Microbiology Topics.Scott Sutton

Li -ting Avo·dab eicro io og al Var"ab"li y

Scott Sutton

"Microbiology Topics" discusses various topics mmicrobiology of practical use in validation and compli­ance. We mtend this column to be a useful resourcefor daily work applications.

Reader comments, questions, and suggestions areneeded to help us fulfill our objective for thiS column.Case studies from readers are most welcome. Pleasesend your comments and suggestiol1.5 to olumncoordinator Scott Sutton at scott.sutton@microbioJ.org or journal coordina1ing editor Susan Haigney atsha igney@advanstar.com.

KEY PINTSThe following key points are discussed in this article:

• Quality control (QC) microbiology test data are sub­ject to significant variability, both avoidable andunavoidable

• Good microbiological procedures, backed by soundmicrobiological practices, can serve to minimizeavoidable variability

• The lab's standard operating procedure (SOP) sys­tem is a powerful tool to describe and documentcompliance with good practice

• The lab should determine critical areas of cover­age for the SOP system to ensure a comprehensiveprogram

• The SOP for a lab test should describe critical param­eters of the test and meet the criteria of regulatoryrequirements and guidance for that procedure. The

documentation of compl iance with these require­ments is both a legitimate good manufacturingpractice (GMP) audit concern and a useful sourceof information for investigations.

• A sound SOP system can serve to minimize avoid­able variability in the microbiology lab

• SOPs may be categorized into testing methods, docu­mentation and SOPs, environmental monitoring,and laboratory support activities

• Training for the members of the lab should be tightlytied to the SOP system, and can support functionalspecialization of staff

• SOPs for each functional area are described• The content of this discussion should serve to bench­

mark your system, guide regulatory compliance, andbe a framework for training

• Considering the SOP system from a functionalperspective links job skills to SOPs and facilitatestracking of revisions

• Controlling variability and avoidable error is criti­cal to successful microbiology laboratory operationbecause microbiology is exquisitely sensitive to per­sonnel performance and techniques.

I NTRODUC1IONMicrobiology in the QC laboratory is subject to vari­ability in the test results, in the samples taken, in themanner in which they are taken (with severe limita­tions in sample size contributing to the problem), and

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ABOUTTHEAUTHORScott Sutton, Ph.D. I~ owner an opl:'r.:Itor of nle MluolJIOlog' fJetiJIJOIl.. iwww.m;cloblol 019),

Which provides SerVI[p', to mierobiology-reiilted Iser s yrOllps Dr Su to/1 fan be redched ill SlOtt

suflon@n lIe loblol.or'~.

50 JOURNAL OF VALIDATION TECllNOLOC\, 'WINTER 2010' m c m

Scott Sutton.

Table: Errors inv Ived in plating.

• Training• Sample handling• Lab operations• Testing methodology• Data handling/reporting/archiving• Investigations.

This method does not correlate to the US Code ofFederal Regulations (CFR) organization, the medicaldevice International Organization of StandardizationISO organization, Pharmaceutical [nspection Con­vention and Pharmaceutical Inspection Co-operationScheme (PICjS), United States Pharma.copeia (USP) Chap­ter <1117>, nor the European Union (EU) organization­al scheme. Each of these general schemes is designedto fit a wide variety of processes and operations. Weneed to focus on a system specific to the microbiol­ogy lab as this environment has unique requirements.Aspects of the lab that are critical to its success (i.e.,control of cultures, media, sample handling, etc.) maynot even playa role in other types of work. Even thissystem will not be optimal for all labs. That is okay;you will not get any functional lab organization thatfollows an external structure in lock-step. However,you must be able to correlate your system to one thatmatches the preferred method ofwhomever is auditingyou at that moment. This article does not go into thevarious organizational schemes; however, it is stronglyrecommended that the reader become fluent in at leastthe US 21 CFR 211 (5), US? Chapter <1117> (6), andthe PIC/S (7) audit guide to serve as a basis for thestructure of a microbiology lab (see the reference sec­

tion for complete references).In general, I prefer a slight variation on the opera­

tional organization scheme listed previously. This

the innate variability of a process heavily dependenton human interaction. This variability is an inescap­able aspect both of the science and of the manner inwhich we do the science. For example, let's look at a;'elatively simple and basic operation such as plating.Jarvis (1) detailed a variety of errors (errors in the sta­tistical sense ofvariabil ity) involved in t his operation(see Table).

