Abstract
Aims
The presence of biological debris on surgical instruments after reprocessing poses potential risks of infection in orthopaedic surgery. While autoclave sterilization is the standard method, its effectiveness in eliminating microbial contamination from residual bone, cement, and fatty tissue remains insufficiently studied. The primary objective was to study in vitro the sterility of retained cement, bone, and fat tissue debris on surgical instruments after autoclave sterilization. The secondary objective was to compare, based on colony-forming units (CFU), which material exhibits greater biofilm formation.
Methods
An in vitro study was conducted to evaluate the sterility of retained biological debris on surgical instruments after autoclave sterilization. The autoclave sterilization procedure was performed at 1 atmosphere pressure (121°C) for 20 minutes. A total of 350 repetitions were performed, including five instruments used as sample specimens (drill bit, cannulated flexible reamer, femoral broach, cannulated screwdriver, and wire passer), with 70 repetitions per specimen. CFU/mL were counted and biofilm morphology was assessed through scanning electron microscopy (SEM).
Results
After autoclave sterilization, no positive cultures were detected in any of the 350 repetitions (0%, 95% CI 0 to 5; n = 0), demonstrating complete absence of viable microorganisms. Due to the lack of microbial recovery, no statistical comparisons between instrument types were performed. We did not find any structural deformity in the mixed biofilms on bone, cement, and stainless steel as observed by SEM.
Conclusion
Autoclave sterilization was effective in eliminating microorganisms from surgical instruments with retained biological debris. Although no apparent structural deformities of microorganisms were observed by SEM in the mixed biofilms, no bacterial growth was detected after sterilization, supporting the microbiological effectiveness of autoclave sterilization even in the presence of retained biological debris. These findings provide microbiological evidence supporting current sterilization practices, and highlight the need for further studies evaluating alternative sterilization methods such as ethylene oxide or plasma.
Cite this article: Bone Joint Res 2026;15(10):1176–1182.
Keywords: Biofilm, Surgical instruments, Periprosthetic joint infection, Sterilization, surgical instruments, biofilms, Microorganisms, reamer, biofilm formation, structural deformities, stainless steel, scanning electron microscopy, orthopaedic surgery, infection
Article focus
To evaluate in vitro the sterility of retained bone, cement, and fatty tissue debris on orthopaedic surgical instruments after autoclave sterilization.
To assess whether different types of retained biological materials differed in their ability to support biofilm formation based only on comparison of colony-forming unit counts.
To examine the ultrastructural characteristics of mixed biofilms on instrument surfaces after sterilization as an additional method for assessing biofilm presence on abiotic- and biotic-type surfaces.
Key messages
Autoclave sterilization effectively eliminated viable microorganisms from surgical instruments despite the presence of retained biological debris.
No bacterial growth was detected across all instrument types and repetitions, precluding differences in contamination risk between materials.
These findings support the microbiological safety of autoclave-sterilized instruments even when complete debris removal is not achieved.
Strengths and limitations
The study employed a high number of repetitions across multiple commonly used orthopaedic instruments.
Combined microbiological culture analysis with ultrastructural evaluation using scanning electron microscopy.
Limitations include the in vitro design and the absence of comparison with alternative sterilization methods.
Introduction
The retention of bone, cement, or fatty tissue debris on surgical instruments represents a challenge during the preparation of the surgical equipment within the operating theatre. Although such debris is generally considered sterile after autoclave sterilization, there is no unanimous scientific evidence in the literature supporting this assumption.1 This situation also generates an economic burden for hospitals, as surgical trays often need to be replaced in order to prepare a new sterile setup.
According to current guidelines, the reprocessing of surgical instruments after use should include the following steps: 1) immersion of the instruments in enzymatic detergent for 20 minutes, 2) manual brushing (first cleaning), 3) placement of the instruments in the surgical tray/s, 4) second cleaning in the sterilization unit, and 5) sterilization.2-6 However, for logistical reasons – often under the responsibility of orthopaedic suppliers – these steps are not always carried out immediately after surgery and may be delayed for 24 to 72 hours. If this delay occurs, it might enhance microbial growth on contaminated surgical instruments.
