Name: Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies
Uploaded: 2023-03-17T12:20:00
Description: Watch this Scientific Journal Video about Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies at JoVE.com

This work was supported by the Intramural Research Program of the National Institutes of Health Clinical Center. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

1. Aseptic practices
<ol>
	<li>Personnel gowning for a cleanroom space 
	NOTE: This procedure is based on initial gowning in an unclassified space, followed by entry into an International Organization for Standardization (ISO) 8 area and then an ISO7 area. This procedure is relevant for laboratories attempting to convert existing space into a cleanroom function. Ideally, all initial gowning would occur in an ISO8 classified space (not in an unclassified space).
	<ol>
		<li>Secure loose hair. Wash ...

Microbial Control and Monitoring Strategies for Cleanroom Environments

<ol>
	<li>Cundell, T., Atkins, J. W., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterility+testing+for+hematopoietic+stem+cells.">Sterility testing for hematopoietic stem cells.</a> Journal of Clinical Microbiology. , (2023).</li><li>United States Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+for+Industry.+Sterile+Drug+Products+Produced+by+Aseptic+Processing+-+Current+Good+Manufacturing+Practice.">Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing - Current Good Manufacturing Practice.</a> United States Food and Drug Administration. , (2004).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;71&gt;+Sterility+Tests+in+USP43-NF38.">USP&lt;71&gt; Sterility Tests in USP43-NF38.</a> United States Pharmacopeia - National Formulary. , (2022).</li><li>England, M. R., Stock, F., Gebo, J. E. T., Frank, K. M., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+evaluation+of+compendial+USP&lt;71&gt;,+BacT/Alert+Dual-T,+and+Bactec+FX+for+detection+of+product+sterility+testing+contaminants.">Comprehensive evaluation of compendial USP&lt;71&gt;, BacT/Alert Dual-T, and Bactec FX for detection of product sterility testing contaminants.</a> Journal of Clinical Microbiology. 57 (2), 01548 (2019).</li><li>Gebo, J. E. T., East, A. D., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+side-by-side+comparison+of+clinical+versus+current+good+manufacturing+practices+(cGMP)+microbiology+laboratory+requirements+for+sterility+testing+of+cellular+and+gene+therapy+products.">A side-by-side comparison of clinical versus current good manufacturing practices (cGMP) microbiology laboratory requirements for sterility testing of cellular and gene therapy products.</a> Clinical Microbiology Newsletter. 43 (21), 181-191 (2021).</li><li>Gebo, J. E. T., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterility+testing+for+cellular+therapies:+What+is+the+role+of+the+clinical+microbiology+laboratory.">Sterility testing for cellular therapies: What is the role of the clinical microbiology laboratory.</a> Journal of Clinical Microbiology. 58 (7), 01492 (2020).</li><li>International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISO+14644-1:2015+Cleanrooms+and+associated+controlled+environments+-+Part+1:+Classification+of+air+cleanliness+by+particle+concentration.">ISO 14644-1:2015 Cleanrooms and associated controlled environments - Part 1: Classification of air cleanliness by particle concentration.</a> International Organization for Standardization. , (2015).</li><li>Parenteral Drug Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDA+Technical+Report+No.+13+Revised+2022+(TR+13)+Fundamentals+of+an+Environmental+Monitoring+Program.">PDA Technical Report No. 13 Revised 2022 (TR 13) Fundamentals of an Environmental Monitoring Program.