The errors in this example might be divided intotwo main types-some that might be considered anavoidable error (e.g., plating error, calculation error)and an unavoidable error (e.g., sampling error, dilu­tion error, distribution error). We cannot eliminateeither type of error in the lab, but the genera I categoryof "unavoidable errors" is not amenable to correctionby training or proper Jab technique. In fac!, some ofthe current practices of the lab, adopted for businesspurposes, may actually increase the effects of this typeof variability. The following are among these unavoid­able errors:

• Insufficient sample numbers• Insufficient number of replicate plates (2, 3, 4)• Difficulties in using living systems (which react

to treatment and growth conditions).

Using microbes in validation studies requires a con­slant and dependable test system, which quite franklydoes not exist. So we are left to do the best we can.

Because we are not likely to be able to test largesample sizes, plate large numbers of replicate platesto increase the precision of plate counts, or do muchto minimize the "unavoidable errors" in our lab, weare left with the avoidable errors. Fortunately, these.-an be affected fairly easily by training and solid lablc:adership. This discussion attempts to guide the readerto how to think about controlling the lab environmentso that the results from microbiological studies are'.ess variable.

IHE or, YSTEMThe key to consistent work in the microbiology lab is asolid SOP system with adequate documentation. Thisseems obvious, but the effects of this requirement arenot always so obvious.

You can break the organization of a logical SOPsystem down several ways. One way is operational,as follows:

• Quality requirements

• Media• Cultures• Equipment

Source of error

S mpling error

Dilution error

Plating error

Distribution error

,alculatian rror

Includes errors due to

WeigilingPipette volumes

Diluent volumesPipette volumes

Pipetting errorCulture medium faultsIncubation faults

Non-randomness af CFUCounting errorsRecording errors

Manual calculationsSoftware errors

JOURNAL OF VALIDATION TECllNOLOGY [WINTER 2010 i 51

Microbiology Topics.

scheme has the advantage, in my mind, of being ame­nable to use as a training organizational tool as wellas a framework for SOP organization. In brief, thelab SOPs are broken into four main areas with severalsubsections, as follows:

• Testing methodologies;; Specific test methods" Validation of test methodsc Investigations

• Documentation and SOP structure• Environmental monitoring (EM) and associated

support'" Viable air.. Non-viable air.. Surface sampling.. Personnel monitoring• Med ia fill support• Qualification offacility after shutdown• Gowning (may share with manufacturing)

• Laboratory support activities... Media., Cultures., Equipment, Safety<: Operations.

WHY I HI 5 H M' U l:FUL?This scheme is useful in regards to training. Training is avery difficult area for the QC laboratory. Aside from thequestions surrounding proficiency testing (which willnot be discussed here), there are real logistical issues withdetermining who should be trained on which SOPs andhow to maintain training as SOPs are revised.

The work in a microbiology lab is performed almostentirely by technicians at the bench. This work is com­plex with some studies lasting only hours while otherswill last more than a month. Throughout, the benchtechnician is handling the material and the cultures as anormal part of the job-a job that is notoriously opera­tor-dependent in nature.

Havingsaid this, how does this organizational image ofa microbiology lab SOP system help in training? A newhire will need to be immediately trained in all the SOPsconcerned with documentation, lab hygiene, and labsafety. The next group ofSOPs will depend on their jobfunction. For example, a technician performing sterilitytests will need additional training in the following:

• Test methods~ Test methods<: Relevant equipment (e.g., operation and mainte­

nance IO&M])

52 JOURNAL OF VALIDATION TtClINOLOGY !WI~l"ER 20101

~ Aseptic technique• Media (e.g., quarantine, handling, and expiry)" Biohazard disposal" Recognition of microbial growth

• Validation.. Method<: Preparation of inocula.

This also encourages different functional specialization.For example, there is no need for the technician workingin the media kitchen to be trained in how to perform anantimicrobial efficacy test. Nor is it particularly efficientfor the bench worker to be running back and forth tothe kitchen to check on media. By separating the jobs,the flow ofwork in the lab is simplified. Major supportfunctions (i.e., media preparation and release, stock cul­ture maintenance, and equipment tracking) can each behandled by a suitably trained manager with backup.

