Research ArticleExperimental Studies
Open Access

Freezing Nitrogen–Ethanol Composite Effectively Eradicates Staphylococcus aureus Biofilm on Prosthetic Surfaces

YU-KUAN LIN, KUN-HAN LEE, CHAO-MING CHEN, SHANG-WEN TSAI, CHENG-FONG CHEN, PO-KUEI WU and WEI-MING CHEN
In Vivo May 2026, 40 (3) 1408-1417; DOI: https://doi.org/10.21873/invivo.14292

Abstract

Background/Aim: Periprosthetic joint infection (PJI) remains one of the most challenging complications after arthroplasty, largely due to biofilm formation on prosthetic surfaces, which protects bacteria from antibiotics and host defenses. This study investigated whether a freezing nitrogen-ethanol composite (FNEC), a semisolid cryogenic material combining liquid nitrogen and ethanol, can effectively eradicate Staphylococcus aureus biofilms on prosthetic components.

Materials and Methods: Biofilms of S. aureus were established on plastic spacers and metallic knee prostheses. Specimens were treated for 15 min with 75% ethanol, liquid nitrogen (LN), or FNEC. Bacterial viability was assessed using LIVE/DEAD fluorescence staining, while eradication efficacy was confirmed through broth inoculation and optical-density (OD600) measurements. Additional indirect-exposure experiments evaluated the contribution of FNEC’s freezing effect, and time-dependent testing determined the minimal effective exposure duration.

Results: FNEC achieved the most complete biofilm eradication across both plastic and metallic surfaces. LIVE/DEAD staining demonstrated widespread cell death with minimal residual viability. Broth cultures from FNEC-treated samples remained clear, with OD600 values indistinguishable from sterile controls, whereas LN and untreated samples showed significant bacterial growth. Indirect experiments confirmed that FNEC’s cryogenic effect alone substantially contributed to bacterial death. Time-course analysis revealed that a 5-min FNEC exposure was sufficient for complete sterilization.

Conclusion: FNEC exhibited potent bactericidal and biofilm-removal capability, outperforming ethanol and LN alone. Its combined chemical and cryogenic effects enabled rapid and complete eradication of S. aureus biofilms within 5 min. These findings support FNEC as a promising adjunctive strategy for intraoperative biofilm management and may facilitate single-stage revision surgery for PJI.

Keywords:

Introduction

Periprosthetic joint infection (PJI) remains one of the most devastating complications following joint arthroplasty. The one-year incidence ranges from 0.25-1.0% after primary total hip arthroplasty and 0.4-2.0% after primary total knee arthroplasty (1, 2) whereas infection rates following revision procedures are considerably higher, reaching 3.2-5.6% (3). Among the causative pathogens, Staphylococcus aureus (S. aureus) is the most prevalent and virulent species implicated in implant-related infections (3). The pathogenesis of PJI is primarily driven by biofilm formation on the prosthetic surface, of which create a viscous, protective extracellular matrix that limits antibiotic penetration, slows diffusion, and enzymatically degrades antimicrobial agents, thereby shielding bacteria from host immunity and drug exposure (4, 5). As a result, infections associated with biofilms are difficult to eradicate, often necessitating combined surgical and antibiotic therapies and sometimes leading to loss of limb function or amputation (6).

The therapeutic approach for PJI largely depends on the depth and chronicity of infection. Deep infections often require prolonged antibiotic therapy, repeated debridement, resection arthroplasty, or one-stage or two-stage revision surgery. Among these, two-stage revision remains the current gold standard, achieving infection control in 85-95% of cases (7, 8). However, this strategy is associated with some drawbacks, including limb shortening, joint contracture, prolonged immobility, and severe bone loss before the second stage, which makes reimplantation technically challenging. Therefore, developing alternative methods that enable effective biofilm eradication and facilitate single-stage revision surgery would represent a significant clinical advance.

