Screening Of Multiple Samples Research Articles

Nanofluidic and monolithic environmental cells for cryogenic microscopy

We present a device capable of combining nanofluidics and cryogenic transmission electron microscopy (cryo-TEM) to allow inspection of water-soluble samples under near-native conditions. The devices can be produced in a multitude of designs, but as a general rule, they consist of channels or chambers enclosed between two electron-transparent silicon nitride windows. With the appropriate design, those devices can allow screening of multiple samples in parallel and remove the interaction between the sample and the environment (no air–water interface). We demonstrate channel sizes from 80 to 500 nm in height and widths from 100 to 2000 μm. The presented fabrication flow allows producing hollow devices on a single wafer eliminating the need of aligning or bonding two half-cavities from separate wafers, which provides additional resistance to thermal stress. Taking advantage of a single-step through-membrane exposure with a 100 keV electron beam, we introduced arrays of thin (10–15 nm) electron-transparent silicon nitride membrane windows aligned between top and bottom (200–250 nm) carrier membranes. Importantly, the final devices are compatible with standard TEM holders. Furthermore, they are compatible with rapid freezing of samples, which is crucial for the formation of vitreous water, hence avoiding the formation of crystalline ice, that is detrimental for TEM imaging. To demonstrate the potential of this technology, we tested those devices in imaging experiments verifying their applicability for cryo-TEM applications and proved that vitreous water could be prepared through conventional plunge freezing of the chips.

Extracellular virulence factors in Bacillus cereus endophthalmitis: methods and implication of involvement of hemolysin BL

Bacillus cereus is a common cause of highly fulminant posttraumatic and metastatic endophthalmitis. Exotoxins or enzymes likely contribute to the severity of the infection, but specific virulence factors have not been identified. We developed two methods for the identification of B. cereus ocular virulence factors. In an in vitro assay that allows screening of multiple samples, retinal toxicity was estimated by measuring the release of lactate dehydrogenase from retinal buttons treated with B. cereus toxins. The results from this assay were confirmed with a sterile endophthalmitis model in which the histopathologic effect of intravitreally injected toxins was assessed. We tested pure hemolysin BL (HBL), a tripartite dermonecrotic vascular permeability factor of B. cereus, and crude exotoxin (CET) preparations, consisting of concentrated, cell-free B. cereus culture supernatant. In the in vitro assay, both CET and HBL caused rapid release of lactate dehydrogenase and retinal disintegration. In vivo, the toxins caused endophthalmitis clinically characteristic of B. cereus within 4 h. Histological changes included rapid retinal necrosis and detachment, choroidal edema, detachment and disruption of the retinal pigment epithelium, and rapid infiltration of polymorphonuclear leukocytes. Neutralization of HBL in CET preparations inhibited toxicity in vitro by 54%, and pure HBL was less toxic than CET with equal HBL contents in both methods. The results suggest that B. cereus ocular virulence is multifactorial and that HBL contributes to virulence.

Detection of the common alpha‐1‐antitrypsin variants by denaturing gradient gel electrophoresis

The well-characterized and highly polymorphic human alpha-1-antitrypsin (AAT) gene was used as a test locus to evaluate the general applicability of denaturing gradient gel electrophoresis (DGGE) for the detection of single base change polymorphisms. We report the resolution of all the major alleles, M1Ala, M1Val, M2, M3, S and Z and the identification of substantial genetic polymorphism in intron 3-4 by this technique. DGGE was found to be a quick and efficient method for the screening of multiple samples for the presence of genetic variation.

Multiple uses of liquepipets in Southern, Northern, and Western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffer within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.