A Sugar‐Containing Basal Medium for Vitamin B₁₂‐Assay with Euglena; Application to Body Fluids*

Abstract

SUMMARY. An improved assay of vitamin B12 is described. The z strain of Euglena gracilis, which grows more vigorously than the bacillaris strain previously used, is recommended. The pattern of B12 specificity of the two strains appears to be the same. A new medium containing sucrose, aspartic acid, glutamic acid, and glycine at pH 3.6 is well buffered and allows luxuriant growth. Vigorous utilization of sugar appears to depend on readily available nitrogen and a Krebs‐cycle component; these requirements are well met by aspartic acid (or asparagine) and glutamic acid. The proposed procedure is especially suitable for the measurement of B12 in blood serum because rise in pH and precipitation of serum proteins during incubation are minimized. Like bacillaris, the z strain allows the distinction between “combined” and “uncombined” B12 in serum. Serum may be an appropriate test material to tell whether such phagotrophs as Peranema can better utilize bound forms of vitamins than can the related osmotrophs. Patterns of B12 requirements and occurrence are discussed as phylogenetic markers.

Ribulose 1,5-Diphosphate Carboxylase Synthesis in Euglena

“…This ineffectiveness has been confirmed by Bovarnick et al (4,5) who reported that chloramphenicol, even at 2 mg/ml, did not prevent the synthesis of NADP-linked triose phosphate dehydrogenase in regreening Euglena. This enzyme is apparently translated on 87S cytoplasmic ribosomes since its synthesis was completely prevented by 15 ,ug/ml of cycloheximide (5). The failure of chloramphenicol to inhibit cell division in Euglena suggests that protein synthesis on mitochondrial ribosomes is not prevented in vivo, possibly because mitochondria are impermeable to this inhibitor (5).…”

“…The D-threo-isomer of chloramphenicol was used exclusively throughout this work (8). The final concentration of inhibitors used was D-threo-chloramphenicol, 1 mg/ml; cyclohemixide, 15 ,g/ml; 5-fluorouracil, 5 mm; rifampicin, 250 ,ug/ml. All NaH'4C03 into acid-stable product was determined exactly as described previously (21).…”

“…Dark-grown cells were obtained by growing Euglena gracilis Klebs strain Z at 25 C, with stirring, in the medium of Hutner et al. (15) in the dark for 4 days. The cells were harvested by centrifugation and were transferred to fresh growth medium from which all carbon sources other than vitamins had been omitted.…”

“…Resting cells were regreened by exposure to continuous white light (6000 lux) after the addition of D-glucose to give a final concentration of 22.5 g/l. Regreening cultures were transferred back into darkness after harvesting the cells by centrifugation and resuspending them in an equivalent volume of Hutner's medium (15).…”

Chlorophyll Formation in Euglena gracilis var. bacillaris: Interference by Analogues of Purines, Pyrimidines and Amino Acids

“…Estimates of growth were made, after suitable dilution of cultures, by the method of Hutner, Bach & Ross (1956), by measuring the turbidity with a Klett-Summerson photoelectric colorimeter with a 66 (640-700 mp) filter. Photosynthetic pigments were routinely determined by extraction of euglenas with 3 x 5.0 ml.…”