelatin agar:  Exoenyzme production

Casein Plates (Milk Plates) contain the major milk protein found in milk and Lipid plates contain Tributyrin.  If an organism can produce the exoenzyme protease/casease( hydrolyzes casein ) or lipase (hydrolyzes tributyriin) then a clear zone will be seen around the growing colony.  Both of these agars are considered differential media's since you can differentiate organisms by there exoenzyme ability.  Organisms that have  protease/casease break the casein down into smaller peptides, polypeptides and amino acids that are more easily transported into the cell for metabolism.  Similarly organism that break down  the fatty tributyrin, a triglyceride that is broken down to glycerol and fatty acids,  into smaller fragments that are more easily used in cellular processes. Organisms that have gelatinase using gelatin as the sources of carbon and can hydrolyse gelatin turning it form a solid to liquid state ate room temperature. 

Casein Hydrolysis

Casein agar inoculated with Pseudomonas aeruginosa (top), Serratia marcesens (lower right), and (lower left).  E. coli grows on casein agar but does not have the protease exoenzyme to break down the casein.

Pseudomonas aeruginosa grown on Casien agar for 72 hours at 37 degree's C to look for the presence of the exoenzyme proteases/casenases. Pseudomonas aeruginosa, casein hydrolysis, as noted by a zone of clearing around the organism, as well as a green diffusable pigment pyocyanin.

Serratia marcesens was grown on Casien agar for 72 hours at 37 degree's C to look for the presence of the exoenzyme proteases/caseases . Serratia marcesens exhibits slight casein hydrolysis as well as red colony pigment prodigiosin. The red pigmentation is normal coloration for Serratia and not a result casein or lipid hydrolysis.

Gelatin Tubes

Top and bottom tubes contain Pseudomonas aeroginosa and Serratia marcesens, both organisms have gelatinase and digest the gelatin in the tubes.  The normally solid tubes are liquid at room temperature after a week of incubation.  The center tube is E. coli which does not have gelatinase and remains solid at room temperature

Pseudomonas P. aeruginosa, Escherichia coli and Serratia marcescens were inoculated into gelatin agar using the stab method and incubated for 1 week at 37 degree’s C. If the organism has gelatinase then it can break down the gelatin (protein) into polypeptides and then amino acids which can then be used in cellular processes. The gelatin tubes are solid and if hydrolyzed by gelatinase become liquid.  Pseudomonas aeruginosa: gelatin is liquid after incubation and cool down (icing)* indicating digestion of gelatin with the exoenzyme gelatinase.
  Escherichia coli: gelatin remains solid, no gelatinase present.
  Serratia marcescens: gelatin is liquid after incubation and cool down indicating digestion of gelatin with the exoenzyme gelatinase. Note: red color is pigment (prodigiosin) produced by Serratia.
* gelatin is liquid at 37 degree’s C so cooling to room temperature or putting the tube in a cup of ice/refrigerating will resolidify gelatin if it has not be hydrolyzed by gelatinase.

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Lipid Hydrolysis

Lipid/Trybutrin agar inoculated with Pseudomonas aeruginosa (top), Serratia marcesens (lower left), and (lower right).  E. coli grows on lipid agar but does not have the lipase exoenzyme to break down the trybutrin.

Pseudomonas aeruginosa grown on Lipid agar for 72 hours at 37 degree's C to look for the presence of the exoenzyme Lipase. Pseudomonas aeruginosa, Lipid hydrolysis, as noted by a zone of clearing around the organism,

Serratia marcesens was grown on Lipid agar for 72 hours at 37 degree's C to look for the presence of the exoenzyme Lipase . Serratia marcesens exhibits lipid hydrolysis as well as red colony pigment prodigiosin.

Starch Agar

This agar contains high molecular weight starch molecules.  If an organism contains the exoenzyeme amylase then a clear zone will appear around the organism.  After growing organisms on the starch agar for 24 hours iodine is added to to plate.  Iodine reacts with the starch in the agar producing a dark brown/purple color.  If the organism is able to break down the starch in the agar then a clear zone will form around the organism.

(A)                  (B)

(A) Bacillus subtilus:  clear zone around the growth after addition of iodine indicates that bacillus contains amylase and can break down the starch present in the agar.

(B).  E coli:  growth os seen but there is no clear zone around the growth, no starch break down.  

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All images copyright T. Sturm