Cryotherapy has been widely used in adjuvant treatment for musculoskeletal tumors, especially with liquid nitrogen (LN) in the surgical bone cavity (9). Despite its efficacy in tumor ablation, conventional LN cryotherapy carries risks such as pathological fracture, skin necrosis, and nerve palsy (10, 11). To mitigate these complications, spray cryotherapy techniques were later developed (12, 13). Beyond oncology, cryotherapy has been explored in other medical fields, including chronic rhinosinusitis. A novel cryogenic material termed freezing nitrogen-ethanol composite (FNEC), using a semisolid mixture of LN and ethanol (3:1 ratio) was developed upon these principles. The semisolid consistency of FNEC allows precise shaping and prevents spillage or overflow to adjacent healthy tissues. Previous in-vitro and clinical studies have demonstrated that FNEC enables effective and safe cryoablation of bone tumors (14). Unlike spray cryotherapy, which is limited to surface treatment, FNEC can conformally coat irregular specimens and provide homogeneous low-temperature exposure.

In the present study, we sought to evaluate whether FNEC could effectively eradicate biofilm formed by pathogenic bacteria on prosthetic surfaces. Using S. aureus to simulate PJI-related biofilms, prosthetic components were preincubated to allow biofilm formation and then treated with FNEC, LN, or 75% ethanol. Bacterial viability was assessed using a LIVE/DEAD assay, and eradication efficiency was validated by optical density measurements following broth inoculation. We hypothesized that FNEC, owing to its combined chemical (ethanol) and physical (cryogenic) effects, would demonstrate superior bactericidal and biofilm-removal properties compared with conventional agents.

Materials and Methods

Bacterial culture and biofilm formation on prosthetic components. Before biofilm eradication experiments, prosthetic components (plastic spacers and metallic knee parts) were coated with S.aureus (strain UAMS-1, ATCC 49230). The bacteria were cultured in 5 ml of tryptic soy broth (TSB) at 37 °C overnight with shaking at 200 rpm. Two milliliters of the bacterial suspension, adjusted to an optical density of 0.1 at 600 nm (OD600), were transferred into a sterile stainless-steel beaker containing 398 ml of TSB supplemented with 5% glucose and 4% sodium chloride. Prosthetic components were fully immersed and incubated for four days at 37 °C without shaking to allow biofilm formation. The culture medium was refreshed daily by replacing 398 ml of supernatant with fresh supplemented TSB.

To confirm biofilm development, samples were stained with Congo Red, which produces a characteristic red discoloration indicative of mature biofilm. After confirming successful biofilm formation, the prostheses were subjected to treatment with 75% ethanol, LN, or FNEC for 15 min (Figure 1). Following treatment, bacterial survival was assessed by inoculating detached biofilms into broth cultures for optical-density measurement, and bacterial viability was visualized using a LIVE/DEAD fluorescence assay.

Figure 1.
Figure 1.

Experimental workflow. S. aureus biofilms were cultured on prosthetic components for 4 days at 37°C. Biofilm-covered specimens were treated with 75% EtOH, LN, or FNEC for 15 min. Bacterial survival was evaluated by broth inoculation and optical-density measurements, and cell viability was assessed with a LIVE/DEAD bacterial viability assay. EtOH: Ethanol, LN: liquid nitrogen, FNEC: freezing nitrogen-ethanol composite.

Biofilm eradication treatments. After biofilm formation, prosthetic components were randomly assigned to treatment groups: (i) 75% ethanol, (ii) LN, or (iii) FNEC, with untreated samples serving as controls. Treatments were conducted in sterile stainless-steel beakers under a biological safety cabinet for 15 min. After exposure, a small portion of each treated biofilm was gently scraped and inoculated into 5 ml of Luria-Bertani (LB) broth, which was incubated at 37 °C for 16 h. Bacterial growth was quantified by measuring optical density at 600 nm using a UV-Vis spectrophotometer (Jasco V-530, Tokyo, Japan). All experiments were performed in triplicate, and mean values were calculated.

