Effects of high pressure on the viability,morphology, lysis,and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.     

Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.     

High hydrostatic pressure inactivated human tumour cells preserve their immunogenicity.

High hydrostatic pressure (HHP) is an established method to inactivate biomolecules and microoganisms. It is routinely used for the sterilization of foodstuff. Recently, new applications as inactivation of microorganisms and tumour cells for bone transplants or for cancer vaccines have emerged. Characterization of the HHP-induced cellular responses are a prerequisite for its clinical use. To this end, we investigated the fate of human cells after HHP by cytofluorometry. We observed that the induction by HHP of cell death is time- and pressure-dependent. Surprisingly, an HHP-treatment of 100 MPa did not reduce viability at any time point. Pressures from 150 to 250 MPa-induced programmed cell death in most cells. However, survivors were observed in long term culture experiments under these conditions. Pressures above 300 MPa immediately induced cell death by necrosis and completely inactivated the cells. In contrast to inactivation by other necrosis inducing treatments like heat, freeze/thaw, or chemical agents, HHP avoids generation of Maillard products and disintegration and lysis of the cells. Instead HHP generates a gelatinised mixture of antigens captured in a distinct and robust particle and maintains their humoral immunogenicity. The high viscosity of the internal matrix of a pressurised cell is reflected by the slow penetration of the low molecular compound propidium iodide and limits the bleeding of antigen before uptake by antigen presenting cells. Taken together, HHP is an alternative method for the inactivation of mammalian cells in clinical settings.     

Ability of high hydrostatic pressure treated plasmids and cells of Escherichia coli to genetically transform

The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic pressure increased the ability of plasmids to transform competent Escherichia coli cells. For pUC18 plasmid, a pressure of 400 MPa, and for pBR322, a pressure of 200 MPa was found to provide the highest transformation efficiency. The DNA duplexes of the two plasmids were found to be the most stable for melting conditions at these pressures. At pressures higher than these, both the stability of the duplex DNA and the transformation efficiency were affected. The stabilizing effect of high hydrostatic pressure on the hydrogen bond may be responsible for the observed increase in transformation efficiency of the pressure-exposed plasmid DNA. The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques. In agarose gel electrophoresis, pressure-treated plasmids (pUC18 at 400 MPa and pBR322 at 200 MPa) consistently showed visibly distinct higher mobility compared to untreated plasmids. Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding as is evident from the reduced intensity of fluorescence of the dye bound pressure-treated DNA. Spectroscopic studies using circular dichroism and Fourier transform infrared (FTIR) spectroscopy also showed significant differences in the absorption profiles of pressure-treated plasmids as compared to an untreated control. These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids. On the other hand, the exposure of competent cells of E. coli to a high hydrostatic pressure of 50 MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.     

High pressure response of fruit jams contaminated with Listeria monocytogenes

High pressure is an alternative to thermal processing and is used to preserve food. Listeria monocytogenes is a bacterium which grows at low temperature, is able to multiply under vacuum, and is responsible for food poisoning. Pressures of 100, 200, 300 and 400 MPa were used for 5, 10 and 15 min at 20 degrees C on pure culture, and on apple and plum jam baby food artificially contaminated with Listeria. Pure culture was also to test pressures of 200, 300, 350 and 400 MPa at 5 degrees C for 30 min. The results were analysed statistically and showed that there were no significant differences between pressures of 100 and 200 MPa at 5, 10 and 15 min. However, at 300 MPa, there were significant differences at 15 min. When the pressure treatment was 400 MPa, significant differences were observed at pressure times of 5, 10 and 15 min. The results were fitted to a linear curve. In pure culture, no viable cells were detected after high pressure treatment of 350 MPa for 30 min at 5 degrees C. The use of low temperature helps to maintain the sensory properties of the product.     

Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients

Background

High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates.

Methodology

Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium.Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy.

Principal Findings

A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms.High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.     

