US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Calibration and Maintenance of pH Meters
SOP Number: EQ-01-07
Date Revised: 11-25-14

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SOP No. EQ-01-07
Date Revised 11-25-14
Page 1 of 9
SOP Number
EQ-01-07
Title
Calibration and Maintenance of pH Meters
Scope
This protocol describes the method for the operation, two point
calibration, and maintenance of the Corning pH meter 430 and the
Corning pH meter 530.
Application
Corning pH meters 430 and 530 are used to determine the pH of
samples, such as media and reagents used in the laboratory. The pH
of media and reagents is a critical attribute of such solutions for the
proper performance of media and reagents.


Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:


Date SOP issued:

Controlled copy
number:

Date SOP withdrawn:


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SOP No. EQ-01-07
Date Revised 11-25-14
Page 2 of 9
TABLE OF CONTENTS
Contents	Page Number
1.
DEFINITIONS
3
2.
HEALTH AND SAFETY
3
3.
PERSONNEL QUALIFICATIONS AND TRAINING
3
4.
INSTRUMENT CALIBRATION
3
5.
SAMPLE HANDLING AND STORAGE
3
6.
QUALITY CONTROL
3
7.
INTERFERENCES
3
8. NON-CONFORMING DATA
3
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
9
14.
FORMS AND DATA SHEETS
9
15.
REFERENCES
9

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SOP No. EQ-01-07
Date Revised 11-25-14
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1. Definitions
Abbreviations/definitions are provided in the text.
2. Health and
Safety
Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study
Director and/or lead analyst should consult the Material Safety Data Sheet for
specific hazards associated with media and reagents.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
As noted below in section 12.
5. Sample
Handling and
Storage
1.	Measure the pH of media and reagents at room temperature (20C to
25C) unless otherwise specified on the Media/Reagent preparation sheet.
For agar based media, the pH is to be taken on a solidified sample (see
section 12.4).
2.	The buffers and reagents (IN HC1 and IN NaOH) are stored adjacent to
the pH meter at room temperature in a secondary containment unit.
3.	Store electrodes for up to one week in 25 mL of pH 7.0 or pH 4.0 buffer.
Never store the electrode in distilled water.
4.	For longer storage periods, remove the bulb protector, fill the wetting cap
with the 3M KC1 fill solution and push the wetting cap onto the tip of the
electrode.
6. Quality Control
For quality control purposes, the required information is documented on the
appropriate form (see section 14).
7. Interferences
1. Rinse the pH meter electrode thoroughly with de-ionized water and blot
dry with Kimwipes before proceeding to the next solution.
8. Non-
conforming
Data
1.	Management of non-conforming data is consistent with SOP ADM-07,
Non-Conformance Reports.
2.	To pass calibration the slope must be slope 100%  5%. If slope is out of
range, the first corrective action is to change the standard pH buffers and
recalibrate. The second step is to change the 3M KC1 fill solution in the
electrode or bring up to volume, then re-calibrate. The final corrective
action is to clean the electrode with the pH Electrode Cleaning Solution
(follow manufacturer's instructions).
3.	Record all corrective action procedures conducted under the notes section
of the pH Meter Calibration Record Form (see section 14).
4.	If the pH meter is not functioning as required, consult the technical
manual to determine the problem and perform corrective action. Notify

