Is Harbor Porpoise (Phocoena phocoena) Exhaled Breath Sampling Suitable for Hormonal Assessments?

Over the last decades, exhaled breath sampling has been established for laboratory analysis in various cetacean species. Due to their small size, the usability of respiratory vapor for hormone assessments was questionable in harbor porpoises (Phocoena phocoena). This pilot study compared three different blow collection devices for their suitability in the field and during laboratory processing: a sterile petri dish covered by a Nitex membrane, as well as sterile 50 mL centrifuge tubes with or without manganese(II) chloride as a stabilizer. Collected exhales varied between three, five or ten, depending on feasibility. Hormones were extracted through an ether mix, followed by centrifugal evaporation and cortisol analysis using an immunoassay. Although close to the lower end of the assay's dynamic range, the ELISA produced results (n = 110, 0.102–0.937 ng/mL). Hence, a simple 50 mL centrifuge tube was determined as the best suited blow collection device, while three consecutive exhales proved sufficient to yield results. These findings are promising regarding the suitability of exhaled breath as a matrix for future endocrine and immune system-related studies in harbor porpoises. If further advanced, blow sampling can become an important, non-invasive tool for studying and monitoring health, stress levels and diseases in harbor porpoises.

New sequential touch method for determining bacterial contact transfer rate from finger to surface

ABSTRACTBacteria can be transferred via surface touch. To evaluate the transfer rate, traditional single-touch methods require measuring the number of bacteria on donor and recipient surfaces, which is typically characterized by high levels of uncertainty. In this study, two concentrations ofStaphylococcus aureusATCC 25923 were inoculated on a clean thumb. For each set of trials, sequential touches were made between the thumb and each of 30 sterile glass slides, and each slide was placed in a sterile petri dish. The transferred bacteria on each slide were directly cultured in situ, and the colony-forming units (CFUs) were counted. The bacterial contact transfer rate was calculated by fitting the series of CFUs with the formula established. The average transfer rate was 12.9% under these conditions. The goodness of fit was compared in terms of the number of slides used in a set of trials and the number of CFUs counted on the slides. The use of more slides in a set of trials allowed more accurate evaluation of the transfer rate. The use of fewer than 20 slides was unacceptable. The high density of CFUs on the slides made counting them difficult, but if fewer than five CFUs were counted in a set of trials, the fit would be significantly influenced. To further evaluate the method, the dermal resident microflora on the thumb were also used to perform contact transfer tests. No statistically significant difference was found in the estimated transfer rate between the standard strain and the resident microflora.IMPORTANCEDiseases can be transferred indoors via the surface route because bacteria and viruses can be transferred to and from the hands when a fomite is touched. Various methods have been used to estimate the bacterial contact transfer rate between hands and surfaces. Evaluated transfer rates have had significant deviations and varied significantly across studies, partially due to the use of the single hand-surface touch method, inefficient hand/surface sampling, and complicated bacteria culture.In this study, the bacterial contact transfer rate was evaluated with a new method involving sequential touches between a donor and a series of recipients. The bacteria on the recipients were cultured in situ without hand/surface sampling, which simplified the process of surface bacteria quantification. The new method significantly reduces experimental complexity, decreases random errors in the data, and provides a new method for understanding microbial transfers between surfaces.

Evaluation of allelopathic potential of safflower genotypes (Carthamus tinctorius L.)

Forty safflower genotypes were grown under normal irrigation and drought stress. In the first experiment, the allelopathic potential of shoot residues was evaluated using the sandwich method. Each genotype residue (0.4 g) was placed in a sterile Petri dish and two layers of agar were poured on that. Radish seeds were placed on agar medium. The radish seeds were cultivated without safflower residues as the controls. The length of the radicle, hypocotyl, and fresh biomass weight and seed germination percentages were measured. A pot experiment was also done on two genotypes with the highest and two with the lowest allelopathic activity selected after screening genotypes in the first experiment. Before entering the reproductive phase, irrigation treatments (normal irrigation and drought stress) were applied. Shoots were harvested, dried, milled and mixed with the topsoil of new pots and then radish seeds were sown. The pots with safflower genotypes were used to evaluate the effect of root residue allelopathy. The shoot length, fresh biomass weight, and germination percentage were measured. Different safflower genotypes showed varied allelopathic potential. The results of the first experiment showed that Egypt and Iran-Khorasan genotypes caused maximum inhibitory responses and Australia and Iran-Kerman genotypes resulted in minimum inhibitory responses on radish seedling growth. Fresh biomass weight had the most sensitivity to safflower residues. The results of the pot experiment were consistent with the results of in vitro experiments. Residues produced under drought stress had more inhibitory effects on the measured traits. Safflower root residue may have a higher level of allelochemicals or different allelochemicals than shoot residue.

