Effect of Glutaraldehyde on Cell Viability, Triphenyltetrazolium Reduction, Oxygen Uptake, and β-Galactosidase Activity in Escherichia coli

Acid (pH 5) and alkaline (pH 8.5) glutaraldehyde solutions were compared for their effects on cell viability, oxygen uptake, and beta-galactosidase activities in Escherichia coli. The action of glutaraldehyde at pH 7 on dehydrogenase activity was also studied. Dehydrogenase activity was inhibited at aldehyde concentrations which had little effect on cell viability. In contrast, oxygen uptake and beta-galactosidase activity took place in cells killed by acid or alkaline glutaraldehyde. The effect of glutaraldehyde on dehydrogenase activity and beta-galactosidase activity of disrupted suspensions was also investigated. The dialdehyde was considerably less inhibitory to these enzyme systems than to those of whole cells, and it is thus feasible that the results with whole cells are a consequence of its interaction with, and strengthening of, the outer cell surface, thereby preventing ready access of substrate to enzyme.     

Studies on the Mechanism of the Sporicidal Action of Glutaraldehyde

S ummary . Low concentrations (0.025–0.125%) of glutaraldehyde inhibited or prevented colony formation by Escherichia coli, Bacillus subtilis and B. pumilis in agar, and inhibited germination of spores of the Bacillus spp. in L-alanine plus D-glucose. Higher concentrations (2%) of glutaraldehyde at pH 8.5 were sporicidal. Pre-treatment of spores with glutaraldehyde lessened release of dipicolinic acid when the spores were subsequently heated at 100°, but not at 121°. Spores treated with glutaraldehyde and then with 0.5 M thioglycollic acid in 6 M urea at 70° were less sensitive to lysis by hydrogen peroxide than spores which had not been exposed to glutaraldehyde. Glutaraldehyde was less effective in preventing peroxide induced lysis if added to spores which had been previously exposed to thioglycollic acid plus urea at 70°. The mechanism of the sporicidal activity of glutaraldehyde is discussed in relation to these findings.     

Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde

G orman , S.P. S cott , E.M. H utchinson , E.P. 1984. Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde. Journal of Applied Bacteriology 56 , 95–102.
Bacillus subtilis spores with altered ionic content were tested for their susceptibility to lysis with lysozyme or sodium nitrite following treatment with glutaraldehyde. The Ca-form was more sensitive to glutaraldehyde (pH 4.0.and pH 7.9) than the untreated or H-form. Removal of spore coat dramatically increased sensitivity of the spore to glutaraldehyde. Pretreatment of spores, the coats of which had been extensively removed, with glutaraldehyde (pH 7.9) reduced the rate of lysis by lysozyme and by sodium nitrite, whereas glutaraldehyde at pH 4.0.had little effect. Glutaraldehyde pretreatment (pH 4.0 and pH 7.9) reduced the amount of hexosamine released by lysozyme but not by nitrite from isolated cortical fragments. Spore protoplasts were more susceptible to 0.01% (w/v) glutaraldehyde at pH 4.0 and isolated spore coats adsorbed alkaline glutaraldehyde more rapidly. These results are discussed in terms of a possible mode of action of glutaraldehyde on the bacterial spore.     

Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.     

Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species

Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C. Different E. coli strains manifested different lysis sensitivities when treated at 45 degrees C. When the lysis rates of E. coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis. The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E. coli cells. A close relation was observed between levels of lysis activity and proteolytic activity. A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E. coli cells. It was concluded that Bacillus proteases played a major role in the observed E. coli lysis.     

Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species.

Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C. Different E. coli strains manifested different lysis sensitivities when treated at 45 degrees C. When the lysis rates of E. coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis. The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E. coli cells. A close relation was observed between levels of lysis activity and proteolytic activity. A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E. coli cells. It was concluded that Bacillus proteases played a major role in the observed E. coli lysis.     

