OCCURRENCE OF FREE AMINO ACIDS — MICROORGANISMS 109 
FREE AMINO ACIDS IN PROTOZOA 
JOHN B. LOEFER anp OTTO H. SCHERBAUM 
Office of Naval Research and California Institute of Technology, Pasadena, Calif. (U.S.A.); 
University of California, Los Angeles, Calif. (U.S.A.) 
Reports on the occurrence of free amino acids (AAs) in protozoa are available for 
only three genera, Vorticella, Paramecium and Tetrahymena. Although arrays of 
bound or protein amino acids (PAAs) have been determined in more cases, investiga- 
tions of this type on protozoa have, indeed, been restricted by the lack of axenic 
culture methods. In a recent review (LOEFER AND SCHERBAUM?) covering both PAAs 
and FAAs, findings were recorded for ten species other than those belonging to Tetra- 
hymena and for six strains of 7. pyriformis, in addition to the results they obtained 
for T. limacis and seven strains of 7. pyriformis. This report summarizes investiga- 
tions on the FAAs reported for protozoa. 
Even in cases where investigators have used axenic cultures, as has been true for 
the reports on FAAs in Tetrahymena (cf. Table I), a comparison of results and evalua- 
tion of data may be difficult for various reasons, such as variation in methods of culture 
or analysis of amino acids. In some cases, quantitative data were not available, or 
if available, were not readily susceptible to comparative interpretation. Hence, it 
will be noted in Table I that records of qualitative results only are reported for Para- 
mecium and Vorticella. 
Comparative results for various strains of Tetrahymena will be noted (Table I) 
after reviewing our own results on T. lémacis and seven strains of T. pyriformis. 
The methods employed had been worked out in connection with earlier studies 
(LOEFER AND SCHERBAUM!; LOEFER AND SCHERBAUM?; SCHERBAUM et al.) and 
details of the procedures are described in these papers. 
Essentially, the method involved use of concentrated washed organisms, axenically 
grown, from stationary-phase cultures. In most cases they were grown at 25°, al- 
though some analyses were also made on material from 10°, 29°, and 35° cultures. 
FAAs were extracted in the manner previously reported (SCHERBAUM ef al.3). The 
method involves principally extraction with hot ethanol (80%). Lipids were removed 
with CHCl, and the aqueous phase evaporated. The dried residue was dissolved in 
10° isopropanol for chromatography. In Figs. 2-6 each chromatogram was spotted 
with material extracted from 10 mg of dried organisms. The descending method of 
development was used with butanol-acetic acid—water for the first, and phenol—water 
for the second dimension. Exposure time for each solvent was approx. 15 h at 29°. 
Color spots following ninhydrin treatment were recorded photographically. F; values 
obtained are indicated in Fig. 1, the standard against which the spots in Figs. 2-6 
may be compared for identification. 
Preliminary work on the quantitative estimation of amino acids had been made 
* Grateful acknowledgement is made to the J. Protozool. for permission to use some previously 
published data and figures. 
References p. 114 
