Like high-functioning food manufacturing facilities, high-functioning food microbiology laboratories should conduct a number of validation and verification activities to demonstrate their processes are under control. Similar to how a food manufacturer must develop and validate its FSMA-required Food Safety Plan, food microbiology laboratories also should conduct a hazard analysis, develop preventive controls for hazards reasonably likely to occur, use monitoring to validate preventive controls and for routine verification activities, develop corrective actions when out-of-spec results are obtained, and keep records for all activities. (In this case, a micro lab “hazard” is the risk of cross-contaminating client samples with out of control microorganisms in the laboratory environment.)
To ensure that laboratory test results are accurate, consider the following:
- Does company management have a conflict of interest in testing programs, and are there protocols in place to mitigate such conflicts?
- Are laboratory employees trained in ethical behavior regarding proper sample collection, testing, and reporting?
- Are there written non-conformance policies?
- Are there undue influences that impact test data integrity?
- Are the methods used fit for their purpose?
Well-performing microbiology laboratories are accredited against ISO17025 standards. The standard includes a focus on yearly employee training, a well-documented laboratory quality system, reason, length, and documentation of planned departures, and regular audits. In addition, USDA provides a laboratory guidance document.
Employees must know the purpose of policies and procedures, the principles of procedures, how to do calculations, understand QC practices, how to keep records, how to correlate test results, and how to keep training documentation. Environmental monitoring can provide useful data points to help validate procedures and routinely verify compliance.
Fit for Purpose
The laboratory physical plant must be fit for purpose. Adequately maintained pest control, lighting, walls, ceilings, and floors are needed. Ensure hot water handwash stations are provided and impervious benchtops for sanitation are used. Air HEPA filtration and positive pressure help prevent laboratory cross-contamination. Regularly conduct air contaminant monitoring and zone environmental pathogen monitoring. Address glove change frequency and adequacy of use. If the laboratory is doing pathogen testing, is it Biosafety Level II compliant?
Individuals trained in proper laboratory cleaning should do laboratory housekeeping on a regular schedule. Care should be taken if these individuals also clean the food facility due to cross-contamination risk. Follow proper standard sanitary operating procedures, with regular environmental monitoring verification conducted for pathogens. Cleaners and sanitizers should be fit for purpose. Check for the presence of cleaning residue on all glassware before use. Keep records of all activities.
All laboratory equipment must also be fit for purpose. Properly maintain such equipment on a regular preventive maintenance schedule and calibrate on a routine appropriate for the equipment. Difficult-to-sanitize equipment should have sanitation standard operating procedures detailing such routine. Usual suspects include pipetors, stomachers, auto preps, balances, and glass/plastic ware. Environmental monitoring of this equipment can verify its sanitary condition before use.
Media performance should be routinely checked. Perform productivity, selectivity, and sterility tests on each medium batch. Statistical process control chart use is advised for quantitative testing. The media preparation autoclave needs to be of suitable size to sterilize media batch sizes in use. In addition, do not use media prep autoclave to decontaminate spent media and contaminated materials. Rather, a separate decontamination room and separate autoclave is preferred.
Media fill volumes should be validated and routinely monitored, including petri plates, dilution blanks, MPN tubes, and slant volume and butt height. Likewise, routinely measure water quality and final media pH. If media sterility is questioned, there may be potential for environmental contamination. Monitoring for this potential will allow for quicker resolution of sterility issues if they occur.
There are many questions that need to be addressed in regard to sample collection. Have employees been trained on aseptic technique? Samples should be held at the appropriate temperature in impervious sample containers. Upon receipt, are samples and tests adequately described, and are the chosen tests fit for purpose? Are samples fit for analysis—what is sample integrity upon receipt and are they held under adequate storage conditions? What is the potential for sample cross-contamination during handling, and is this potential routinely measured through environmental monitoring?
Is the laboratory accredited to ISO 17025 standards to perform tests? Can lab personnel describe and perform methods? Is lab capable of performing sample preparation, such as thawing, composite pooling, and experience with difficult matrices (e.g., large samples, complex samples, antimicrobial ingredients)? How is sample uniformity ensured (e.g., mixing by blending, stomaching, by hand)? Is the laboratory using appropriately validated methods (e.g., AOAC, FDA, FSIS, AFNOR, MicroVal), and are they validated for the matrices of interest? Can the lab fully justify and validate using non-standard methods?