</a> Parenteral Drug Association, Inc. , (2022).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;1116&gt;+Microbiological+Evaluation+of+Cleanrooms+and+Other+Controlled+Environments+in+USP43-NF38+2S.">USP&lt;1116&gt; Microbiological Evaluation of Cleanrooms and Other Controlled Environments in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li><li>Guinet, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+study+on+incubation+conditions+for+environmental+monitoring+and+aseptic+process+simulation.">Multicenter study on incubation conditions for environmental monitoring and aseptic process simulation.</a> PDA journal of pharmaceutical science and technology. 71 (1), 43-49 (2017).</li><li>Gordon, O., Berchtold, M., Staerk, A., Roesti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+incubation+conditions+for+microbiological+environmental+monitoring.">Comparison of different incubation conditions for microbiological environmental monitoring.</a> PDA Journal of Pharmaceutical Science and Technology. 68 (5), 394-406 (2014).</li><li>Anders, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+qualification+of+an+automated+incubator+and+colony+counter+for+environmental+and+bioburden+applications+in+pharmaceutical+microbiology.">Multisite qualification of an automated incubator and colony counter for environmental and bioburden applications in pharmaceutical microbiology.</a> PDA Journal of Pharmaceutical Science and Technology. , (2022).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;1072&gt;+Disinfectants+and+Antiseptics.">USP&lt;1072&gt; Disinfectants and Antiseptics.</a> United States Pharmacopeia - National Formulary. , (2020).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;1071&gt;+Rapid+Microbial+Tests+for+Release+of+Sterile+Short-Life+Products:+A+Risk-Based+Approach+in+USP43-NF38+2S.">USP&lt;1071&gt; Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk-Based Approach in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PUSP&lt;1223&gt;+Validation+of+Alternative+Microbiological+Methods+in+USP43-NF38+2S.">PUSP&lt;1223&gt; Validation of Alternative Microbiological Methods in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li><li>Parenteral Drug Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDA+Technical+Report+No.+33,+Revised+2013+(TR+33)+Evaluation,+Validation+and+Implementation+of+Alternative+and+Rapid+Microbiological+Methods.">PDA Technical Report No. 33, Revised 2013 (TR 33) Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods.</a> Parenteral Drug Association, Inc. , (2013).</li><li>Putnam, N. E., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+study+identifies+a+sensitive,+low-risk,+closed-system+model+for+detection+of+fungal+contaminants+in+cell+and+gene+therapy+products.">Comprehensive study identifies a sensitive, low-risk, closed-system model for detection of fungal contaminants in cell and gene therapy products.</a> Journal of Clinical Microbiology. 59 (11), 0135721 (2021).</li><li>Pharmaceutical Inspection Convention &amp; Pharmaceutical Inspection Co-operation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+Good+Manufacturing+Practice+for+Medicinal+Products+Annexes+(PE++009-16+(Annexes)).">Guide to Good Manufacturing Practice for Medicinal Products Annexes (PE 009-16 (Annexes)).</a> Pharmaceutical Inspection Convention &amp; Pharmaceutical Inspection Co-operation Scheme. , (2022).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;825&gt;+Radiopharmaceuticals+-+Preparation,+Compounding,+Dispensing,+and+Repackaging+in+USP43-NF38+2S.">USP&lt;825&gt; Radiopharmaceuticals - Preparation, Compounding, Dispensing, and Repackaging in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li></ol>

