Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed

AIMS: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. METHODS AND RESULTS: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>10(8) active fluorescent units). CONCLUSIONS: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. SIGNIFICANCE AND IMPACT OF THE STUDY: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.     

Transcription of the Campylobacter jejuni cell division gene ftsA

Abstract Long-lived radioactive waste will be buried several hundred meters below ground in metal canisters surrounded by a buffer of compacted bentonite. Sulfate-reducing bacteria present in the bentonite may induce canister corrosion by production of hydrogen sulfide. Here we show that survival of sulfate-reducing bacteria in bentonite depends on the availability of water and that compacting a high quality bentonite to a water activity ( a _w) of 0.96 was lethal for the species investigated.     

In situ bacterial colonization of compacted bentonite under deep geological high-level radioactive waste repository conditions

Subsurface microorganisms are expected to invade, colonize, and influence the safety performance of deep geological spent nuclear fuel (SNF) repositories. An understanding of the interactions of subsurface dwelling microbial communities with the storage is thus essential. For this to be achieved, experiments must be conducted under in situ conditions. We investigated the presence of groundwater microorganisms in repository bentonite saturated with groundwater recovered from tests conducted at the Äspö underground Hard Rock Laboratory in Sweden. A 16S ribosomal RNA and dissimilatory bisulfite reductase gene distribution between the bentonite and groundwater samples suggested that the sulfate-reducing bacteria widespread in the aquifers were not common in the clay. Aerophilic bacteria could be cultured from samples run at ≤55°C but not at ≥67°C. Generally, the largely gram-negative groundwater microorganisms were poorly represented in the bentonite while the gram-positive bacteria commonly found in the clay predominated. Thus, bentonite compacted to a density of approximately 2 g cm^?3 together with elevated temperatures might discourage the mass introduction of the predominantly mesophilic granitic aquifer bacteria into future SNF repositories in the long run.     

Analysis of copper corrosion in compacted bentonite clay as a function of clay density and growth conditions for sulfate‐reducing bacteria

Aims: To investigate the relationships between sulfate‐reducing bacteria (SRB), growth conditions, bentonite densities and copper sulfide generation under circumstances relevant to underground, high‐level radioactive waste repositories. Methods and Results: Experiments took place 450 m underground, connected under in situ pressure to groundwater containing SRB. The microbial reduction of sulfate to sulfide and subsequent corrosion of copper test plates buried in compacted bentonite were analysed using radioactive sulfur (³⁵SO₄^2?) as tracer. Mass distribution of copper sulfide on the plates indicated a diffusive process. The relationship between average diffusion coefficients (D_s) and tested density (ρ) was linear. D_s (m² s^?1) = ?0·004 × ρ (kg m^?3) + 8·2, decreasing by 0·2 D_s units per 50 kg m^?3 increase in density, from 1·2 × 10^?11 m² s^?1 at 1750 kg m^?3 to 0·2 × 10^?11 m² s^?1 at 2000 kg m^?3. Conclusions: It is possible that sulfide corrosion of waste canisters in future radioactive waste repositories depends mainly on sulfide concentration at the boundary between groundwater and the buffer, which in turn depends on SRB growth conditions (e.g., sulfate accessibility, carbon availability and electron donors) and geochemical parameters (e.g., presence of ferrous iron, which immobilizes sulfide). Maintaining high bentonite density is also important in mitigating canister corrosion. Significance and Impact of the Study: The sulfide diffusion coefficients can be used in safety calculations regarding waste canister corrosion. The work supports findings that microbial activity in compacted bentonite will be restricted. The study emphasizes the importance of growth conditions for sulfate reduction at the groundwater boundary of the bentonite buffer and linked sulfide production.     

