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- Chiamaka T. Umah
- Ashleigh Riegler
- Mishi Bhushan
- Danielle Scarborough-Carr
- Elizabeth King
- Jamie Hutchinson
- Megan H. Amerson-Brown⇑
- University of Alabama at Birmingham
- University of Alabama at Birmingham
- Baylor Scott and White Medical Center, Baylor College of Medicine
- Department of Pathology, University of Alabama at Birmingham Health Service Foundation
- Department of Pathology, University of Alabama at Birmingham Health Service Foundation
- Department of Pathology, University of Alabama at Birmingham Health Service Foundation
- Department of Pathology, University of Alabama at Birmingham
- Address for Correspondence: Megan H. Amerson-Brown
, Department of Pathology, University of Alabama at Birmingham, mamersonbrown{at}uabmc.edu
ABSTRACT
Anaerobic bacteria are critical pathogens implicated in a variety of human infections, yet their recovery from clinical samples remains challenging because of their fastidious nature and sensitivity to oxygen. Establishing adequate and effective methods to recover clinically relevant obligate anaerobic organisms and reduce the recovery of facultative aerobic organisms from anaerobic cultures is critical for the clinical diagnostic value of anaerobic culture, laboratory workload, and workflow. This study describes a quality improvement (QI) project that focused on a simple, yet effective way to improve the recovery of anaerobes in clinical cultures through alteration of methods for initial incubation at specimen processing. This included the addition of single-use anaerobic bags for initial incubation prior to batched culture storage in large anaerobic jars to improve recovery of obligate anaerobes by minimizing exposure of the organisms to nonanaerobic conditions during initial processing. Our data showed that reducing the interval between specimen setup and the establishment of anaerobic conditions, by implementing the use of anaerobic bags, significantly improved recovery of obligate anaerobes by 22.22%. The findings from this QI project highlight that using a combination of methods for anaerobic culture can improve workflow and time to anaerobic conditions resulting in better recovery of anaerobic organisms.
- CL - control limit
- LCL - lower CL
- MALDI-TOF - matrix-assisted laser desorption ionization time-of-flight
- QI - quality improvement
- UABHCM - University of Alabama at Birmingham Hospital Clinical Microbiology Laboratory
- UCL - upper CL
INTRODUCTION
Anaerobic bacteria can be categorized according to oxygen tolerance into 3 major groups: facultative anaerobes that can grow with or without the presence of oxygen, obligate anaerobes that require an oxygen-depleted environment for growth, and aerotolerant anaerobes that can grow in an oxygen-present environment even though they do not use it.1 Organisms classified as obligate anaerobes can have varying tolerance to oxygen exposure, even among the same genera and species, contributing to phenotypic diversity and presenting challenges in clinical recovery.2,3 Anaerobes are integral components of the human microbiome and play an essential role in maintaining physiological homeostasis. When mucosal barriers are disrupted, anaerobic organisms may invade sterile body sites and act as opportunistic pathogens. In addition to their role in infectious diseases, several anaerobes have been associated with noninfectious disease processes. For example, Clostridium septicum and Fusobacterium spp have been associated with colorectal malignancies.4⇓-6
Anaerobic bacteria can cause a wide range of clinically significant infections including abscesses, gas gangrene, and those of the abdominal, skin and soft tissue, urogenital, and respiratory tract. Risk factors for anaerobic infections include malignancies, immunosuppressive or immunocompromising conditions, gynecological or gastrointestinal surgeries, and chronic wounds, such as decubitus ulcers. These infections are often polymicrobial and occur owing to heterogenous spread. Notably, the distinction between monomicrobial and polymicrobial infection is clinically important because treatment protocols can differ.7⇓⇓-10
Despite their clinical relevance, anaerobic organisms are often underrecovered in clinical cultures because of suboptimal sample collection, transport, and inconsistent incubation workflows.2,9,11⇓⇓-14 Inefficient or delayed establishment of anaerobic conditions reduces the recovery of obligate anaerobic pathogens and increases the likelihood of false-negative results. Therefore, optimizing the recovery of clinically relevant anaerobes in the laboratory is critical to the diagnosis and effective treatment of these infections, especially of otherwise sterile body sites.2,8,11,14⇓-16
Clinical guidance on acceptable specimen types and overall culture conditions for samples subjected to anaerobic culture are well defined in the Clinical Microbiology Procedures Handbook12; however, the culture workflow can be highly variable among different clinical microbiology laboratories. Many high-volume clinical laboratories use incubation systems like the Anoxomat jar system intended to handle a large volume of anaerobic cultures by batching numerous plates together rather than single-culture incubation systems like the GasPak system, designed for simultaneous culture of only a few plates. However, in these large throughput systems, culture plates are often held in large jars or chambers until enough accumulate for batch processing into anaerobic conditions, introducing delays in the establishment of an oxygen-free environment. Single-use anaerobic bag systems have long been shown to provide a rapid, cost-effective method to quickly establish anaerobic conditions.8,17,18 As part of this initiative, we used a hybrid approach to improve the recovery of anaerobic organisms with minimal workflow interruption. Notably, this approach used anaerobic bags for immediate incubation of individual or small batch cultures at initial processing followed by transition to large anaerobic jars for continued culture workup after initial 48-hour culture review. The development, implementation, and outcomes of this process are described, including a significant improvement in the recovery of obligate anaerobes in clinical anaerobic microbiology cultures.