1 ESTINEach major type oftest performed will have an associatedSOP. This SOP should list critical pieces of equipment .Training will necessitate familiarity with the O&M SOPfor each critical piece of equipment. The test will alsolist specific organisms to be used (ifappropriate), neces­sitating training in relevant culture SOPs. Finally, eachSOP will list required media, necessitating training inrelease, and expiry requirements for the relevant media(how do you determine which media can be used foryour test?). Finally, the test may require training in thedepartment's SOP on how to count colony forming unit(CFU) on plates, and on the lab's methods ofhandlingbasic math operations (e.g., rounding, significant figures,loglo conversions, etc.).

In addition, each test method SOP should be accompa­nied by an SOP on how to validate the method. This usu­ally consists ofdemonstrating suitable microbial recoveryfrom samples spiked into the sample or into a neutralizingbroth (see USPChapter <1227». Specific tests may haveadditional validation requirements depending on theregion (e.g., sterility tests have additional requirementsby rlc/s over those recommended in USP <1227> andrequired in the harmonized sterility test).

In addition to the validation SOP, it may be prudentto develop an SOP on how to handle failing or question­able results. This SOP can be specifiC to the test methodor a more general one that provides specific instructionfor every test type. This is important, as the company's

out-of-specification (OOS) and corrective and preventa­tive action (CAPA) procedures will almost certainly bedirected at the analytical chemistry group or manu-

facturing group, and be completely inappropriate tothe microbiology lab. Further, an investigation intoa putative sterility test failure will be fundamentallydifferent from that of a putative failure of the anti­microbial effectiveness test. There will, of course, bedements in common, but they are defined by theirdifferences.

A separate category of testing involves microbialidentification. This group should include basic tests(e.g., Gram's stain, spore stains, biochemical reac­tions, and the use of selective/differential media) aswell as more advanced methods such as the O&M ofproprietary identification equipment and their use inmicrobial identification. Since any potential inves­ligation may require accurate identification, some(ontrols on the different stages of identification areadvisable. These include the following:

• Streaking the sample for pure, monoclonal micro­bial culture

• Controls on the Gram stain to ensure accurateresult

• Controls on the identification run to ensureaccuracy.

)0 1I 1E 'TA I • U AND PTRUCTURE

It is frequently useful to have an SOP on what a goodlest method SOP should include. Each data sheet forthe test method should include sufficient informationt determine the culture used (tracing back to the ini­1ial receipt from the national stock culture), all criticalpieces of equipment used, all buffer and media lotsIlsed, time and date of activities, who performed them,and the date all information was reviewed. This is in. ddition to the actual data for the test (i.e., dilution1.1ctor and CFU/plate for plate count methods).

This then bri ngs up the question of proactive docu­mentation. Compendial tests specify the critical testparameters. These might include temperatures ofincubation, time of incubation, handling, proficiencyrequirements, etc. At a minimum, the test documen­tation should clearly indicate that all the specifiedparameters detailed in the test method were met.,his includes documentation that the technician wastrained, the equipment was in repair and calibrated,c' nd all cond itions of the test were met. The readershould understand that although this discussion ofI roactive documentation is occurring in one particu­

lar section, it is a general GMP requirement-youmust always be able to document that the test was[lerformed correctly.

Scott Sutton.

A benefit of this practice is that it wiJl cut down on themost egregious source ofvariabil ity in the microbiologylab-technician creativity. Microorganisms are livingcreatures and respond to stimuli. IfTechnician A handlesthem in one fashion and Technician B in another, itshould not be surprising that recorded test data will beinconsistent. This is not a problem with the microor­ganisms, nor is it a problem with the technician; it is aproblem with the lab leadership.

Consistent execution ofstrong microbiological practic­es in the lab is the key responsibi lity of the lab leadership,and the most obvious measure of its competence. Thisshould be evident through the data documentation andobservation of the lab operation. Thorough and carefullyprepared documentation can also be an extremely usefultool for self-audits. One way to approach this task mightbe to take a particular activity that has been problematicand pull out all relevant documents (e.g., SOP, regula­tory requirements, regulatory guidance, industry grouptechnical reports). With this information, create a chartofcritical parameters for that test as described by the SOPincluding temperatures, incubation times, or any otherspecific instructions. Repeat this exercise for the taskusing each relevant supporting document. Now you areready to answer the following two questions:

• Does your SOP satisfy critical parameters for thattest as described in USP, FDA guidance, or other sup­porting documentation? It is a good idea to writethis up as a white paper in preparation for beingasked this question during an audit.