Biomechanical properties of articular cartilage after high hydrostatic pressure treatment]

AIM: Reconstruction of bone defects due to malignant tumors can be realized by several methods. Up to now, two methods, irradiation and autoclaving, are available for extracorporeally devitalizing resected tumor-bearing osteochondral segments. Previous investigations have shown that human normal and tumor cells in culture were irreversibly impaired when subjected to extracorporeal high hydrostatic pressure (HHP) of 350 MPa. The aim of this study was to examine the biomechanical and immunohistochemical properties of cartilage after exposure to HHP MATERIALS AND METHODS: Osteochondral segments of bovine femoral condyles were exposed to pressure of 300 and 600 MPa (n=20 each). Biomechanical and biological properties of untreated and treated segments were evaluated by repetitive ball indention testing and immunohistochemical labelling aggrecan, link protein and collagen II. The contralateral segments served as untreated control. RESULTS: No significant alterations concerning stiffness and relaxation of osteochondral segments even after 600 MPa were observed. Immunohistochemically, staining was positive in all cases and no differences in the labeling pattern of proteoglycanes were observed between untreated and HHP-treated specimens. CONCLUSION: These findings give hope that HHP eventually will be used as a new gentle way of treating resected cartilage and bone without alteration of biomechanical properties to inactivate tumor cells in order to allow autologous reimplantation.     

Characterization of a Listeria monocytogenes Scott A isolate with high tolerance towards high hydrostatic pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30 degrees C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H(2)O(2) compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Effect of high pressure on the reduction of microbial populations in vegetables

Resistance of micro-organisms to high pressure is variable and directly related to extrinsic and intrinsic factors. Pressures of 100, 200, 300, 350 and 400 MPa were applied at 20°C for 10 min and at 10°C for 20 min using strains of Gram-positive and Gram-negative bacteria, moulds and yeasts, as well as spores of Gram-positive bacteria. The results showed that at pressures of 100 and 200 MPa, decreases in microbial populations were not significant, whereas the populations of all the micro-organisms tested decreased considerably at a pressure of 300 MPa. A pressure of 300 MPa at 10°C for 20 min was required to completely reduce the population of Saccharomyces cerevisiae , and a pressure of 350 MPa was needed to reduce most of the Gram-negative bacteria and moulds. The Gram-positive bacteria were more resistant, and pressures of 400 MPa were unable to completely reduce their populations. The different pressures employed had little effect on the initial numbers of spores. The initial populations of viable aerobic mesophiles and moulds and yeasts in vegetables (lettuce and tomatoes) decreased 1 log unit at pressures of 300 MPa and above under both sets of experimental treatment conditions. However, treatment at that pressure also resulted in alterations in the organoleptic properties of the samples. In the tomatoes, the skin loosened and peeled away, though the flesh remained firm, and colour and flavour were unchanged. The lettuce remained firm but underwent browning; flavour was unaffected. In vegetables use of moderate pressures in combination with other treatment conditions would appear to be required to reduce the populations of contaminating micro-organisms while avoiding the undesirable alterations in organoleptic properties that take place at 300 MPa.     

Characterization of a Listeria monocytogenes Scott A Isolate with High Tolerance towards High Hydrostatic Pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H₂O₂ compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Effect of high hydrostatic pressure on murine norovirus in Manila clams

Aims: Eating raw or insufficiently cooked bivalve molluscs contaminated with human noroviruses (NVs) can result in acute cases of gastroenteritis in humans. Manila clams (Ruditapes philippinarum) are particularly prone to exposure to NVs due to the brackish environment in which they are farmed which is known to be susceptible to human faecal contamination. High hydrostatic pressure processing (HHP) is a food treatment technique that has been shown to inactivate NV. Methods and results: In this study we investigated the ability of HHP to inactivate murine norovirus (MNV‐1), a recognised surrogate for NV, in experimentally contaminated manila clams. Pools of contaminated live clams were subjected to hydrostatic pressure ranging from 300 to 500 MPa for different time intervals of between one and 10 min. The trial was repeated three times, at monthly intervals. Conclusions: Virus vitality post‐treatment was assessed and the data obtained indicates that the use of high hydrostatic pressures of at least 500 MPa for 1 min was effective in inactivating MNV‐1. Significance and Impact of the Study: HHP results to be an effective technique that could be applied to industrial process to obtain safe Manila clams ready to eat.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

