Sporicidal action of alkaline glutaraldehyde: factors influencing activity and a comparison with other aldehydes

The sporicidal efficacy of glutaraldehyde (2% w/v) was investigated under various conditions. Numerous factors influenced its activity: method of spore production, inherent spore resistance characteristics, alkalination, storage time and storage temperature. The sporicidal action of 2% alkaline glutaraldehyde at room temperature was compared with that of other aldehydes and commercially available formulations. Cidex (glutaraldehyde) and Sporicidin (glutaraldehyde + phenol full strength) were the most effective, followed by 8% (w/v) formaldehyde and 10% (v/v) Gigasept, a formaldehyde-containing product. Five per cent (v/v) Gigasept and 10% (w/v) glyoxal also had good sporicidal activity, though that of Sporicidin (1:16) was poor. No activity was observed with 10% (w/v) butyraldehyde.     

Effect of sodium hydroxide and two proteases on the revival of aldehyde-treated spores of Bacillus subtilis

Spores of Bacillus subtilis were subjected to a 24 h exposure (22 C) to vaious commercial and non-commercial aldehyde formulations Subsequent treatment with sodium hydroxide (15 min, 22 C)enabled the recovery of injured spores as determined by enumeration on nutrient agar. Greatest revival was obtained with spores treated with glyoxal, followed by 2% alkaline glutaraldehyde, Sporicidin and 10% Gigasept. Revival was dependentupon neutralization of residual aldehyde with 2% (w/v) glycine prior to alkali treatment. Two percent alkaline glutaradehyde-treated spores were also exposed to to proteases, proteinase K and pronase. No revival was observed. The results are discussed in terms of the mechanism of action of the aldehydes used, with particular emphasis on glutaradehyde.     

Author index volume 65

Spores of Bacillus subtilis 168 were apparently fully inactivated by exposure to 2% (w/v) glutaraldehyde for 20 h but a few spores could be revived by further treatment with 10-100 mM NaOH. A similar effect was found with spores from a range of Bacillus species. A minimum concentration of 5% (w/v) glutaraldehyde was required to prevent the alkali-induced reactivation. The implications of these results for the use of glutaraldehyde as a sporicidal agent are discussed.     

A Quantitative Evaluation of the Antifungal Properties of Glutaraldehyde

Fungistatic data were obtained from measurements of mycelial growth of several fungal species in the presence of acid or alkaline glutaraldehyde. Alkaline glutaraldehyde in concentrations > 0·1% (w/v) prevented growth of all species examined while 0·5% (w/v) acid glutaraldehyde was necessary to achieve this effect. Further fungistatic data were obtained using a Coulter Counter method to measure spore swelling. Fungicidal determinations resulted in a 99·99% reduction in viable count after 90 min contact with 0·5% (w/v) alkaline glutaraldehyde. A considerable drop in spore production was also observed after treatment with alkaline glutaraldehyde.     

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde

Aims: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. Methods and Results: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality‐controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. Conclusions: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time‐duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. Significance and Impact of the study: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde‐based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.     

Temperature-Induced Changes in the Sporicidal Activity and Chemical Properties of Glutaraldehyde

Freshly prepared 2% acid and alkaline glutaraldehyde solutions were stored at 4, 20, and 37 C. At intervals, samples were removed and changes in pH, ultraviolet spectrum, and sporicidal activity (against Bacillus pumilus spores) were recorded. Alkaline solutions stored at 4 C showed little changes in these properties, whereas such solutions stored at 37 C became turbid and showed a decrease in pH, marked changes in ultraviolet spectrum, and an almost complete loss of sporicidal activity. Intermediate results were obtained with alkaline solutions stored at 20 C. In contrast, acid 2% glutaraldehyde solutions (initial pH 3.5) showed comparatively few changes in their properties. Treatment of spores with freshly prepared glutaraldehyde solutions (0.5%) at temperature above 40 C reduced the effect of pH on sporicidal activity.     

