Microbial Samples

Other pattern recognition strategies have been used for bacterial identification and data interpretation from mass spectra. Bright et al. have recently developed a software product called MUSE, capable of rapidly speciating bacteria based on matrix-assisted laser desorption ionization time-of-flight mass spectra.13 MUSE constructs a spectral database of representative microbial samples by using single point vectors to consolidate spectra of similar (not identical) microbial strains. Sample unknowns are then compared to this database and MUSE determines the best matches for identification purposes. In a... 

The environment shall be sampled during normal operations to allow collection of meaningful data. Microbial sampling should be performed... 

Environmental microbial monitoring and analysis of data by qualified personnel will permit the status of control to be maintained in clean rooms and other controlled environments. The environment should be sampled during normal operations to allow for the collection of meaningful data. Microbial sampling should occur when materials are in the area, processing activities are ongoing, and a full complement of operating personnel is on site. 

There are regulations in place to cover the collection of microbes in Japan, but not the collection of higher plants and animals. Since 1968, efforts have been underway to conserve microbial samples and deposit them for patent application in the International Patent Organism Depository (IPOD)53 within the Patent Office. Protocols and their review are conducted by IPOD and forms are available online. No distinction is made between academic and... 

Pressurized LLE using acetic anhydride for simultaneous acylation was applied to the analysis of phenolic pollutants, sterols and carboxylic acids in environmental and microbial samples . ... 

Products Prescreening analysis and characterization of microbial samples. Plans to extend and develop its library of organisms and characterized samples so that a database associated with chemical data can be made available for industry. 

Avecia identified approximately 60 microorganisms with amidase activity capable of resolving racemic amines [17, 18]. Arthrobacter species predominated in the list of microorganisms identified. The kinetic resolution of N acetyl 1 aminoindanol 35 by a freeze dried microbial sample (BH2 NI amidase) allowed access to (1S,2R) N acetyl 1 aminoindanol 35 in high enantiomeric excess (96%). This compound is a key intermediate in the synthesis of Merck s HIV protease inhibitor Crixivan 37 (indin avir) (Figure 14.12). 

Microbial samples are collected from washes 1 and 5, using a sterile basin wash procedure, which entails subjects washing their hands with the bland soap in a polyethylene container, such as a plastic freezer storage bag, containing 1 liter of sterile water. After washing, the wash water is well mixed and sample aliquots are plated on tryptic soy agar. The agar plates are incubated at 30-35°C for approximately 48 hours. 

Normally, 10-20 subjects are recruited for each product evaluated. A control product is sometimes used. During a washout period of at least 7 days, subjects are not permitted to use antimicrobial products or expose their hands to known compounds that have antimicrobial properties. The remaining study procedures are identical to those of the healthcare personnel handwash previously described, but the total number of washes is 5-10, with glove juice samples collected at baseline and after wash 1 and wash 5, and after wash 10 if 10 washes are used. The microbial sample counts taken after the test washes are then compared to the baseline values. 

Chemical components characteristic of specific nticroorganisms or nticrobial groups are usually enclosed in or part of a polymeric cellular matrix. Such nonvolatile and intractable biological samples present some difficulties for their direct characterization by GC, MS, or GC/MS. For these techniques to be useful, chemical components characteristic of the microbial sample must be released intact or a related compound must be generated before GC or MS analysis. Depolymerization is usually performed off-line by acid hydrolysis, methanolysis, sapoifification, or other reactions. One or more chemical derivatization steps might then be employed to produce volatile and thermally stable derivatives suitable for GC or MS. 

When analytical pyrolysis is applied to a microbial sample, depolymerization and volatilization of bacterial components are accomplished simultaneously. The volatile thermal products may be separated on-line by capillary GC with flame ionization detection (Py-GC-FlD), separated by GC and detected by MS (Py-GC/MS), or detected directly by MS (Py-MS). In contrast to derivatization-based methods and like other direct approaches, microbial characterization by analytical pyrolysis requires miiumal sample pretreatment and short total analysis times. 

Knowledge of chemical composition with respect to relevant carbohydrate, amino acid, or fatty acid chemical markers is often critical for proper interpretation of the pyrolysis of a microbial sample. Sometimes this information is already available in the literature regarding specific bacteria. More often, however, complementary analyses for that particular class of components by an independent GC, GC/MS, or spectroscopic method may be needed. 

To prepare a microbial sample for analysis, an amount of the lyophilized powder is weighed and added to a measured volume of water to give a solution of known concentration (e.g., 10 Itg/ftl). The solution should be vortexed or sonicated to produce a homogeneous suspension before sampling on the pyrolysis sampling device. 

When a resistively heated filament pyrolyzer (e.g., the Pyroprobe from CDS Analytical, Inc., Oxford, PA) is used, the sample may be placed directly on a platinum filament or in a quartz tube or boat inside a platinum coil. In either case, the placement of sample with respect to the sampling tube or the ribbon should be the same for all samples. For liquid sample suspensions placed on a ribbon or in a coil, the solvent is evaporated prior to pyrolysis. Solid microbial samples can be sandwiched between quartz wool plugs inside the quartz sampling tube so as to reduce extraneous nonvolatile material from leaving the sampling tube during pyrolysis. With quartz, the sample never comes into direct contact with the pyrolyzer filament, as it does when sample is coated directly on a thin ribbon filament. Ribbon filaments sometimes exhibit a memory effect (particularly with polar components), are harder to clean, and typically have a shorter lifetime. Quartz tubes may be reused after cleaning. 

When pyrolysis is applied to a microbial sample, a complex mixture of thermal degradation products is produced. Analytical pyrolysis is often performed in a fingerprinting mode using sophisticated pattern recognition methods. However, if the chemical basis of pattern differences is not defined, pyrograms are so complex... 

Strategies implementing LC-MS and LC-MS/MS can extend the number of proteins accessible in the microbial proteome for the purpose of strain-level differentiation. In top-down proteomics, the microbial sample is rrstrally processed in... 

Various strategies have been developed for the enrichment of microbial samples. The enrichment methods involving non-covalent, covalent interactions, and immunoassays are discussed in this section. In general, affinity enrichment steps include binding of targets, washing, and elution. A general scheme is provided in Fig. 3.2. Microbial cells/markers are isolated and concentrated after the incubation of the sample solution with affinity probes. The enriched cells are lysed or directly mixed with a matrix-assisted laser desorption/ionization (MALDI) matrix solution and subjected to mass spectrometry (MS) analysis. Moreover, biomarkers obtained from the enriched cells may be concentrated and separated prior to MS analysis... 

Strong organic acids, bases, alcohols, and detergents/srrrfactants have been employed in microbial sample preparatiorts, due to their high efficiency in disrapting membranes and solubilizing proteins. Different eell types require different buffer formulations. Conditions such as pH, salt concentration, and temperature are eon-sidered to be important parameters in the sample preparation protocols. 

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