There are several critical areas in this protocol, including the maintenance of aseptic technique and unidirectional airflow within cleanrooms and the BSCs. Best practices include moving slowly and deliberately to minimize turbulence. Aseptic manipulations should be performed from the side of the product, not from above. Closed system processing and the use of terminally sterilized raw materials are recommended. Speaking in critical areas and leaning against walls or equipment should be avoided. Similarly, unnecessary to...

Implementing a strong microbial monitoring program through environmental monitoring (EM), process monitoring, and product sterility testing is a regulatory requirement for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) in cellular therapy laboratories1. Additionally, the United States Food and Drug Administration (FDA) expects that the laboratory performing the quality control (QC) testing of the product should also employ facilities and controls comparable to those used for aseptic filling operations2. 
This protocol has four main sections: 1) Aseptic practices, including personnel gowning, cleaning, and staging of materials; 2) EM, including viable air and surface cultures and non-viable particle air monitoring; 3) process monitoring, including settling plates and gloved fingertip sampling; and 4) product sterility testing via the compendial United States Pharmacopeia (USP) &lt;71&gt; method3 or the NIH Alternative Sterility Testing Method4. When used together, these measures can be an effective method for ensuring that a facility remains in a state of control.
The techniques described here are not novel; however, current standards from regulators and professional organizations lack detail, which has led to an absence of microbial monitoring or the implementation of non-standardized practices, particularly in academic centers where on-site manufacturing and product sterility testing are emerging at a rapid rate1,5,6. This protocol can be used as a guide to create a microbial monitoring and control program that meets regulatory requirements when used in conjunction with end-user validation and risk assessments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20-25&#176;C Incubator</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>30-35&#176;C Incubator</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Alcohol-based hand sanitizer</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>BacT/ALERT Dual-T instrument</td>
			<td>BioMerieux Industry</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beard cover</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Biosafety cabinet (BSC)</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Cleanroom shoes</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Fluidthioglycollate medium (FTM)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>U84</td>
			<td>USP</td>
		</tr>
		<tr>
			<td>Handheld cleaning mop</td>
			<td>Contec</td>
			<td>2665LF</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>iFA+ BacT/ALERT bottle</td>
			<td>Biomerieux</td>
			<td>412990</td>
			<td></td>
		</tr>
		<tr>
			<td>iFN+ BacT/ALERT bottle</td>
			<td>Biomerieux</td>
			<td>412991</td>
			<td></td>
		</tr>
		<tr>
			<td>Isokinetic head</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Laser particle counter</td>
			<td>TSI Incorporated</td>
			<td>9500-01</td>
			<td></td>
		</tr>
		<tr>
			<td>LpH III</td>
			<td>Steris</td>
			<td>1S16CX</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Non-sterile bouffant</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Non-sterile gloves</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Non-sterile shoe covers</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Non-sterile sleeve covers</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Peridox RTU</td>
			<td>Contec</td>
			<td>CR85335IR</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic bag</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Sabouraud Dextrose Agar with Lecithinase and Tween (SABLT)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>P595</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Sabouraurd Dextrose Agar (SAB)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>W565</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Safety glasses</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Scrubs (top and bottom)</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Spor-Klenx RTU</td>
			<td>Steris</td>
			<td>6525M2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 70% isopropyl alcohol (IPA)</td>
			<td>Decon CiDehol</td>
			<td>8316</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol wipe</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Sterile boot covers</td>
			<td>Kimberly Clark</td>
			<td></td>
			<td>Cat# varies based on size</td>
		</tr>
		<tr>
			<td>Sterile coveralls</td>
			<td>Kimberly Clark</td>
			<td></td>
			<td>Cat# varies based on size</td>
		</tr>
		<tr>
			<td>Sterile face mask</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Sterile hood</td>
			<td>Kimberly Clark</td>
			<td></td>
			<td>Cat# varies based on size</td>
		</tr>
		<tr>
			<td>Sterile low-lint wipes</td>
			<td>Texwipe</td>
			<td>TX3210</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile mop cleaning pads</td>
			<td>Contec</td>
			<td>MEQT0002SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile sleeve covers</td>
			<td>Kimberly Clark</td>
			<td>36077</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile spreading rod</td>
			<td>Fisher Scientific</td>
			<td>14665231</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Tacky mats</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar (TSA)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>W570R</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar with Lecithinase and Tween (TSALT)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>P520R</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth (TSB)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>U46</td>
			<td>USP</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>Vesphene III</td>
			<td>Steris</td>
			<td>1S15CX</td>
			<td></td>
		</tr>
		<tr>
			<td>Viable air sampler</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>BAS22K</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
	</tbody>
</table>

microbial control and monitoring strategies for cleanroom environments and cellular therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP&lt;71&gt;) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies  

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

Clinical microbiology labs are being increasingly asked to assist with sterility testing as cellular therapies emerge as frontline treatment options. This project aims to provide clear instructions for implementing microbial monitoring and testing strategies applicable to any CGMP setting with a particular focus on academic and clinical laboratories. Gold standard microbial testing and CGMP relies on traditional culture-based methods, which are slow.While newer technologies exist, implementation is often limited by the regulatory requirement to conduct rigorous end-user validations to demonstrate non-inferiority. We hope that future work will help establish newer technologies into the mainstream for CGMP testing. There is no standardization between programs in CGMP.While a regulatory framework is provided, microbial monitoring and testing strategies are unique to each facility and rely on a risk-based approach and end-user validation. This can be confusing for establishing new CGMP programs in the clinical setting. Our group aims to bring awareness of CGMP to the clinical setting.We have published large-scale studies highlighting the advantages and pitfalls of blood culture systems for sterility testing as well as review articles demonstrating major differences between the clinical and CGMP microbiology labs and areas for consideration if implementing sterility testing in the clinical setting.

The expected results are described in Table 1. The EM data should be reviewed and followed up with an appropriate investigation and response to action, alert, or ISO limit excursions. If an excursion occurs for non-viable particles, one should proceed as per ISO 14644-Annex A, sec A.5.57. If the excursion can be attributed to an immediately identifiable abnormal occurrence, the original sampling results should be documented, a note should be added to disregard the original results...