An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range

AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.     

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study’s aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin’s sterilization potential Bacillus subtilis var. niger spore strips were treated with 0–10 % genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100–100:0), and treatment duration (24–72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63–10 % genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63 % genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100 % PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin’s effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.     

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.     

Assesssment of sporicidal efficacy

On the basis of their activity against bacterial spores, chemical agents can be divided into two groups: Group A contains chemicals that are bactericidal and sporistatic but not sporicidal, e.g. phenols, cresols, parabens, quaternary ammonium compounds and biguanides; Group B contains those, such as glutaraldehyde, chloride-releasing agents, ethylene oxide and hydrogen peroxide, that show both bactericidal and sporicidal activity, although much higher concentrations may be needed to achieve the latter effect.Sporistatic activity can be examined by various methods, e.g. (i) determining minimum inhibitory concentrations, (ii) detecting by phase-contrast microscopy the concentrations needed to inhibit germination or outgrowth, (iii) measuring spectrophotometrically effects on germination or outgrowth. Information thereby obtained is of considerable importance in food microbiology. Sporicidal efficacy can be tested against spores in liquid medium or suspended on appropriate carriers. Factors influencing activity such as concentration, pH, temperature and the presence of organic matter need to be assessed. The principles of evaluating sporicidal activity are essentially the same as for determining bactericidal activity. The main difference is that a treated spore has to pass through complex stages (germination and outgrowth) before a vegetative cell, and eventually a colony, is produced, if a conventional counting method is employed. It is essential that adequate quenching of residual activity is achieved and that recovery conditions are such as to permit viable but damaged spores the opportunity to revive. A recently developed procedure utilises bioluminescence as a means of determining sporicidal efficacy.     

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO₃ (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K₂HPO₄ and 0.06% (w/v) MgSO₄. It was possible to increase the productivity of dextranase to 41.8 units ml⁻¹ in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.     

Sporicidal Activity of Alkaline Alcoholic Saturated Dialdehyde Solutions

During a search for a sporicidal agent to equal or surpass formaldehyde in activity, a group of saturated dialdehydes ranging from two to six carbons was discovered. These dialdehydes exerted a surprisingly high degree of activity in isopropanol buffered with a carbonate or bicarbonate. Without the proper buffer, practically no activity was observed. A solution of 1% glutaraldehyde, 0.3% NaHCO₃, and 70% isopropanol sterilized stainless-steel penicylinders contaminated with standardized numbers of four spore types in a shorter time than did commercially available 8% formaldehyde solution. Both glyoxal and glutaraldehyde in isopropanol destroyed spores in a relatively short time. Care was taken to eliminate bacteriostasis. Various tests were performed to evaluate accurately experimental and formaldehyde solutions.     

Interfering mechanism of sodium bicarbonate on spore germination of Bacillus stearothermophilus

Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO₃) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO₃ was attributed to the release of HCO⁻₃ and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO₃ was mainly due to bicarbonate. As NaHCO₃ increased from 0·3% to higher concentrations, the effect of HCO⁻₃ reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm⁻¹) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na⁺ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm⁻¹) and of amide II (1546 cm⁻¹) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO₃ in enhancing the sporicidal effect of glutaraldehyde is discussed.     

α-Amylase immobilization on gelatin: Optimization of process variables

α-Amylase was extracted and purified from soybean seeds to apparent homogeneity by affinity precipitation. The homogeneous enzyme preparation was immobilized on gelatin matrix using glutaraldehyde as an organic hardener. Response surface methodology (RSM) and 3-level-3-factor Box–Behnken design was employed to evaluate the effects of immobilization parameters, such as gelatin concentration, glutaraldehyde concentration and hardening time on the activity of immobilized α-amylase. The results showed that 20% gelatin (w/v), 10% glutaraldehyde (v/v) and 1 h hardening time yielded an optimum immobilization of 82.5%.     

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.     