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SOP No. EQ-01-07
Date Revised 11-25-14
Page 4 of 9
media prep staff, and if the problem cannot be determined or corrected,
call for service.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
1.	Do not stir the sample while the instrument is reading, as this may
provide inaccurate readings.
2.	Do not allow the 3M KCL fill solution to run dry. Add 3M KC1 fill
solution whenever the level falls more than 25 mm below the fill hole.
Replace the 3M KC1 fill solution at least once a month.
3.	Always remove the wetting cap and the fill hole plug during calibration
and measurements. Replace the fill hole plug in between use.
4.	Do not leave the electrode in organic solvents as the tip and body may be
damaged.
5.	Discard used buffer solutions daily. Do not reuse.
11. Special
Apparatus and
Materials
1.	Corning pH Meter #1: Model 430 (Serial No. 005166)
2.	Corning pH Meter #3: Model 530 (Serial No. 301887)
3.	Corning pH Meter #4: Model 530 (serial No. 304953)
4.	Electrodes:
5.	Three '3 inl' Combo w/RJ (Epoxy body) (Corning, now Nova Analytics,
Catalog No. 76436). Electrode performance specifications
(Manufacturer's claim): the pH range 0.0 -14.0 and the temperature range
0 - 100C. The electrodes are identified as #5, 10, and 12. These
electrodes are used for pH measurement of liquids.
6.	Two Flat-Surface (Epoxy Body) with Replaceable Junction Electrode
(Corning Catalog No. 476286). Electrode performance specifications
(Manufacturer's claim): the pH range is 0.0-14.0 and the temperature
range is 0-100C. The electrodes are identified as # 3 and # 4. These
electrodes are used for pH measurement of solid media.
7.	One Semi-Micro Glass Body with Refillable Junction Combo Electrode
(Corning Catalog No. 476156). Electrode performance specifications
(Manufacturer's claim): the pH range is 0.0-14.0 and the temperature
range is 0-100C. Electrode is identified as #9. The electrode is used for
measuring pH of small aliquots of media (< 1 mL).
8.	Calibration Buffers: pH 4.0, pH 7.0 and pH 10.0.
9.	Fill solution: 3M KC1 solution.

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SOP No. EQ-01-07
Date Revised 11-25-14
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10.	Storage Solutions: 3M KC1 solution for capping OR pH 4.0 or pH 7.0
buffer for buffer immersion.
11.	pH Electrode Cleaning Solution.
12.	Ceramic replacement junction.
13.	IN Sodium Hydroxide Solution (NaOH).
14.	IN Hydrochloric acid (HC1).
12. Procedure and
Analysis
1.	Calibrate the pH meter at least once on the day of use as outlined in
section 12.1 and 12.2. A two point calibration is required.
2.	The standard buffers used are pH 4.0, pH 7.0, and pH 10.0. Dispense a
small amount of the required buffers into a smaller container for
calibration.
3.	Use two standard pH buffers for calibration.
4.	Use pH buffers for calibration one time only. After calibration buffers are
discarded as per manufacturer's recommendations.
5.	Use two standard pH buffers to determine the pH of the medium or
reagent. If the final pH of the sample falls between pH 7.0 and 10.0, use
the pH 7.0 and 10.0 buffers for calibration. If the final pH of the sample
falls between pH 4.0 and 7.0, use the pH 4.0 and 7.0 buffers for
calibration.
6.	The expiration dates of the buffers are recorded on the upper part of the
pH Meter Calibration Record Form (see section 14). Record the date the
buffers are changed under Notes on the form.
7.	Check the level of the 3M KC1 fill solution every day when in use to
ensure that it is within 25 mm of the filling hole. Bring to level if needed.
Record under Notes any adjustments made with the fill solution on the pH
Meter Calibration Check Record Form.
12.1 Two-point
calibration pH
Meter Model
430
a.	Rinse the probe tip with de-ionized water and blot dry with
Kimwipes, then place the tip of the electrode into the pH 7.0 buffer
and press "Cal."
b.	The pH meter will automatically read the endpoint when the reading
is stable. The appropriate buffer symbol will appear on the display.
Record the pH value on the pH Meter Calibration Check Record
Form (see section 14).
c.	Rinse the tip of the electrode with de-ionized water and blot dry with
Kimwipes.
d.	Place the tip of the electrode in the second calibration buffer (either