• Does your documentation capture sufficient detailto provide witness that you performed the test in amanner that met all critical parameters identified?This question is critical for GMP and in preparationfor any potential investigation.

Investigations in the microbiology lab are extremelydifficult, not only due to the detective work involvedbut also because very few labs adequately documentthe work performed. If a problem must be investigateddays or weeks after the event, there likely will be veryIittle actual material to investigate. The most useful toolin an investigation is the available GMP documenta­tion. In my experience, this is the area in which mostlabs are deficient. In these cases, investigations will endwith an inconclusive determination of the root causefor a particular problem. This will occur due to thenature of the discipli ne (see discussion on variabi Iity).

More comprehensive documentation will allow forbetter investigations into events that happened daysor weeks earlier.

JOURNAL Of V,\UDATION TECIINOLOGY iWINTER 20101 53

Microbiology Topics.

ENVIRONMENTAL MONITORING ANDASSOCIATED SUPPOREnvironmental monitoring (EM) activities are oftenseparated from other microbiology testing due totheir complexity. In addition to the obvious issues ofsampling, equipment used for sampling, gowning, andaseptic technique, this area will also be responsiblefor trending of environmental monitoring data, mediafill support, and disinfectant qualification.

Many organizations split off the EM group frommicrobiology altogether. This is, in my opinion, amistake. It clearly is a huge role for a microbiologydepartment. A competent, technically qualified man­ager should be able take care of the range of require­ments. The fragmentation of the EM group from themicrobiology group serves on ly to separate the sa mpleacquisition and data analysis functions from the incu­bation and plate reading/data recording functions.This sets up a situation that encourages avoidance ofresponsibility for unwelcome results. In addition, itlimits the opportunity of the lab head to shift resourc­es in times of greatest need. If the analysts cannotperform EM sampling, they cannot be used if needed.If the EM technicians are not part of microbiology,they cannot help in the testing lab. Splitting the func­tions into two departments requires the company tohire two competent microbiologists with experienceto lead the groups. As this is extremely unlikely tohappen, one group inevitably is weaker than the otherand discrepancies in microbiological techniques creepinto the procedures of the two groups, unfortunatelyleading to conflict or apathy. Finally, the temptationwill be strong for each group to use its own SOPs forcommon tasks.

SOPs unique to this area might include not onlysampling techniques for air, surface and personnel,but also sample handling and transport, incubation,and whatever trending and data handling proceduresare needed. In addition, specific consideration mightbe given to media fill support activities, qualificationof the facility after shutdown, and gowning proce­dures and qualification (these may be shared withmanufacturing).

LABORATORY UPPOR1 ACTIVITIESThis area is probably the most misunderstood area ofthe microbiology lab, especially among senior man­agement. Part of the problem is that laboratory sup­

port activities are financial overhead to the lab. It isa very tempting target when the lab management isinstructed to reduce spending. However, this is prob-

.34 JOURNAL OF VALlDAnON TECIINOLOGY iWINTER 20ID!

ably one of the worst places to tighten the budgetarybelt as all work depends on these functions beingperformed properly.

MediaThe activities that need SOP coverage here include thereceipt and acceptance of incoming dehydrated andprepared media, its quarantine, growth promotionconfirmation (which may require training in culturesand preparation of inocula), and media release foruse. In add ition, the mixing and steril ization of in­house media, establish ment of its ex pi ry dati ng, andlabeling of all media are important. All lab workerswho perform testing that involves microbial growthmedia will require relevant training in how to iden­tify usable media, even if they are not trained in itspreparation.

In add ition to the direct SO Ps on med ia receipt,preparation, and release, there are supporting SOPson relevant equipment O&M procedures. Particularattention must be given to autoclaves (sterilizers), vali­dated sterilization cycles, and load configuration.

CulturesThe integrity of the culture collection is critical tothe QC microbiology lab. This begins with receiptof the culture from the national stock collection andprocedures in place to confirm the identity and purityof the sample. SOPs should be in place to governreceipt, quarantine, quality check, release, and seedlot technique. Many of these functions can be com­bined into the seed lot technique method. See PMFNewsletter 13 for further discussion (8).