Bacterial viability staining. Bacterial viability was assessed using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA). SYTO® 9 and propidium iodide (PI) dyes were mixed at a 1:1 ratio, and 3 μl of the dye mixture was added to 1 ml of phosphate-buffered saline (PBS). One milliliter of this staining solution was applied to each specimen and incubated in the dark for 15 min at room temperature. After gentle PBS washes, samples were observed under an inverted fluorescence microscope. Live bacteria emitted green fluorescence (SYTO 9), while dead bacteria with compromised membranes stained red (PI). Each treatment was examined in triplicate.

Statistical analysis. All statistical analyses and graphs were performed and generated using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). Data are presented as mean±standard deviation (SD). Differences among experimental groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value <0.05 was considered statistically significant.

Results

Eradication of biofilm on plastic spacers by FNEC. Biofilm-covered plastic spacers exhibited a strong red coloration before treatment. After 15 min of exposure, the 75% ethanol and LN groups showed only partial color reduction, whereas the FNEC-treated group displayed marked elimination of the red biofilm layer (Figure 2). These findings indicate that FNEC achieved superior surface decontamination compared with ethanol or LN alone.

Figure 2.
Figure 2.

Biofilm eradication on plastic spacers. Plastic spacers were incubated with S. aureus for 4 days to induce biofilm formation (red staining). After treatment with 75% EtOH, LN, or FNEC, color reduction reflected biofilm removal. (a) Untreated, (b-b’) EtOH, (c-c’) LN, (d-d’) FNEC. The FNEC group showed near-complete biofilm clearance. EtOH: Ethanol, LN: liquid nitrogen, FNEC: Freezing nitrogen-ethanol composite.

Removal of biofilm from metallic knee prostheses by FNEC. To confirm that FNEC is effective on metallic components, knee prosthesis surfaces were cultured with S. aureus and then treated on one side (right) with the same protocols. The 75% ethanol group showed partial clearing of red biofilm, the LN group showed minimal change, whereas the FNEC-treated surface demonstrated near-complete biofilm removal (Figure 3).

Figure 3.
Figure 3.

Biofilm removal on knee prosthetic metal surfaces. Knee components were cultured with S. aureus for 4 days and then half-treated on the right side with 75% EtOH, LN, or FNEC. (a) Untreated, (b-b’) EtOH, (c-c’) LN, (d-d’) FNEC. Yellow dashed lines delineate the treated region. The FNEC-treated area demonstrated complete biofilm eradication. EtOH: Ethanol, LN: liquid nitrogen, FNEC: freezing nitrogen-ethanol composite.

Bacterial viability analysis after FNEC treatment. LIVE/DEAD fluorescence imaging showed that untreated biofilms exhibited only green fluorescence (live bacteria). Following 75% ethanol treatment, overlapping green and red signals (yellow) indicated extensive bacterial death. The LN group also showed mixed viability with double-positive, indicating that many bacteria remained alive. In contrast, the FNEC-treated group showed nearly complete red and yellow fluorescence, signifying widespread cell death (Figure 4).

Figure 4.
Figure 4.

Bacterial viability assay on plastic spacers. LIVE/DEAD staining showing live bacteria (green, SYTO 9), dead bacteria (red, PI), and overlapping signals (yellow). (a-c) Untreated, (d-f) 75% EtOH, (g-i) LN, (j-l) FNEC. Extensive bacterial death was observed in the 75% EtOH and FNEC groups. Scale bars=50 μm. PI: Propidium iodide; PEtOH: ethanol, LN: liquid nitrogen, FNEC: freezing nitrogen-ethanol composite.

Validation of biofilm eradication by broth culture. To verify bacterial viability after treatment, portions of biofilm were inoculated into fresh broth. The untreated and LN-treated groups produced turbid broths, confirming active bacterial growth, whereas the 75% ethanol and FNEC groups remained clear (Figure 5a and b). Quantitative spectrophotometric analysis confirmed that optical density (OD600) was significantly lower in the FNEC and 75% ethanol groups compared with untreated and LN groups (p<0.001) (Figure 5c).

Figure 5.
Figure 5.