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SOP No. EQ-01-07
Date Revised 11-25-14
Page 6 of 9

pH 4.0 buffer or pH 10.0 buffer) and press "Cal".
e.	The pH meter will automatically read the endpoint when the reading
is stable and the appropriate buffer symbol will appear on the
display.
f.	The pH meter will briefly display the electrode slope value. Record
the pH and slope values on the pH Meter Calibration Check Record
Form. If the slope is less than 95% or greater than 105%, refer to the
troubleshooting section of the Corning pH meter 430 instructions
manual (see section 15).
g.	Rinse the electrode with de-ionized water and blot dry with
Kimwipes. Confirm the calibration by reading the pH of the pH 7.0
buffer a second time. Record the pH value on the pH Meter
Calibration Check Record Form.
h.	Press "Mode" one time to switch to temperature mode. Record the
temperature value on the pH Meter Calibration Check Record Form.
i.	Press "Mode" two more times to return to pH mode.
12.2 Two-point
calibration pH
Meter Model
530
a.	Prior to use, verify that the pH meter is set at Auto endpoint as
indicated by a letter "A" in the upper left hand corner of the display.
If the letter "A" is not indicated, press the "Auto" button until it is
displayed. After the endpoint is reached, brackets will appear around
the A (e.g. [A]). The temperature and slope will appear at the bottom
of the display. When selecting the buffer for calibration, the selected
buffer pH will also appear at the bottom of the display, but once the
calibration begins, the pH of the buffer will disappear.
b.	To calibrate, rinse the probe tip with de-ionized water and blot dry
with Kimwipes, then place the tip of the electrode into the pH 7.0
buffer and press "Cal" one time so that "pH 7" and "Cal 1" appear.
c.	The pH meter will automatically read the endpoint when the reading
is stable. The appropriate buffer symbol will appear on the display.
Record the pH value on the pH Meter Calibration Check Record
Form.
d.	Rinse the tip of the electrode with de-ionized water and blot dry with
Kimwipes.
e.	Place the tip of the electrode in the second calibration buffer. If pH
4.0 buffer is used, press "Cal" one time so that "pH 4" and "Cal 3"
appear. If pH 10.0 buffer is used, press "Cal" two times so that "pH
10" and "Cal 3" appear.
f.	The pH meter will automatically read the endpoint when the reading

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SOP No. EQ-01-07
Date Revised 11-25-14
Page 7 of 9

is stable and the appropriate buffer symbol will appear on the
display. The display will show the electrode slope value. Record the
pH value and slope on the pH Meter Calibration Check Record Form.
g.	If the slope is less than 95% or greater than 105%, refer to the
troubleshooting section of the Corning pH meter 530 instructions
manual (see section 15).
h.	Rinse the electrode with de-ionized water and blot dry with
Kimwipes. Confirm the calibration by reading the pH of the pH 7.0
buffer a second time. Record the pH value and temperature on the
pH Meter Calibration Check Record Form.
12.3 Liquid
Sample pH
reading
a.	Measure the pH of the liquid sample prior to autoclaving and verified
after autoclaving.
b.	Measure the pH of the liquid sample at room temperature unless
otherwise specified on the Media/Reagent preparation sheet.
c.	Place the tip of the rinsed and dried electrode in the sample and press
read to start the measurement. The decimal point will flash while the
electrode is reading.
Note: Do not stir the sample while the instrument is reading. Add
acid or base if any pH adjustments are necessary and mix sample
throughly by stirring. Once sample is mixed stop stirring the
sample prior to re-reading the pH. Repeat this process until the
desired pH has been reached.
d.	When the pH reading is stable, record the initial pH of the solution
on the Media/Reagent preparation sheet (see SOP MB-10, Media and
Reagents Used in Efficacy Testing).
e.	If the pH of the liquid falls outside of the desired range, adjust the pH
of the tempered sample using the specified acid and base solutions
and record the amount used on the Media/Reagent preparation sheet.
f.	Record the adjusted pH of the solution on the Media/Reagent
preparation sheet.
g.	Measure the pH of the sample after autoclaving. Let sample tube
adjust to room temperature prior to pH verification. Follow steps
12.2 c-d.
h.	Record the temperature of the sample.
i.	For pH model 430, press "Mode" one time to switch to temperature
mode. The display will show the temperature in C. Record the
temperature at which the pH of the solution was measured on the