It has been demonstrated that biofilm formation begins within two to six hours after bacterial adhesion into an inert surface. In an in vitro study, Smith et al7 showed that cannulated drill bit containing retained bone, after 180 minutes of exposure to Bacillus cereus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA), yielded B. cereus in cultures obtained following autoclave sterilization. Similarly, Wellington et al8 investigated drill bits used in surgery and found that 97% of the samples were sterile despite the presence of cement remnants, with only one positive case for Corynebacterium spp.
According to the Hospital Italiano de Buenos Aires orthopedic operating room records for 2023 and 2024, a total of 5,927 and 4,996 surgeries were performed, respectively, in which inadequately cleaned surgical instruments were reported in 139 (2.5%) and 52 (1%) cases, respectively. Undoubtedly, this issue resulted in substantial economic loss for both the institutional and overall healthcare system, besides inconvenience for patients, as surgeries may be delayed or postponed until the following day(s) or weeks, depending on waiting lists. An example of some inadequately cleaned surgical instruments can be seen in Figure 1.
Fig. 1.
An example of some inadequately cleaned surgical instruments. a) Flexible drill bit with accumulated debris. b) Femoral broach with retained bone.
In this context, further scientific evidence is required regarding the potential for contamination of insufficiently cleaned surgical instruments after sterilization, so that surgeons can make appropriate decisions regarding their use or definitive disposal.
The primary objective was to study in vitro the sterility of retained cement, bone, and adipose tissue debris on surgical instruments after autoclave sterilization. The secondary objective of this study was to compare, based on colony-forming units (CFUs), which material exhibits greater biofilm formation.
Methods
Specimens
To recreate a scenario with inadequately cleaned surgical instruments, we used: 1) polymethylmethacrylate (PMMA) bone cement (in the drill bit); 2) cancellous bone (in the femoral broach); 3) adipose tissue (on the cannulated screwdriver and wire passers); and 4) adipose tissue and bone (in the cannulated flexible reamer) (Figure 2).
Fig. 2.
An example of different surgical instruments used (top row) and the simulation of their inadequate cleaning process using different materials depending on the instrument type (bottom row). a) Polymethylmethacrylate (PMMA) bone cement on drill bit. b) Cancellous bone in the femoral broach. c) Adipose tissue on cannulated screwdriver and d) wire passer. e) Adipose tissue and bone in flexible reamer.
This study was approved by the Comité de Ética de Protocolos de Investigación (CEPI) of Hospital Italiano de Buenos Aires (IRB00010193, protocol #10,403) and follows the Declaration of Helsinki.9 Cancellous bone and adipose tissue were obtained from scheduled procedures in our hospital, specifically primary hip arthroplasty. All primary hip arthroplasty patients’ cases received one preoperative gram of cefazolin as prophylaxis before incision, routinely. Antibiotic-free surgical cement (Stryker Simplex Bone Cement) was prepared exclusively in laboratory conditions for the purpose of the experiment. To standardize the amount of bone and cement retained on surgical instruments, we analyzed samples retrieved from the hospital’s operating theatre (when the instruments contained residual bone or cement from a previous surgery). The mean weight of retained bone on the femoral broach after sterilization was 400 mg, and on the cannulated screwdriver/drill bit was 200 mg. For cement, the mean retained weight was 100 mg. We were unable to measure the actual weight of retained adipose tissue on different types of surgical instruments after sterilization due to its melting at high temperatures. To simulate the worst-case scenario with the presence of bone/cement/fat residues on surgical instruments, we used 380 mg of bone in the cannulated flexible reamer and 750 mg in the femoral broach. Triturated cancellous bone and adipose tissue were weighed using an electronic scale and introduced into the cannulated flexible reamer using Adson tissue forceps. In the case of wire passers and cannulated screwdrivers, the amount of fat added on the surface of each was approximately 180 mg and 300 mg, respectively. For cement preparation, liquid PMMA was obtained by mixing 40 g of powder monomer with 20 ml of catalyst. A total of 1 ml3 of liquid cement was applied onto the surface of a standard drill bit with a syringe. All procedures were performed under sterile conditions using gloved hands.
