Whole bacteria

In this section we summarise the manner in which i -metals. Fig. 6, and where possible specifically the platinum complexes of concern here, interact with biological molecules. Some radio-tracer studies have been carried out on the distribution of platinum complexes in whole bacteria grown in media inocculated with the metal ion. The results are summarised in Table 11. It is noteworthy that the bacteriocidal complex [PtClg]2- was taken up almost entirely by the cytoplasmic protein whereas the filamentous forming neutral species, [Pt(NHs)2Cl4], was... 

Holland, R. D. Wilkes, J. G. Sutherland, J. B. Persons, C. C. Voorhees, K. J. Lay, J. O. Rapid identification of intact whole bacteria based on spectral patterns using matrix assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 1996,10,1227-1232. 

An interesting variation on the whole-cell MALDI approach was recently reported in a study aimed more at FTMS than TOF MS, but the results are nevertheless interesting and important to users of both methods for analysis of bacteria 40. Wilkins s group showed both MALDI-TOF and MALDI-FTMS spectra of whole bacteria grown on isotopic media depleted in C13 and N14. Because most bacterial identification protocols involve a culture step prior to analysis, it is possible to manipulate the sample based on control of the growth media. For mass spectral analysis manipulation of the isotope profile... 

Notwithstanding the aforementioned difficulty in detecting specific target proteins other than the types normally observed in the taxonomic fingerprints from whole bacteria MALDI spectra (i.e., ribosomal proteins), some other target proteins and protein-like materials have been studied directly from whole cells. For example, Lantibiotics, antimicrobial peptides secreted by Gram-positive bacteria have been detected directly from whole bacteria by MALDI-TOF MS.51 The lantibiotics nisin and lacticin 481 were detected from whole cells and crude supernatants. Surprisingly, better results were reported from whole cells than the supernatants. In this study the presence of variants... 

Williams,T. L. Andrzejewski, D. Lay, J. O., Jr. Musser, S. M. Experimental factors affecting the quality and reproducibility of MALDI TOF mass spectra obtained from whole bacteria cells. J. Am. Soc. Mass Spectrom. 2003,14, 342-351. 

Reschiglian, P. Zattoni, A. Cinque, L. Roda, B. Dal Piaz, F. Roda, A. Moon, M. H. Min, B. R. Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 2004, 76,2103-2111. 

Hindre, T. Didelot, S. Le Pennec, J.-P Haras, D. Dufour, A. Vallee-Rehel, K. Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl. Environ. Microbiol. 2003, 69,1051-1058. 

The enormous temperatures attained on resistively heated sample holders can also be used to intentionally enforce the decomposition of non-volatile samples, thereby yielding characteristic pyrolysis products. Pyrolysis mass spectrometry (Py-MS) can be applied to synthetic polymers, [54] fossil biomaterial, [55] food [56] and soil [57] analysis and even to characterize whole bacteria. [58]... 

In most instances, either for mitochondrial suspensions or whole bacteria, ApH is less negative than -0.5 unit making a contribution of, at most, -30 mV to Ap. The exception is found in the thylakoid membranes of chloroplasts (Chapter 23) in which protons are pumped into the thylakoid vesicles and in which the internal pH falls dramatically upon illumination of the chloroplasts.185 The ApH reaches a value of -3.0 or more units and Ap is 180 mV, while Em remains 0. Reported values of Em for mitochondria and bacteria range from -100 to -168 mV and Ap from -140 to -230 mV.172 179 Wilson concluded that Em for actively respiring mitochondria, using malate or glutamate as substrates,... 

Phospholipid-derived fatty acids are often used to identify bacteria by capillary GC analysis after liquid solvent extraction, concentration steps, and chemical derivatization to their methyl esters. Our initial investigations attempted to extract the intact phospholipids, but no significant recoveries were achieved using pure C02. Even if SFE conditions were developed that could extract intact phospholipids, an additional derivatization step would be required before GC analysis of the fatty acid components. For these reasons, chemical derivatization/SFE was investigated in an effort to eliminate the lengthy conventional liquid solvent extractions as well as to combine (and shorten) the extraction and derivatization steps. The derivatization/SFE procedure was performed on samples of whole bacteria using 0.5 mL of 1.5% TMPA in methanol. The static derivatization step was performed for 10 minutes at 80°C and 400 atm C02, followed by dynamic SFE for 15 minutes at a flow rate of ca. 0.5 mL/min of the pressurized C02. Extracts were collected in ca. 3 mL of methanol and immediately analyzed by capillary GC without any further sample preparation. 

Cholera vaccines are produced by inactive bacteria and are administered subcutaneously, intramuscularly, or intradermally. Cholera vaccines should not be administered intradermally in children less than 5 years of age. The vaccination is particularly indicated for people living in highly endemic areas, as well as laboratory and medical personnel exposed to Vibrio cholerae. Diphtheria tetanus pertussis (DTP) vaccine can be made either as toxoids or inactivated whole bacteria. Hemophillus influenzae vaccine is a bacterial polysaccharide conjugated to proteins and is given as one intramuscular dose. A booster dose is not recommended. This vaccine is given to all children in cases such as plenia and other at-risk condiUons. 

As the typical molecule in Gram-negative bacteria, lipid A can be used to detect the bacteria. Detection methods need to be rapid and sensitive therefore, a few rapid methods for lipid A micro-extraction from whole bacteria have been developed. 

Fig. 8.2. Stages of enzyme screening. Microorganisms are grown in flasks or deep-well microplates. Separation of single clones is achieved either by FACS or by plating on agar plates and subsequent colony picking into microplates. Finally, assays are carried out in microplate formats using whole bacteria or cell extracts. Alternatively, bacterial clones can be directly screened on the level of colonies on indicator agar plates or during FACS.

The use of enzymes in drinking-water treatment has several advantages over the use of whole bacteria. Enzymes often have high specificities for substrate, so there is better control over the compound removed and the product produced. The enzyme concentration in the water is controlled by the plant operator. Thus there is no dependence on the growth rate of the bacteria. This can be particularly important when the compound targeted for removal appears only sporadically in the raw water, such as taste and odor compounds. Bacteria require an incubation time to acclimate to the substrate and grow, but enzymes may be added as needs require. 

K. J. Lay, J.O., Jr. Rapid Identification of Intact Whole Bacteria Based on Spectral Patterns Using Matrix-Assisted Laser Desorption/Ionization With Time-of-FlightMass Spectiometry, Rapid Commun. Mass Spectrom. 10(10), 1227-1232 (1996). 

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