Encapsulation of R. planticola Rs-2 from alginate-starch-bentonite and its controlled release and swelling behavior under simulated soil conditions

The plant growth-promoting bacteria (PGPR) Raoultella planticola Rs-2 was encapsulated with the various blends of alginate, starch, and bentonite for development of controlled-release formulations. The stability and release characteristics of these different capsule formulations were evaluated. The entrapment efficiency of Rs-2 in the beads (capsules) was more than 99%. The diameter of dry beads ranged from 0.98 to 1.41 mm. The bacteria release efficiency, swelling ratio, and biodegradability of the different bead formulations were enhanced by increasing the starch or alginate contents, but were impeded by higher bentonite content. The release kinetics of viable cells from capsules and the swelling ratio of capsules were studied in simulated soil media of varying temperature, moisture, pH, and salt content. The release of loaded Rs-2 cells and swelling of capsules are greatly affected by moisture, temperature, pH and salt content of the release medium. The release of viable Rs-2 cells from capsules was positively associated with the swelling properties of the capsules. The release of Rs-2 cells occurred through a Case II diffusion mechanism. In summary, this work indicates that alginate-starch-bentonite blends are a viable option for the development of efficient controlled-release formulations of Rs-2 biofertilizer, and which could have a promising application in natural field conditions.     

Inactivation of Salmonella Typhimurium and Listeria monocytogenes using high-pressure treatments: destruction or sublethal stress?

AIMS: To investigate potential resuscitation of Listeria monocytogenes and Salmonella Typhimurium after high hydrostatic pressure treatments. METHODS AND RESULTS: Pressure treatments were applied at room temperature for 10 min on bacterial suspensions in buffers at pH 7 and 5.6. Total bacterial inactivation (8 log(10) CFU ml(-1) of bacterial reduction) obtained by conventional plating was achieved regarding both micro-organisms. Treatments at 400 MPa in pH 5.6 and 600 MPa in pH 7 for L. monocytogenes and at 350 MPa in pH 5.6 and 400 MPa in pH 7 for S. Typhimurium were required respectively. A 'direct viable count' method detected some viable cells in the apparently totally inactivated population. Resuscitation was observed for the two micro-organisms during storage (at 4 and 20 degrees C) after almost all treatments. In the S. Typhimurium population, 600 MPa, 10 min, was considered as the treatment achieving total destruction because no resuscitation was observed under these storage conditions. CONCLUSIONS: We suggest a delay before performing counts in treated samples in order to avoid the under-evaluation of surviving cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The resuscitation of pathogen bacteria after physical treatments like high hydrostatic pressure has to be considered from the food safety point of view. Further studies should be performed in food products to study this resuscitation phenomenon.     

Distribution of sulphur-cycle bacteria in the Ems-Dollard estuary

Summary A quantitative study of the sulphur cycle in the tidal flat-sediments of the Eems-Dollard estuary was started by determining the distribution of two physiologically different groups of bacteria: sulphate-reducing and sulphide-oxidizing bacteria. Viable counts of these bacterial groups were determined by most probable number techniques.The highest numbers of aerobic sulphide-oxidizing bacteria were found in the upper 2 cm of the sediment. A rapid decrease was observed with increasing depth.The anaerobic sulphate-reducing bacteria showed different distribution-patterns with depth. Frequently high numbers of these bacteria were found above the redox-discontinuity-layer. This may be attributed to the presence of anaerobic micro-pockets in this largely aerobic top-layer of the sediment.The horizontal distribution of the sulphide-oxidizing bacteria appeared to be highly correlated with sediment parameters such as organic carbon and clay content of the sediment. The sulphate-reducing bacteria showed only a small linear correlation with these parameters.By means of polyfactor-analysis mathematical models were made with bacterial numbers as the dependent variables and with some environmental parameters as independent variables. The parameters used in this models could explain the variance of the viable counts for approximately 70%. The clay content of the sediment and the number of sulphate reducing bacteria appeared to determine to a large extent the variance in numbers of sulphide-oxidizing bacteria. There are indications that a great deal of the sulphide-oxidizing bacteria might be mixotrophic.For the explanation of the variance in numbers of sulphate-reducing bacteria the most important parameters were the clay content of the sediment, the number of aerobic heterotrophic bacteria and temperature (or season). Therefore the numbers of these organisms were varying throughout the year. It is assumed that the heterotrophic bacteria supply the sulphate-reducing bacteria with organic substrates.     