Watch this Scientific Journal Video about Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies at JoVE.com

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical ...

microbial-control-monitoring-strategies-for-cleanroom-environments

Research

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Bioengineering

Gowning for a Cleanroom Space and Donning for a Biological Safety Cabinet

To begin, change into long-sleeved scrub top or short-sleeved scrub top with non-sterile sleeves, full-length scrub pants, and cleanroom shoes with shoe covers on. Sanitize hands with an alcohol-based hand sanitizer, and don non-sterile gloves, a beard cover if applicable, and a non-sterile bouffant. Remove the shoe covers, and step onto the tacky mat in front of the International Organization for Standardization or ISO-8 anteroom door.After discarding the shoe covers, decontaminate the gloves and door handle with 70%sterile isopropyl alcohol before entering the ISO 8 anteroom. Gather a sterile hood, face mask, coveralls, boot covers, and safety glasses. After decontaminating the gloves again, don safety glasses and stage the materials onto a clean surface.Don a new pair of non-sterile shoe covers over the dedicated cleanroom shoes, step onto the tacky mat, and enter the secondary ISO 7 anteroom, remaining on the dirty side of the demarcation line. Don the sterile hood and the sterile coverall, ensuring the neck of the hood is tucked inside the coverall to create a complete seal. Don the secondary sterile boot covers and decontaminate the gloves between each donning step before entering the ISO 7 space.To don into the biological safety cabinet, don sterile sleeves over the coverall, using thumb loops if present. Don sterile gloves over the existing gloves, ensuring the cuff of the glove extends over the sterile sleeve before entering the biological safety cabinet.

Settling Plates, Gloved Fingertip Sampling, and Sterility Testing by Direct Product Inoculation

Place the labeled TSA and SAB settling plates near the work area but in an area that will not interfere with testing. Document the sampling start time. Aseptically, open both plates, placing the lids on a clean surface in the biological safety cabinet away from the work area for up to four hours to prevent the agar from drying out.At the end of the processing close the lids of the plates and document the sampling stop time. Loosely, bag the plates and incubate the cultures. For gloved fingertip sampling, obtain two labeled TSALT plates.Aseptically remove the lid of the plates and place it on a clean work surface away from the sampling area. Gently roll the pad of each finger and thumb on the surface of the plate. Use sufficient force to make slight depressions on the agar without cracking the agar surface.Aseptically place the lid back onto the plate. After sanitizing the hands at the completion of sampling, loosely bag the plates and incubate the cultures. For direct inoculation, following the United States Pharmacopeia method, place the TSA and SAB settling plates in the biosafety cabinet.After preparing the cabinet and staging the materials, document the sampling start time. Obtain the test article and the associated tryptic soy broth and fluid thioglycollate medium bottles. If the bottles have a septum, remove the protective caps from each bottle and wipe the septum with a sterile alcohol wipe.Vortex the test article to ensure a homogenous suspension and an inoculate the bottles with a validated volume of the test article using a sterile syringe and a hypodermic needle. Wipe the septum with a sterile alcohol wipe and insert a sterile venting unit to allow for air exchange during incubation. Repeat the steps for each test article of that session.Aseptically close the TSA and SAB settling plates and document the sampling end time before performing the gloved fingertip sampling. Wipe the gloves with 70%sterile isopropyl alcohol, loosely bag the plates and incubate the cultures. Transfer all the test materials out of the biological safety cabinet.Similarly, for direct inoculation, following the NIH alternative sterility testing method, place the TSA and SAB settling plates in the biosafety cabinet and document the sampling start time. Obtain the test article and the associated IFA Plus labeled, IFN Plus labeled and SAB plate. Remove the protective caps from the IFA Plus and IFN Plus bottles and wipe the septum with a sterile alcohol wipe.After vortexing the test article, inoculate the bottles with validated volume of the test article using a sterile syringe and a hypodermic needle and wipe the septum again. Incubate the bottles on the BACT/ALERT Dual T instrument. Using a pipetter, inoculate the SAB plate with a validated amount of the test article and streak for isolation using a sterile loop.Let stand for 10 minutes to ensure the sample does not run onto the plate lid when inverted for incubation Place each SAB plate inoculated with a test article into a plastic bag. Tie them loosely. Aseptically close the TSA and SAB settling plates.Document the sampling end time on the plates, and perform the gloved fingertip sampling. After sampling, wipe the gloves with 70%alcohol. Loosely bag the plates after wiping the gloves with alcohol and incubate the cultures.A TSA air settling plate illustrating a single colony of a contaminant cultured during passive air process monitoring in the biological safety cabinet is shown here. The TSALT surface sampling plate with three colony forming units of two distinct colony morphologies is shown in this figure. The TSALT surface culture with a single colony on the plate's edge indicates poor aseptic handling during the sampling process.The correct method for obtaining gloved fingertip samples using the largest surface area of each finger or thumb is shown in this image. The incorrect process where only the fingertip is sampled is shown here. An example of mold balls visible to the naked eye that failed to be automatically detected by the BACT/ALERT system is shown here.Based on these findings, terminal visual inspection of all BACT/ALERT bottles and the addition of the SAB plate for fungal culturing using the NIH alternative sterility testing method is recommended.