Laboratories must have quality control procedures for monitoring the validity of tests. The resulting data must be recorded in such a way that trends are detectable and, where practicable, statistical techniques must be applied to the reviewing of the result. Good laboratory procedures include the use of quality control samples with each sample batch to demonstrate the test worked properly.
A daily process control system, or the use of a non-pathogenic microorganism sample of a known quantified amount, must be plated on a daily basis for those assays being performed. Positive controls are those that include the target microbe to see if the method is working that day or if there are interfering matrix substances. Negative controls use non-target microbes to assess for method discrimination, while sterility controls use blank samples to ensure that media and materials are sterile.
If you are a food manufacturer, is it wise to do pathogen testing in house? Doing so may be a significant biosecurity risk. Without positive controls, a lab is doing faith-based microbiology.
Separate personnel and limit culture access to prevent cross-contamination. Implement policies that govern glove and lab coat use. Understand the risk associated with the lab location in relation to food production. Care must be taken in staging positive control samples in relation to other samples to avoid cross-contamination.
Running a lab requires constant use of measurements that utilize instruments traced to national or international standards. For example, temperature hold precision adequacy is extremely important when doing coliform/E. coli testing at 44.5 or 45.5 degrees Celsius. Sample and media pH values are routinely measured, with precise adjustments sometimes necessary. Pipet and pipetor fill volumes should be periodically calibrated. Balance calibration is necessary to ensure proper sample and consumable weights. Keep records and validation of correction factors.
Integrity of analytical result data should be maintained, and laboratory information management systems secured through password protection. For physical records, labs need a hand error correction policy. To protect against unauthorized access, back up data and log off unattended computer terminals. Scrutinize results for correlation with other results and analyze all lab QC before result release.
Math errors are a common problem. Quantitative microbiology is difficult! Dilution problems are challenging! Counting rules are insanely complicated! Just as self-taught brain surgery is discouraged, don’t do the math yourself: Hire a trained microbiologist, and use a sophisticated laboratory information management system to do calculations. Regularly check performance by subscribing to a check sample proficiency program. Use prepared culture pellets to make spiked controls.
A records retention program should include how to identify, collect, record, index, file, and access records. Additional information on record storage, maintenance, and destruction must be available.
In a world that’s far from perfect, laboratories will make mistakes, or a client will challenge results. Lab errors can happen, so a full discussion of the occurrence with the client is best.
Such complaints should have a formal recording structure that details who is responsible for dealing with complaint or error. The document should detail how to conduct a root cause investigation, identify the cause of the failure, detail corrective and preventive actions, and validate effectiveness. If the issue is serious or a common reoccurrence, conduct a reassessment of lab standard operating procedures along with routine verification.
If a client insists on retesting, a subsequent negative pathogen test result does not negate a previous positive. Because samples may not be uniformly homogenous and the analyte in question may not be uniformly distributed in a lot, an out-of-spec result is not always associated with laboratory error. Clear articulation is needed to justify retesting.
Because environmental monitoring is an effective assessment tool to determine if the laboratory environment is fit for purpose, testing for pathogens and amplicon in the pathogen handling portion of the lab will provide data points showing how the risk of cross-contamination is being managed. Eurofins advocates using a zone approach to laboratory EMP.
For example, lower-risk areas in the lab include media preparation and materials supply storage. The sample reception area can be greater risk if samples are high count or have a history of pathogen detections, such as sponges, swabs, or raw meats and poultry. In such cases, designated sample receiving and sample preparation areas should be used and considered higher risk.
Because the goal of a food micro lab is to cultivate large numbers of microbes (indicators, spoilers, or pathogens), downstream areas where such high-count materials are handled should be considered high risk. For example, plate counting, enrichment transfers, pathogen detections, positive control handling, and waste disposal are considered greatest risk. Indicator (aerobic plate count, coliform count, E. coli count, Enterobacteriaceae count, yeast and mold count) environmental sampling may show surfaces in the laboratory that have been poorly cleaned and sanitized. Direct swabbing of employee hands, gloves, and lab coats can inform personal hygienic practices and conformance.
The value of laboratory EMP testing is most realized when clients blame the laboratory for out-of-spec results, including elevated indicator counts or positive pathogen detections. One major way to rebut such claims is to rely on routine laboratory quality control data points. Such data can help argue that laboratory cross-contamination is not the mostly likely cause of the out-of-spec result.
Dr. Marshall is chief scientific officer at Eurofins Microbiology Laboratories, Inc. Reach him at 970-217-6854.