A fluorescence-based method for the detection of adhesive properties of lactic acid bacteria to Caco-2 cells

AIMS: The ability of probiotic micro-organisms to adhere to the intestinal surface is regarded as a substantial advantage in terms of bacteria persistence in the gastrointestinal tract. The aim of the present study was the development of a method based on fluorescent staining of bacteria and subsequent spectrofluorimetric detection to quantify the adhesion of several strains of Lactobacillus and Bifidobacterium to Caco-2 cells. METHODS AND RESULTS: Lactic acid bacteria strains were subjected to fluorescent staining using the viable probe carboxyfluorescein diacetate and subsequently incubated on Caco-2 monolayers. The adhesion of the micro-organisms was determined by spectrofluorimetry following the lysis of the attached bacterial cells and expressed as adhesion percentage. The values obtained for the micro-organisms tested ranged from 4% for Bifidobacterium infantis Bi1 to 10% for a Bifidobacterium mixture containing three different strains. CONCLUSIONS: In the present study we successfully applied fluorescent labelling and fluorimetric detection to investigate the adhesive properties of some Lactobacillus and Bifidobacterium strains and a Bifidobacterium mixture to Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The results proved that fluorescent labelling is suitable for adhesion studies and provides a reliable and safer alternative to radioactive labelling.     

Culture-dependent comparison of microbial diversity in deep granitic groundwater from two sites considered for a Swedish final repository of spent nuclear fuel

Site selection for a spent nuclear fuel (SNF) repository required analysis of microbial abundance and diversity at two Swedish sites, Forsmark and Laxemar-Simpevarp. Information about sulphate-reducing bacteria (SRB) was required, as sulphide could corrode copper SNF canisters. Total number of cells (TNC) and ATP were analysed, and plate counts and most probable number (MPN) analyses were conducted using eight media based on different electron donors and acceptors for specific microorganism physiological groups. Groundwater chemical composition and E(h) were analysed; sampling depths were 112-978 m below sea level. TNC was 5.5 × 10(3) to 4.7 × 10(5) cells mL(-1), correlating with ATP concentrations. Culturability in TNC percentage was 0.01-35.9, averaging 5.12. Culturable numbers varied greatly between sample positions and uncorrelated with depth. SRB were found in 29 samples and were below detection in three; the MPN of SRB correlated negatively with E(h), as did the MPN of acetogens. Data indicated that microbial sulphate reduction was ongoing in many sampled aquifers; published stable isotope data and modelling results supported this observation. The sites did not differ significantly, but the large data range suggested that analysis of more samples would enable detailed evaluation of microbial processes and their relationship with geochemical information.     

Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus

Aims: The ability to distinguish between viable and/or infectious micro-organisms and inactivated cells is extremely important for correctly performing microbial risk assessments. In this study, we evaluated whether propidium monoazide (PMA)-qPCR could distinguish between viable and nonviable bacteria and viruses. Methods and Results: A PMA-qPCR combined assay was applied to viable and inactivated bacteria (Escherichia coli and Bacillus subtilis) and viruses (MS2 and murine norovirus [MNV]). PMA, a DNA-intercalating agent, in combination with PCR was better able to distinguish between viable and nonviable bacteria and viruses than conventional PCR. Conclusions: These results suggest that a combined PMA-qPCR assay can be used to measure the viability of bacterial cells and bacteriophage MS2, but not MNV. Significance and Impact of the Study: PMA-qPCR could potentially be used to measure the viability of some micro-organisms, including virus. However, a thorough evaluation should be performed prior to measuring the viability of micro-organisms by PMA-qPCR in a quantitative way.     

