Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

Pentose phosphate pathway in rat colonic epithelium

The colonic cells of the large intestine are one of the most proliferative tissues of the animal body. The pentose pathway has an essential role in cell division and growth being the only pathway forming ribose 5-P necessary for all nucleotide and nucleic acid sunthesis. The pentose pathway may also provide reducing potential as NADPH for biosynthesis and C-3- C-8 glycolyl compounds. The maximum catalytic capacities of the reactions of the non-oxidative pentose pathway for the conversion of ribose 5-P to hexose and triose phosphates by the proximal and distal colon under feeding and starvation regimes are among the highest in the animal body. The qualitative presence of the oxidative pentose pathway was assessed by measurement of the C-1/C-6 ratio value of 1.67-1.82. Enzymes of the F-type and L-type pentose pathways are present in colonocytes and their maximum catalytic activities in colonocyte cytosol are reported. The contribution of the F-type pentose cycle to the total glucose metabolism of colonocytes, measured by the specific yield method, is negligibly low (approximately 1.5%). Colonic epithelial cells use glucose at a high rate (7.1 +/- 0.33 mumol min-1g-1 dry wt) and 79% of the glucose is converted to lactate. Arabinose 5-P has an intermediary role in the formation of keto pentose, sedoheptulose and hexose phosphates from ribose 5-P by colonocyte cytosol. The intermediary and reaction products of [1-13C] ribose 5-P dissimilation by colonocytes is investigated by 13C NMR spectroscopy. The 13C positional isotope distributions show labelling of C-1 and C-3 of hexose 6-phosphates consistent with either the theoretical predictions of the F-type pentose pathway or of the activities of exchange reactions catalysed by transketolase and/or transaldolase. Measurements of exchange reactions showed that the C-1/C-3 labelling of these compounds is mostly, if not wholly, attributable to exchange catalysis by these group transferring enzymes. The results suggest that the F-type PC has little role in the glucose metabolism of colonocytes and pentose phosphate formation may thus occur by a contribution (approx 20% of the total glucose metabolism) by the alternate L-type pathway.     

The Tc5 Family of Transposable Elements in Caenorhabditis Elegans

We have identified Tc5, a new family of transposable genetic elements in the nematode Caenorhabditis elegans. All wild-type varieties of C. elegans that we examined contain 4-6 copies of Tc5 per haploid genome, but we did not observe transposition or excision of Tc5 in these strains. Tc5 is active, however, in the mut-2 mutant strain TR679. Of 60 spontaneous unc-22 mutations isolated from strain TR679, three were caused by insertion of Tc5. All three Tc5-induced mutations are unstable; revertants result from precise or nearly precise excision of Tc5. Individual Tc5 elements are similar to each other in size and structure. The 3.2-kb element is bounded by inverted terminal repeats of nearly 500 bp. Eight of the ten terminal nucleotides of Tc5 are identical to the corresponding nucleotides of Tc4. Further, both elements recognize the same target site for insertion (CTNAG) and both cause duplication of the central TNA trinucleotide upon insertion. Other than these similarities to Tc4, Tc5 is unrelated to the three other transposon families (Tc1, Tc3 and Tc4) that transpose and excise at high frequency in mut-2 mutant strains. Mechanisms are discussed by which four apparently unrelated transposon families are all affected by the same mut-2 mutation.     

Use of the polymerase chain reaction and 16S rRNA sequences for the rapid detection of Brochothrix spp. in foods

Oligonucleotide primers were designed against rRNA sequences to give a DNA-based PCR assay for the rapid identification/detection of Brochothrix spp. The PCR products could be confirmed by hybridization to an internal oligonucleotide probe. The method successfully and sensitively detected/identified these organisms in pure cultures but was of limited value as a detection method because the detection sensitivity, in relation to conventional plate counts, varied and the assay sensitivity was reduced in the presence of staphylococci. Furthermore, sensitivity was also lost when the assay was applied directly to meat samples. However, a separation step using a lectin (from Agaricus bisporus ) immobilized on magnetic beads prior to the PCR assay, allowed the direct detection of low numbers (> 10 cfu g^-1) of Brochothrix in meat samples within a working day.     

Infectivity of the Santa Lucia (El Salvador) strain of Plasmodium falciparum to different anophelines.

Anopheles freeborni mosquitoes were much more heavily infected with the Santa Lucia strain of Plasmodium falciparum from coastal El Salvador than were any of the other species tested. Of 5 strains of A. albimanus examined, the most heavily infected was the CA-109A and the least was the Melara, both of which come from coastal El Salvador. Of the exotic anophelines, the A. maculatus was infected at a slightly higher level than was the A. balabacensis. The incidence of highly infected individual mosquitoes was greatest in the Panama-Escobal strain of A. albimanus from the Republic of Panama; the incidence was lowest in the Melara strain from El Salvador. All strains of A. albimanus developed infected salivary glands, but the A. freeborni and A. maculatus mosquitoes appeared to develop infected glands more effeciently. Infection rates in A. freeborni mosquitoes were highest if mosquitoes were fed on Aotus trivirgatus monkeys between the 19th and 25th days of patent gametocytemia.