US Environmental Protection Agency Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Monitoring of Laboratories for Airborne Contaminants Standard Operating Procedures (SOPs) Office of Pesticide Programs SOP Number: QC-02-04 Date Revised: 09-09-10 ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 1 of 7 EPA/OPP MICROBIOLOGY LABORATORY ESC, Ft. Meade, MD Standard Operating Procedure for Monitoring of Laboratories for Airborne Contaminants SOP Number: QC-02-04 Date Revised: 09-09-10 Initiated By: Date: / / Print Name: Technical Review: Date: / / Print Name: Technical Staff QA Review: Date: / / Print Name: QA Officer Approved By: Date: / / Print Name: Branch Chief Effective Date: / / Controlled Copy No.: Withdrawn By: Date: / / ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 2 of 7 TABLE OF CONTENTS Contents Page Number I.0 SCOPE AND APPLICATION 3 2.0 DEFINITIONS 3 3.0 HEALTH AND SAFETY 3 4.0 CAUTIONS 3 5.0 INTERFERENCES 3 6.0 PERSONNEL QUALIFICATIONS 3 7.0 SPECIAL APPARATUS AND MATERIALS 3 8.0 INSTRUMENT OR METHOD CALIBRATION 3 9.0 SAMPLE HANDLING AND STORAGE 3 10.0 PROCEDURE AND ANALYSIS 4 II.0 DATA ANALYSIS/CALCULATIONS 4 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 5 13.0 QUALITY CONTROL 5 14.0 NONCONFORMANCE AND CORRECTIVE ACTION 5 15.0 REFERENCES 5 16.0 FORM AND DATA SHEET 6 ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 3 of 7 1.0 SCOPE AND APPLICATION: 1.1 This SOP describes a method for determining the occurence (number and type) of airborne microorganisms in the laboratory. This procedure is based on aspects of references 15.1 and 15.2. Additional attributes have been added to detect airborne contamination in specific environments. 2.0 DEFINITIONS: 2.1 CFU = Colony Forming Unit 2.2 BSC = Biological Safety Cabinet 2.3 TSA = Tryptic Soy Agar 2.4 TSB = Tryptic Soy Broth 2.5 NB = Nutrient Broth 2.6 SDA = Sabouraud Dextrose Agar 3.0 HEALTH AND SAFETY: Not applicable 4.0 CAUTIONS: None 5.0 INTERFERENCES: 5.1 Building construction, power outages and equipment maintenance may cause transient aberrant counts (see 14.0). These events should be considered in interpreting results of the air testing and efficacy tests conducted during that time. Note these events in the comments section of the Air Monitoring Record Form (see 16.1). 5.2 Media must pass sterility assessment in advance of use. 6.0 PERSONNEL QUALIFICATIONS: 6.1 Personnel are required to be knowledgeable of the procedures in this SOP. Documentation of training and familiarization with this SOP can be found in the training file for each employee. 7.0 SPECIAL APPARATUS AND MATERIALS: None 8.0 INSTRUMENT OR METHOD CALIBRATION: Not applicable ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 4 of 7 9.0 SAMPLE HANDLING AND STORAGE: Not applicable 10.0 PROCEDURE AND ANALYSIS: 10.1 Summary: In this method, general growth media are exposed to the environment to monitor the occurrence of airborne microorganisms (e.g., bacteria, mold and yeast). This is a passive air sampling method. Petri plates containing TSA and/or SDA are exposed to the environment for a specific period of time at various sites in a laboratory (sample locations include bench tops, incubators and biosafety cabinets etc). The uncovered plates are exposed to the environment for 15-60 minutes followed by incubation for 2 to 7 days. Following incubation, colonies on the plates are counted and recorded. 10.2 The test can be performed on an as needed basis or at least once a year for all laboratories. 10.3 Label TSA and /or SDA plates in accordance with their locations where they are placed in a laboratory (room #, specific sites in a lab, etc). 10.4 Place the plates at desired locations and remove the covers sequentially at 15-30 second intervals. 10.5 Sequentially replace the covers after 15-60 minutes of exposure time. Record exposure time on the Air Monitoring Record Form 10.6 Incubate the plates at 36±1°C (for TSA) or at 24±1°C (for SDA) for 2 to 7 days. Plates can be wrapped in parafilm to prevent dehydration during extended period of incubation (> 48 hrs). Plates may be observed daily for growth. 