Effects of temperature, pH, water activity and CO2 concentration on growth of Rhizopus oligosporus NRRL 2710

AIMS: To investigate the effects of temperature, pH, water activity (aw) and CO2 concentration on the growth of Rhizopus oligosporus NRRL 2710. METHODS AND RESULTS: Hyphal extension rates from mycelial and spore inocula were measured on media with different aw (approximately 1.0, 0.98 and 0.96) and pH (3.5, 5.5 and 7.5) incubated at 30, 37 or 42 degrees C in atmospheres containing 0.03, 12.5 or 25% (v/v) CO2. The effects of environmental conditions on hyphal extension rate were modelled using surface response methodology. The rate of hyphal extension was very sensitive to pH, exhibiting a pronounced optimum at pH 5.5-5.8. The hyphal extension rate was less sensitive to temperature, aw or CO2, exhibiting maximum rates at 42 degrees C, a(w) approximately 1.0 and 0.03% (v/v) CO2. CONCLUSIONS: The fastest hyphal extension rate (1.7 mm h(-1)) was predicted to occur at 42 degrees C, pH 5.85, a(w) approximately 1.0 and 0.03% CO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is the first to model the simultaneous effects of temperature, pH, aw and CO2 concentration on mould growth. The information relates to tempe fermentation and to possible control of the microflora in Tanzanian cassava heap fermentations.     

Immobilization and kinetics of catalase onto magnesium silicate

Bovine liver catalase was immobilized covalently with glutaraldehyde, or glutaraldehyde+3-aminopropionic acid as a spacer, onto magnesium silicate. The coupling time was determined as 2 h for immobilization. The pH and temperature optima as well as the changes in the kinetics (K_m, V_max, E_a) of the immobilized catalase was observed and discussed. Immobilized catalase preparations showed higher storage stabilities than free catalase. The half-life of free catalase, catalase immobilized via glutaraldehyde and catalase immobilized via glutaraldehyde+spacer were calculated as 2, 55 and 10 days at room temperature and 4, 85 and 107 days at 5 °C, respectively. The operational stability of the catalase immobilized via glutaraldehyde was higher than the catalase immobilized via glutaraldehyde+spacer. The remaining activity of the catalase immobilized via glutaraldehyde was about 90% and that of the catalase immobilized via glutaraldeyde+spacer was about 30% after 20 cycles of batch operation.     

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

Proteolytic degradation of cold-water fish gelatin solutions and gels

The stability of cold-water fish gelatin (FG), both in solution and in the gel phase, has been studied as function of both temperature and exposure towards novel proteases of marine origin. A 1% (w/v) FG solution was readily degraded by such proteases above 20 degrees C, which was expected since FG at this temperature is a random coil molecule lacking the protective triple helical structure found in collagen. The dynamic storage modulus for a 10% (w/v) FG gel increased monotonically at 4 degrees C. Ramping the temperature to 6, 8 or 10 degrees C led to a drastic reduction in G', but an apparent partial recovery of the network (increasing G') was observed with time at all temperatures. In the presence of proteases, a lower storage modulus was observed. At constant 4 degrees C, an apparent maximum value was reached after curing for 2h followed by a decrease in G' indicating protease activity. Ramping of temperature in the presence of proteases led to an even more drastic reduction in G' and no recovery of structure was observed with time. In this case, the overall rheological behaviour is a complex function of both thermal influence as well as proteolytic activity. In an endeavour to quantify the effect of the presence of proteolytic enzymes on the gelatin network, rheological investigation were undertaken where the dynamic storage moduli were recorded on different 10% (w/v) FG samples that had been acid hydrolysed to yield different average molecular weights. A significant reduction in storage modulus for average molecular weights below 50 kDa was found. This critical molecular weight most probably reflects the on-set of a regime where shorter chain lengths prevent percolation due to an increase in the loose end and sol fraction as well as a reduction in the average length of the pyrrolidine-rich regions reducing the number of possible junction zones.