Bacterial strains
Microorganisms commonly found in the environment and those frequently involved in bone infections were used, including: 1) MRSA ATCC 25923; 2) B. cereus (IIMCS collection - 14); and 3) P. aeruginosa ATCC 27853. All strains were obtained through a donation from the institutional Microbiology Department (IIMCS, Universidad del Salvador, Buenos Aires, Argentina).
Sterility and biofilm controls
Positive control: the surgical instruments contaminated with MRSA, B. cereus, and P. aeruginosa biofilm, which had not gone through the sterilization procedure and had undergone vortex agitation for colony liberation from the instruments’ surfaces. This presented approximately 10^8 CFU/ml of MRSA, B. cereus, and P. aeruginosa combined.
Negative control: inert material without MRSA, B. cereus, or P. aeruginosa biofilm, which did undergo autoclave sterilization.
Experimental procedure
The assays were carried out in the Laboratory of Microbiology (IIMCS) of Universidad del Salvador (Buenos Aires, Argentina). Cement was aseptically applied to the surface of the sterile surgical drill bit using a syringe, while bone was placed onto the femoral broach using Adson tissue forceps. Similarly, adipose tissue was introduced onto cannulated screwdrivers, wire passers, and a cannulated flexible reamer, along with bone tissue in the last one. The instruments were incubated in tryptic soy broth (TSB) in the presence of MRSA, P. aeruginosa, and B. cereus for 48 hours. The instrument was then removed and washed with sterile saline solution for three minutes.
Subsequently, the surgical set was sterilized in an autoclave at 121° for 20 minutes (Arcano; Model: LS - B50L; Series NO. 16 L - 0056). The sterilized instruments were placed into flasks containing saline solution for biofilm detachment by vortexing for three minutes (2,800 revolutions per minute). Samples were collected with a micropipette and plated onto cystine lactose electrolyte deficient (CLED) agar. CFU were analyzed after 24 to 72 hours of incubation. All experiments were performed in triplicate, in accordance with previous literature.10
Scanning electron microscopy evaluation
The five surgical instruments were aseptically sectioned into smaller pieces inside a laminar flow hood using a Medtronic MR8 saw with MC254 metal cutter for SEM analysis. Both biofilm-negative samples (negative controls) and biofilm-positive samples (positive controls) of instruments containing bone, cement, and fat residues were prepared and analyzed.
Samples were fixed in 3% paraformaldehyde, dehydrated through increasing concentrations of ethanol (50%, 70%, 80%, 90%, 100%), followed by critical point drying, and then coated with a 20 nm layer of gold and palladium. Samples were examined using a Zeiss GeminiSEM 360 scanning electron microscope (Zeiss, Germany).
Statistical analysis
A sample size calculation was performed for the descriptive question of the proportion of bone-containing surgical instrument samples testing positive. It was expected that the proportion of positive samples would be 0%, with a 95.7% confidence interval. Thus, at least 70 samples of each material were required to achieve a confidence interval width of 5%. Sample size calculation was based on the Clopper-Pearson formula,11 and performed using Stata v. 16 (StataCorp, USA).
Results
A total of 350 repetitions were performed, corresponding to 70 repetitions for each of the specimens included in the study: 1) drill bit, 2) cannulated flexible reamer, 3) femoral broach, 4) cannulated screwdriver, and 5) wire passer.
At the end of the repetitions, the proportion of positive cultures in the five specimens analyzed was 0% (95% CI 0 to 5; n = 0). In other words, none of the 70 repetitions performed for each specimen yielded positive cultures.
Regarding the secondary objective, comparisons between specimens were not carried out, since no CFUs were recovered in any of the repetitions.
