Bacteria labeling

Recently the direct use of bacterial thymine for the synthesis of phage DNA has been demonstrated by infection with Tfir" of bacteria labeled with C -thymi-dine and with N in the other nitrogenous components (GoUub-Banks, personal communication). 

Microbiological procedures which exploit the ability of bacteria and photosynthetic algae to incorporate exogenous labeled precursors such as 14CO2, SO%, and 32pQ3- [ can be used to label complex molecules in cells such as proteins (qv) and nucleic acids (qv), which are then processed to give labeled constituents such as uniformly labeled C-amino acids, C-nucleotides, C-fipids, LS-amino acids, etc (8). 

The inhibition of Streptococcus mutans adherence to hydroxyapatite with combinations of alkyl phosphates and nonionic surfactants was tested. Seven alkyl phosphate derivatives and three nonionic surfactants were examined for their ability to inhibit the adherence of 3H-labeled cells of S. mutans to hydroxyapatite treated with buffer or parotid saliva. No compound by itself effectively hindered binding of bacteria to hydroxyapatite. A combination of certain of the alkyl phosphates, notably a disodium phosphate of 1-octadecanol, and nonionic surfactant at a 1 1 molar ratio gave a strong inhibition of S. mutans adherence. Treatment with this combination resulted in 98% reduction of adherence. Adsorption of the two types of surface-active agents alone and in combinations was studied using 14C-labeled agents. Electrophoretic measure-... 

An important development has been the isolation of bacteria that were able to degrade phenan-threne that was sorbed to humic acid material (Vacca et al 2005). Enrichment was carried ont with PAH-contaminated soils using phenanthrene sorbed to commercial hnmic acid. Only the strains isolated from this enrichment were able to carry ont degradation of C-labeled phenanthrene, and this exceeded by factors of 4-9 the amonnt estimated to be available from the aqneons phase alone. It was snggested that specially adapted bacteria might interact specifically with natnrally occnrring colloidal material. 

A site at the Agricultural Experimental Station (Ithaca, NY) was treated in microcosms with C-labeled glucose, phenol, caffeine, and naphthalene. Levels of C02 were measured to assess utilization of the substrates, and the populations analyzed by separating the C-labeled DNA by density centrifugation, followed by PCR amplification and sequencing of 16S rRNA (Padmanabhan et al. 2003). Populations contained relatives to a range of bacteria that varied with the substrate. Only relatives of Acinetobacter were found in all samples, and for caffeine only Pantoea. 

It is worth drawing attention to the significance of other issues. In natural ecosystans, other microorganisms including bacteria are almost always present and it has been shown that, in experiments using [7,10- C]benzo[a]pyrene, incubation for 215 d mth Bjerkandera sp. strain BOS55 alone resulted in the formation of 13.5% C02 and 61% of labeled metabolites in the... 

Both H-thymidine incorporation and radiolabeled leucine incorporation techniques have been recently used to determine bacterial activity and growth in the rhizosphere of barley seedling (28), Bacteria were initially released from the rhizosphere using homogenization and centrifugation before adding the labeled substrates. The cell incorporation rate was twice as high in the rhizosphere than in bulk soil. In addition, both the leucine and thymidine incorporation rates increased with the distances from the root tip (28). 

The presence of microorganisms in the rhizosphere has been shown to increa.se root exudation (58,61-65). This stimulation of exudation has been shown to occur in the pre.sence of free-living bacteria such as Azospirillum spp. and Azotobacter spp. (66,67) and in the presence of symbiotic organisms such as mycorrhizae (68,69). Increased root exudation has also been shown to be species-specific for example Meharg and Killham (65) found that metabolites produced by Pseudomonas aeru/ inosa stimulated a 12-fold increase in C-labeled exudates by perennial ryegrass. However, under the same conditions, metabolites from an Arthro-bacter species had no effect on root exudation. 

Loosen the caps on the test tubes about half way and place them all in the labeled 250-mL beaker. Place the specimens in a warm location in the classroom. CAUTION Do not touch bacteria cultures. 

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