Lysis of Vibrio succinogenes by ethylenediamine-tetraacetic acid or lysozyme

Wolin, M. J. (University of Illinois, Urbana). Lysis of Vibrio succinogenes by ethylenediaminetetraacetic acid or lysozyme. J. Bacteriol. 91:1781-1786. 1966.-Cell suspensions of Vibrio succinogenes are lysed by ethylenediaminetetraacetic acid (EDTA) or lysozyme. Lysis occurs at alkaline pH and is prevented by 0.15 m NaCl or KCl or 0.3 m sucrose. The addition of 10(-3)m Mg(++), 10(-3)m spermine, or 10(-2)m Ca(++) prevents lysozyme lysis, and 10(-4)m spermine prevents EDTA lysis. EDTA lysis leads to the formation of a cell ghost, and lysozyme lysis leads to the formation of an empty round body. Freezing and thawing of cells permits lysozyme attack which is not prevented by the protective agents mentioned above. Much of the cell protein, and almost all of the nucleic acids, are released from the cells during EDTA lysis. Treatment of frozen-thawed cells with lysozyme at neutral pH does not cause release of more than 50% of the cell protein and 60% of the nucleic acids of the cells.     

Differentiation of the effects of lethal pH and water activity: food safety implications

The influence of a second lethal stress (SLS) was investigated when populations of Escherichia coli M23 OR.H- were exposed to either a low water activity (aw) of 0.90 or a pH of 3.50 after 24 h at 25 degrees C. Regardless of the initial stress, E. coli M23 OR.H- populations initially demonstrated biphasic inactivation kinetics consisting of a rapid first phase of death followed by a slower second phase. When cultures initially exposed to aw 0.90 experienced an SLS of pH 3.50, a second rapid inactivation period was observed before a subpopulation of more resistant cells emerged. This subpopulation was able to persist for approximately 50 h after imposition of the SLS. In contrast, E. coli M23 OR.H- cells first exposed to a pH of 3.50 were inactivated rapidly to levels below the limits of detection upon imposition of an SLS of aw 0.90. It is hypothesized that pH stress constitutes a large energy drain on the cell and subsequently sensitizes it to other environmental constraints requiring expenditure of metabolic energy.     

Transport capacity, alkaline phosphatase activity and protein content of glutaraldehyde-treated cell forms of Escherichia coli.

Studies on the comparative transport capacity of various cell forms of Escherichia coli suggest that glutaraldehyde acts only in the outer regions of the cell envelope and to such an extent that transport of alpha-aminoisobutyric acid is reduced by 50%. Alkaline phosphatase activity in whole cells was severely impaired in the presence of alkaline glutaraldehyde and in NaCl-washed cells both acid and alkaline glutaraldehyde (0.01%) caused approximately 80-90% reduction in enzyme activity in 10 min. Protein content was reduced by only 10-15% with this concentration of glutaraldehyde, and cell volume decreased by the same extent. These results are discussed in terms of the mode of action of the disinfectant.     

Maintenance of a neutral cytoplasmic pH is not obligatory for growth of Escherichia coli and Streptococcus faecalis at an alkaline pH

Both Escherichia coli and Streptococcus faecalis grew in an alkaline medium containing 100 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). Our data suggested that CCCP functioned as a protonophore at a high pH and that the proton-motive force was dissipated almost completely under such conditions. The pH gradient measured with dimethyloxazolidine-2,4-dione and acetylsalicylic acid was very small in both bacteria at a high pH above 8, and was not affected significantly by the addition of CCCP. Based on these findings, we propose that the maintenance of neutral cytoplasmic pH is not obligatory for the growth of E. coli and S. faecalis in an alkaline medium.     

Solubility and antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli as influenced by pH

The antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli was evaluated at concentrations from 50 to 10 000 microgram ml-1 and pH levels from 5.5 to 8.0. The minimum inhibitory concentrations decreased with increasing pH. Protamine inhibited E. coli at all pH values while L. monocytogenes was inhibited at pH 6.5 and above. The antimicrobial efficacy of protamine decreased in the presence of negatively charged gelatine B but remained almost unchanged with addition of the positively charged gelatine A. Binding studies showed that the amount of protamine adsorbed to culture media components in tryptic soy broth and bacterial cells increased with increasing pH values. The increased efficacy of protamine at alkaline pH may be explained on the basis of an increase in electrostatic affinity for the cell surface of target cells. E. coli produced a protamine-degrading enzyme, however, was still susceptible to protamine.     

Expression of outer membrane proteins in Escherichia coli growing at acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.     

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

Microbial volatile organic compound emissions from Stachybotrys chartarumgrowing on gypsum wallboard and ceiling tile

In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.     

