Scanning Fluorometer Research Articles

Preliminary Evaluation of Mixed Dyes for Fingerprinting Non-Fluorescent Bacteria

Abstract A rapid scanning fluorometer is used to obtain fluorescence spectral data characteristic of bacteria which do not readily produce fluorescent pigments. The data is generated in the form of an array of fluorescence intensity as a function of multiple excitation and emission wavelengths. Fluorescence spectral properties are introduced into the bacterial specimen using multiple dye mixtures for staining. The characteristic fluorescent fingerprints are believed to occur by preferential adsorption and reaction of bacterial cellular components with the individual dyes of the mixture. The procedure is found to be rapid and reproducible for the bacteria examined.

Identification of fluorescent Pseudomonas species.

Fluorescent pseudomonads may be identified through examination of the fluorescence "profiles" of diffusible pigments released into the growth medium. The profiles are obtained in the form of an emission-excitation matrix, the elements of which correspond to fluorescence intensity as a function of multiple exciting and emitting wavelengths. Use of a rapid scanning fluorometer to acquire these data provides a relatively fast method for identification of the microorganisms after incubation. This procedure can potentially be expanded for the identification of other microorganisms.

Fluorescent tracer method for protein SH groups. II

An improved fluorescent tracer technique for protein SH groups is described using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The direct measurement with a scanning fluorometer of the fluorophore-labeled proteins separated by SDS polyacrylamide gel electrophoresis achieved a simple, precise, and sensitive determination of the SH group of the order of picomoles per band. In addition, the direct recording of peaks enabled us to analyze more complex protein systems, compared with our previously reported method using an extraction step from gel slices. As an application, we compared the reactivity of the SH groups of proteins in glycerinated muscle fibers under two conditions, in rigor and in contraction.

Spectrofluorescent detection of malonaldehyde as a measure of lipid free radical damage in response to ethanol potentiation of spinal cord trauma

AbstractStudies of the role of free radical damage to the spinal cord following a 400 g‐cm impact have suggested an increase in at least one free radical product, malonaldehyde, 24–36 hr post injury. To investigate further the role of free radical lipid peroxidation in degeneration of the spinal cord following injury, a study of specific lipid fluorescence (SLF) indicative of the double Schiff‐base adduct formed by a reaction between malonaldehyde and cellular components was carried out in the presence of ethanol, a known potentiator of free radical lipid peroxidation. The study was carried out in cats who received a 200 g‐cm impact 3 hr to 23 days prior to sacrifice. Half of the impacted animals received ethanol, 5 ml/kg, prior to injury. These animals were rendered paraplegic, whereas the nonethanol treated animals were neurologically intact. Controls consisting of laminectomies alone or laminectomies with ethanol but without injury were also studied. Spinal cord segments at the impact or laminectomy site were minced and extracted with chloroform‐methanol, cleared by centrifugation, and examined in a scanning fluorometer with excitation maximum at 360 nm and emission maxima at 420, 440, 450, and 460 nm. SLF was minimal in cats 3 hr and 1 day post injury, but markedly increased at 3 days. By 5 days, background levels were again found in all groups. SLF in the alcohol‐pretreated impact animals rose to a peak at 7 days, followed by a decline to background by 10 days. The presence of SLF supports a role for free radical lipid peroxidation in the degenerative changes in the spinal cord following injury. The findings of two peaks of SLF activity suggest two different sites of damage. One site, found acutely after injury, appears in all groups and was associated with reversible changes, while the other site is associated with later changes and chronic paraplegia only. The two sites could be the gray and white matter.

A fluorometric assay of proteins on Kieselguhr plates using sodium hypochlorite and thiamine

A simple, rapid, and sensitive method for the fluorometric assay of proteins is described in this communication. Proteins are spotted on a Kieselguhr G plate and N-chlorinated by spraying with a solution of sodium hypochlorite. After removal of excess hypochlorite by aeration, the plate is sprayed with a solution of thiamine. The fluorescence of thiochrome, formed by the oxidation of thiamine with the N-chlorinated proteins, is then measured with a scanning fluorometer. Interfering substances including DNA are readily removed by the development of the plate with 2 m sodium chloride prior to the assay. The standard curves were linear in the range of 0.1 to 1.0 μg/spot of proteins, and the fluorescence intensity deviated only slightly among proteins.

A new fluorometric method for the determination of spermidine and spermine in tissues by thin layer chromatography

A new fluorometric method is described for the rapid, sensitive analysis of spermidine and spermine in animal tissues. The polyamines added with N-3-aminopropylheptane-1, 7-diamine as internal standard were chromatographed on a silica-gel sintered-glass plate in a solvent system of n-BuOH-AcOH-Pyridine-H 2O, and the chromatograms were developed with an acetone solution of fluorescamine. Determination, using a scanning fluorometer, was performed by measuring peak area ratio with respect to the internal standard. The precision and accuracy of the method were established, and the limits of determination were of the order of 100 pmoles.

Microassay of proteins on membrane filter in the nanogram range using cycloheptaamylose-dansyl chloride complex

A protein assay is described in which the sample is spotted on a Sartorius membrane filter and dansylated with cycloheptaamylose-dansyl chloride complex dissolved in aqueous urea solution. The fluorescence intensity of the spot on the membrane was directly measured by a scanning fluorometer. This assay is rapid, simple, highly reproducible, and linear from 0.05 to 4 μg of protein. Very dilute solutions of protein can also be determined by filtering off the sample on the membrane filter in the presence of 0.1 m MgCl 2 and dansylating the protein collected on the membrane. There is no interference either by commonly used reagents such as Tris, citrate, guanidine, and glycine, or by naturally occurring amines and amino acids.

Fluorometric assay of protein on a membrane filter using fluorescein isothiocyanate dissolved in cycloheptaamylose-guanidine system

A sensitive, specific, and simple method for the microassay of protein on a membrane filter is described. In the present study, fluorescein isothiocyanate (FITC) was found to be readily soluble in cycloheptaamylose solution in 8 M guanidine and the resultant mixture was employed in the fluorescent staining of a protein spotted on a membrane filter. Reagent: Five milligrams of FITC is dissolved in 0.5 ml of 8 M guanidine hydrochloride containing 100 mg cycloheptaamylose. Procedure: A sample solution containing 0.5 μg to 4 μg of protein is made 0.1 M with respect to magnesium chloride prior to the assay. When the volume of the sample solution is not more than 2 μl, the sample is spotted with the aid of micropipet on Sartorius Membranfilter SM 113 of 10 mm wide. When the volume is in the range of 2 μl to 2 ml, the protein is collected by filtering the sample solution through the membrane filter employing the suction device illustrated in Fig. 1. The spotted membrane is immersed into the reagent mentioned above and allowed to stand for 30 min at room temperature. The membrane is then rinsed three times with agitation for each 1 min in 0.1 M sodium tetraborate. The fluorescence intensity of the spot is measured with Yamato Scanning Fluorometer, Model SFR-21. Results: The standard curve for bovine serum albumin was linear in the range of 0.5 μg to 4 μg. Amines, amino acids, and other substances of low molecular weight tested did not affect the assay. The reagent herein described may also be applicable to the fluorescent labeling of protein in solution.