New possibilities for bacterial cytochemistry: light microscopical demonstration of beta-galactosidase in unfixed immobilized bacteria.

The possibility of applying light microscope cytochemical techniques in order to determine the identity and physiological state of microbes, particularly bacteria, has received little attention in recent years. The technical obstacles have perhaps been thought too great and the potential rewards too small. In order to demonstrate the feasibility of the cytochemical approach to problems in microbiology, an indoxyl method was developed for the demonstration of beta-galactosidase activity in unfixed bacteria. Cells were immobilized on 3-aminopropyltriethoxysilane-treated glass, permeabilized by air drying, then incubated in an indoxyl beta-D-galactopyranoside substrate plus a ferri-ferrocyanide reaction mix. Specific enzyme activity was demonstrated in Escherichia coli and a strain of Bacillus subtilis containing the LacZ gene. In the former, activity was inducible both before and after immobilization. These findings indicate that the basic prerequisites for light microscopical demonstration of bacterial intracellular activities, i.e. immobilization without disruption and reagent access without loss of localization, can now be fulfilled. Further development of this approach is desirable because it allows rapid demonstration of specific microbial activities without an intervening period of in vitro cultivation, thus avoiding the time delays and adaptive changes associated with propagation on laboratory media.     

Polyamine cytochemistry

Summary o-Phtalaldehyde (OPT) reacts with a number of biologically important molecules, including the polyamines, spermidine and spermine. By systematically varying reaction conditions with respect to temperature, pH, concentration and length of exposure to the reagent, using both model systems and tissues, we have succeeded in constructing a cytochemical OPT-method specific for spermidine and spermine. The method detects cell types known to contain these polyamines, including growing and neoplastic cells. The staining pattern obtained with the OPT method is identical to that obtained with the formaldehyde-fluorescamine (FF) technique recently shown to be specific for spermidine and spermine. In contrast to the FF technique, the OPT method can be used for staining suspensions of isolated cells and may hence be employed in studies using fluorescence-activated cell sorting (FACS). Preliminary such studies show a pronounced decrease in cellular OPT-induced fluorescence, paralleled by a decrease in content of polyamines, after treatment with the polyamine biosynthesis inhibitor -difluoromethylornithine (DFMO). In contrast, cells simultaneously treated with DFMO+spermidine show pronounced increases in their spermidine content and parallel increases in their OPT-induced fluorescence. Availability of methods selectively demonstrating polyamines at the cellular and subcellular level is expected to aid our understanding of polyamine functions in normal growth and cancer.     

Lanthanide chelates as new fluorochrome labels for cytochemistry

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.     

Acrolein as a fixative for enzyme cytochemistry.

Since acrolein can penetrate more quickly and deeply into tissue blocks than glutaraldehyde, the possibility of the use of this aldehyde as a prefixative in enzyme cytochemistry was reinvestigated. At low concentrations, acrolein preserves the activities of the enzymes investigated, including those of glucose-6-phosphatase, which is known as one of the most vulnerable to aldehyde fixation; thus, acrolein is usable in enzyme ultracytochemistry. Enzyme activities are also preserved in tissues fixed with acrolein and glutaraldehyde combined. The rapid penetration of acrolein enables fixation in larger tissue blocks and provides greater freedom in specimen selection, especially important advantages when encountering heterogeneous materials as in pathology.     

Diaminobenzidine cytochemistry in cryo-ultramicrotomy for the detection of peroxidases

Summary Rat erythrocytes and lymphoid cells were fixed by glutaraldehyde and encapsulated in bovine serum albumin plus rabbit IgG globulins for cryo-ultramicrotomy. A technical procedure is described by which endogenous peroxidases of erythrocytes in ultrathin frozen sections were detected by hydrogen peroxide and diaminobenzidine as hydrogen donor. Modifications of this classical cytochemical procedure proved also useful in cryo-ultramicrotomy for immunoperoxidase labeling of antigenic determinants in rabbit IgG globulins which have been crosslinked within the supporting matrix of cells.Supported by grants of the Deutsche Forschungsgemeinschaft (SFB 136, publ. No. 18) Bonn, Federal Republic of Germany