Influence of copper-alloying of austenitic stainless steel on multi-species biofilm development

AIMS: To investigate the bactericidal influence of copper-alloying of stainless steel on microbial colonization. METHODS AND RESULTS: Inhibition of bacterial adherence was investigated by monitoring (192 h) the development of a multi-species biofilm on Cu-alloyed (3.72 wt%) stainless steel in a natural surface water. During the first 120 h of exposure, lower numbers of viable bacteria in the water in contact with copper-containing steel relative to ordinary stainless steel were observed. Moreover, during the first 48 h of exposure, lower colony counts were found in the biofilm adhering to the Cu-alloyed steel. No lower colony or viable counts were found throughout the remainder of the experimental period. CONCLUSION: The presence of Cu in the steel matrix impedes the adhesion of micro-organisms during an initial period (48 h), while this bactericidal effect disappears after longer incubation periods. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of Cu-alloyed stainless steels for bactericidal purposes should be restricted to regularly-cleaned surfaces.     

Atrazine degradation by encapsulated Rhodococcus erythropolis NI86/21

AIMS: To develop an encapsulation procedure for Rhodococcus erythropolis NI86/21 and demonstrate its use as a slow-release inoculant for reducing atrazine levels in aquatic and terrestrial environments. METHODS AND RESULTS: Alginate encapsulation procedures were developed for the atrazine-degrading bacteria R. erythropolis NI86/21. Several bead amendments, including bentonite, powdered activated carbon (PAC) and skimmed milk (SM), were evaluated for slow release of R. erythropolis NI86/21 and efficacy of atrazine degradation. All bead types demonstrated a capacity to degrade atrazine in basal minimal nutrient buffer whilst continually releasing viable bacterial cells. We found that the addition of bentonite hastened cell release whilst SM sustained cell viability in bead formulations. Reducing the percentage of SM to 1% (w/v) resulted in faster rates of atrazine degradation in both liquid and soil, and was found to prolong cell survival upon bead storage. Limited oxygen transfer affects the capacity of the encapsulated R. erythropolis cells to degrade atrazine. CONCLUSIONS: Degradation studies have demonstrated the efficacy of R. erythropolis encapsulated cells to degrade atrazine in amended liquid and soil. However, in their current formulation, the wet alginate-based beads are impractical for field application because of their poor cell viability during storage. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21-encapsulated cells have the potential to reduce atrazine residues in a number of soil and water environments, possibly ensuring the continued registration and use of atrazine in agriculture by minimizing or eliminating nontarget effects.     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

Some aspects on the role of bacteria in deep water ecosystems

Summary Some remarks on the role of bacteria in deep lakes will be presented with an emphasis on their functioning in the carbon cycle in such ecosystems.The metabolic intensity of most lakes is regulated to a large extent at the primary producer level. Aerobic and anaerobic decomposition by heterotrophic bacteria of the unstable ultimate products of photosynthesis results in the production of carbon dioxide and methane. Bacterial growth occurs at the expense of energy released by the flow of electrons from donors to acceptors. Typical electron acceptors for bacterially mediated reactions are oxygen, nitrate, sulphate and carbon dioxide. When oxygen is used as electron acceptor, the highest amount of energy is released, while the lowest is released when carbon dioxide is used. These reactions are mediated biologically, and the chemical reaction sequence is paralleled by an ecological succession of microorganisms: aerobic heterotrophs, denitrifiers, fermenting bacteria, sulphate reducers, and methane producers. The presence of oxygen is inhibitory to the organisms mediating the last reactions (STUMM, 1966; McCARTY, 1972), and this explains the succession of micro-organisms concomittant with the decrease in redox potential.Both labile and refractory fractions of the pelagial dissolved organic matter can directly be utilized largely by heterotrophic bacteria. At the onset of the summer stratification of Lake Vechten a high concentration (numbers 10⁹ bact/L) of heterotrophic bacteria has been detected in the lower water layers (CAPPENBERG, 1972). The reason for this increase may be a release of nutrients from the mud. During stratification the water layers above the mud become anaerobic owing to the metabolic activity of the bacterial flora and the chemical oxygen demand of the mud. In summer time the highest numbers of heterotrophic bacteria are found in the metalimnion, where organic matter accumulates due to a lower relative rate of sedimentation caused by an increase in viscosity and density.Furthermore it was found that sulphate-reducing bacteria which are capable of reducing sulphates to sulphides using sulphate as terminal electron acceptor, were observed in the hypolimnion only at the time of maximal stratification. During stagnation, gradually decreasing sulphate concentrations are found in the hypolimnion, correlated with increaseing cell numbers of sulphate reducers. As no sulphate could be detected in the mud of Lake Vechten as well, we may conclude that the sulphate concentration limits the number of these bacteria, as can be predicted for similar aquatic environments.Usually the sulphate reducers are found in bottom deposits and are an important group of bacteria of the non-methanogenic populations in mud. Summarizing the biological methane production and its subsequent oxidation by methane-oxidizing bacteria, we may conclude that these processes can be important factors functioning in the carbon cycle in deep fresh-water ecosystems.     