Phospholipid metabolism by phagocytic cells. Phospholipases A2 associated with rabbit polymorphonuclear leukocyte granules

Polymorphonuclear leukocytes obtained from sterile peritoneal exudates in rabbits contain two phospholipid-splitting activities (phosphatidylacylhydrolases EC 3.1.1.4), one most active at pH 5.5 and the other between pH 7.2 and 9.0. Hydrolysis of phospholipid was demonstrated using Escherichia coli labeled during growth with [1-(14)C]oleate and then autoclaved to inactivate E. coli phospholipases and to increase the accessibility of the microbial phospholipid substrates. The acid and alkaline phospholipase activities are both membrane bound, calcium dependent, and heat stable, and they appear to be specific for the 2-acyl position of phospholipids. Evidence was also obtained suggesting that the E. coli envelope phospholipids with oleate in position 2 are more readily degraded than those with palmitate. The two activities are associated with azurophilic as well as specific granules (obtained by zonal centrifugation) and with phagosomes (isolated after ingestion of paraffin particles by the granulocytes). Phospholipase A activities at pH 5.5 and pH 7.5 degrade the two major phospholipids of E. coli, phosphatidylethanolamine and phosphatidylglycerol, to the same extent, but the phospholipase activity at acid pH does not hydrolyze micellar dispersions of phosphatidylethanolamine. By contrast, phospholipase A(2) activity at pH 7.5 degrades both types of phosphatidylethanolamine substrates. Heparin and chondroitin sulfate inhibit phospholipase activity at pH 5.5 but have little effect on activity at pH 7.5. All detergents tested inhibited phospholipase activity, and both activities are inhibited by reaction products, free fatty acid and lysophosphatidylethanolamine. This product inhibition is only partially prevented by addition of albumin. Supernatant fractions of granulocyte homogenates contain a heat-labile inhibitor of granule phospholipase activity at pH 7.5. Boiling the fraction not only removes the inhibition but actually results in stimulation of hydrolysis at pH 7.5 as well as pH 5.5. These granule-associated phospholipase A activities of polymorphonuclear leukocytes differ in several of their properties from granule or lysosomal phospholipases of other phagocytic cells.     

Expression of alpha2,8/2,9-polysialyltransferase from Escherichia coli K92. Characterization of the enzyme and its reaction products

The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides G(D3) and G(Q1b) were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both alpha2,9- and alpha2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAcalpha2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with alpha2,9- and alpha2,8-linkages in vitro and in vivo.     

Enzyme-linked immunosorbent assay of Escherichia coli O157:H7 in surface enhanced poly(methyl methacrylate) microchannels

A novel surface treatment method was developed to enhance polymer-based microchannel enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157:H7 detection. By applying an amine-bearing polymer, poly(ethyleneimine) (PEI), onto poly(methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups were introduced to PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that the alkali condition is preferable for PEI attachment onto the PMMA surface. The amine groups on the PMMA surface were then functionalized with glutaraldehyde, whose aldehyde groups served as the active sites for binding the antibody by forming covalent bonds with the amine groups of the protein molecules. This surface modification greatly improved antibody binding efficiency and the microchannel ELISA for E. coli O157:H7 detection. Compared with untreated PMMA microchannels, approximately 45 times higher signal and 3 times higher signal/noise ratio were achieved with the PEI surface treatment, which also shortened the time required for cells to bind to the microchannel surface to approximately 2 min, much less than that usually required for the same ELISA carried out in 96-well plates. The detection in the microchannel ELISA only required 5-8 cells per sample, which is also better than 15-30 cells required in multi-well plates. With the high sensitivity, short assay time, and small reagent consumption, the microchannel ELISA can be economically used for fast detection of E. coli O157:H7.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Studies on the Mode of Action of Glutaraldehyde on Escherichia coli

S ummary . Glutaraldehyde was readily taken up by Escherichia coli cells with an increase in solutions buffered to pH 7·9; it was paralleled by a corresponding increase in bactericidal activity. Attempts to desorb glutaraldehyde from the cells indicated that the drug molecules were firmly bound. Inhibition of synthesis of macromolecules was demonstrated. Cell walls of E. coli exhibited a much reduced rate of hydrolysis following treatment with glutaraldehyde.