Twelve-well culture plate for the efficient collection and culture of human oocytes and embryos.

OBJECTIVE: Our goal was to assess a 12-well oocyte collection and embryo culture plate for use in the IVF laboratory. DESIGN: This was a prospective nonrandomized study. SETTING: The setting was a university in vitro fertilization program. PATIENTS: Eighty-four consecutive infertility couples presenting for IVF were studied. MAIN OUTCOME MEASURES: The main outcome measure was pregnancy (delivered). RESULTS: A 34% delivered pregnancy rate per retrieval was attained using the 12-well collection and culture plate without the use of expensive culture media and special serum supplementation. CONCLUSIONS: The use of a 12-well plate for oocyte collection and embryo culture provides a simple, economical, efficient, and effective means of producing human embryos during in vitro fertilization. This system is capable of supporting high rates of ongoing and delivered pregnancies.

Intrauterine insemination. Effect of wash media on pregnancy rates and sperm motility.

Testyolk Buffer has been shown to enhance sperm penetration in hamster penetration assays and increase fertilization of oocytes in in vitro fertilization. Based on these findings, we compared pregnancy rates and sperm motility in intrauterine inseminations done with sperm samples washed and resuspended in Ham's F10 as compared with Testyolk buffer. Charts were reviewed retrospectively from 1,098 husband and donor intrauterine inseminations performed at the University of Florida. Data were analyzed using life table analysis and the curves compared with the Mantel-Haenszel statistical test. In addition, sperm motility in fresh sperm was observed in samples incubated in Testyolk or Ham's F10, with motility counts performed at 0, 6 and 24 hours. Four hundred ninety-two Testyolk cycles and 579 Ham's F10 cycles were compared, with cumulative pregnancy rates at one year of 53% and 44%, respectively (P = .58). With donor sperm, 229 cycles with Testyolk and 314 cycles with Ham's F10 had cumulative pregnancy rates of 68% and 48%, respectively (P = .52). With husband insemination, 264 Testyolk and 253 Ham's F10 cycles had pregnancy rates of 37% and 35%, respectively (P = .23). Fresh sperm motility in 22 samples compared at 0, 6 and 24 hours in Ham's F10 (76%, 67.8%, 56.6%) versus Testyolk (76%, 67.7%, 58.8%) revealed no significant differences. There was also no difference in total motile sperm inseminated and postwash motility in 1,098 samples with Testyolk versus Ham's F10. This study demonstrates that there is no enhanced pregnancy rate or increased sperm motility when sperm are treated with Testyolk Buffer instead of Ham's F10.

Use of whey for production of exocellular polysaccharide by a mutant strain of Xanthomonas campestris.

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain of Xanthomonas campestris lac+ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium.

The possibilities of changing the production of exocellular polysaccharides of a mutant Mycobacterium strain.

By second-step mutagenesis and treatment with N-methyl-N'-nitro-N-nitrosoguanidine a mutant strain of Mycobacterium sp. V-649 producing a glucan extracellular polymer and another new streptomycin-resistant mutant were prepared. This mutant strain formed more than 100% first-rate (1.0-1.2%) exocellular polysaccharide. Treatment with 1% dimethyl sulfoxide during submerged cultivation of the mutant strain did not increase the production of the extracellular polysaccharide.

Composition of intracellular soluble proteins in Mycobacterium "rubrum" and its mutants.

The composition of intracellular soluble proteins in a parental strain of Mycobacterium "rubrum" and its mutants was studied by polyacrylamide gel gradient electrophoresis. The composition of the protein fractions of the parental and the mutant strains was similar, they differed in one protein only. The presence of a 60 kDa protein should be stressed since its increased amount may be connected with a different intensity of the production of pigments in the strains studied.

Effect of Tween 80 and dimethyl sulfoxide on biosynthesis of L-lysine in regulatory mutants of Corynebacterium glutamicum.

By using dimethyl sulfoxide or Tween 80 (1 or 0.2%), the production of L-lysine was increased by 20-28 and 23-25%, respectively, in regulatory mutant strains of Corynebacterium glutamicum. The stimulation observed is supposed to be caused by influencing cellular surface structures.

Gene manipulation in mycobacteria.

Gene manipulation in mycobacteria developed in two phases. In the first phase genes of mycobacteria were transferred into cells of E. coli and Streptomyces lividans. In the second phase, heterologous genes were transferred into mycobacteria either with a shuttle plasmid or hybrid plasmids. A prerequisite for successful gene manipulation in mycobacteria was a thorough understanding of plasmids in mycobacteria. Construction of recombinant DNA molecules contributed not only to the fact that mycobacteria did not remain outside the mainstream of modern genetic research but also to their present practical importance.

Production and characteristics of the exocellular polysaccharide of a mutant Mycobacterium strain.

Mutant strains of Mycobacterium sp. V-649 producing highly mucous colonies on a solid cultivation medium were prepared after treatment with N-methyl-N'-nitro-N-nitrosoguanidine and production of the exocellular polysaccharide was tested. The strains were cultivated in media with suitable sugar sources under submerged conditions. It was found that Mycobacterium sp. V-649/15 produces a maximum of 15-19% polymer after a 5-6-d cultivation. Gas chromatography indicated that the exocellular polysaccharide produced by this strain is of glucan type.

Mycobacteria in the light of modern genetics development.

It is generally assumed that genetic research of mycobacteria is delayed as compared with other, more commonly used, bacterial models, particularly in the field of genetic transfers. In the field of mutagenesis the problems have been studied to such an extent that replication maps of the chromosome of M. phlei and M. tuberculosis H37 Rv have already been constructed and a new model of the cell cycle of bacteria exhibiting a slow growth rate has been worked out. When the problems of mycobacterial genetics are looked upon in the light of gene manipulations it may be concluded that mycobacteria belong to a few models whose genes are used for cloning and that problems of practical significance will be studied by means of the most modern approaches.