Screening of Fungal Isolates Against Diamondback Moth Larvae, 1995

Fifty-five isolates of four fungi, Beauveria bassiana (Balsamo) Vuillemin, Fursarium sp.,Metarhizium anisopliae (Metschnikoff) Sorokin and Paecilomyces farinosus (Holm ex SF Gray) Brown & Smith, were obtained from the USDA-ARS Collection of Entomopathogenic Fungal Cultures (Ithaca, NY). Isolates were subcultured once after retrieval from liquid nitrogen storage. Cultures were maintained on Subouraud dextrose agar plus 2% yeast extract (SDAY) at 24° Cunder a photoperiod of 15:9 h (L:D) for 14 to 21 d. Conidia were scraped from the culture surface and suspended in 0.01 % Tween 80 by vortexting for 2 min. Suspensions were filtered through 16 layers of sterile cheesecloth to remove conidial clumps and hyphal fragments. Spore concentrations were estimated using a hemacytometer and then adjusted to 312,500 spores/ml. Three to 7 isolates were screened against isolate ARSEF 4543 as a standard derived from a single infected DBM larva collected in Ontario County, NY, in 1994. First instars of DBM maintained on wheat germ diet. Fifteen 2nd instars were placed 2–3 h prior to inoculation in a petri dish containing filter paper moistened with deionized water and incubated at 4° C. Dishes were assigned randomly to treatments with 4 replicates each for each isolate. Insects were inoculated at room temperature (–22° C) using a Bergergon spray tower equipped with a Spraying Systems 2850 noz-zle, calibrated to deliver 312.5 spores/cm2 using 5 ml suspension at a concentration of 312,500 spores/ml. DBM larvae treated only with the suspending medium (0.01% Tween 80) served as checks. (Mortality in the checks was minimal and no evidence of fungal infection was detected.) After spraying the insects, two discs of DBM larval diet (Bioserv Premix) were placed in each dish which was then sealed with parafilm and incubated at 25° C in darkness for 24 h. Thereafter, the insects were transferred to a sterile petri dish with fresh diet and incubated at 25° C under a photoperiod of 15:9 h (L:D). Larvae were monitored daily and diet was replaced as needed for 7 d. Dead larvae were transferred to petri dishes containing a piece of moistened filter paper. Mycosis was confirmed by observing characteristic mycelial eruption from cadavers and subsequent sporulation. To confirm viability of spore suspensions and dosage, 5 ml of each was sprayed on SDAY plates which were incubated at 25° Cunder a photoperiod of 15:9 h (L:D) for 3 d until identifiable fungal colonies were counted. To estimate the potency of each isolate relative to the standard (ARSEF 4543), the percentage mortality due to fungus infection was divided by the percentage mortality due to infection by the standard within that experiment. Average survival times were also estimated for all fungus-killed larvae for each isolate from records of the number of days from inoculation to death.

Neustaedter. Modification of the Frank-Goldberger method for the determination of estrogenic substances in the blood (Endocrinologia Los Angelos, V. 2 (. XL. 1936)

A syringe is taken from the cubital vein 40 cubic meters. cm of blood and place it in a sterile Petri dish, in which 30 to 40 g of dehydrated sodium sulfate are poured, mix with a glass rod until a thick mass is obtained. The dried mass is transferred into a mortar and turned into powder. If the mixture does not thicken, then add more sodium sulfate. The powder is transferred to an Erlenmeyer flask and poured over 100 cubic meters. cm of ether, is mixed in a scupper for 20 minutes. Then the flask is placed on a special shelf that gives it a 45 tilt. The top layer of liquid is decanted and centrifuged twice in a row. The centrifuged ether is evaporated to dryness. The lipoid precipitate is dissolved in 6 cubic meters. cm of gasoline, to which 0.6 cu. see olive oil. The gasoline is allowed to evaporate. The olive oil, into which the lipoid extract has passed, is sterilized in an autoclave at 15 pounds of pressure for 15 minutes. When placed in a dark bottle with a ground-in stopper, this extract can be stored for several days. It is administered in fractionated doses to a sexually mature castrated mouse. On the first day, three injections are given at four hour intervals. The next day, two, at the same intervals. The extract is injected into the mouse under the skin of the back. Starting the next day, vaginal swabs are prepared for 4 consecutive days, twice a day. The smears are stained with 1% thionine.