MATERIALS AND METHODS
Data Recovery and Analysis
Six months of anaerobic culture data generated by the University of Alabama at Birmingham Hospital Clinical Microbiology Laboratory (UABHCM) were retrospectively evaluated: data from 3 months prior to implementation of the new anaerobic processing workflow (January through March 2022) and 3 months following implementation (January through March 2023). The revised process was implemented in October 2022, allowing for a stabilization period before collection and analysis of postimplementation data. The pre- and postimplementation months were intentionally matched to control for the seasonal variation in organism prevalence, ordering practices related to staffing patterns, and trainee turnover throughout the academic year.
Culture data from the pre- and postimplementation periods were compared to evaluate the following outcomes: recovery of total organisms, recovery of aerobes, recovery of obligate and facultative anaerobes, and recovery of organisms from repeat cultures ordered on the same patient from the same anatomical site. Anaerobic body fluid cultures and blood cultures were excluded from this analysis and quality improvement (QI) initiative because these were collected directly into anaerobic blood culture bottles prior to laboratory processing and therefore were not subjected to the incubation protocol changes. Organism identification was achieved by standard laboratory practices, primarily through analysis on Vitek mass spectrometry matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF; Biomerieux, Durham, NC) or Bruker biotyper MALDI-TOF (Billerica, MA).19⇓-21 A total of 56 species were isolated, identified, and classified as obligate or facultative anaerobes (Supplemental Table 1).
Differences in organism recovery
Sample Collection for Anaerobic Cultures
Clinical specimens were collected by the patient care teams following standard protocol and sent to the microbiology laboratory immediately after collecting, either in a sterile container for tissue samples or in Amies medium if an eswab was used in the collection process. On receipt at the UABHCM, samples were processed for anaerobic culture within 2 hours (Figure 1).
Old vs new process. A visual representation of the process used in this study. The left side of the diagram shows the old process, whereas right side shows the new process implemented in late 2022.
Original Process for Anaerobic Cultures
On receipt at the UABHCM, the specimens were plated onto 3 media types: Centers for Disease Control and Prevention agar (Remel, Lenexa, KS), bile esculin/laked kanamycin vancomycin agar biplate (Remel), and phenylethyl alcohol agar (Remel). Following inoculation, the plates were placed into a large-capacity Anoxomat anaerobic jar system (Advanced instruments, Norwood, MA). When the jars were full or at the end of every 8-hour shift, they were sealed and processed to establish anaerobic conditions. Inoculated plates remained in the sealed jars for 2 days before being opened for routine culture workup. At that time, the plates were examined for growth. After the initial 48-hour culture read, samples that required additional incubation for organism recovery, isolation, or identification were transferred and batched into a large-capacity anaerobic jars according to laboratory bench assignments and processed following standard protocol. Cultured organisms were subsequently tested for aerotolerance and identified according to institutional laboratory protocols.
New Process for Anaerobic Cultures
The new anaerobic culture process used the same specimen-plating protocol as the original process; however, following inoculation, the agar plates were immediately placed into individual anaerobic bags with disposable anaerobic-generating catalyst sachets (Gaspak, Beckton Dickinson, Sandy, UT) to create and maintain the anaerobic conditions (Figure 1). The plates were subsequently incubated in these anaerobic bags for 2 days, after which, they were removed from anaerobic conditions and worked up following standard laboratory processes. Notably, no changes were made to the subsequent workup of cultures. After the initial 48-hour culture read, samples that required additional incubation for organism recovery, isolation, or identification were transferred and batched into the large-capacity anaerobic jars and processed following standard protocol. This return to established batched incubation ensured that observed differences were attributed solely to the initial sample incubation process.