Enumeration of faecal coliforms from recreational coastal sites: evaluation of techniques for the separation of bacteria from sediments

AIMS: To identify the most efficient techniques for the separation of micro-organisms from coastal sediments and, using these techniques, to determine the concentration of faecal indicator organisms in recreational coastal water and sediment. METHODS AND RESULTS: Sediment samples were taken from a range of recreational coastal sites and subjected to various physical techniques to separate micro-organisms from sediment particles. Techniques investigated included manual shaking, treatment by sonication bath for 6 and 10 min, respectively, and by sonication probe for 15 s and 1 min, respectively. The use of the sonication bath for 10 min was the most successful method for removing micro-organisms from sediment particles where sediments consisted mainly of sand. When sediments contained considerable proportions of silt and clay, however, manual shaking was most successful. Faecal coliforms were then enumerated by membrane filtration in both water and sediment from three recreational coastal sites, chosen to represent different physical sediment characteristics, over a 12-month period. Faecal coliform concentrations were generally greater in sediment compared with overlying water for all samples. This was most evident in sediment consisting of greater silt/clay and organic carbon content. CONCLUSIONS: This study demonstrated the importance of sediment characteristics in determining the most efficient method for the separation of micro-organisms from coastal sediments. Sediment characteristics were also found to influence the persistence of micro-organisms in coastal areas. SIGNIFICANCE AND IMPACT OF THE STUDY: Recreational coastal sediments can act as a reservoir for faecal coliforms; therefore, sampling only overlying water may greatly underestimate the risk of exposure to potentially pathogenic micro-organisms in recreational waters.     

Triclosan-bacteria interactions: single or multiple target sites?

AIMS: To investigate the inhibitory and lethal effects of triclosan against several micro-organisms at different stages of their phase of population growth. METHODS AND RESULTS: Triclosan minimum inhibitory concentrations against several test organisms were determined in broth and agar using standard protocols. The bisphenol effect on bacterial population growth kinetics was studied using the Bioscreen C microbial growth analyser. Finally, the efficacy of triclosan on phases of bacterial growth was determined using a standard suspension test. The duration of the lag phase for all micro-organisms tested was increased by bisphenol in a concentration-dependent manner. The population growth kinetics of the micro-organisms was also altered after biocide exposure. At higher concentrations, triclosan was bactericidal regardless of their phase of population growth, although population in stationary phase and particularly, washed suspensions, were more resilient to the lethality of triclosan. This lethal activity was concentration and contact time dependent, and in some instances, bactericidal activity of bisphenol was observed within 15 s. CONCLUSIONS: Low concentrations of triclosan affected the growth of several bacteria, while higher concentrations were bactericidal regardless of the bacterial phase of population growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Here, we presented clear evidence that the interaction of triclosan with the bacterial cell is complex and its lethality cannot be explained solely by the inhibition of metabolic pathways such as the enoyl acyl-reductase. However, the inhibition of such pathways cannot be ruled out as part of the lethal mechanism of the bisphenol at a low bactericidal concentration.     

