Standard Operating Procedure PCM SOP 1-1. Microscope Calibration, Preparation and Analysis of Air Samples by Phase Contrast Microscopy (PCM)

Transcription

1 Standard Operating Procedure PCM SOP 1-1 Microscope Calibration, Preparation and Analysis of Air Samples by Phase Contrast Microscopy (PCM) Scope and Purpose: The scope and purpose of this standard operating procedure is to document the procedures involved in the calibration, preparation and analysis of air samples by Phase Contrast Microscopy (PCM) according to the current NIOSH 7400 Method (Analysis of Asbestos and Other Fibers by PCM), Procedures For Laboratory and Field Activities: Alignment Procedures 1. Turn the power switch on and adjust the voltage control dial until the proper light intensity is reached (should be set between the levels of 7 and 10). 2. Turn the revolving nose piece and rotate the objectives until the 40X objective is in place. 3. Place the microscope slide that contains an amount of background loading (a reference slide is recommended) on top of the mechanical stage. While viewing the slide through the eyepiece, focus on the slide using the fine and coarse adjustment knobs. 4. Once focused, close the field iris diaphragm. Turn the stage condenser slowly up or down until a polygonal ring (either blue to magenta in color) appears as shown below: If the image appears to be uncentered from the field of view, center with the centering screws. 5. Open the diaphragm so the polygonal ring disappears from the field of view. Page 1 of 6 Revised 2015

2 6. Remove the right ocular lens and insert telescopic ocular into its place. View into the telescopic ocular and focus. Two rings will be seen; one dark and one light as seen below: Adjust rings with the phase centering screws until the rings are centered one above the other as seen below: Remove the telescopic ocular and replace the right ocular. Record calibration information in the microscope logbook. 7. Test the resolution of the microscope and analysts using the HSE/NPL Phase Contrast Test Slide (see SOP 1-2 for details and timetable for recalibration of the test slide) on each day client/field samples are analyzed or if microscope was moved or serviced. 8. The Walton-Beckette Graticule must be measured once for the graticule using a stage micrometer (see SOP 1-3 for details and timetable for recalibration of the micrometer slide) on each day the client/field samples are analyzed. The area of the graticule must be documented in the microscope logbook. 9. Clean the microscope with canned/aerosol air. The microscope is now ready for use. 10. Any problems or concerns with the QuickFix must be immediately reported to the Laboratory Director or the QC Coordinator and the sample preparation is stopped until the problem is resolved. The issues with the QuickFix shall be recorded on the IEA Lab Reagent Blank Result Log in the Comments section. 11. Rotometers are calibrated according to the steps spelled out in the NIOSH 582e manual, located in the PCM Laboratory, before each summer session begins. The rotometers are kept in the PCM laboratory and checked out during use. Rotometer calibration is noted in Equipment Section of the laboratory equipment spreadsheet and on each rotometer. Sample Reception: 1. When samples are received for analysis by laboratory personnel, the chain-of-custody form shall be signed and dated. Page 2 of 6 Revised 2015

3 2. The sample cassette(s) and chain-of-custody form(s) are inspected to determine if they pass rejection criteria (see SOP 1-4 for details). If the samples fail, the appropriate person(s) is notified that the samples cannot be prepared and analyzed, the reason for the rejection is noted on the chain of custody form, and the samples will not be analyzed until corrective actions are taken. If samples pass rejection criteria, the analyst is able to proceed to the prepare samples. 3. The Chain of Custody and samples are entered into the IEA Laboratory Information Management System (LIMS), where the uniques samples are stored and a log of samples can be produced. At this point the general information is entered into the LIMS, such as client, building where samples were taken, and what each samples unique sample ID number is. Sample Preparation: 1. For Field Analysis, the environmental conditions must be taken into account before the samples can be prepared and analysis is done, some of these include: good ventilation for the use of the QuickFix acetone vaporizer, clean preparation area, a safe environment, and an area that can be secured. It is encumbent upon the field analyst to ensure and maintain a clean area to prep and analyze samples, if at any time this cannot be maintained, a new area must be found for the sample preparation and analysis. 2. Samples are laid out in numerical order on the prep table. QuickFix acetone vaporizer is turned on and allowed to warm up (vaporizer is ready for use when light turns on). 3. Analyslides are set out in front of the sample cassettes and labeled with the project number, sample number, analyst s initials and analysis date. 4. Grasp the air cassette firmly at its base on the prep table. Depending on the type of cassettes, either the white label is cut with a disposable scalpel at the top of the cassette or the white sticker label is peeled back. With a twisting motion, remove the top of the air cassette. Remove the filter from the cassette with a pair of clean forceps and place in the analyslides according to the corresponding project number and sample number. 5. Any sample filters found torn, wet, overloaded with debris or if the filters show signs of tampering, these filters are placed in their analyslide, and the comments noted on the chainof-custody form. These samples will not be prepared past this point and the project manager will be informed about why the samples could not be analyzed. 6. After samples have been placed in their respective analyslide, frosted microscope slides are labeled with project number and sample number. Slides must be free of dust before use. 7. Remove the cover from the first sample analyslide and cut a pie-shaped portion of the filter ( 1 / 6 to 1 / 4 of the total area of the sample filter). The filters are cut in a rocking motion using a clean surgical scalpel to avoid tearing. Place the filter section on the center of a labeled frosted microscope slide (sample side up) with the tip of the wedge facing the frosted section of the slide. 8. The sample slide shall be placed onto the slide holder of the QuickFix acetone vaporizer (sample filter should be under the injector port of the vaporizer). Inject approximately 250µl of acetone into the injector port of the vaporizer where it will vaporize onto the slide (repeat Page 3 of 6 Revised 2015