Statistical Analysis
Power analysis was performed using ClinCalc online sample size calculator with α = .05 and β = .80 that determined that a minimum of 1500 cultures each would be needed for the pre-and postimplementation periods.22 A total of 4016 cultures were analyzed as part of the 6-month evaluation period. Statistical comparison of culture results was done using GraphPad Prism 10 (Dotmatics, Boston, MA). Categorical comparisons among study periods were performed by Fisher exact t-test or Mann–Whitney U test based on data normality. Statistical significance defined as P values of .05 or less. Control charts to assess variability were created using the QI Macros extension (KnowWare International Inc, Denver, CO) to Microsoft Excel.
RESULTS
Old vs New Process
The total number of positive cultures were compared from samples collected from January to March of 2022 (pre-QI; 2320 total cultures) with those collected from January to March of 2023 (post-QI; 1696 total cultures; Figure 2). A significant increase in the percentage of cultures, which were positive for any bacterial growth in 2023 compared with 2022 (11.38% vs 14.92%; P = .0010), was observed, indicating improved recovery of all organisms in these cultures following QI implementation (Figure 2). Notably, further analyses of these cultures (Table 1) demonstrated higher mean values in 2023, with a 60.87% mean difference in recovery of obligate anaerobes between 2022 and 2023 suggesting improved anaerobic organism recovery following process changes. This increased organism recovery in post-QI was observed in cultures of specimens collected from sterile as well as those from nonsterile sites, further supporting the indication of improved total organism recovery with processing method changes independent of sample type. Tables 1 and 2 demonstrate an overall increase in the recovery of obligate anaerobes from 2022 to 2023. The large SD indicates increased variability of results across specimens. This variability is consistent with the heterogeneous nature of clinical samples, which can differ in microbial load, specimen source, and handling. Collectively, these findings suggest that although the intervention enhanced overall recovery, it also captured increased variability in the total number of organisms recovered from each culture.
Improvement in percent positivity. The bar chart shows the percent positivity of total cultures in 2022 and 2023. Using a Fisher exact text, the percentage of positive cultures in 2023 was significantly higher than the percentage of positive cultures in 2022. ***P = .0010.
Difference in organism recovery among first cultures from patients
Improvement in Total and Obligate Anaerobe Recovery
To evaluate which organisms were driving the observed total increased number of organisms recovered post-QI, comparative analyses were performed on positive cultures from the preimplementation (2022) and postimplementation (2023) periods using the first culture ordered per site per patient to minimize sampling bias (Table 2). Despite fewer total cultures ordered in 2023, total organism recovery was improved significantly with a mean percentage difference of 14.81% (P = .0009) compared with 2022. Among positive cultures, this improved recovery in 2023 was attributed to a 22.22% increase in obligate anaerobes (P = .004; Table 2; Figure 3). Notably, although a 13.33% increase in recovery of facultative anaerobes was observed post-QI compared with pre-QI, the difference in mean recovery was not significant (P > .05). Likewise, and despite the observed 6.45% decrease in recovery of aerobes in post-QI compared with pre-QI, the difference in mean recovery of aerobes between these periods was found not to be significant (P > .05).
Comparison of facultative and obligate anaerobe percent recovery. Obligate anaerobes (gray bar) and facultative anaerobes (black bar) percentage recovery in 2022 and 2023 study periods are shown. (A) First cultures analysis. (B) Repeat cultures analysis. The repeat cultures analysis is consistent with the results of the first cultures analysis, showing an increase in anerobic organism recovery in 2023, more significantly in the obligate anaerobic organism recovery.
Reproducibility of Repeat Cultures
To assess the consistency and reproducibility of organism recovery following the workflow modification, repeat cultures ordered per patient were analyzed. Repeat cultures were defined as any cultures collected after the first culture within the study period from congruous sample sources. A total of 2192 repeat cultures were included in a separate analysis comprising 1235 cultures collected over a 3-month period in 2022 and 957 cultures collected over a comparable 3-month period in 2023. Although the findings were not statistically significant (P > .05), the percent recovery of obligate and facultative anaerobes among all repeat cultures increased from 9.60% and 3.50% in 2022 to 17.60% and 4.70% in 2023, respectively. This trend is consistent with the observed improvements in anaerobic organism recovery in first cultures following implementation of the revised laboratory process (Figure 3A and 3B).