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SOP No. EQ-01-07
Date Revised 11-25-14
Page 8 of 9

Media/Reagent preparation sheet. Press "Mode" two more times to
return to pH mode.
j. For pH model 530, the temperature will display at the same time as
the pH. The display will show the temperature in C. Record the
temperature at which the pH of the solution was measured on the
Media/Reagent preparation sheet.
12.4 Solid sample
pH reading
(media
containing
agar)
a.	Measure the pH of the agar sample prior to autoclaving and verified
after autoclaving.
b.	Measure the pH of the agar sample as a solid.
c.	Place an aliquot (approximately 5-10 mL) into a petri dish or small
weigh boat and let solidify; keep the remainder of the media between
45-50C (in case the media needs to be adjusted). After the media is
solidified, use the Flat-Surface (Epoxy Body) with Replaceable
Junction Electrode to measure the pH of the media (see section 15).
d.	Place the electrode on the surface of the solidified media and press
"Read" to start the measurement. The decimal point will be flashing
whenever the electrode is reading.
e.	When the pH reading is stable, record the pH of the media on the
Media/Reagent preparation sheet (see SOP MB-10, Media and
Reagents Used in Efficacy Testing).
f.	If the pH of the agar falls outside of the desired range as specified on
the media/reagent preparation sheet, adjust the pH of the tempered
liquid agar (prior to autoclaving) using the specified acid and base
solutions. Discard any media that cannot be adjusted.
g.	Repeat steps 12.4 a-f as needed. Record the adjusted pH of the
solution on the Media/Reagent preparation sheet.
h.	The pH of some media (e.g. Trypticase Soy Agar) is tested after
autoclaving. Follow the instructions on the media/reagent
preparation sheet.
i.	Place an aliquot (approximately 5-10 mL) of the medium into a petri
dish or small weigh boat, let solidify; then take the pH of the
solidified agar. Record this reading on the Media/Reagent
preparation sheet.
j. The Flat-Surface electrode is incapable of reading the temperature of
the sample and will always read 25C. Record N/A on the
temperature section of the Media/Reagent preparation sheet.
12.5 Electrode
a. Clean the electrode when the slope falls below 95% or is greater than

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SOP No. EQ-01-07
Date Revised 11-25-14
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Cleaning and
Maintenance
105%.
b.	Test the flow of the junction by letting the electrode hang in the air
for 1-2 hrs. A proper flowing junction will have KC1 salt crystals
forming on it. If none appear, review the following suggestions for
cleaning.
c.	Soak the tip of electrode in the pH Electrode Cleaning Solution for 1-
2 hrs. Rinse the electrode with de-ionized water and soak in pH 7.0
buffer until stable.
d.	For oil contamination: Carefully clean the tip of the electrode using
a cotton swab soaked with alcohol or acetone. Rinse the electrode
with de-ionized water and soak in pH 7.0 buffer until stable.
e.	If cleaning does not improve junction flow, the junction will be
replaced. To replace, simply pull out the clogged junction, rinse the
electrode barrel with de-ionized water, and replace with a clean
junction. Then refill electrode with the 3M KC1 fill solution up to the
fill hole plug (see section 15).
f.	After cleaning and maintenance activities, check the calibration of
the pH meter and record on the pH Meter Calibration Form.
g.	Record cleaning and maintenance activities under notes on the pH
Meter Calibration Record Form.
13. Data Analysis/
Calculations
Not Applicable
14. Forms and Data
Sheets
Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
pH Meter Calibration Record Form EQ-01-07 F1 .docx
15. References
1.	Corning Electrodes Information sheet for pH combination 3-in-l
electrodes.
2.	Corning Labware & Equipment- Electrodes: How to Maintain the
Premium Performance of Your Corning Electrodes.
3.	Instruction manual for Corning pH meter 430.
4.	Instruction manual for Corning pH meter 530.
5.	Corning Electrodes Information pamphlet for flat surface electrodes.

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