In addition to the seed lot technique out to theworking cultures, a specific SOP may need to be inplace for preparation of the inocula for the varioustests (although this might be included in the testmethod SOPs).

It is frequently found to be useful to have two orthree individuals in the lab responsible for mainte­nance of the culture collection. This relieves othersof trying to keep up with the procedures, and allowsthe specialists to trade off responsibilities in a rota­tion schedu Ie.

EquipmentI have found equipment to be sufficiently involvedthat it requires a dedicated worker (and backup) for

the same reasons cited herein for media and cultures.Someone needs to maintain the equipment master files(i.e., vendor qualifications, manuals, certifications,

etc.), track preventative maintenance (PM) schedulesfor critical equipment, review performance logs, andensure autoclave cycle records are maintained.

Each critical piece of equipment should have anO&M SOP that is sufficiently detailed so that testprocedure SOPs will not need to describe how to usethe equipment but can simply reference appropriateO&M SOPs. Obviously, qualification to perform aparticular test would require proficiency in all relevantequipment SOPs.

Finally, many pieces of equipment in the microbi­ology lab have additional requirements beyond thestandard PM scheduled work. Equipment designed to

maintain temperature (e.g., incubators, refrigerators,lnd cold rooms, and water baths) must be monitoredto document compliance. In addition, equipmentthat is used to house "dirty" samples (i.e., incubators,{efrigerators, water baths, etc.) must be cleaned regu­larly to minimize the potential for contamination.The method and frequency of this cleaning shouldbe described by the SOP and documented.

Lab SafetyMany companies have lab safety requirements. Thesemight involve the requirement to maintain avail­able material safety data sheets (MSDS) (i.e., what:n do in case of spi lis, fi res, earthquake, tornado, orother natural disasters). They may also cover acids,bases, flammables, toxins, equipment, etc. In termsof equipment there is a real need to address the useof autoclaves and compressed gasses in the microbi­ology lab.

An additionaL and somewhat unique, require­ment for the microbiology Jab is to have a bio-safetymanual prepared and ready to handle at least risklevel 2 microorganisms. This is not difficult whenbasic good laboratory practices (i.e., no mouth pipet­ting, lab coats, use of containment hoods for opera­tions leading to aerosols, etc.) are performed. It isimportant to formalize these requirements to avoidmisunderstandings.

lab OperationsThis is somewhat a catch-all category ofSOPs. It isn'tlhat the activities are unimportant, but rather thatl ey are so basic to the operation of the lab that allparties are involved.

Control of Incoming Samples and Materials.The lab should have an SOP governing how to logincoming samples for testing, how to track date-on­test, date-off-test, and report date. In addition, the

Scott Sutton.

lab should have a general procedure on acquisitionand acceptance of perishable consumables.

Documentation Concerns. These can includeversion control on data sheets, data entry into labnotebooks and the laboratory information manage­ment system (L1MS), and record retention for differentdocuments. All should be described by an SOP.

Training and Proficiency Requirements. AnSOP should exist for all job functions in the lab. Thisshould describe the job function in a manner thatallows easy categorization of the SOP to meet the jobrequirements. If job responsibilities exist for whichthere is no SOP, an SOP should be written. Thisallows SOP training to be assigned by job functionand allows easy identification of technicians whoneed retraining when an SOP is revised.

A system should be in place to demonstrate thetechnician's proficiency in activities critical to theirjob. This system is, of course, described by an SOP.The system should identify critical skills needed, recer­tification periods, and methods of initial certificationand recertification.

Laboratory Hygiene. SOPs should be in place todescribe the cleaning and sanitization of the labora­tory benches at the beginning and end of each day,the general state of t he lab, and expectations of thelab environmental monitoring program ifrequired.The preparation and expiry dating of sanilizers shouldbe part of this procedure.

The hygiene expectations of the workers should alsobe addressed. Requirements for clean clothes andbod ies, closed-toed shoes, clean lab coats, gloves, andother personal protective equipment (prE) as requiredshou ld be part of the stated expectations as shou Idthe proper use of hand washing equipment.

Biohazardous Waste Disposal. There shouldbe a procedure or procedures for decontaminationand disposal of biohazardous waste.