Control Charts
To further evaluate the influence of the process, change on the overall organism recovery and variability over time, total positive cultures, and obligate anaerobes recovered counts were plotted against time, in 7-day increments, using X (process average) and R control charts (process variability; Figure 4). For overall organism recovery (X chart), assessed by total positive cultures (Figure 4A), the control limit (CL) for 2022 was determined to be 25.4 (upper CL [UCL] 50.9/lower CL [LCL] 0.1), which increased to 36.6 in 2023 (UCL 92.5/LCL 19.3). By further evaluation of the variability in recovery (R chart; Figure 4B), an increase in organism variability was also observed in 2023 compared with 2022 (CL 21.0 vs CL 9.5). Comparison of obligate anaerobe recovery demonstrated an increase in both total recovery (X chart) and process variability (R chart) from 2022 to 2023, with recovery (Figure 4C) CL2022 18.8 (UCL 45.2/LCL 7.5) to CL2023 of 29.1 (UCL 81.4/LCL 23.2) and variability (Figure 4D) CL2022 9.8 (UCL 32.4/LCL < 0) to CL2023 of 19.7 (UCL 64.3/LCL< 0). These upward shifts in the X chart and R chart CLs signify that the 2023 period had increased average organism recovery as well as increased variability in organism recovery as the result of the process change.
Control charts indicating a higher average and variance owing to process change. Data were plotted for a 7-day timeframe of the datasets used during our study periods. The data indicate an increase in (A) the average of the total number of positive cultures, (B) variance in total positive cultures, (C) average strict anaerobes recovered, and (D) variance of the total strict organisms recovered. All graphs show an increase in the CL from 2022 to 2023 and a widening of the range in the UCL and LCL between 2022 and 2023 data. CL, control limit; LCL, lower CL; UCL, upper CL.
Anaerobic Organisms’ Diversity
Further analysis of the organisms recovered in cultures pre- and post-QI revealed expanded diversity in the postimplementation period. A broader diversity of anaerobic organisms was observed that vary widely in taxonomy, metabolic capacity, growth kinetics, and oxygen tolerance (Figure 5; Supplemental Table 1). Figure 5 shows a heatmap with visible intensification among many obligate anaerobes in the postimplementation window, supporting that the process changes enhanced recovery of obligate anaerobes while suppressing incidental aerobic organisms from anaerobic cultures. Compared with 2022, in 2023, there was an increase in recovery of Actinotignum spp, Anaerococcus spp, Bacteroides spp, Bilophila spp, Clostridium spp, Finegoldia spp, Fusobacterium spp, Paeniclostridium spp, Parabacteroides spp, Parvimonas spp, Peptoniphilus spp, Peptostreptococcus spp, Prevotella spp, and Veillonella spp. There was also a reduction in aerobic organism recovery including fungal organisms isolated from anaerobic culture. Isolates that could not be identified to the species level by MALDI-TOF were reported only to the genus level based on morphology and biochemical results.8,9,23,24
Heatmap showing a number of organisms identified in the study period with improved recovery in obligate anaerobes, especially in the Clostridium spp, Bacteroides spp, Prevotella spp, Fusobacterium spp, and Bilophila spp. Red indicates a highly recovered organism, whereas purple indicates a lower, not recovered organism.
DISCUSSION
There are multiple commercially available systems to support anaerobic cultivation in the clinical microbiology laboratory, including pouches, jars, boxes, and anaerobic chambers. Greater variability is expected in studies involving clinical microbiology specimens because of the inherent heterogeneity of sample sources and handling conditions. Clinical specimens originate from diverse anatomic sites and are frequently polymicrobial, with variable organism burden and composition, all of which contribute to inconsistent recovery rates and increased dispersion of results.12,25⇓⇓-28 Regardless of the platform, the viability of anaerobic organisms recovered is highly dependent on minimizing oxygen exposure during specimen collection and handling and while evaluating cultures for growth and organism identification. Because anaerobes exhibit variable oxygen tolerance, even transient exposure can impair recovery; this is particularly true for obligate anaerobes. Studies have linked lower microbial recovery/organism yield, longer detection times, and reduced overall diagnostic effectiveness to delays in the processing and/or incubation of specimens in the laboratory. Timely establishment of anaerobic conditions is therefore a critical determinant of culture sensitivity and diagnostic yield.8,12,15,21,29⇓⇓⇓-33
An additional benefit of optimized anaerobic culture techniques is the suppression of aerobic organism growth in anaerobic cultures resulting in improved analytical specificity of the cultures. Rapid transition to anaerobic conditions suppresses aerobic overgrowth within the anaerobic cultures, facilitating a clearer pathogen recovery and culture interpretation. Aerobic organisms, when clinically relevant and if present, are typically recovered in parallel aerobic cultures, reducing redundant workups and supporting more efficient laboratory workflows.34 These operational advantages align with the broader goal of delivering accurate and clinically actionable microbiologic data within meaningful turnaround times.
