An investigation of enumeration and DNA partitioning in Bacillus subtilis L-form bacteria.

Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units (cfu)). The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers. Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts. In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance. The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.

Glucose translocation and metabolism in the rat jejunum perfused in once-through mode in vitro.

Simultaneous once-through luminal and vascular perfusions with glucose of rat jejunum in vitro were used in an attempt to resolve conflicting reports on glucose metabolism in jejuna perfused in once-through mode in vivo and in recirculation mode in vitro. Results include: (a) respiration rates during each perfusion, (b) CO2 production from absorbed glucose, (c) absolute rates of glucose absorption from the lumen into the epithelium, (d) absolute rates of glucose translocation from lumen to vascular bed, (e) absolute rates of lactate excretion into the two perfusion media, (f) recoveries of absorbed glucose, (g) the distribution of radioactivity among translocated glucose and its metabolites. About 50% of the total lactate originates in the glucose extracted from the vascular medium during concomitant absorption and translocation of luminal glucose. The results suggest what further experiments need to be done. Results from recirculation perfusions in vitro cannot contest or confirm the results quoted from experiments conducted with once-through perfusions in vivo.

Lactate is not a major product of glucose metabolism in the respiring jejunum perfused in the recirculation mode in vitro.

Yields of lactate from glucose absorbed from the luminal medium and extracted concurrently from the vascular medium of the perfused rat jejunum in vitro were half those previously reported from comparable glucose concentrations in the recirculated perfusion media. These low yields were obtained by maintaining perfusion medium flow rates and pressures pharmacologically at values found in vivo; permitting normal peristalsis; and restoring the depleted glyceric acid-2,3-bisphosphate and reduced deformability of out-dated blood-bank erythrocytes to normal physiological values before they were added to the vascular perfusion medium and equilibrated at 37 degrees C with air/CO2. Respiration rates are recorded for the first time in each perfusion experiment. A substantial fraction of glucose consumed in this and all earlier recirculation perfusion experiments in vitro has still to be accounted for.

The vascularly and luminally perfused small intestine in vitro: dissection technique and model system.

The quantitative study in vitro of a wide range of intestinal functions is best achieved by perfusing the intestine simultaneously by the luminal and vascular routes. A comprehensive description is given of the dissections involved in preparing a selected segment of jejunum for such simultaneous perfusions. Attention is drawn to a number of occasional aberrant vascular structures which require special attention if successful perfusions are to be established. It is essential to restore the ATP and diphosphoglycerate concentrations of erythrocytes, and to assess their deformability, before incorporating them in the vascular perfusion medium. The perfusion equipment, the perfusion media and the general conduct of a perfusion experiment, are described.