Plate Count Procedures and Basic Math. Thistype of SOP is designed to standardize common prac­tices in the lab. The plate count SOP seems silly untilyou realize that the CFU/plate recorded by the techni­cian is reallyonly an estimation of the CFU, and thatestimate is immediately interpreted further (9). It isimportant to establish some consistency in this mostbasic function in the lab.

The basic math SOP is also critical. This shouldaddress topics such as rounding, significant figures,

loglo conversions, and the deduction ofCFU/mL fromthe dilution and the CFU/plate. This might also bea good place to define what the lab means when an

JOURNAL Of VAUIJAHON TECHNOLOGY iWINTER 2010 i SS

RE LR N1. Jarvis, B., "Statistical Aspects of tile Microbiological Exami­

nation of Foods," Progress in Indusc. Microbiol. 21, Elsevier

ScientifIC Publishers B.v. Amsterdam, 1989.

2. Eisenhart, C. and r. Wilson, "Statistical Methods and Con­

trol in Bacteriology," Baa Rev. 7:57-137, 1943.

3. Stearman, RL., "Statistical Concepts in Microbiology: Bace

Rev. 19:160-215, 1955.

4. IIstrup, D., "Statistical Methods in Microbiology," Clin Mi­

crobial Rev 3(3)219-226,1990.

5. FDA, "Amendments to the Current Good Manufacturing

Practice Regulations for finished Pharmaceuticals/21 CFR

Pans 210 and 211," Federal Register 73(174):51919 - 51933,

2008.

6. USP, USP Chapter <1117>, "Microbiological Best Labo­

ratory Practices (in-process revision)," Pharm Forum

35(4):945-951,2009.

7. PI CIS. PI 023-2 Aide-Memoire: Inspecrion of Phamlaceutical

Quality Control Laboratories, 2007.

8. Pharmaceutical Microbiology Forum, PMF Newsletter, Vol­

ume 13, #11, MicrobiologyForum.org, November 2007.

9. Pharmaceutical Microbiology Forum, PMF Newsletter, Vol­

ume 12, #9, MicrobiologyForum.org, September 2006.

JVT

icrobiology Topics.

SOP states "S-day incubation period" vs. "l20-hourincubation period."

CON. I NThis article has attempted to describe an SOP systemfor the QC microbiology lab in a regulated industry.This is not presented as the only SOP system possible,or even that it will be sufficient to your particularneeds. It should, however, serve as a starting pointor assist with benchmarking your system. A goodSOP system should serve as guidance to regulatorycompliance and be useful as a framework for training.This article focused on the training aspect rather thantrying to link the SOP system to regulatory require­ments (although it is the author's belief that the twoare by no means exclusionary, only that regulatorycompliance is not the focus of this article).

By looking at the SOP system from a functionalperspective we can easily group media, stock culture,equipment, and documentation requirements to testactivity, making the creation of "job skills" relativelystraightforward. This, in turn, simplifies the assign­ment of SOPs to individuals based on their job func­tions and simplifies tracking of individuals affectedby SOP revisions.

The importance of controlling variability (alsoreferred to as minimizing "avoidable error") in themicrobiology lab cannot be overstated. Microbiol­ogy as a discipline is inherently variable, with a busi­ness culture that results in increasing some aspectsof this variability (usually in an effort to minimizeoverhead and labor costs). In addition, microbiologyis exquisitely sensitive to operator effects. A strongand coherent SOP system coupled with aggressivetraining and enforcement will minimize at least theavoidable variability in data from the lab for testingand validation work.

<i6 JOURNAL OF VALIDATION Tr:C/lNOLOGl' WINTER 2010

ARTICAPA

CFRCFU

EM

EUGMP

ISO

O&M

OOS

PIClS

QCSOP

USP

lE ACRONYM LIS 'INCCorrective Actions and Preventive Actions

Code of Federal Regulations

Colony Forming Unit

Environmental Monitoring

European Union

Good Manufacturing Pr etice

Int rnation I Organization of

Sland rdization

Operation and Maintenance

Out-af-Specification

Pharmaceutical Inspection Convention and

Pharmaceuticallnspeetion Co-operation

Scheme

Quality Control

Standard Operating Procedure

United States Pharmacopeia

wth m