Confirmation of biofilm eradication by broth culture. (a) Schematic of knee metal prosthesis treated with 75% EtOH, LN, or FNEC. (b) Broth inoculated with treated biofilm showed turbidity in untreated and LN groups but clear solutions in EtOH and FNEC groups. (c) Optical density analysis (mean±SD). ***p<0.001; ****p<0.0001; ns: not significant. EtOH: Ethanol, LN: liquid nitrogen, FNEC: freezing nitrogen-ethanol composite; SD: standard deviation.

Indirect treatment confirms the sterilizing effect of FNEC. To distinguish chemical versus cryogenic effects, indirect exposure experiments were performed on biofilm-coated femoral head components. The untreated and 75% ethanol groups produced opaque broths, while LN and FNEC treatments-applied externally-resulted in markedly clearer broths (Figure 6a, b). Quantitative analysis showed significantly reduced absorbance in the LN and FNEC groups compared with controls (p<0.05 and p<0.0001, respectively). The OD600 of the FNEC group was indistinguishable from sterile broth, confirming complete biofilm eradication (Figure 6c).

Figure 6.
Figure 6.

Indirect treatment and validation of FNEC efficacy. (a) Schematic of biofilm-coated femoral head component indirectly exposed to 75% EtOH, LN, or FNEC. (b) Broth inoculation revealed clear solutions after LN and FNEC exposure. (c) OD600 quantification. *p<0.05; ****p<0.0001; ns: not significant. EtOH: Ethanol, LN: liquid nitrogen, FNEC: freezing nitrogen-ethanol composite.

Determination of optimal FNEC exposure time. To define the minimal effective exposure duration, FNEC treatment was applied for 3, 5, 10, and 15 min (Figure 7a). Broth inoculation revealed persistent turbidity after 3 min, whereas treatments of ≥ 5 min yielded clear broths (Figure 7b). Quantitative OD600 analysis confirmed that 5-min exposure was sufficient for complete sterilization, with no statistical difference compared with the sterile broth control (Figure 7c).

Figure 7.
Figure 7.

Time-dependent effect of FNEC treatment. (a) Experimental design showing FNEC exposure for 3, 5, 10, and 15 min. (b) Broth clarity after inoculation indicated complete sterilization with ≥ 5-min treatment. (c) OD600 analysis confirmed optimal eradication at 5 min. *p<0.05; **p<0.01; ****p<0.0001; ns: not significant. FNEC: Freezing nitrogen-ethanol composite.

Discussion

PJI remains one of the most challenging complications for orthopaedic surgeons. Although two-stage revision arthroplasty is considered the current gold standard for chronic PJI, this approach carries several shortcomings, including the need for multiple procedures, prolonged immobilization, and increased patient morbidity. Moreover, severe bone loss and soft-tissue contracture between stages may complicate reimplantation and compromise functional recovery. Identifying alternative strategies that can achieve comparable eradication rates while enabling single-stage revision would substantially improve patient outcomes and reduce healthcare burden.

In this study, we evaluated the efficacy of a novel cryogenic agent-FNEC-for eliminating Staphylococcus aureus biofilm on prosthetic materials. Our results demonstrated that FNEC treatment effectively eradicated bacterial biofilm from both plastic spacers and metallic prosthetic components in vitro. By contrast, both 75% ethanol and LN achieved only partial eradication. The LIVE/DEAD assay confirmed that FNEC induced widespread bacterial death, and broth culture analysis verified complete loss of viability after a minimum of 5-min exposure. These findings suggest that FNEC combines the bactericidal properties of ethanol with the cryogenic effects of extreme cold, resulting in enhanced biofilm destruction.

The eradication of biofilm is essential for successful PJI management. Biofilm confers mechanical protection and impedes antibiotic penetration, allowing bacterial persistence despite systemic therapy (4). Although ethanol is known to possess broad antimicrobial activity, its efficacy is concentration- and exposure-dependent. Ethanol solutions between 60-90% could disrupt bacterial membranes and denature proteins (15-17), and 70% ethanol has been shown to sterilize Staphylococcus epidermidis-infected vascular catheters in animal models (18-20). However, ethanol treatment alone is limited by incomplete penetration into dense biofilm matrices, potential tissue toxicity, and reversible inhibition of bacterial sporulation (21, 22). Our data corroborate these findings-while 75% ethanol induced significant bacterial death, residual viability was still detected in some biofilms.

