US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Systems Check for the Beckman (DU Series 500) Spectrophotometer SOP Number: EQ-04-05 Date Revised: 11-30-11 ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 1 of 9 Initiated By: Technical Review: QA Review: Approved By: EPA/OPP MICROBIOLOGY LABORATORY ESC, Ft. Meade, MD Standard Operating Procedure for Use and Maintenance of the Beckman DU 520 Spectrophotometer SOP Number: EQ-04-05 Date Revised: 11-30-11 Print Name: Print Name: Technical Staff Print Name: QA Officer Print Name: Branch Chief Date: / / Date: / / Date: / / Date: / / Effective Date: / / Controlled Copy No.: Withdrawn By: Date: / / ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 2 of 9 TABLE OF CONTENTS Contents Page Number 1.0 SCOPE AND APPLICATION 3 2.0 DEFINITIONS 3 3.0 HEALTH AND SAFETY 3 4.0 CAUTIONS 4 5.0 INTERFERENCES 4 6.0 PERSONNEL QUALIFICATIONS 4 7.0 SPECIAL APPARATUS AND MATERIALS 5 8.0 INSTRUMENT OR METHOD CALIBRATION 5 9.0 SAMPLE HANDLING AND STORAGE 5 10.0 PROCEDURE AM) ANALYSIS 5 11.0 DATA ANALYSIS/CALCULATIONS 7 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 7 13.0 QUALITY CONTROL 7 14.0 NONCONFORMANCE AND CORRECTIVE ACTION 7 15.0 REFERENCES 7 16.0 FORMS AM) DATA SHEETS 8 ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 3 of 9 1.0 SCOPE AND APPLICATION: 1.1 This protocol describes the procedure for verifying the systems check for the Beckman DU 520 Spectrophotometer. 1.2 This protocol describes the procedure for determining the Absorbance (A, in optical density units O.D.) and Percent Transmittance (%T) of microbial suspensions at a wavelength of 650 nm. 2.0 DEFINITIONS: 2.1 Sampling module = The Single Cell Module (part no. 517605) outfitted with the Standard Cell Holder (part no. 517611) which holds a standard 1 cm cuvette. 2.2 Absorbance A vs. % Transmittance %T= Absorbance is the measure of the quantity of light that a sample neither transmits nor reflects and is proportional to the concentration of a substance in a solution. The absorbance scale is logarithmic. The transmittance of a sample is the ratio of the intensity of the light that has passed through the sample to the intensity of the light when it entered the sample (T = Iout / Iin )• The transmittance is displayed as a percentage on an analog scale. To convert between the absorbance and transmittance scales, use the equation: A = -log (%T! 100) The numbers obtained for both A and %T are unitless as both are derived from a ratio between the incident light and the transmitted light, so the units cancel. Absorbance refers to the absorbance of light by molecules which is NOT what is observed when placing a microbial sample in a spectrophotometer. Light that doesn't reach the detector has not been absorbed but rather scattered as a result of turbidity. Because of this, the term used to describe the absorbance value is Optical Density O.D. 3.0 HEALTH AND SAFETY: 3.1 All manipulations of test organisms (i.e., microbial suspensions) are required to be performed in accordance with biosafety practices stipulated in SOP MB-01, Lab Biosafety. All M. bovis (BCG) manipulations are performed in a Biosafety Level 3 isolation laboratory (i.e., room B202 or room B207). 3.2 Cuvettes containing microbial suspensions must be capped prior to removal from the BSC. The use of a cuvette rack for transporting capped cuvettes ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 4 of 9 within the laboratory is recommended. For transporting microbial suspensions in capped cuvettes between laboratories, place the cultures in secondary containment. 4.0 CAUTIONS: 4.1 Maintain an ambient temperature of 10-40°C for proper instrument operation. Leave at least a 15 cm clearance at the top and on all sides for air circulation. Keep the air vents under the instrument clean and free of materials that might obstruct the air flow. 4.2 The instrument includes both the visible and UV range. Cuvettes made of silica or quartz are required for use in the UV region. Pyrex (glass) or plastic cuvettes can be used in the visible region (e.g., 650 nm). Only semi-microcells that have back-masked sidewalls should be used. To accommodate the use of non-masked plastic cuvettes, a SLIT (part no. 517678) has been installed on the single cell holder. It may remain in place with other cuvettes (e.g., silica, quartz, Pyrex, or plastic) without affecting spectrophotometer performance. 4.3 Allow the instrument to warm up for a minimum of 15 minutes before taking any sample readings. 4.4 Lenses are located on both sides of the sampling module. Wipe lenses with a soft lint-free and oil free cloth to clean. Do not use organic solvents such as acetone to clean the lenses. 4.5 Keep the inside of the Sampling module clean and dry. 4.6 During system startup and initialization, the analyst must remain present during the entire system check of the instrument and must either hear the three beeps or observe the "pass" statement on the machine in order to verify that the instrument has passed the system check. 5.0 INTERFERENCES: 5.1 The entire sampling module must be kept clean. If it becomes dirty, wipe it clean with soap and water and a soft cloth. 6.0 PERSONNEL QUALIFICATIONS: ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 5 of 9 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: 7.1 Beckman DU Series 520 General Purpose Spectrophotometer: wavelength range 190-1100 run, wavelength accuracy ±0.1 nm, wavelength repeatability 0.1 run, photometric readout -0.3 to 3.0A or 0.1-200.0% T, photometric accuracy ± 0.005A (IA at 546.1 nm), stray light ± 0.05% Tmaximum at 220 nm and 320 nm, scanning speed 40-1000 nm/min. 7.2 Cuvettes - semi-micro plastic disposable cuvettes: 12.5x12.5x45 mm, capacity of 1.5 - 3 mL, 10 mm light path. To the right is an image of both a standard cuvette and a semi- micro cuvette. The triangular arrow at the top of each cuvette indicates the optical path orientation. 