muramidase and formamide

Aspects of the fluorescence in situ hybridization (FISH) method for the detection of clinically important bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, were investigated for optimization.. Various approaches to optimizing the FISH procedure were taken and different methods were compared. To save time, hybridization and washing buffers were prepared beforehand and stored at -20 °C and mixed to their final formamide and NaCl concentrations just before use. The use of 50-ml tubes for hybridization incubation reduced drying out, reagent wastage, and reaction times.. A two-step permeabilization FISH assay was developed that used phosphate-buffered saline as a buffer for lysostaphin. It could detect bacteria with DNA probes conjugated to fluorophores with a higher signal intensity and the less expensive biotinylated DNA probes with minimal cell lysis in 1 hr.. The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules.

Topics: Analysis of Variance; Escherichia coli; Formamides; Histocytological Preparation Techniques; In Situ Hybridization, Fluorescence; Lysostaphin; Molecular Diagnostic Techniques; Muramidase; Sodium Chloride; Staphylococcus aureus; Staphylococcus epidermidis; Streptavidin

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.)

Topics: Animals; Chickens; Circular Dichroism; Dimethyl Sulfoxide; Dimethylformamide; Ethylene Glycol; Formamides; Methanol; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Solutions; Solvents

1. The cell walls of Corynebacterium tritici contain much carbohydrate and their mucopeptide contains diaminobutyric acid instead of lysine or diaminopimelic acid. They are resistant to lysozyme. 2. The residue after extraction with hot formamide contains only about 10% less carbohydrate but is attacked by lysozyme. Lysozyme also slowly attacks cell walls treated with fluorodinitrobenzene and more rapidly cell walls that have been N-acetylated. 3. All these processes block the free gamma-amino groups of diaminobutyric acid present in the untreated cell wall. Hot formamide introduces formyl groups, as shown by its ability to make formylglycine and diformyl-lysine under the same conditions. 4. N-Formyl groups are also introduced into the cell walls of Micrococcus lysodeikticus by hot formamide, but this change increases only slightly their already great sensitivity to lysozyme. N-Acetylation also increases sensitivity to lysozyme.

Topics: Bacillus megaterium; Carbohydrates; Cell Physiological Phenomena; Cell Wall; Chromatography; Corynebacterium; Formamides; Micrococcus; Muramidase; Nitrobenzenes; Peptidoglycan; Pharmacology; Polysaccharides, Bacterial; Research; Solvents