Cryotherapy using LN has been widely applied in musculoskeletal oncology and its cytotoxicity is mediated by intracellular ice formation, osmotic shock, and vascular stasis. However, direct application of LN can cause local complications such as fractures, skin necrosis, and neuropathy (10, 11). Moreover, several studies have reported that a fraction of microorganisms can survive cryogenic exposure, particularly in non-sterile or protective environments (21, 23, 24). Indeed, Postgate and Hunter observed approximately 35% bacterial survival after freezing without cryoprotectants (23). Contamination of LN storage systems has also been implicated in cross-infection events, supporting our observation that LN alone was insufficient for sterilization and may even pose biohazard risks (25).

FNEC, by contrast, offers several distinct advantages. First, its semisolid phase allows it to conformally coat irregular prosthetic surfaces, ensuring uniform thermal contact and avoiding spillage to adjacent healthy tissues. Second, the ethanol component provides chemical denaturation and membrane disruption, while the cryogenic temperature (−136 °C) ensures irreversible cell injury through rapid freezing. Third, FNEC can induce non-lethal injury cascades leading to delayed bacterial death, as described by Ray et al. (26). Our indirect-contact experiments further confirmed that the cryogenic effect itself contributes significantly to bacterial inactivation. These synergistic mechanisms likely account for the superior performance of FNEC compared with ethanol or LN alone. Furthermore, previous reports have shown that rapid freezing enhances the bactericidal effect of aminoglycoside antibiotics via increased membrane permeability, suggesting potential additive benefits when FNEC is combined with antibiotic therapy (27).

Collectively, these findings indicate that FNEC treatment achieves effective biofilm eradication within five min of exposure while minimizing the limitations of conventional cryotherapy or chemical disinfection. Given its established clinical safety in cryoablation of bone tumors (14)–including clinical scenarios at high risk for PJI following large-scale prosthetic reconstruction after tumor resection (28)–FNEC may represent a feasible adjunct for single-stage PJI revision, either as an intraoperative surface sterilization step or as part of combined debridement and reimplantation protocols.

This study has several limitations. First, all experiments were conducted in vitro using a single S. aureus strain, which may not fully reflect the polymicrobial and host-modulated environment of clinical periprosthetic joint infection. Sterilization efficacy was not evaluated in the presence of organic load, such as synovial fluid, blood, or serum proteins, which may attenuate antimicrobial effects in vivo. Second, although FNEC demonstrated effective biofilm eradication on prosthetic surfaces, its effects on surrounding soft tissues, bone, and implant surface properties that may influence subsequent biological interactions under clinical conditions remain unknown (29). In addition, the impact of FNEC exposure on implant surface characteristics and long-term material integrity was not assessed. Finally, standardized protocols for intraoperative handling and exposure parameters of FNEC have yet to be established. Further studies incorporating protein-rich environments, polymicrobial models, and in vivo validation are warranted.

Conclusion

FNEC, a semisolid composite of LN and ethanol, demonstrated strong bactericidal activity and effectively eradicated S. aureus biofilms on prosthetic surfaces. The combination of chemical denaturation and cryogenic injury resulted in complete sterilization after a 5-min treatment. These results suggest that FNEC may serve as a promising adjuvant for biofilm eradication and potentially enable single-stage revision surgery for PJI.

Footnotes

  • Authors’ Contributions

    YKL and KHL helped write the manuscript and collect the data; CMC, SWT, CFC, and WMC designed the study and helped revise the manuscript; YKL and KHL analyzed the data; YKL and PKW designed and arranged this study. All authors proofread the final version of the manuscript.

  • Conflicts of Interests

    The Authors have no relevant financial or non-financial interests to disclose.