Pigmentation changes in brevibacteria induced by mutagenic treatment.

Inducible pigmentation changes were observed in pigmented strains of Brevibacterium sp. M27 and B. flavum treated with N-methyl-N'-nitro-N-nitrosoguanidine. The highest frequency of induction was reached already at a survival of 30-40% with the maximal yield of 6-10%. As compared with the initial yellow colour, three new pigmentation types, viz. white, pink and orange, were observed. The yellow pigmented parent strains are most resistant to the lethal effects of UV radiation. By selecting pigmented mutants of all types on media containing antibiotics it was possible to obtain strains that were resistant either to tetracycline or to streptomycin. Auxotrophic pigmented mutants were also isolated. In multiple mutant strains of Brevibacterium sp. M27 a number of strainsexhibited a changed L-lysine production. In some strains the production was variable, whereasother strains did not produce L-lysine at all and stains with a limited production of other amino acids were also detected.

Induction of mutants resistant to antibiotics in Brevibacterium flavum.

The optimum conditions for the induction of mutants resistant to antibiotics in Brevibacterium flavum ATCC 14067 were determined. UV irradiation at the energy fluence of 6.5 kJ/m2 and N-methyl-N'-nitro-N-nitrosoguanidine (1 mg/mL) at pH 6.0 were used for the induction of mutants. Mutant strains resistant to rifampicin, oleandomycin, streptomycin and erythromycin were prepared.

Genetic transfers in Brevibacterium sp.

A positive genetic transfer by protoplast fusion was obtained in auxotrophic mutants Brevibacterium sp. M27 his and Brevibacterium sp. M27 arg. Transformation and protoplast fusion with liposomes (as genetic transfers in intact cells and their protoplasts by both the chromosomal and plasmid DNA) did not lead to transfer of the markers followed.

Cell cycle in Mycobacterium phlei.

The initial replication region of the chromosome on the replication map of M. phlei constructed by means of sequential mutagenesis in synchronous populations was accurately determined. By following the time shift of the replication moment of the genes bac and met in the control culture and in the culture with the initial inhibition of DNA synthesis by nalidixic acid the start of replication of the chromosome was determined at 15 min before replication of the gene ile. On the basis of the results obtained a scheme of the cell cycle in M. phlei was proposed. Intervals C and D depend on the generation time, become prolonged independently of each other and assume the whole cycle. The ratio C/(C + D) equals to 0.56 and the interval D has a value of 0.76 of the interval C. The mutual ratio of the intervals C : D is 1.3 : 1.0. The obtained results make it possible to form the assumption about mutual ratios between the chromosome replication and cell division in bacteria exhibiting slow growth rates.

Use of nalidixic acid for constructing the replication map of the Mycobacterium phlei chromosome.

Nalidixic acid was used for describing more accurately the terminal replication region of the Mycobacterium phlei chromosome. Cell division in synchronized cultures was not sensitive to this acid any more between 185-190 min, i.e. about 10 min after replication of the ser gene, the last of 24 genes of the replication map described so far. The replication of the chromosome was controlled by determining the position of the bac gene. Microscopic studies in phase contrast of the cells that were subjected for long time periods to nalidixic acid treatment at a bactericidal concentration showed elongated cells. The electronmicroscopic observation showed that a portion of the population influenced by nalidixic acid lyses, whereas other cells remain intact and resemble control cells.

Light and scanning electron microscopic studies of oxamide urolithiasis in rats.

The formation of urinary oxamide stones was induced in rats by feeding them oxamide mixed with powdered rat chow. The structure of these stones and the changes in the rat renal papillary structure following oxamide administration were studied using bright field and polarizing light microscopy as well as scanning electron microscopy. Oxamide appeared in the papillary collecting ducts, pelvis, ureters and urinary bladder of the rats in the form of yellow spherulitic units composed of dendritic crystals. Oxamide stones in turn were aggregates of these spherulites. Our results indicate the renal stone formation started with the crystallization of oxamide in the tubular lumina of the collecting ducts of the papillae. Crystal formation in the tubules was associated with epithelial necrosis. Some of the crystals became attached to injured epithelium, thus impeding urinary flow. The attachment of the crystals resulted in their retention in the renal tubules. These oxamide deposits then grew or aggregated to form stones. The formation of oxamide deposits in the ducts of Bellini resulted in dilatation, compression of the epithelium and destruction of the papillary urothelium. These factors resulted in the deformation of the papillary tip of the kidney.

The completion of the replication map of the Mycobacterium phlei chromosome.

New facts about the replication map of Mycobacterium phlei chromosome are summarized. Replication positions of two genes located in marginal regions of the replication map, ile close to the origin and ser near the terminus, were determined. Known positions of replication of some genes were defined with more precision within 2.5--5-min intervals using the method of sequential mutagenesis in synchronized cultures (leu, met, bac, pyr, stm, tet, cyc, his). Replication positions of genes responsible for the biosynthesis of thiamine and resistance to tetracycline and vancomycin were further identified. The contemporary replication map contains replication positions of 24 genes.

Mutagenic effect of n-methyl-N-nitrosourea on Mycobacterium phlei.

N-Methyl-N-nitrosourea was used to induce auxotrophic, scotochromogenic and isonicotinic acid hydrazide resistant mutants in Mycobacterium phlei and its effect was compared with that of nitrosoguanidine. Seventeen auxotrophic mutants requiring amino acids or vitamins and 52 scotochromogenic mutants with orange colonies were induced. The frequency of isonicotinic acid hydrazide-resistant mutants increased by two orders of magnitude.