超高压对植物乳杆菌能量代谢影响的研究

以植物乳杆菌ATCC8014为试材,研究超高压对其能量代谢的影响。建立了用氯化碘硝基四唑紫测定ATCC8014的INT代谢还原活性的比色法。用比色法测定了超高压对ATCC8014的INT代谢还原活性与葡萄糖利用的影响。试验结果表明,150~250MPa作用15min在MRS琼脂培养基上随着压力的增大菌落数显著降低,INT代谢还原活性降低显著,葡萄糖的利用变化不明显;超过300MPa后,葡萄糖的利用才显著降低;400MPa处理15min,尽管在MRS琼脂培养基上菌落数低于检测限,INT代谢还原活性为0%,而葡萄糖的利用能力仍为对照组的56.1%,超高压作用下ATCC8014的灭活与INT代谢还原活性的降低的相关性较好。说明ATCC8014的细胞膜上参与葡萄糖的吸收和运输的酶、糖酵解的酶与调节系统比三羧酸循环的酶与调节系统较耐压。三羧酸循环比糖酵解对超高压敏感,三羧酸循环的抑制是超高压灭活其的重要原因,这为了探讨超高压杀灭植物乳杆菌的机制提供了一定的理论依据。     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.     

High hydrostatic pressure increased stability and activity of immobilized lipase in hexane

Lipases are important to high value product synthesis, modification, and enhancement. However, they are often unstable above 40 °C. While most current applications of high hydrostatic pressure (HHP) are for inactivating deleterious enzymes, there is evidence that HHP can stabilize and increase activity of some enzymes. This study examines the apparent kinetics of immobilized lipase-catalyzed synthesis of isoamyl acetate at HHP in hexane. HHP reduced thermal inactivation of lipase by up to 152% after 4 h at 80 °C and 400 MPa when compared to incubations at low pressure. No significant differences were found in activation energy (E_a) at different pressures, irrespectively of the pressurization and heating sequence, and were between 35.7 ± 3.5 and 47.8 ± 8.2 kJ mol^?1, depending on the method. In all methods utilized, activity at 63.5 and 80 °C at 400 MPa was greater (from about 20 to 96% increase) than at low pressure. Activity increased by 110% at low pressure versus a 239% increase at 350 MPa when the temperature was increased from 40 to 80 °C. Increasing pressure up to 350 MPa increased lipase activity while pressures greater than 350 MPa maintained or decreased lipase activity. Activation volume (ΔV^≠) appeared negative between ambient pressure and 200 MPa in contrast to a positive ΔV^≠ between 300 and 600 MPa. Apparent ΔV^≠ was 14.3 ± 1.7 or 15.2 ± 2.2 cm³ mol^?1 at 40 or 80 °C, respectively, between 300 and 500 MPa.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.     

Successful disinfection of femoral head bone graft using high hydrostatic pressure

The current standard for sterilization of potentially infected bone graft by gamma irradiation and thermal or chemical inactivation potentially deteriorates the biomechanical properties of the graft. We performed an in vitro experiment to evaluate the use of high hydrostatic pressure (HHP); which is widely used as a disinfection process in the food processing industry, to sterilize bone grafts. Four femoral heads were divided into five parts each, of which 16 were contaminated (in duplicate) with 10⁵–10⁷ CFU/ml of Staphylococcus epidermidis, Bacillus cereus, or Pseudomonas aeruginosa or Candida albicans, respectively. Of each duplicate, one sample was untreated and stored similarly as the treated sample. The remaining four parts were included as sterile control and non-infected control. The 16 parts underwent HHP at the high-pressure value of 600 MPa. After HHP, serial dilutions were made and cultured on selective media and into enrichment media to recover low amounts of microorganism and spores. Three additional complete femoral heads were treated with 0, 300 and 600 MPa HHP respectively for histological evaluation. None of the negative-control bone fragments contained microorganisms. The measured colony counts in the positive-control samples correlated excellent with the expected colony count. None of the HHP treated bone fragments grew on culture plates or enrichment media. Histological examination of three untreated femoral heads showed that the bone structure remained unchanged after HHP. Sterilizing bone grafts by high hydrostatic pressure was successful and is a promising technique with the possible advantage of retaining biomechanical properties of bone tissue.