8.0 INSTRUMENT OR METHOD CALIBRATION: 8.1 The Beckman DU 520 spectrophotometer is sent out to a private, ISO 17025 accredited vendor on an annual basis for certification. 9.0 SAMPLE HANDLING AND STORAGE: 9.1 For sample readings, microbial suspensions should be aliquotted into cuvettes in the biological safety cabinet (BSC) and capped prior to removal from the BSC. Cuvettes should remain capped at all times once removed from the BSC. Never leave cuvettes containing microbial suspensions in the cell holder once a reading has been taken. 10.0 PROCEDURE AND ANALYSIS: 10.1 SYSTEM MAINTENANCE. The Beckman DU 520 Spectrophotometer contains internal diagnostic tests to aid in self-diagnosis, and a system check to validate the performance of the instrument. 10.1.1 Each time the instrument is powered up, a series of diagnostic tests is performed automatically to ensure operation of major system components. This procedure, which takes two minutes, checks the memory, voltage, systems, offset correction, lamp alignment, and the wavelength calibration. The instrument contains programs for ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 6 of 9 checking wavelength accuracy, bandwidth, photometric noise, stray light, photometric accuracy, photometric repeatability and photometric stability. 10.1.2 When all tests are complete, the main menu is displayed. The screen briefly displays "Pass" to denote that each of these areas is functioning properly. 10.2 SYSTEM USE. The spectrophotometer has been programmed with analysis parameters for determining the %T (or A) of microbial suspensions at a wavelength of 650 nm. 10.2.1 Turn on the instrument making sure the sampling module lid is closed and no cuvettes are in the single cell holder. Allow the instrument to initialize and warm up. Record results of the diagnostic check on the Beckman DU 520 Spectrophotometer - Systems Check Record Form (see 16.0). NOTE: Analyst must remain in the room during the system check of the instrument (see cautions section 4.6). Beckman DU 520 Spectrophotometer - Systems Check Record 10.2.2 To recall the program, with the Main Menu displayed, press the [USER PROGRAM] soft key to display a listing of all user programs. Use the scroll keys to highlight the %T @ 650 nm program. Press [ENTER] to run the program. 10.2.3 For single wavelength analysis, the use of a blank is required. Take a blank reading on a cuvette of the growth medium (or appropriate background liquid). Fill the blank and sample cuvettes approximately half way to a level just above the window shoulder (23 mm). Once filled, cap the cuvettes gently being careful to avoid touching the cuvettes below the shoulder. 10.2.4 Prior to inserting a cuvette into the sample holder, check that no air bubbles are present; gently tap to remove if necessary. Using a clean, dry, and lint free towel, gently wipe the cuvette sides oriented in the optical path (indicated with an arrow - see 7.2 above) and firmly position the cuvette in the sample holder. 10.2.5 Insert the Blank first and press the [BLANK] soft key. ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 7 of 9 10.2.6 Remove the Blank and insert the sample cuvette (with bacterial suspension). Press the [SAMPLE] soft key. 10.2.7 The %T will be immediately displayed on the screen. To toggle between %T and Absorbance, press the indicated soft key. 10.2.8 For multiple sample readings, it is advisable to take a blank reading between samples. 11.0 DATA ANALYSIS/CALCULATIONS: None 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT: 12.1 The calibration certificates are filed and maintained in notebooks in a secured file cabinet in the 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. 12.2 Data will be recorded promptly, legibly and in indelible ink on the Systems Check Record Form (see 16.0). Completed forms are archived in notebooks kept in secure file cabinets in the file room D217. Only authorized personnel have access to the secured files. Archived data are subject to OPP's official retention schedule contained in SOP ADM-03, Records and Archives. 13.0 QUALITY CONTROL: 13.1 Annual certification records are maintained in notebooks in D217. 13.2 For quality control purposes, the required information is documented on the appropriate form (see 16.0). 14.0 NONCONFORMANCE AND CORRECTIVE ACTION: 14.1 If any function does not pass or if any error messages are displayed, refer to section 17, Troubleshooting in the Operating Instructions (see ref. 15.1) and, if necessary, contact the manufacturer for assistance. Record corrective actions on the Systems Check Record Form under "Comments." Take spectrophotometer out of service if troubleshooting does not resolve the issue. Arrange for service by a qualified company or vendor. 15.0 REFERENCES: ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 8 of 9 15.1 Operating Instructions: BeckmanDU Series 500 Spectrophotometer. Beckman Instructions 517640-AC, April 1998. 16.0 FORMS AND DATA SHEETS: 16.1 Beckman DU 520 Spectrophotometer - Systems Check Record ------- SOP No. EQ-04-05 Date Revised 11-30-11 Page 9 of 9 Beckman DU 520 Spectrophotometer - Systems Check Record OPP Microbiology Laboratory Date/Initials Pass/ Fail Results Comments ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ~ PASS ~ FAIL ------- |