  • Funding

    The Authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received January 3, 2026.
  • Revision received January 31, 2026.
  • Accepted February 13, 2026.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

References

  1. ↵
    1. Peersman G,
    2. Laskin R,
    3. Davis J,
    4. Peterson M
    : Infection in total knee replacement: a retrospective review of 6489 total knee replacements. Clin Orthop Relat Res 392: 15-23, 2001. DOI: 10.1097/00003086-200111000-00003
    OpenUrlCrossRefPubMed
  2. ↵
    1. Blom AW,
    2. Taylor AH,
    3. Pattison G,
    4. Whitehouse S,
    5. Bannister GC
    : Infection after total hip arthroplasty. The Avon experience. J Bone Joint Surg Br 85-B(7): 956-959, 2003. DOI: 10.1302/0301-620x.85b7.14095
    OpenUrlCrossRefPubMed
  3. ↵
    1. Montanaro L,
    2. Speziale P,
    3. Campoccia D,
    4. Ravaioli S,
    5. Cangini I,
    6. Pietrocola G,
    7. Giannini S,
    8. Arciola CR
    : Scenery of Staphylococcus implant infections in orthopedics. Future Microbiol 6(11): 1329-1349, 2011. DOI: 10.2217/fmb.11.117
    OpenUrlCrossRefPubMed
  4. ↵
    1. Jolivet-Gougeon A,
    2. Bonnaure-Mallet M
    : Biofilms as a mechanism of bacterial resistance. Drug Discov Today Technol 11: 49-56, 2014. DOI: 10.1016/j.ddtec.2014.02.003
    OpenUrlCrossRefPubMed
  5. ↵
    1. Doub JB,
    2. Parmiter D,
    3. Brantner CA,
    4. Moshyedi M,
    5. Hughes M,
    6. Kolevar M,
    7. Johnson A
    : The location of biofilms on chronic prosthetic joint infections and the ramifications for clinical practice. Arthroplast Today 25: 101314, 2024. DOI: 10.1016/j.artd.2023.101314
    OpenUrlCrossRefPubMed
  6. ↵
    1. Visperas A,
    2. Santana D,
    3. Klika AK,
    4. Higuera-Rueda CA,
    5. Piuzzi NS
    : Current treatments for biofilm-associated periprosthetic joint infection and new potential strategies. J Orthop Res 40(7): 1477-1491, 2022. DOI: 10.1002/jor.25345
    OpenUrlCrossRefPubMed
  7. ↵
    1. Pangaud C,
    2. Ollivier M,
    3. Argenson JN
    : Outcome of single-stage versus two-stage exchange for revision knee arthroplasty for chronic periprosthetic infection. EFORT Open Rev 4(8): 495-502, 2019. DOI: 10.1302/2058-5241.4.190003
    OpenUrlCrossRefPubMed
  8. ↵
    1. Uchiyama K,
    2. Takahira N,
    3. Fukushima K,
    4. Moriya M,
    5. Yamamoto T,
    6. Minegishi Y,
    7. Sakai R,
    8. Itoman M,
    9. Takaso M
    : Two-stage revision total hip arthroplasty for periprosthetic infections using antibiotic-impregnated cement spacers of various types and materials. ScientificWorldJournal 2013: 147248, 2013. DOI: 10.1155/2013/147248
    OpenUrlCrossRefPubMed
  9. ↵
    1. Marcove RC
    : A 17-year review of cryosurgery in the treatment of bone tumors. Clin Orthop Relat Res 163: 231-234, 1982. DOI: 10.1097/00003086-198203000-00035
    OpenUrlCrossRefPubMed
  10. ↵
    1. Meller I,
    2. Weinbroum A,
    3. Bickels J,
    4. Dadia S,
    5. Nirkin A,
    6. Merimsky O,
    7. Issakov J,
    8. Flusser G,
    9. Marouani N,
    10. Cohen N,
    11. Kollender Y