4 process if necessary for slide to become clear). Allow slide to dry (usually 3-5 seconds), and remove slide from the slide holder of the vaporizer. Place one drop of Triacetin on the dried filter and cover immediately with a clean glass coverslip. Coverslip should be placed onto the slide in a hinge-like manner to minimize air bubbles. 9. Using a permanent marker, outline the area of the filter on the back side of the microscope slide. Place slides on the microscope slide tray and allow to sit for at least ten minutes before being analyzed. 10. Repeat steps 6 through 8 until all samples have been prepped. 11. After all samples have been prepped, place all analyslides with the remaining filters in a labeled, plastic bag with a zip-lock top and archive. These samples are to be kept out of direct light and extreme temperatures to minimize sample breakdown. Sample Analysis: 1. Type the information into the Laboratory Information Management System (LIMS). The following information must be included and typed into the LIMS (If the electronic LIMS is not available, hand write the information into the PCM Field Worksheet Form and return form to laboratory for entry): - Project number, project name, brief project description and sampling date on top of form - Sample number for the project - Sample description (I=inside, O=outside, H=HEPA, C=clearance, P=personal, AR=adjacent response) - Comments/Location section - Sample time, flow rate and correction factor of the rotameter - Volume of sample(s) - Maximum sample fields to be read - Fiber density - Reporting Limit 2. Each analyst analyzes one (1) reference slide before any client samples are analyzed. The reference result is recorded in the reference entry form and on the bottom of the PCM Worksheet Form, if used. 3. Prepared sample slides are analyzed according to the A Counting Rules as described in the NIOSH 7400 Method (see the current NIOSH 7400 method for details). Sample raw counts are placed on PCM Worksheet Form, if used, and into the LIMS system under Raw Counts section. 4. Calculations for fibers per cubic centimeter (f/cc), 95% confidence interval and reporting limit for each sample are computed by the LIMS. 5. When a field blank s fiber density is less then the reporting limit, less than 7 f/mm 2 (<7 f/mm 2 ) is written instead of calculated number. Page 4 of 6 Revised 2015

5 6. Analysts records the date of analysis in the LIMS and signs their name on the Chain of Custody. Copies of results are given to the proper personnel, and originals are placed in the appropriate areas of the laboratory for quality control analysis. 7. The slides are placed in a slide storage box and the slide box and the associated paperwork are placed in the quality control area of the laboratory. 8. PCM samples are read daily according to a first come first serve basis. If several sets of samples are received at the same time and have the same turn-around time for results, the sets are to be read in the following order of importance: 1. Clearance samples 2. Duration samples 3. Background samples 4. Personal samples Quality Assurance Procedures: 1. Each analyst must perform replicate analysis on at least ten percent (10%) of their total number of slides analyzed on any particular day. A different analyst may perform duplicate analysis on at least five percent (5%) of the total number of samples analyzed by any one analyst. Replicate and duplicate results are entered into their appropriate section of the Laboratory Information Management System (LIMS) 2. Field Blank Samples must be prepped in the same manner as field samples and the counts must be entered/written into the PCM Worksheet. Field blank samples must come from the same box of cassette s that the field sample came from. 3. Laboratory blanks are prepared once per day when client samples are analyzed. A clean, labeled microscope slide is placed into the acetone vaporizer with no air filter. Acetone is vaporized on the slide and dried. It is then removed from the vaporizer and a drop of Triacetin is placed onto the slide along with a clean glass coverslip. Approximately 100 fields are analyzed as a check for contamination of sample reagents. Contamination of the reagents or consumables must be defined as excess debris if the mean is greater than or equal to 5 fibers per 100 graticule fields. If contamination is discovered, reagents and consumables should be replaced under laboratory management s supervision. Results are recorded in the appropriate logbook. 4. Air samples are collected and analyzed annually in the PCM Laboratory or in the sample prep area or more frequently if conditions of the laboratory change. Once analyzed, any sample results with a result of 0.02 f/cc or less is considered acceptable, and no corrective action needs to be taken. If the result is higher than the 0.02 f/cc value, the source of the contamination must be determined and the situation rectified. Sample result information is recorded in the appropriate logbook. Page 5 of 6 Revised 2015

6 Author: Timothy Froehlig Revision Number: 11 Date Created: 8/29/95 Approved by: Date: References: NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 7400 Method, Asbestos and Other Fibers by PCM, 15 August NIOSH 582 Equivalent Course Manual Institute for Environmental Assessment. Olympus CHS/CHT Biological Microscope Instructional Manual. Page 6 of 6 Revised 2015