The visual confirmation of biofilm that was developed during the culture process on surgical instruments was performed using SEM. We did not observe any structural deformities or complete destruction of microorganisms in the mixed biofilms on bone, fat, cement, and stainless steel after sterilization using a standard autoclave (Figure 3).
Fig. 3.
Scanning electron microscopy (SEM) images of mixed biofilm on the surface of a) metal without autoclave sterilization, showing the mixed biofilm; b) metal after autoclave sterilization, showing the normal biofilm structure; c) bone without autoclave sterilization, showing the presence of biofilm; and d) bone after autoclave sterilization, showing the biofilm. EHT, electron high tension; WD, working distance.
Discussion
In our study, using a qualitative microbiological method, we demonstrated the complete absence of viable microorganisms in debris retained on contaminated surgical instruments. These findings suggest that if any instrument with inadequate cleaning is detected when opening the trays, the surgeon could still use it without the need to postpone the intervention or request its arthroplasty. However, there were not observed by SEM, as reported by other authors, any structural deformities or complete destruction of microorganisms in the mixed biofilms (B. cereus, MRSA, and P. aeruginosa) on bone, fat, cement, and stainless steel. The presence of a rigid cell wall in both Gram-positive and Gram-negative bacteria is an important factor limiting deformation caused by heat (121°C). This could be different in bacteria lacking rigid cell walls, such as Mycoplasma spp., Ureaplasma spp., and L-form bacteria. It should be clarified that SEM visualization does not allow assessment of bacterial viability, as it provides only morphological information. As the biofilm image did not suggest any morphological deterioration after autoclave sterilization in our study, the conclusions were based only on CFU culture results.
In the study conducted by Smith et al,7 cannulated drills with biological debris experimentally contaminated with B. cereus, P. aeruginosa, and S. aureus were sterilized using a standard autoclave cycle. Viable B. cereus was recovered from one instrument after 180 minutes of prior exposure to the bacteria. In the study published by Wellington et al,8 surgical instruments with visible debris were set aside for six months. The majority (97%) of macroscopic debris found on orthopaedic instruments after sterile reprocessing showed no bacterial load. The most common contaminant was bone tissue, and the most frequently affected instruments were surgical trays and cannulated drills. Bacterial growth (Corynebacterium spp.) was detected in only one case. Our methods differ from Smith et al and Wellington et al, since we used steam autoclave sterilization at 133°C for 20 minutes instead of four minutes (Wellington et al), without the need for a 40-minute dry time (Smith et al).
The 2018 International Consensus Meeting (ICM) on Musculoskeletal Infection did not establish specific recommendations regarding the presence of debris on inadequately cleaned surgical instruments during surgery preparation.1 However, it did address the possibility of reusing a previously infected prosthesis as a spacer during the first stage of revision surgery, employing intraoperative autoclave sterilization. Several studies have demonstrated that the reuse of autoclave-sterilized prosthetic components may be a viable option.12-19 Nevertheless, this practice poses potential legal implications and lacks a defined standard regarding the optimal autoclave sterilization time, with controversy remaining over the sterility achieved in infected prosthetic components because of the scarcity of studies directly evaluating this technique.
Lyons et al20 performed swab cultures on six retrieved femoral components before and after a 45-minute autoclave cycle at 121°C. The procedure successfully eliminated most bacterial species, both in planktonic form and in biofilm, from the smooth cobalt-chrome (CoCr) surfaces. Subsequently, the six sterile components were inoculated with different microorganisms and retested, with no bacterial growth detected after autoclaving. SEM analysis of the inoculated samples also showed a marked reduction in biofilm. However, this study only used 24-hour biofilms. In contrast, Leary et al21 reported that autoclaving at 121°C for 30 minutes was insufficient to eradicate S. aureus or Staphylococcus epidermidis biofilms on CoCr discs, but that prior treatment with brushing and 4% chlorhexidine gluconate achieved complete eradication. In a more recent study, Williams et al22 evaluated different autoclave temperature and time combinations to eradicate mono- and polymicrobial biofilms matured for eight days. While ten minutes at 132°C were sufficient to render all biofilms non-viable according to cultures, residual biofilm was still detected on titanium materials. The clinical relevance of this non-viable biofilm remains unclear, especially when extrapolating results from titanium materials to CoCr implants used in autoclave-sterilized prosthetic components. Brushing with 4% chlorhexidine gluconate, as proposed by Leary et al,21 may overcome this potential limitation.
