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The addition of 2% Trypticase to a minimal salts-glucose plating medium increased the bacterial count of frozen and thawed suspensions ofEscherichia coli 451B cells, even when precautions were taken to remove toxic trace elements from the plating diluent. Hydrolysis of the Trypticase with HCl or H2 SO4 reduced its count-increasing activity. Treatment of the H2 SO4 hydrolysate with a cation-exchange resin greatly improved its capacity to replace Trypticase. Addition of a mixture of amino acids approximating the composition of casein also increased the plate count when added at a level equivalent to 0.1% casein, but at 2% it depressed the count. Tests of amino acids in the mixture revealed that aspartic acid could replace Trypticase completely as a supplement to the basal medium. When added at a level of 2.5 mm , aspartic acid doubled and occasionally tripled the plate count of a suspension of frozen and thawed cells. Glutamic acid, alanine, and to a lesser extent certain other amino acids also showed a capacity to increase the count. Cysteine was without significant effect. Serine and other amino acids depressed the count. None of the amino acids or other supplements affected the count of suspensions of cells that had not been frozen. The effect of adding aspartic acid, cysteine, or Trypticase to the basal medium on the bacterial count of suspensions of various strains ofE. coli, Aerobacter aerogenes, Serratia marcescens , and two species ofPseudomonas after freezing was examined. The response to the supplements was unique for each organism.

Capacity of Aspartic Acid to Increase the Bacterial Count on Suspensions of Escherichia coli After Freezing