Resuspension Method Research Articles

Resuspension method for road surface dust collection and aerodynamic size distribution characterization

Traffic-generated fugitive dust is a source of urban atmospheric particulate pollution in Beijing. This paper introduces the resuspension method, recommended by the US EPA in AP-42 documents, for collecting Beijing road-surface dust. Analysis shows a single-peak distribution in the number size distribution and a double-peak mode for mass size distribution of the road surface dust. The median diameter of the mass concentration distribution of the road dust on a high-grade road was higher than that on a low-grade road. The ratio of PM 2.5 to PM 10 was consistent with that obtained in a similar study for Hong Kong. For the two selected road samples, the average relative deviation of the size distribution was 10.9% and 11.9%. All results indicate that the method introduced in this paper can effectively determine the size distribution of fugitive dust from traffic.

Resistance of antivenom proteins to foaming-induced denaturation

Reconstitution of lyophilized antivenom is usually accomplished by gentle swirling for sometimes as much as 45 min. Gentle resuspension is employed in order to avoid foaming the antivenom, which could have a variety of consequences including unfolding and denaturation of the protein, thus rendering it inactive. However, foaming of antivenom might cause only a small portion of the total protein to unfold or the protein may refold as foaming subsides. In this report, the effects of intentional severe foaming of three antivenom preparations are tested. Samples of gently resuspended antivenoms were subject to severe foaming by mechanical means, then tested for precipitation by examining the amounts of protein remaining in solution, for structural changes by circular dichroism spectroscopy and for activity changes by ELISA. In all cases, either no or minimal changes in antivenom properties were observed. These results indicate that severe intentional foaming does not substantially affect the properties of the antivenoms. It may be possible to employ more vigorous resuspension methods without affecting the efficacy of the antivenom. This could lead to a significant decrease in the time between envenomation and administration of antivenom.

Evaluation of detection methods for Legionella species using seeded water samples

South African laboratories are currently using various methods in a non-standardised approach to detect Legionella species in environmental samples. In an attempt to provide guidelines for the development of a standard method, a number of currently available detection methods were evaluated, using seeded samples of sterile and non-sterile tap water, cooling water and make-up water. The samples were seeded with a type strain of L. pneumophila serogroup 1 (American Type Culture Collection 33152). The effect of sample concentration by centrifugation and membrane filtration followed by either vortex or sonication for resuspension of organisms was studied. Three currently available culture methods were evaluated: the International standard method (ISO/DIS 11731), the Australian standard method (AS 3896 - 1991) and a locally-developed adaptation of the most probable number method (MPN). In addition, the direct immunofluorescence test and a commercially available latex agglutination test kit were included in the evaluations. The usefulness of treatment with acid or heat prior to culture was also compared. Our results indicated that concentration by membrane filtration using nitro-cellulose filters with a pore size of 0.45 ?m, followed by sonication for 10 min, would be the most appropriate concentration and resuspension method for the samples. In the absence of sample pretreatment with acid or heat, organism recovery from sterile seeded samples on BCYE ranged from 85.9 - 98.7%. However, in the non-sterile samples, these figures dropped to 8.1 - 38.5%. Sample pretreatment resulted in a further loss of at least 50% of organisms in all the samples, regardless of the pretreatment method or culture medium used. In general, the ISO and AS methods were more appropriate than the MPN method for organism recovery from sterile seeded samples. However, for the nonsterile samples, the MPN method yielded better recovery. WaterSA Vol.27(4) 2001: 523-528

An efficient biofilm removal method for bacterial cells exposed to drinking water

Reliable quantification of microbial growth in a drinking water environment has typically been difficult, primarily due to the development of thin, patchy biofilms. Therefore, initial sampling and resuspension procedures become critical to the subsequent biomass determination. Biofilm cells attached to polycarbonate coupons of an annular reactor (AR) have typically been removed by aseptically scraping the coupon surface with a sterile utility knife. The advantage of this method is its simplicity, however, scraping often compromises sterility and is highly subject to individual variation. The purpose of this research was to develop a method that could remove and resuspend biofilm cells efficiently, consistently and with a good recovery rate. This paper presents a comparison of removal and resuspension methods. Three methods used to remove cells from the polycarbonate coupons include: scraping with a utility knife, swabbing and stomaching. In addition, four methods were selected for cell resuspension and involved the use of a tissue blender, vortex, stomacher and a sonicator. Of the removal methods examined, stomaching consistently yielded the highest number of culturable and total bacterial cells, ranging from two to four times more cells than scraping procedures. For one experimental set, the number of colonies enumerated by heterotrophic plate count (HPC) from the stomacher ranged from 1.0–1.7×10 6 CFU/cm 2, whereas numbers obtained using the scraping method were 4.6–4.9×10 5 CFU/cm 2. It was found that the number of HPCs recovered with stomaching was significantly greater at the 5% level than that obtained using the scraping method. Similarly, for cell resuspension, stomaching provided the highest enumeration. Once removal was achieved, sonication also provided good resuspension. An analysis of variance showed that, compared to the resuspension step, the removal step is more significant at the 5% level to the recovery of biofilm cells. The stomacher has the unique advantage of combining cellular removal and resuspension into a single step. This method was, therefore, selected as an ideal method for recovery of biofilm cells. Subsequent optimization measures using the stomacher showed that sterile deionized water was a suitable diluent for recovering cells from a drinking water environment. At normal speed (i.e., 230 rpm±5%), the optimal stomacher run length for maximum cell removal was 2 min.

Medium-dependent sporulation resulting from a mutation in the spollAB gene of Bacillus subtilis

Summary: The SpollAB protein of Bacillus subtilis is an anti-sigma factor that controls the release of -F in the prespore during sporulation. A missense mutation, spollAB22, in the N-terminal coding portion of spollAB was isolated previously on the basis of conferring increased -F activity on agar plates. We present the results of experiments further characterizing the phenotypic effects of the spollAB22 mutation. The mutation severely impairs spore formation on nutrient agar (NA) plates. Surprisingly, however, cultures induced to sporulate in liquid medium by the resuspension method showed little or no reduction in spore formation, and gene expression was more or less normal. The effects on sporulation on NA can be suppressed by mutations in the spollAC gene encoding -F. The implications for our understanding of the regulation of -F and -G activities, and the nature of the apparent medium dependence of sporulation in the mutant, are discussed.

Influence of Alkali Metal Cations on the Germination of Spores of Wild-type and gerD Mutants of Bacillus subtilis

Spores of gerD mutants of Bacillus subtilis prepared on a rich medium were defective in their germination responses to l-alanine, to a mixture of l-asparagine, d-glucose, d-fructose and KC1 (AGFK), and to Penassay broth. They showed an increased response and sensitivity to lalanine when monovalent cations were also present. When prepared by a resuspension method, the spores had an improved germination response to l-alanine, even in the absence of these cations, but showed no such improvement in response to AGFK. No gross differences were found between wild-type and gerD spores in heat or chemical resistance, dipicolinic acid level or morphology. It is proposed that an alternative pathway of germination involving monovalent cations exists in both wild-type and gerD spores.