As strengths, our study is the first to investigate the presence of polymicrobial biofilm on the surface of surgical instruments with different biological residues and PMMA. In terms of limitations, we did not evaluate other sterilization methods, such as ethylene oxide or plasma sterilization. Also, extrapolation of findings from in vitro models of biofilm formation to clinical settings is limited. Therefore, the use of an in vitro model may represent a limitation, as it does not fully replicate the complexity of in vivo conditions. In this study we used the real washing, cleaning, and sterilization steps that surgical instruments currently employ at our institution. We acknowledge that these procedures may differ at other institutions with different methods and resources.
In conclusion, a complete absence of bacterial contamination was demonstrated after autoclave sterilization of the surgical instruments, indicating that the sterilization process was effective in eliminating the microbial load in all specimens evaluated. SEM analysis confirmed the presence of disintegrated biofilm residues on bone, cement, and on the stainless steel surface of the instruments.
Author contributions
D. Semeshchenko: Conceptualization, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing
A. Farinati: Conceptualization, Investigation, Writing – original draft
I. A. Huespe: Formal analysis, Methodology, Software, Supervision
HIBA Hip Surgery Unit: Conceptualization
J. Iviglia: Conceptualization, Investigation, Methodology, Project administration, Resources
M. A. Buttaro: Conceptualization, Supervision
P. A. Slullitel: Conceptualization, Methodology, Project administration, Resources, Supervision
Funding statement
The authors received no financial or material support for the research, authorship, and/or publication of this article.
ICMJE COI statement
Each author certifies that he or she has no commercial associations (e.g. consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.
Data sharing
All data generated or analyzed during this study are included in the published article and/or in the supplementary material.
Acknowledgements
We gratefully acknowledge the valuable expertise and contributions of Martin Cornut, Professor of Histology and Cell Biology and Human Embryology, Faculty of Medicine, Universidad del Salvador, Buenos Aires, Argentina, for his valuable expertise and contributions, which were instrumental in making this research possible.
ChatGPT (OpenAI, USA) was used to assist with language editing and to improve the clarity and readability of the manuscript. All AI-assisted text was reviewed and approved by the authors, who take full responsibility for the final content.
HIBA Hip Surgery Unit
Fernando Comba, MD, Gerardo Zanotti, MD, Agustín Facundo Albani-Forneris, MD, Carlos Martín Lucero, MD. ‘Sir John Charnley’ Hip Surgery Unit, Institute of Orthopaedics ‘Carlos E. Ottolenghi’, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina.
Ethical review statement
This study was approved by the Comité de Ética de Protocolos de Investigación (CEPI) of Hospital Italiano de Buenos Aires (IRB00010193; protocol #10,403), and was conducted in accordance with the Declaration of Helsinki.
Open access funding
The authors report that the open access funding for their manuscript was self-funded.
© 2026 Semeshchenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (CC BY-NC-ND 4.0) licence, which permits the copying and redistribution of the work only, and provided the original author and source are credited. See https://creativecommons.org/licenses/by-nc-nd/4.0/
Contributor Information
Daniyil Semeshchenko, Email: daniyil.semeshchenko@hospitalitaliano.org.ar.
Collaborators: Fernando Comba, Gerardo Zanotti, Agustín F. Albani-Forneris, and Carlos M. Lucero
Data Availability
All data generated or analyzed during this study are included in the published article and/or in the supplementary material.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data generated or analyzed during this study are included in the published article and/or in the supplementary material.