10.7 Count colonies and record the number of colonies per square foot. The number of organisms which settle in 15 minutes of exposure on a petri dish is equivalent to that for 1 sq. foot. 10.8 Presumptive identification of contaminants may be determined by use of Gram stain and/or Vitek® analysis. Note: It may be necessary to use a specific growth medium and incubation conditions if one is attempting to identify the presence of a more fastidious microbe. 11.0 DATA ANALYSIS/CALCULATIONS: 11.1 Determine the number of CFUs per 15 x 100 mm plate (up to 300 CFU) per 15 minute period (or multiply with the factor if the exposure time is more than 15 minutes, e. g., the number of CFUs be multiplied with 2 if the exposure time is 30 minutes (Ref. 15.2). ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 5 of 7 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT: 12.1 Data will be recorded promptly, legibly and in indelible ink on the Air Monitoring Record Form (see 16.0). Completed forms are archived in notebooks kept in secured file cabinets in file room D217. Only authorized personnel have access to the secured files. Archived data is subject to OPP's official retention schedule contained in SOP ADM-03, Records and Archives. 13.0 QUALITY CONTROL: 13.1 The OPP Microbiology Laboratory conforms to 40 CFRPart 160, Good Laboratory Practice Standards. Appropriate quality control measures are integrated into each SOP. 13.2 For quality control purposes, the required information is documented on the appropriate forms (see 16.0). 14.0 NONCONFORMANCE AND CORRECTIVE ACTION: 14.1 If contamination exceeds 15 colonies/plate/15 minute (Ref 15.2) or a laboratory base line on the basis of past historical data, possible contamination sources will be investigated. In order to minimize or remove the contamination source, the laboratory will undergo a decontamination process that will consist of general cleaning and the application of an antimicrobial product, if necessary, in specific areas. Following this process, the air monitoring procedure will be repeated and operation in a laboratory may be suspended until the problem is resolved. 14.2 If a plate that corresponds to a location inside a BSC demonstrates unacceptable contamination (15 colonies/plate/15 minute or a laboratory base line on the basis of past historical data), the BSC will not be used and the facility maintenance staff will be informed of the situation. If necessary, decontamination procedures (e.g. disinfectant application) will be conducted in the BSC. The monitoring test will be repeated for the affected BSC. The BSC will not be used until the air monitoring indicates acceptable level of microbial counts. 14.3 If any unusual air handling events take place (e.g., significant laboratory air flow changes, construction within the facility), air monitoring may be conducted at any time to verify the quality of the lab's air according to procedures in section 10.0 of this SOP (the test will be conducted only on the affected laboratory). 15.0 REFERENCES: 15.1 Bordner, R.H., Winter, J.A., & Scarpino, P.V., eds. 1978. Microbiological Methods for Monitoring the Environment, Water, and Wastes. EPA 600/8-78- ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 6 of 7 017, Part IV Quality Assurance, U.S. Environmental Protection Agency, Cincinnati, Ohio. 15.2 Eaton, A.D., Clesceri, L.S., Rice E. W. eds. 2005. Standard Methods for the Examination of Water and Wastewater, (Page 9-4), 21st Edition. American Public Health Association, American Water Works Association, Water Environment Federation. 16.0 FORM AND DATA SHEET: 16.1 Air Monitoring Record Form ------- SOP No. QC-02-04 Date Revised 09-09-10 Page 7 of 7 16.1 Air Monitoring Record Form OPP Microbiology Laboratory SOP QC-02 INFORMATION SHEET Test Performed By: Date/Init. Incubation Date and Time Start: Stop: Exposure Time for Plates: TSA Prep. No.: SDA Prep. No.: Test Results Read By: Date/Init. Location TSA Plate # CFU/Plate SDA Plate # CFU/Plate Room Site Description 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 Comments ------- |