    : Fifteen years of bone tumor cryosurgery: A single-center experience of 440 procedures and long-term follow-up. Eur J Surg Oncol 34(8): 921-927, 2008. DOI: 10.1016/j.ejso.2007.11.001
    OpenUrlCrossRefPubMed
  11. ↵
    1. Veth R,
    2. Schreuder B,
    3. Van Beem H,
    4. Pruszczynski M,
    5. de Rooy J
    : Cryosurgery in aggressive, benign, and low-grade malignant bone tumours. Lancet Oncol 6(1): 25-34, 2005. DOI: 10.1016/s1470-2045(04)01710-3
    OpenUrlCrossRefPubMed
  12. ↵
    1. Greenwald BD,
    2. Dumot JA,
    3. Horwhat JD,
    4. Lightdale CJ,
    5. Abrams JA
    : Safety, tolerability, and efficacy of endoscopic low-pressure liquid nitrogen spray cryotherapy in the esophagus. Dis Esophagus 23(1): 13-19, 2010. DOI: 10.1111/j.1442-2050.2009.00991.x
    OpenUrlCrossRefPubMed
  13. ↵
    1. Greenwald BD,
    2. Dumot JA,
    3. Abrams JA,
    4. Lightdale CJ,
    5. David DS,
    6. Nishioka NS,
    7. Yachimski P,
    8. Johnston MH,
    9. Shaheen NJ,
    10. Zfass AM,
    11. Smith JO,
    12. Gill KR,
    13. Burdick JS,
    14. Mallat D,
    15. Wolfsen HC
    : Endoscopic spray cryotherapy for esophageal cancer: safety and efficacy. Gastrointest Endosc 71(4): 686-693, 2010. DOI: 10.1016/j.gie.2010.01.042
    OpenUrlCrossRefPubMed
  14. ↵
    1. Wu PK,
    2. Chen CF,
    3. Wang JY,
    4. Chen PC,
    5. Chang MC,
    6. Hung SC,
    7. Chen WM
    : Freezing nitrogen ethanol composite may be a viable approach for cryotherapy of human giant cell tumor of bone. Clin Orthop Relat Res 475(6): 1650-1663, 2017. DOI: 10.1007/s11999-017-5239-3
    OpenUrlCrossRefPubMed
  15. ↵
    1. McDonnell G,
    2. Russell AD
    : Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 12(1): 147-179, 1999. DOI: 10.1128/CMR.12.1.147
    OpenUrlAbstract/FREE Full Text
    1. Chambers ST,
    2. Pithie A,
    3. Gallagher K,
    4. Liu T,
    5. Charles CJ,
    6. Seaward L
    : Treatment of Staphylococcus epidermidis central vascular catheter infection with 70% ethanol locks: efficacy in a sheep model. J Antimicrob Chemother 59(4): 779-782, 2007. DOI: 10.1093/jac/dkl542
    OpenUrlCrossRefPubMed
  17. ↵
    1. Morton HE
    : The relationship of concentration and germicidal efficiency of ethyl alcohol. Ann N Y Acad Sci 53(1): 191-196, 1950. DOI: 10.1111/j.1749-6632.1950.tb31944.x
    OpenUrlCrossRefPubMed
  18. ↵
    1. Trujillo R,
    2. Laible N
    : Reversible inhibition of spore germination by alcohols. Appl Microbiol 20(4): 620-623, 1970. DOI: 10.1128/am.20.4.620-623.1970
    OpenUrlCrossRefPubMed
    1. Pérez-Granda MJ,
    2. Barrio JM,
    3. Muñoz P,
    4. Hortal J,
    5. Rincón C,
    6. Rabadán PM,
    7. Pernia MS,
    8. Bouza E
    : Ethanol lock therapy (E-Lock) in the prevention of catheter-related bloodstream infections (CR-BSI) after major heart surgery (MHS): a randomized clinical trial. PLoS One 9(3): e91838, 2014. DOI: 10.1371/journal.pone.0091838
    OpenUrlCrossRefPubMed
  20. ↵
    1. Mokha JS,
    2. Davidovics ZH,
    3. Samela K,
    4. Emerick K