Effect of copper on the degradation of phenanthrene by soil micro-organisms

AIMS: The effect of copper on the degradation by soil micro-organisms of phenanthrene, a polycyclic aromatic hydrocarbon, was investigated. METHODS AND RESULTS: Inert nylon filters were incubated in the soil for 28 days at 25 degrees C. Each filter was inoculated with a soil suspension, phenanthrene (400 ppm), copper (0, 70, 700 or 7000 ppm) and nitrogen/phosphorus sources. The filters were assessed for phenanthrene degradation, microbial respiration and colonization. Phenanthrene degradation proceeded even at toxic copper levels (700/7000 ppm), indicating the presence of phenanthrene-degrading, copper-resistant and/or -tolerant microbes. However, copper at these high levels reduced microbial activity (CO2 evolution). CONCLUSION: High levels of copper caused an incomplete mineralization of phenanthrene and possible accumulation of its metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of heavy metals in soils could seriously affect the bioremediation of PAH-polluted environments.     

The assessment of airborne bacterial contamination in three composting plants revealed site-related biological hazard and seasonal variations

AIMS: The purpose of this study was to evaluate the degree of bacterial contamination generated by three Italian composting plants (1, 2 and 3) in two different seasons and to assess the health risk for the employees. METHODS AND RESULTS: Aerosols samples were collected with an agar impact sampler. Several plant sites and external upwind and downwind controls were examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram-negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to isolate pathogenic bacteria. The levels of total mesophilic and thermophilic micro-organisms ranged between 33 and >40,000 CFU m(-3) in plant 1, 39 and 18,700 CFU m(-3) in plant 2 and 261 and 6278 CFU m(-3) in plant 3. Strains of Escherichia coli, Staphylococcus aureus and Clostridium perfringens were also found. CONCLUSIONS: The plants monitored in this study have proved to be sources of aerosolized bacteria. The activities involving mechanical movement of the composting mass and the indoor activities were of greatest potential risk. In all the studied plants, a statistically significant dependence was found between the bacterial contamination and the season for some or almost all the analysed parameters, but a clear seasonal trend could not be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides broad evidence of bacterial aerosol dispersion and site-related biological hazards that may be useful to the regional government to implement regulations on worker safety in composting plants.     

Effect of Bentonite on Compacted Clay Landfill Barriers

Compacted clay barriers (liner and cap) are one of the most important components of municipal waste landfills. On-site soils are generally used to construct the clay barriers as long as they can be compacted to standard specifications, including hydraulic conductivity. Wherever the available on-site soils are not suitable to be used for constructing clay barriers, soils amended with bentonite are commonly used. This article presents the results of an experimental study conducted on compacted clay-bentonite mixtures to develop data on the effects of bentonite on engineering properties of compacted clay-bentonite mixtures. Clay-bentonite mixtures with bentonite contents of up to 7% were tested to determine consistency using Atterberg limits, moisture-density relationships using standard Proctor compaction tests, shear strength using unconfined compression tests, hydraulic conductivity using triaxial flexible-wall hydraulic conductivity tests, and consolidation properties using one-dimensional consolidation tests. Unconfined compression tests were also performed with 11% bentonite content. The laboratory test results indicated that liquid limit, plastic limit, and plasticity index increased linearly with increased amount of bentonite. The addition of bentonite resulted in decreased maximum dry unit weight but the optimum moisture content increased slightly. Unconfined compression strength of compacted clay-bentonite mixtures increased linearly with an increase in the amount of bentonite. Hydraulic conductivity of compacted clay-bentonite mixtures decreased nonlinearly with increased amount of bentonite, but a linear relationship was observed between logarithm of hydraulic conductivity and bentonite content. The compression index increased slightly from 0 to 3% bentonite content but increased nonlinearly beyond 3% bentonite content, whereas the swelling index of clay-bentonite mixtures has been observed to increase approximately linearly with increase in the amount of bentonite.