The findings demonstrate that reducing the interval between specimen receipt and anaerobic incubation is associated with significantly improved recovery of anaerobic organisms and that this can be achieved in a cost-effective system with minimal changes to workflow. The marked increase in obligate anaerobe recovery is consistent with established susceptibility to oxygen exposure and supports the premise that preanalytical timing is a modifiable determinant of culture performance. Improved recovery of anaerobic pathogens has a direct clinical implication in that improved recovery of anaerobic pathogens strengthens diagnostic confidence and may inform more precise antimicrobial selection, including appropriate anaerobic coverage. In addition, higher culture sensitivity may reduce reliance on adjunct molecular testing in cases in which false-negative cultures could otherwise obscure clinically relevant pathogens.
This intervention was designed to address preanalytical workflow rather than incubation duration, reflecting evidence that most clinically significant anaerobes demonstrate detectable growth within the standard 48-hour incubation window when optimal environmental conditions are achieved.35,36 Following implementation, increased recovery of both facultative and obligate anaerobes and a reduction in aerobic isolates recovered from anaerobic cultures was observed. Although the increase in facultative anaerobes did not reach statistical significance, their metabolic flexibility provides a mechanistic rationale for more consistent growth that was observed across both processes.37 The concurrent reduction in aerobes supports improved environmental selectivity, reinforcing the effectiveness of expedited anaerobic incubation.
Collectively, these findings highlight preanalytical handling as a critical, yet often underrecognized, contributor to anaerobic culture performance. Importantly, the observed improvements were achieved through workflow optimization rather than additional instrumentation or resource intensification, underscoring the feasibility of implementation in routine clinical settings.
LIMITATIONS
This QI initiative was conducted at a single center, and laboratory-specific workflow characteristics may limit generalizability. The retrospective comparative design introduces the possibility of misclassification bias; however, adherence to standardized laboratory protocols in the clinical setting and direct extraction of grouped culture data from the laboratory information system mitigated this risk. In addition, there was not a side-by-side comparison study performed on each sample to show that there was improved recovery within each sample using this new process. Although these data would be beneficial, it was not feasible at UABHCM at the time of this initiative. A formal cost or resource use analysis was not performed, which may be relevant for institutions evaluating anaerobic culture workflow modification such as the one described in this study. Future multicenter evaluations incorporating operation and economic metrics would further clarify scalability and impact.
CONCLUSION
Using multiple incubation methods can optimize preanalytical workflow, minimizing the time to anaerobic incubation. In our quality initiative, this process change resulted in significantly improved recovery of both obligate and facultative anaerobic organisms. These findings reinforce the central role of timely environmental controls critical to preserving organism viability and maximizing diagnostic yield. Implementation of streamlined anaerobic processing represents a practical, scalable strategy to enhance anaerobic culture performance without significant changes in workflow, additional capital equipment, and minimizing procedural complexity. Adoption of similar workflow strategies may improve the recovery of anaerobic organisms from clinical specimens in culture that informs antimicrobial decision-making and reinforce the microbiologic basis of clinical care.
- Received May 13, 2026.
- Accepted May 19, 2026.
American Society for Clinical Laboratory Science
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Keywords
- CL - control limit
- LCL - lower CL
- MALDI-TOF - matrix-assisted laser desorption ionization time-of-flight
- QI - quality improvement
- UABHCM - University of Alabama at Birmingham Hospital Clinical Microbiology Laboratory
- UCL - upper CL
- anaerobiosis
- culture techniques
- specimen handling
- workflow
- quality improvement
- clinical microbiology