    : Effects of ethanol lock therapy on central line infections and mechanical problems in children with intestinal failure. JPEN J Parenter Enteral Nutr 41(4): 625-631, 2017. DOI: 10.1177/0148607115625057
    OpenUrlCrossRefPubMed
  21. ↵
    1. Moses V
    : Tricarboxylic acid cycle reactions in the fungus Zygorrhynchus moelleri. Microbiology 13(2): 235-251, 1955. DOI: 10.1099/00221287-13-2-235
    OpenUrlCrossRefPubMed
  22. ↵
    1. Yasuda-Yasaki Y,
    2. Namiki-Kanie S,
    3. Hachisuka Y
    : Inhibition of Bacillus subtilis spore germination by various hydrophobic compounds: demonstration of hydrophobic character of the L-alanine receptor site. J Bacteriol 136(2): 484-490, 1978. DOI: 10.1128/jb.136.2.484-490.1978
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Postgate JR,
    2. Hunter JR
    : On the survival of frozen bacteria. J Gen Microbiol 26(3): 367-378, 1961. DOI: 10.1099/00221287-26-3-367
    OpenUrlCrossRefPubMed
  24. ↵
    1. Wade HE,
    2. Lovett S
    : Polynucleotide phosphorylase in ribosomes from Escherichia coli. Biochem J 81(2): 319-328, 1961. DOI: 10.1042/bj0810319
    OpenUrlFREE Full Text
  25. ↵
    1. Bielanski A
    : A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and to related new reproductive technologies. Biotechnol Adv 15(3-4): 633-656, 1997. DOI: 10.1016/s0734-9750(97)00041-4
    OpenUrlCrossRefPubMed
  26. ↵
    1. Ray B,
    2. Janssen DW,
    3. Busta FF
    : Characterization of the repair of injury induced by freezing Salmonella anatum. Appl Microbiol 23(4): 803-809, 1972. DOI: 10.1128/am.23.4.803-809.1972
    OpenUrlCrossRefPubMed
  27. ↵
    1. Zhao Y,
    2. Lv B,
    3. Sun F,
    4. Liu J,
    5. Wang Y,
    6. Gao Y,
    7. Qi F,
    8. Chang Z,
    9. Fu X
    : Rapid Freezing enables aminoglycosides to eradicate bacterial persisters via enhancing mechanosensitive channel MscL-mediated antibiotic uptake. mBio 11(1): e03239-19, 2020. DOI: 10.1128/mBio.03239-19
    OpenUrlCrossRefPubMed
  28. ↵
    1. Nakamura T,
    2. Sakai T,
    3. Tsukushi S,
    4. Kimura H,
    5. Wasa J,
    6. Hosono K,
    7. Izubuchi Y,
    8. Kozawa E,
    9. Nagano A,
    10. Asanuma K,
    11. Sudo A,
    12. Nishida Y
    : Clinical outcome in patients with high-grade soft-tissue sarcoma receiving prosthetic replacement after tumor resection of the lower extremities: Tokai Musculoskeletal Oncology Consortium study. In Vivo 37(6): 2642-2647, 2023. DOI: 10.21873/invivo.13372
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Akbas O,
    2. Reck L,
    3. Jahn A,
    4. Hermsdorf J,
    5. Stiesch M,
    6. Greuling A
    : Effect of different sandblasting parameters on the properties of additively manufactured and machined titanium surfaces. In Vivo 39(3): 1767-1785, 2025. DOI: 10.21873/invivo.13979
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Freezing Nitrogen–Ethanol Composite Effectively Eradicates Staphylococcus aureus Biofilm on Prosthetic Surfaces
YU-KUAN LIN, KUN-HAN LEE, CHAO-MING CHEN, SHANG-WEN TSAI, CHENG-FONG CHEN, PO-KUEI WU, WEI-MING CHEN
In Vivo May 2026, 40 (3) 1408-1417; DOI: 10.21873/invivo.14292
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords