POLICIES AND PROCEDURES MANUAL

CLIA #01D0665512

Sharon P. Massingale, Ph.D., HCLD(ABB)

Laboratory Director

Prepared by:
Bureau of Clinical Laboratories
Quality Management Division
February 2012
 ALABAMA
COUNTY HEALTH DEPARTMENT
LABORATORY SYSTEMS
(ACHDLS)

Table of Contents

Glossary of Laboratory Terms..…..……………………………………………….………………1

Quality Management….…….…………………………..…….…………….……………….…….3

Laboratory Personnel in County Health Departments…………………………………………….8

Laboratory Safety………………………………….……………………………………………..15

Miscellaneous Equipment………………………………………………………………………..19

Waived Tests……………………………………………………………………………………..25

Brightfield Microscopy……………………………………………………………………….….29

Vaginal Wet Mount………………………………………………………………………………38

Rapid Plasma Reagin (RPR) 18-MM Circle Card Test………………………………………….52

Darkfield Microscopy…..…………….….…..……………………….………….…….………...62

Darkfield Examination for the Detection and Identification of Treponema pallidum…………..67

 Glossary of
Laboratory Terms
 GLOSSARY OF TERMS

1. ANALYTE - A substance or constituent for which the laboratory performs testing.

2. ACCURACY - State of quality of being accurate; closeness of test results to the true value
and implies freedom from error; also referred to as bias.

3. CALIBRATION - Process by which the readings obtained from an instrument or other

measuring device in an analytical process are related to known concentrations.

4. CALIBRATOR - A material, solution, or lyophilized preparation designed to be used in

calibration. The values or concentrations of the analytes of interest in the calibration material
are known within limits ascertained during its preparation and determined by the use of an
analytical method of stated reliability.

5. CLIA - The Clinical Laboratory Improvement Amendments of 1988.

6. CONTROL - Essentially identical specimens of stable materials, usually similar in

composition and physical properties to natural specimens, subjected to the same analytical
process for surveillance control (monitoring) to estimate the performance characteristics
(accuracy, precision) of the analytical process. The control materials are designed to be used
in the quality control process and are not used for calibration purposes. The concentration of
the analytes of interest in the control material is known within limits ascertained during its
preparation and confirmed in use.

7. KIT - All components of a testing system (or unit) that are packaged together.

8. LABORATORY - A facility for the biological, microbiological, serological, chemical,

immunohematological, hematological, biophysical, cytological, pathological, or other
examination of materials derived from the human body for the purpose of providing
information for the diagnosis, prevention, or treatment of any disease or impairment of, or of
the assessment of the health of human beings. These examinations also include procedures
to determine, measure, or otherwise describe the presence or absence of various substances
or organisms in the body. Facilities only collecting and/or preparing or only serving as a
mailing service and not performing testing are not considered laboratories.

9. MEAN - A number that represents an entire set of numbers, determined for the set in any
number of ways; average.

10. PERFORMANCE CHARACTERISTIC - A property of a test that is used to describe the

quality, e.g., accuracy, precision, analytical sensitivity, analytical specificity, reportable
range, reference range, etc.

11. PERFORMANCE SPECIFICATION - A value or range of values for a performance

characteristic established or verified by the laboratory that is used to describe the quality of
patient results.

ACHDLS – Glossary of Terms 1 Revised February 2012

12. PRECISION - State or quality of being precise; freedom from inconsistency or random
error; closeness with which repeated determinations agree with each other and implies
freedom from variation; also referred to as reproducibility.

13. PREVENTIVE MAINTENANCE - A program of scheduled inspections of laboratory

instruments and equipment resulting in minor adjustments or repairs for the purpose of
delaying or avoiding major repairs and emergency or premature replacements.

14. PROFICIENCY SURVEY - A program in which specimens of quality control material are
periodically sent to members of a group of laboratories for analysis and comparison of each
laboratory’s results with those of other laboratories in the group through some central
organization. Participation in a proficiency survey does not replace the day-to-day quality
control process of an individual laboratory.

15. QUALITY MANAGEMENT - Sum of all those activities in which the laboratory is
engaged to ensure that information generated by the laboratory is correct. Quality
management is not restricted to the development and retention of quality control charts but
rather includes all aspects of laboratory activities that affect the results produced, from the
choices of methods, to the monitoring of instruments, to the education of personnel, to the
handling of specimens, and to the reporting of results. The true purpose of quality
management activities is to determine how correct, or incorrect, the results emanating from
the laboratory are, and to allow those managing the laboratory to determine whether or not
the lab is fulfilling its function satisfactorily.

16. QUALITY CONTROL - A procedure performed to measure and maintain quality of

performance in the analytical laboratory through measurement of the variability against pre-
established criteria specifications, correction as indicated, and documentation. The major
purpose of these programs is to establish analytical goals and to assist in their achievement.

17. REPORTABLE RANGE - The range of test values expected for a designated population of
individuals.

18. SAMPLE - In proficiency testing, means the material contained in a vial, on a slide, or other
unit that contains material to be tested by proficiency testing program participants. When
possible, samples are of human origin.

19. STANDARD DEVIATION - The most common measure of statistics, measuring how
widely spread the values in a data set are dispersed.

20. TARGET VALUE - For quantitative tests, means either the mean of all participant
responses after the removal of outliers (those responses greater than three (3) standard
deviations from the mean) or the mean established by definitive or reference methods
acceptable for use by the National Reference System for the Clinical Laboratory (NRSCL)
by the National Committee for Clinical Laboratory Standards (NCCLS).

ACHDLS – Glossary of Terms 2 Revised February 2012

Quality Management
 QUALITY MANAGEMENT
Quality management (QM) is an ongoing process encompassing all facets of the laboratory’s
technical and nontechnical functions. This includes patient preparation and specimen acquisition
(preanalytical), test analysis or examination (analytical), and test result reporting (postanalytical).
QM also extends to the laboratory’s interactions with and responsibilities to patients, physicians,
and the Alabama Department of Public Health.

I. General Quality Management Policies

A. Quality in the entire test system is of foremost importance.

B. All laboratory personnel must be trained properly, commensurate with their positions,
duties, and responsibilities.

C. The Alabama County Health Department Laboratory Systems (ACHDLS) will maintain
a quality control system to assure continued precision and accuracy of laboratory
results.

D. The ACHDLS will participate in the Centers for Medicare & Medicaid Services (CMS)
approved proficiency testing (PT) program.

II. The Quality Management Program

The policies and procedures of the quality management program will be approved by the
laboratory director when first written, with notation of approval by signature and date. The
technical consultants will review the policies and procedures on a regular basis. If a policy
or procedure requires a change, a new policy or procedure will be written, approved by the
laboratory director, and distributed to testing sites. The old policy or procedure will be
retained in a file at the Bureau of Clinical Laboratories, County Assistance Section, for a
minimum of two years.

III. Components of the ACHDLS Quality Management Program

A. Patient Test Management

The laboratory director, with the assistance of the technical consultants, will monitor
and evaluate laboratory information recorded in the Complete Health Record (the
patient chart used in the Alabama county health departments). Chart review will be
conducted and documented at least annually with records maintained in the County
Assistance Section of the Bureau of Clinical Laboratories. Any errors in
documentation will be addressed and brought to the attention of appropriate personnel.

B. Procedure Manuals
A written procedure manual containing procedures for all activities of the ACHDLS
will be maintained and readily available at all times to personnel in each testing site.

ACDHLS - Quality Management 3 Revised February 2012

 The manual will be reviewed at least biannually by the laboratory director and/or the
technical consultants.

C. Quality Control (QC) Assessment

1. Quality control records will be reviewed annually by a technical consultant. This
review is conducted electronically and during onsite visits to all county health
department sites and is documented on the County Visit Worksheet.
Documentation of these reviews will be maintained in the County Assistance Office
of the Bureau of Clinical Laboratories.
2. Quality control specimens are tested randomly in all county health department
laboratories to assess the waived testing being performed. These control results are
documented and maintained in the County Assistance office as well.

D. Training Assessment (See Quality Management Training Schedule on pages 6-7)

1. The technical consultants are responsible for assessing, performing, and
documenting the training and competency of the testing staff of the ACHDLS. All
laboratory testing, under CLIA, are categorized as waived or nonwaived tests.
a. Waived tests performed by the county health department laboratories:
Hemoglobins by HemoCue Method, Occult Bloods, Pregnancy Test, Strep A
Rapid Test, and Urine Dipstick. All tests are to be performed per product insert
and no competency required for testing staff.
b. Nonwaived tests performed by the county health department laboratories:
Darkfield Microscopy, Rapid Plasma Reagin (RPR), and Wet Prep Microscopy.
Workshop trainings and competency precedes county health department
laboratory staff performing stated testing.
Note: All nonwaived testing staff must provide a copy of their college diploma
during a CLIA inspection.
2. New testing personnel must have competency twice during their first year of
training, then annually thereafter.
3. Nonwaived workshop trainings are presented twice a year at the Bureau of Clinical
Laboratories.

E. Proficiency Testing
ACHDLS will participate in a CMS-approved PT program. PT results will be reviewed
by testing personnel and technical consultants, verifying that all information is correct
and complete before they are mailed, faxed, or e-mailed to the PT program.
Investigations of unsatisfactory PT results (performance that does not result in 100% of
acceptable responses for each analyte, including graded, ungraded, and unregulated
analytes) will be documented by testing personnel and reviewed by technical
consultants and laboratory director.

F. Comparison of Test Results and Method Validation

Tests performed at multiple sites will be evaluated once a year. Any test performed for
which PT is not available will be verified at least once a year, and the results will be

ACDHLS - Quality Management 4 Revised February 2012

 reviewed and evaluated by the laboratory director or his/her designee. The results of
these evaluations are documented and maintained in the County Management Section
for each site.

G. Relationship of Patient Information to Test Results

Laboratory personnel will monitor test requests for appropriateness to the patient’s age,
sex, and diagnosis. If any test request or result appears inappropriate, proper
consultation should be obtained.

H. Personnel Assessment
An ongoing evaluation of all testing personnel will be conducted through use of
proficiency testing results, review of quality control records, Complete Health Records,
observation and annual competency evaluations for nonwaived testing procedures.
These include wet preps, RPRs, and darkfield microscopy. If during competency
evaluations an employee is found to be incompetent for one or more procedures, the
employee will not be able to perform the test(s). If (s)he is still not competent after
reevaluation, the employee cannot perform that testing until (s)he has attended training
for that procedure and deemed competent.

I. Communications
Problems that occur as a result of breakdowns in communication between testing
personnel and the authorized individual who orders or receives the results of tests will
be documented. In addition, corrective actions taken to resolve problems and minimize
communication breakdowns will be documented.

J. Complaint Investigations
Investigation of complaints will be made and corrective actions, when necessary, will
be taken. Documentation will be maintained.

K. Quality Management Review with Staff

Quality issues (e.g., unacceptable PT results, ways to improve the quality of testing,
problems identified with QC) will be brought to the attention of ACHDLS personnel
through use of official memorandums and technical bulletins. Documents will also be
maintained in the Quality Management Office.

L. Recordkeeping in the Laboratory

1. CLIA’88 regulations require that all laboratory records be retained for at least two
years. Also check the archives record retention schedule for record retention and
destruction. The office manager in each county health department should have a
copy of this document.
2. Examples of laboratory records include:
a. Temperature charts used to document refrigerator, freezer, incubator, or room
temperatures.
b. Quality control records.
c. Proficiency testing records.
d. Documentation of employee competency.

ACDHLS - Quality Management 5 Revised February 2012

 e. Equipment maintenance.
f. Patient test results [e.g., county health records (CHR) or patient logs].
g. Quality management records.
3. Records of laboratory testing must include:
a. Patient identification.
b. Date of testing.
c. Test performed.
d. Test results, with units of measure if applicable.
e. Time of testing (required on CHR).
f. Initials of person performing the test (nonwaived testing only).
4. Documentation of specimens unacceptable for testing will be maintained in the
patient’s CHR.

M. Laboratory Log
1. The Laboratory Log is designed to assure follow-up on laboratory tests referred
from the county health department to other laboratories. The Laboratory Log is
also a method to track when a specimen leaves the county health department and
the date the report is received. This tracking system for referred specimens is
required by the Clinical Laboratory Improvement Amendments of 1988 (CLIA’88),
which is a federal regulatory program governing all laboratory testing.
2. This tracking system will include patient name, identification number, date of
service, tests referred, and date the report was received. See “Alabama County
Health Department Laboratory Systems Laboratory Log”.
3. Instructions for the Laboratory Log:
a. Place the page number in the space provided.
b. Enter the date in the space provided.
c. Place the label (PHALCON) indicating the patient identity on the sheet. Be
sure to correct the service date if labels were preprinted.
d. Indicate the referred tests on the lines provided. Use 1 line per test (i.e., For
multiple testing such as TV, indicate numbers 1, 5 and 18). If you have more
than 6 tests per patient, use the next 6 lines and mark out the space for the label.
A list of numbers corresponding to tests most commonly referred is printed at
the bottom of the log. Any other tests should be written.
e. Indicate the date the referred laboratory test report is received in the CHD.
f. Have a system devised to locate any test results not received in a timely manner.

IV. Training Schedule

A. Classes
1. Rapid Plasma Reagin (RPR): One-day class for the macroscopic screen for syphilis.
2. Darkfield Microscopy for the Detection and Identification of Treponema pallidum:
Two-day class to include Brightfield Microscopy, Darkfield Microscopy and
Identification of Treponema pallidum.
3. Vaginal Wet Mount: Two-day class to include Brightfield Microscopy and Vaginal
Wet Mount.

ACDHLS - Quality Management 6 Revised February 2012

 B. Reminders
1. Lecture information and procedures on all of the above classes are found in the
Alabama County Health Department Laboratory Systems Policies and Procedures
Manual as well as on the Bureau of Clinical Laboratories’ website.
2. A competency test is administered after each training session followed by a second
competency test in approximately 5-6 months. Thereafter, annual competency
testing is administered.
3. All classes are held in the training laboratory at the Bureau of Clinical Laboratories
in Montgomery.
4. Class size is limited to 12 participants per session.
5. If an employee has previous testing experience (vaginal wet mount, darkfield
microscopy, and/or RPR) at another agency, a request can be made for him/her to
take the competency test after reviewing the Alabama County Health Department
Laboratory Systems Policies and Procedures Manual. If a score of 80% or greater
for vaginal wet mount and/or darkfield microscopy is achieved, (s)he can perform
the test without training from Quality Management. A score of 90% with all
reactives identified is passing for RPR.
6. Classes are approved for nursing continuing education (CEs).

C. Faculty
1. Jane Duke
2. Charlene Thomas

D. Training Schedule

Class Date CE Hours

RPR Spring 5.5 contact hours
Darkfield Microscopy Spring 5.1 contact hours + 6 contact
hours
Vaginal Wet Mount Spring 3.75 contact hours + 5.25
contact hours

RPR Fall 5.5 contact hours

Darkfield Microscopy Fall 5.1 contact hours + 6 contact
hours
Vaginal Wet Mount Fall 3.75 contact hours + 5.25
contact hours

ACDHLS - Quality Management 7 Revised February 2012

 ALABAMA COUNTY HEALTH DEPARTMENT LABORATORY SYSTEMS LABORATORY LOG Page_________ Date______________________
The Laboratory Log is designed to assure follow-up on all laboratory tests referred from the county health department to outside laboratories. This log is also a mechanism to track
when a specimen leaves the CHD and if the report is received in a timely manner.
PHALCON LABEL TESTS DATE PHALCON LABEL TESTS DATE
REFERRED RECEIVED REFERRED RECEIVED

1.GC 2.PAP 3.Syphilis EIA 4.HIV 5.Chlamydia 6.MCH-FP 7.Urine C&S 8.PKU 9.Galactosemia 10.CAH 11.T4 12.TSH 13.Abn Hgb 14.Lead 15.SGOT 16.CBC 17.TB Profile 18.TV
ACHDLS - Quality Management Any other referred test must be written in. Revised 4/2012
Laboratory Personnel
in County Health
Departments
 LABORATORY PERSONNEL IN COUNTY HEALTH DEPARTMENTS

The Clinical Laboratory Improvement Amendments of 1988 (CLIA’88) require the designations
of laboratory director, clinical consultant, technical consultant, and testing personnel for the
Alabama County Health Department Laboratory Systems. A current list of individuals
functioning as the laboratory director, technical consultant, and clinical consultants is included at
the end of this section. Testing personnel are persons in the local county health departments who
perform any laboratory testing (e.g., nurses, clinic nurse aides, nurse practioners, physicians,
disease intervention specialists, laboratory technicians, and nutritionists).

I. Responsibilities of Laboratory Director

The laboratory director is responsible for the overall operation and administration of the
laboratory, including the employment of personnel who are competent to perform test
procedures, record and report test results promptly, accurately, and proficiently and for
assuring compliance with the applicable regulations. Some responsibilities may be
reapportioned to the technical consultants; however, the laboratory director remains
responsible for assuring that all duties are properly performed.

The laboratory director must be accessible to provide onsite, telephone, or electronic

consultation as needed. The laboratory director must:

A. Ensure testing systems used for each of the tests performed provide quality laboratory
services for all aspects of test performance, which include preanalytic, analytic, and
postanalytic phases of testing.

B. Ensure that the physical plant and environmental conditions of the laboratory are
appropriate for the testing performed and provide a safe environment in which
employees are protected from physical, chemical, and biological hazards.

C. Ensure that test methodologies selected have the capability of providing the quality of
results required for patient care.

D. Verification procedures are adequate to determine the accuracy, precision, and other
pertinent performance characteristics of the method.

E. Laboratory personnel are performing test methods as required for accurate and
reproducible results. (This responsibility has been delegated to the technical
consultants.)

F. Ensure that the laboratory is enrolled in a CMS approved PT program for the testing
performed and that:
1. Proficiency testing (PT) samples are tested as required by CLIA’88 regulations.
2. Results are returned within the time frames established by the PT program.

ACHDLS - Laboratory Personnel in CHD 8 Revised February 2012

 3. All proficiency testing reports received are reviewed by the appropriate staff to
evaluate the laboratory’s performance and to identify any problems that require
corrective action.
4. An approved corrective action plan is followed when any PT results are
unacceptable or unsatisfactory. (This responsibility has been delegated to the
technical consultants.)

G. Ensure that quality control and quality management programs are established and
maintained to assure quality of laboratory services and to identify failures in quality as
they occur. (This responsibility has been delegated to the technical consultants.)

H. Ensure the establishment and maintenance of acceptable levels of analytical

performance for each test system. (This responsibility has been delegated to the
technical consultants.)

I. Ensure that reports of test results include pertinent information required for
interpretation. (This responsibility has been delegated to the technical consultants.)

J. Ensure consultation is available to the laboratory’s clients on matters relating to the

quality of the test results reported and their interpretation concerning specific patient
conditions. (This responsibility has been delegated to the technical consultants.)

K. Employ a sufficient number of laboratory personnel who possess appropriate education

with either experience or training to provide consultation, properly supervise and
accurately perform tests and report test results in accordance with CLIA’88 regulations.

L. Ensure that prior to testing patients’ specimens, all personnel have the appropriate
education and experience, receive the appropriate training for the type and complexity
of the services offered, and have demonstrated that they can perform all tests reliably to
provide and report accurate results. (This responsibility has been delegated to the
technical consultants.)

M. Ensure that policies and procedures are established for monitoring testing personnel
who conduct preanalytical, analytical, and postanalytical phases of testing to assure that
they are competent and maintain their competency to perform test procedures and
report test results promptly and proficiently, and whenever necessary, identify needs for
remedial training or continuing education to improve skills.

N. Ensure that an approved procedure manual is available to all personnel responsible for
any aspect of the testing process. (This responsibility has been delegated to the
technical consultants.)

O. Specify in writing the responsibilities and duties of each consultant and person
engaged in the performance of the preanalytic, analytic, and postanalytic phases of
testing that identifies which examinations and procedures each individual is authorized
to perform, whether supervision is required for specimen processing, test performance,

ACHDLS - Laboratory Personnel in CHD 9 Revised February 2012

 or results reporting, and whether consultant or director review is required prior to
reporting patient test results. (This responsibility has been delegated to the technical
consultants.)

II. Responsibilities of Technical Consultant

The technical consultant is responsible for the technical and scientific oversight of the
laboratory. The technical consultant is not required to be onsite at all times testing is
performed; however, (s)he must be available to the laboratory on an as needed basis to
provide consultation via onsite, telephone, or electronically. In addition to the laboratory
director responsibilities delegated to the technical consultant, other responsibilities are:

A. Selection of test methodology appropriate for clinical use of the test results.

B. Evaluation of testing facilities at any time to ensure testing personnel and procedures,
including the precision and accuracy of each test and test system, are in compliance
with CLIA ’88 Regulations.

C. Enrollment and participation in a CMS approved proficiency testing program

commensurate with the services offered.

D. Establishing a quality control program appropriate for the testing performed, establish
parameters for acceptable levels of analytic performance and ensuring that these levels
are maintained throughout the testing process from the initial receipt of the specimen
through sample analysis and reporting of test results.

E. Resolving technical problems and ensure that remedial actions are taken when test
systems deviate from the laboratory’s established performance specifications and
ensuring that test results are not reported until all corrective actions have been taken
and the test system is functioning properly.

F. Identifying training needs and assuring that each individual performing tests receives
regular in-service training and education appropriate for the type and complexity of the
laboratory services performed.

G. Evaluating the competency of all testing personnel and assuring that the staff maintains
its competency to perform test procedures and report results promptly, accurately, and
proficiently. The procedures for evaluation of the competency of the staff must
include, but are not limited to:
1. Direct observation of routine test performance, including patient preparation, if
applicable, specimen handling, processing and testing.
2. Monitoring the recording and reporting of test results.
3. Reviewing intermediate test results or worksheets, quality control records, PT
results, preventive maintenance and function checks.

ACHDLS - Laboratory Personnel in CHD 10 Revised February 2012

 4. Assessment of test performance through testing previously analyzed specimens,
internal blind testing of samples or external proficiency samples, and assessment of
problem solving skills.

H. Evaluating and conducting the performance of employees responsible for moderate

complexity testing at least semi-annually during the first year the employee tests patient
specimens. Thereafter, evaluations must be performed at least annually unless test
methodology or instrumentation changes, in which case, prior to reporting patient
results, the employee’s performance must be reevaluated to include the use of the new
test methodology or instrumentation.

III. Responsibilities of Clinical Consultants

A. Be available to provide clinical consultation to the laboratory’s clients.

B. Be available to assist clients in ensuring that appropriate tests are ordered.

C. Ensure that reports of test results include pertinent information for specific patient
interpretation.

D. Ensure that consultation is available and communicated to clients on matters related to

the quality of test results and interpretation of results concerning specific patient
conditions.

IV. Responsibilities of Testing Personnel

Testing personnel performing waived and nonwaived tests are responsible for specimen
processing, test performance, and for reporting test results. Employees perform only those
nonwaived tests that are authorized by the laboratory director (or his/her designee) and
require a degree of skill commensurate with the individual’s education, training or
experience and technical abilities.

Each employee performing nonwaived testing must:

A. Follow the laboratory’s procedures for specimen handling and processing, test analyses,
reporting and maintaining records of patient results.

B. Maintain records demonstrating that PT samples are tested in the same manner as
patient samples.

C. Adhere to the laboratory’s quality control policies and document all quality control
activities (e.g., instrument and procedural calibrations and maintenance).

D. Follow the laboratory’s established corrective action policies and procedures whenever
test systems are not within the laboratory’s established acceptable levels of
performance.

ACHDLS - Laboratory Personnel in CHD 11 Revised February 2012

 E. Be capable of identifying problems that may adversely affect test performance or
reporting of test results and either must correct the problems or immediately notify the
technical consultant or clinical consultant.

F. Document all corrective actions taken when test systems deviate from the laboratory’s
established performance specifications.

V. Laboratory Supply Evaluation

The Laboratory Supply Evaluations are used to ensure that laboratory reagents, media, test
kits, and other laboratory supplies are maintained in adequate volumes and are used before
the expiration dates. See “Laboratory Supply Evaluation”.

A. It is the responsibility of the clinic supervisor/coordinator or designee to perform

reviews and document the observations.

B. It is the responsibility of the clinic supervisor/coordinator to investigate problems.

C. “Laboratory Supply Evaluation” documentation is to be compiled and retained in the

clinic by the clinic supervisor/coordinator for two years. See “ACHDLS - Quality
Management Section III.L”.

D. Technical consults will examine the “Laboratory Supply Evaluation” documentation

during visits or may request copies be sent to their offices periodically.
VI. Monthly Laboratory Reviews

Monthly Laboratory Reviews are used to monitor and investigate potential noncompliance
in the clinic’s Quality Management System. See “Monthly Laboratory Equipment and
Supply Review”.

A. It is the responsibility of the clinic supervisor/coordinator or designee to perform these

reviews and document observations at the end of each month.

B. It is the responsibility of the clinic supervisor/coordinator to investigate problems.

C. Monthly laboratory review documentation is to be compiled and retained in the clinic

by the clinic supervisor/coordinator for two years. See “ACHDLS - Quality
Management Section III.L”.

D. Technical consults will examine laboratory monthly review documentation during visits
or may request copies be sent to their offices periodically.
VII. Competency Evaluations

ACHDLS - Laboratory Personnel in CHD 12 Revised February 2012

 Employees responsible for nonwaived testing in the County Health Departments (CHD)
will not perform testing until they have been certified by the laboratory director as being
competent to perform testing. Competency will be evaluated at least semiannually during
the first year the employee tests patient specimens. Thereafter, evaluations will be
performed annually for each laboratory test a person performs. Competency evaluations
can be performed onsite by technical consultants (TC) or in conjunction with approved
workshops. Documentation of all activities will be maintained in the County Assistance
Office.

The competency evaluation process will also be used to assess training needs. As needs are
identified, training will be provided to testing personnel.

VIII. Criteria Used for Competency Evaluation

A. Criteria have been defined for evaluating the competency of testing personnel.

B. All phases of laboratory testing (preanalytic, analytic, and postanalytic) will be

reviewed. Evaluation methods include direct observation, record review, and testing
unknown specimens.
1. Direct observation - Personnel will be observed performing testing, QC, reporting,
function checks, and maintenance to determine if written procedures are followed.
2. Record review - Quality control and maintenance records will be reviewed for
accuracy and completeness. In addition, documentation of “out of control results”
and corrective actions taken to correct problems will be reviewed to assess problem
solving skills. Patient records will be reviewed to determine if test reports include
initials of testing personnel, date and time of testing.
3. “Unknowns” - In addition to participation in PT programs, “unknowns” (previously
analyzed specimens, internal blind testing samples or pictures), will be used to
evaluate testing personnel. Testing personnel should maintain a passing score of
80% or above with the exception being RPR which requires a score of 90% or
greater and all reactives discerned.

C. Evaluation activities are documented on Competency Evaluation forms specific to the

procedure being evaluated. Completed forms are maintained by the County Assistance
Section of the Quality Management Division and are filed according to the employee’s
name. Folders are then grouped by Public Health Areas and filed alphabetically.

D. Certificates are presented to personnel who successfully complete competency

evaluations. Copies of these certificates are placed in the employee’s personnel file.

IX. CLIA’88 Training Requirements of Testing Personnel

CLIA’88 Regulations require that individuals perform only those nonwaived tests that
require a degree of skill commensurate with the individual’s education, training or
experience, and technical abilities. Elements included in training must ensure that each
individual has the skills required for:

ACHDLS - Laboratory Personnel in CHD 13 Revised February 2012

 A. Collecting specimens properly, including patient preparation, and if applicable,
labeling, handling, preservation or fixation, processing or preparation, transportation
and storage of specimens.

B. Implementing all standard laboratory procedures.

C. Performing each test method and properly using instruments.

D. Performing preventive maintenance, troubleshooting and calibration procedures related

to each test performed.

E. Working knowledge of reagent stability and storage.

F. Being aware of the factors that influence test results.

G. Assessing and verifying the validity of patient results through the evaluation of QC
sample values prior to reporting patient test results.

X. Delegation of Responsibilities

A. The Laboratory Director of the Alabama CHDLS is Sharon P. Massingale, Ph.D.

B. Technical Consultants are:

1. Jane Duke.
2. Charlene Thomas.

C. The Clinical Consultant is the Laboratory Director, Sharon P. Massingale, Ph.D.

ACHDLS - Laboratory Personnel in CHD 14 Revised February 2012

 Monthly Laboratory Equipment and Supply Review
County Health Department___________________________________ Year ____________

By signing and initialing this form, the staff member attests that he/she has reviewed all equipment according to the
requirements stated in the Alabama County Health Department Laboratory Systems Policies and Procedures Manual.

Printed Name: ________________________________ Signature: ________________________________ Initials: ___________

Printed Name: ________________________________ Signature: ________________________________ Initials: ___________
Printed Name: ________________________________ Signature: ________________________________ Initials: ___________
Printed Name: ________________________________ Signature: ________________________________ Initials: ___________
Printed Name: ________________________________ Signature: ________________________________ Initials: ___________

Laboratory Supply Evaluation: Laboratory Supply Evaluation has been started and all laboratory testing kits and supplies are
within acceptable dates. Monthly date and initials below denote compliance to the above statement. Make use of the back of this form to document
problems, noncompliance issues, and comments.
Lab Supply JAN FEB MAR APR MA
AY JU
UNE
Evaluation
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

Refrigerators: Annual Temperature Chart has been started for the current calendar year with temperatures documented every day the
clinic is open. Temperatures are within acceptable range, corrective action performed/documented, and no food is present. Monthly date and
initials below denote compliance to the above statement. Make use of the back of this form to document problems, noncompliance issues, and
comments.
Refrigerators JAN FEB MAR APR MA
AY JU
UNE
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

ADPH/BCL/QM/ Rev 12-11 Page 1 of 4

 Monthly Laboratory Equipment and Supply Review
County Health Department___________________________________ Year ____________

HemoCue: HemoCue Maintenance Chart and Annual Temperature Chart have been started for the current calendar year with maintenance/
temperatures documented each day of use. Daily/monthly cleanings have been documented. Corrective action performed/documented when
instrument malfunctions. Monthly date and initials below denote compliance to the above statement. Make use of the back of this form to
document problems, noncompliance issues, and comments.
HemoCue JAN FEB MAR APR MA
AY JU
UNE
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

Centrifuge: Centrifuge Preventive Maintenance has been started for the current calendar year, a cover is provided, and monthly cleanings
have been documented. Monthly date and initials below denote compliance to the above statement. Make use of the back of this form to
document problems, noncompliance issues, and comments.
Centrifuge JAN FEB MAR APR MA
AY JU
UNE
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

Wet Mount Microscope: Microscope Maintenance Chart has been started for the current calendar year, a cover is provided, and daily
/ monthly cleanings have been documented. Wet Mount supplies (KOH, saline, ph paper, lens paper and cleaner) are present and not
contaminated. The last annual cleaning date is also documented. Monthly date and initials below denote compliance to the above statement.
Make use of the back of this form to document problems, noncompliance issues, and comments.
WM Microscope JAN FEB MAR APR MA
AY JU
UNE
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

ADPH/BCL/QM/ Rev 12-11 Page 2 of 4

 Monthly Laboratory Equipment and Supply Review
County Health Department___________________________________ Year ____________

RPR Rotator: RPR Rotator Preventive Maintenance has been started for the current calendar year. All daily, monthly, and
quarterly cleanings/checks have been documented. Monthly date and initials below denote compliance to the above statement. Make use of the
back of this form to document problems, noncompliance issues, and comments.
RPR Rotator JAN FEB MAR APR MA
AY JU
UNE
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

Darkfield Microscope: Microscope Maintenance Chart has been started for the current calendar year, a cover is provided, and daily/
monthly cleanings have been documented. Darkfield supplies (immersion oil, lens paper and cleaner) are present and not contaminated. The
last annual cleaning date is also documented. Monthly date and initials below denote compliance to the above statement. Make use of the back
of this form to document problems, noncompliance issues, and comments.
Darkfield JAN FEB MAR APR MA
AY JU
UNE
Microscope
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

Incubator: Incubator Preventive Maintenance and Annual Temperature Chart have been started for the current calendar year with
maintenance/temperatures documented each day of use. Temperatures are within acceptable range and corrective action performed/
documented. Climate control bags are present. Monthly date and initials below denote compliance to the above statement.
Make use of the back of this form to document problems, noncompliance issues, and comments.
Incubator JAN FEB MAR APR MAY JUNE
Date / Initials
JULY AUGUST SEPT OCT NOV DEC
Date / Initials

ADPH/BCL/QM/ Rev 12-11 Page 3 of 4

 Monthly Laboratory Equipment and Supply Review
County Health Department___________________________________ Year ____________

County Clinic Personnel

Date: _____________________________

CHD: ______________________________________ Site: _________________________________

Contact: ________________________________________

List ALL personnel performing laboratory testing, including nurses, clinic aids, nutritionist, and disease intervention
specialist. This form should be completed whenever there is staff change. When changes occur, please update the form
and send a copy to the Quality Management Office, Bureau of Clinical Laboratories in Montgomery.

Name Title

ADPH/BCL/QM/ Rev 12-11 Page 4 of 4

Laboratory Safety
 LABORATORY SAFETY

Laboratory workers will be aware of safety at all times and protect themselves and others.
Further information may be found in the recent publications of the Alabama Department of
Public Health’s Infection Control Guidelines Manual and Bloodborne Pathogens Exposure
Control Plan.

I. Biological Hazards

A. Use protective coverings when handling blood or other body fluids which may contain
blood or mucous membranes. For example:
1. Cover street clothes with a lab coat.
2. Protect eyes, nose, and mouth with a full face shield or safety goggles and mask.
3. Wear gloves.
4. Wear closed toed shoes.

B. Wash hands thoroughly after removing gloves, handling laboratory specimens, and
before leaving the laboratory area.

C. Remove protective clothing and gloves before leaving the lab area to prevent
contamination of other work areas.

D. Do not eat, drink, smoke, apply cosmetics or handle contact lenses in the laboratory area.

E. Avoid hand contact to mouth, nose and eyes while working in the laboratory area.
Keep pencils and instruments away from mouth.

F. Keep immunizations and skin tests up to date: Hepatitis B vaccine, tuberculin skin test
(chest x-ray as indicated), etc.

G. Treat all specimens as potentially infectious.

1. Take care not to spill specimens or create aerosols.
2. Use extreme care when removing rubber stoppers to avoid spatters and aerosols.

H. Handle infectious waste separately from non-infectious waste.

1. Dispose of all sharp objects properly into a puncture resistant container. Do not
recap, break, or otherwise manipulate contaminated needles.
2. Seal tightly and dispose of properly full puncture-resistant containers.
3. Disinfect contaminated disposables before discarding. (e.g., Soak in 1:10 dilution
of household bleach before discarding).
4. The best solution to the disposal of medical waste is to contract with a medical
waste management company.

I. Practice centrifuge safety.

1. Never open the lid while the centrifuge is in operation. Allow the centrifuge to
completely stop before opening to avoid contact with broken sample tubes and to

ACHDLS - Laboratory Safety 15 Revised February 2012

 avoid injuring hands/arms.
2. Clean up all spills immediately with an appropriate disinfectant.

J. Clean up biological spills as soon as possible. Follow the steps listed below.
1. Use puncture resistant gloves for sharp objects involved in the spill.
2. Absorb the spill with paper towels or other appropriate absorbent material. Using
two pieces of stiff cardboard as shovels, carefully scoop the soaked towels and
debris into an appropriate biohazard waste container.
3. Clean the site with soap and water until all visible material has been removed.
4. Disinfect the area with a freshly prepared 1:10 bleach solution or other appropriate
disinfectant. This may be done by covering the area with absorbent paper towels
and flooding the towels until they are “glistening wet.” Allow the disinfectant to set
briefly, then blot up the disinfectant soaked towels and place into an appropriate
waste container.
Note: Bleach solution should be prepared fresh each day of use because it loses its
germicidal effect upon prolonged sitting.
5. Rinse the area well with water.

II. Chemical Hazards

A. Know the chemical with which you will be working. Consult the Material Safety Data
Sheet (MSDS) from the manufacturer before you begin to use the chemical to
determine the physical and health hazards associated with it. For example:
1. Does it contain hazardous ingredients?
2. What are its physical/chemical characteristics?
3. Are there fire and explosion hazards?
4. Are there health hazards?
5. What are the first aid procedures?
6. What should be done if the chemical is spilled?
7. How should the chemical be stored?

B. Know how to protect yourself when using chemicals.

1. Contain the chemical within the work area.
a. Cover the work area with absorbent paper towels.
b. Choose a work area with adequate ventilation.
2. Maintain a barrier between the potentially hazardous chemical and yourself by
wearing protective clothing such as a lab coat, safety goggles, gloves, etc.
3. Be prepared to safely clean up accidental spills. (Consult the MSDS.)
4. Assess the size of the spill, e.g., What is the exposure potential during clean up?
Large spills may have an added risk of serious inhalation exposure.

C. Large spill example: A gallon jug of bleach was dropped and the side split, spilling the
entire contents on the floor.
1. This spill could create enough toxic vapors to possibly damage the lungs of the
individual exposed long enough to clean up the spill.
2. Evacuate the area.

ACHDLS - Laboratory Safety 16 Revised February 2012

 3. Open the doors and windows to ventilate the area.
4. Limit the exposure time of each individual by having several people take turns
containing and absorbing the spill.

D. Small spill example: A small puddle of bleach landed on the counter while measuring it
out for a 1:10 solution.
1. The spill is very small and easy to manage. Possible skin irritation would be the
hazard most likely encountered.
2. Maintain the spill within the area where the accident occurred.
3. Wear protective clothing as applicable.
4. Use absorbent towels to contain, carefully wipe up, and properly dispose of the
debris.
5. Rinse the area well to remove all traces of the chemical. (In some instances, such
as strong acids or bases, this would involve a neutralizing agent).

III. Mechanical Hazards

A. Use proper lifting techniques, putting the weight on the leg muscles to avoid back
strain.

B. Avoid frayed electrical cords and overloaded outlets to prevent an electrical fire.

C. Any electrical equipment that produces a “tingle” when touched should be disconnected
and sent for repair.

D. Keep electrical cords coiled and away from sink areas to prevent instrument
displacement and electrical shock.

E. Use caution signs to prevent falls when floors are wet.

F. Keep walkways free of articles such as pencils and paper clips that could cause unsure
footing.

G. Avoid exposed sharp corners such as open drawers and cabinet doors.

H. Avoid carrying sharp objects without protective coverings.

I. FOLLOW ALL LOCAL FIRE AND SAFETY CODES.

IV. References

A. Balows, Alvert. Manual of Clinical Microbiology, 5th Edition, American Society of

Microbiologist, Washington, D.C., 1991.

B. Baron, Ellen Jo and Finegold, Sydney M. Bailey and Scott’s Diagnostic Microbiology,
8th Edition, Mosby Co., St. Louis Co.

ACHDLS - Laboratory Safety 17 Revised February 2012

 C. Kent, P.T. and Kubica G.P. Public Health Mycobacteriology: A Guide for the Level
III Lab., Centers for Disease Control PHS,HHS, Atlanta, GA, 1985.

D. Kentucky Department for Health Services, Division of Laboratory Services.

Recommended Laboratory Safety Measures for County Health Departments/Centers,
1987.

E. Kentucky Labor Cabinet, Division for Education and Training. Hazard

Communication Program, 1988.

F. National Committee for Clinic Laboratory Standards. Protection of Laboratory

Workers from Infectious Disease Transmitted by Blood, Body Fluids, and Tissue,
Volume 9, Number 1, 1989.

ACHDLS - Laboratory Safety 18 Revised February 2012

Miscellaneous
Equipment
 MISCELLANEOUS EQUIPMENT
NOTE: ANY EQUIPMENT NOT IN USE IS TO BE PLACED IN STORAGE.

I. General Purpose Centrifuges

A. In general, centrifuges are used as filtration and/or packing devices for the separation of
components within a liquid or from a liquid medium, e.g., separating plasma or serum
from the cellular components of blood. Adequate and proper separation without
damage to the components depends upon proper spin (centrifugal force produced by the
speed and length of the radial arm and the time the material is spun). Proper balance,
lubrication, and rotor function are also important in producing the desired result.
Ensuring proper instrument function with regular maintenance is absolutely essential.

B. General purpose centrifuges place a centrifugal force upon fluids to separate out
cellular components. The centrifugal forces increase with the radial arm length and
increasing speed. Usually the radial arm length is fixed, so most centrifuges increase
centrifugal forces by increasing the speed. Speeds of 5,000 revolutions per minute
(RPMs) are generally adequate for most laboratory purposes. Normograms for
calculation of relative centrifugal force (RCF) are available. The RCF can be
calculated by using the following formula:
RCF = 28.38 (r) [n/1000]2
Where r = rotor radius in inches
n = revolutions per minute (RPM)
RCF is expressed in gravities (g)
The RCF is important to know because different centrifuges with different rotor radii
will result in different RCFs when the RPM is equal.

C. Components of a centrifuge include:

1. The chamber which encloses the head and centrifuge tubes.
2. A cover.
3. The shaft and rotor (which turns the head).
4. The motor and drive assembly which impart the force to the shaft and rotor to create
the desired centrifugal force.
5. A control panel with a power switch.

The centrifuge may also have a braking device, speed controls, a timer, or a fuse. Some
larger centrifuges may be equipped with a tachometer.

When tubes of blood are subjected to the proper centrifugal force, the serum and clot
separate. If a serum “separator” is in the tube, it will come to reside between the serum
and the cells and cover the cells completely to prevent further cell-serum contact. This
must be performed without damage to the cellular components to prevent release of
materials such as potassium from the cells. For routine serum separations, clotted
blood is centrifuged for 10 minutes at an RCF of 850 to 1000g.

ACHDLS - Miscellaneous Equipment 19 Revised February 2012

 D. Function Checks: See “Centrifuge Preventive Maintenance”
1. RPM – RPMs will be checked with a tachometer annually and after repairs. The
manufacturer’s instructions should be followed when operating the tachometer.
Documentation of RPM checks will be maintained.
2. Timer – If the centrifuge is equipped with a timer, it will be checked at least
annually against a standard time source. The results must be recorded and any
correction factors posted on the unit. If a correction factor is necessary, the time
should be checked against multiple times (e.g., 1 minute through 10 minutes) and
corrective factors posted for each time. When used to check a constant packed cell
volume, multiple times will be checked to the point (and beyond) where constant
packing is achieved to ensure that the constant packing will always be obtained
even though the timer might not be functional to the desired degree.
3. Speed Control – If the centrifuge has a speed control, settings must be checked,
verified, and performed at the same time the RPMs are checked. All regularly used
speeds should be checked. The readings must be recorded along with any of the
settings. If correction factors are critical, the correction factors will be posted with
the settings.

E. Maintenance
Always unplug the centrifuge from its electrical source before conducting preventive
maintenance, cleaning, and/or inspection. Use only recommended cleaners.
1. Spillage – Every spill will be cleaned up at the time of its occurrence to avoid
materials getting into the mechanism. Biological substances will be handled with
universal precautions. Be particularly cautious when cleaning up broken glass.
2. Unusual previously unnoted noises and/or vibrations – Noises and vibrations should
be listened/looked for with each use of a centrifuge. Appropriate steps will be taken
to determine the cause if they are noted. Damage to the centrifuge and/or the user
may result if vibrations or unusual noises are allowed to continue.
3. Cleaning the exterior and interior – The exterior should be cleaned each day of use.
Any spills will be cleaned up immediately. The chamber should be cleaned
monthly with soap and water followed by an adequate rinse. An appropriate
germicidal and virucidal (with minimal residual) disinfectant-type solution should
be used as the rinse. Gaskets must be washed and checked for wear and/or defects.
When tube shields and/or cups are removed and cleaned, make certain that all
rubber cushions and shields are replaced to maintain proper balance.
4. Inspection of the head, head shaft, and coupling – Inspect these components for
evidence of wear, cracks in fitting, corrosion, uneven wear, and/or signs of fatigue.
Immediately replace any part found to be unacceptable.
5. Rotor balance – Follow the manufacturer’s instructions to check the rotor balance
when any unusual vibrations occur.
6. Brushes, bearings, and commutators – Brushes will be checked as indicated in the
manufacturer’s manual. Replacement is generally indicated if they have worn
down by 5/16 to ½ inch or to within ¼ inch of spring depending upon the motor.
New brushes must slide freely in the holder but yet maintain spring tension to keep
them in good contact with the commutator.

ACHDLS - Miscellaneous Equipment 20 Revised February 2012

 a. The commutator must be checked for scratches and/or dirt. Check for anything
that impedes brush contact. In normal operation, a fine brush of sparks is seen
where the brushes contact the commutator. If this is excessive, the commutator
may need cleaning and/or smoothing. Seek help from an electrical service. The
commutator and brush holders must be kept free of oil, dust, and dirt to prevent
electrical arcing.
b. Always replace the centrifuge brushes oriented as you found them. The curved
surfaces of the brush must be oriented to match the curved surface of the
commutator prior to insertion. After new brushes have been installed, allow the
centrifuge to operate a few hours so the brushes may get accustomed prior to
routine use. Keep spare brushes available.
7. Power supply – Refer to the manufacturer’s instructions for a lubrication schedule.
Some centrifuges may not need further lubrication.
8. Gaskets, seals, mounts, and lubricants – Observe these for signs of excessive wear,
corrosion, and/or fatigue. Rubber gaskets and mounts may be lightly coated with a
light petroleum jelly.

II. Thermometers

Two basic types of thermometers are generally used in the clinical laboratory: bimetallic
and mercury column.

A. Bimetallic thermometers have a bimetallic element dampened with silicone and

completely enclosed in a stainless steel tube. The element changes its shape with
temperature, and this mechanical movement is connected to a dial that indicates
temperature. These thermometers do not break as easily as mercury column
thermometers but are usually not as precise. For this reason, they are acceptable for
monitoring refrigerators where high precision is not needed but where breakage of
thermometers is common. No maintenance is required for this type of thermometer
other than a periodic check of accuracy. If the thermometer is out of calibration,
discard it.

B. Mercury column thermometers have a relatively thin-walled cylindrical bulb of

mercury sealed to a length of capillary tubing. An increase in temperature causes the
mercury in the bulb to expand and rise higher in the capillary. A temperature range and
proper use depend on the type of thermometer available.

1. Periodically inspect each thermometer carefully for cracks in the capillary or bulb.
Check the mercury column for separations. If separations are found, the column
may be reunited by using one of the following methods:
a. Immerse the thermometer bulb in an ice-salt mixture until all mercury is drawn
into the bulb. Hold the thermometer in a vertical position, tap it gently to
dislodge gas bubbles, and allow it to warm at room temperature.
b. Hold the thermometer vertically in your fist so that the bulb is in the center of
the palm of your hand. If the mercury begins to move, continue until column is
reunited.

ACHDLS - Miscellaneous Equipment 21 Revised February 2012

 c. Firmly hold the thermometer horizontally at arm’s length and swing it down-
ward in a circular motion. Do not use a snapping movement. For thermometers
with ring tops, attach a string and carefully swing the thermometer in a circular
motion.
d. If all else fails, carefully warm the bulb over a low burner flame. Tilt the
thermometer slowly back and forth so that the mercury is gradually forced into
the expansion chamber. Set the thermometer in a vertical position for cooling.
The column should recede united.
2. After successfully reuniting the mercury column, check the accuracy of the
thermometer as outlined in the following procedure.

C. Calibration of thermometers
1. When placed in service, the accuracy of each thermometer will be checked against
an NIST-traceable thermometer. Mercury column thermometers should keep their
calibration unless the mercury separates; after reuniting the mercury column, the
thermometer should be calibrated. Bimetallic thermometers will need calibration
periodically. Correction factors should be noted on the thermometer and on the
temperature chart. Recorded temperatures will be those obtained after the
correction factor has been used. Thermometers with a correction factor greater than
1°C are not acceptable and will not be used.
2. Because of the cost of an NIST thermometer, thermometers checked against an
NIST will be used to calibrate laboratory thermometers. NIST-traceable
thermometers may be purchased from suppliers of laboratory equipment.
a. Obtain an NIST-traceable thermometer (formerly referred to as an NBS-
traceable thermometer).
b. Place the thermometer being calibrated and the NIST-traceable in the
refrigerator or incubator in which the thermometer will be routinely used. If the
thermometer is to be used for obtaining room temperatures, simply place the
NIST-traceable thermometer next to the thermometer to be calibrated. Allow
time for the temperature to stabilize.
c. Read the temperature on both thermometers.
d. Record readings on the “Thermometer Calibration Log”, and make note of the
correction factor, if any.
e. If the correction factor is greater than +1°C, the thermometer will be replaced
with one that reads within +1°C of the NIST-traceable.
f. Correction factors will be noted on the thermometer and on temperature charts.
g. When recording temperatures, the true temperature (thermometer reading + the
correction factor) will be recorded.

D. Placement of thermometers
Refrigerators and incubators may have “cold spots” or “hot spots”. Windows and air
vents may cause these same conditions in rooms. Therefore, it is important to place
thermometers in locations which reflect the most consistent temperature of the area.
Periodically changing locations of thermometers for short periods of time is useful in
locating “cold spots” and “hot spots” so that these areas can be avoided.

ACHDLS - Miscellaneous Equipment 22 Revised February 2012

III. Room Temperature: See “Annual Temperature Chart”

When performing RPR testing, the room temperature should be 23°C - 29°C (74°F - 84°F).
The Hemocue requires a room temperature of 18°C to 30°C (64°F to 86°F). The room
temperature of areas where reagents are stored will be maintained with the temperature
range defined by the reagent manufacturer. This range is noted on the package insert.

IV. Laboratory Refrigerators

Biological or chemical materials in conjunction with food and drinks designed for human
consumption will not be stored in the same refrigerator.

Note: Frost-free refrigerators will not be used if reagents or materials are temperature
sensitive.

A. Function Checks
1. Read and record internal temperature – The desired temperature range is 2°C to 8°C
(36°F-46°F).
a. A thermometer should be placed inside the refrigerator in a location where it is
protected and easily read without disturbing.
b. A temperature chart will be attached to the front or side of each refrigerator.
c. Temperatures will be checked and recorded on the appropriate chart each day
the clinic is open.
d. If the temperature is not within the range of 2°C to 8°C, actions will be taken to
remedy the problem (e.g., adjust the thermostat).
i. If the thermostat is adjusted, recheck the temperature.
ii. If the temperature is still not within the acceptable range and adjusting the
thermostat has not solved the problem, move the contents of the refrigerator
to another refrigerator and contact a repairman.
iii. Dispose of materials which could have been damaged (e.g., items which were
frozen were stored for a long period of time at temperatures above 8°C).
iv. Record corrective actions on the temperature chart.
2. Check door gasket – Ensure that the door gasket seals properly when the door is
closed. If the door does not seal properly and gasket appears to be adequate, the
refrigerator may not be level.

B. Maintenance
1. Defrost unit – If the refrigerator is not frost-free, defrost the unit approximately
every three months. If frost buildup is a problem, defrosting may be required more
frequently. Follow the manufacturer’s instructions for defrosting.
2. Periodic cleaning – The interior should be decontaminated periodically. The
exterior will be cleaned, when necessary, with a damp cloth and mild soap.

V. Microbiology Incubators

ACHDLS - Miscellaneous Equipment 23 Revised February 2012

 The purpose of a microbiology incubator is to promote the growth of microorganisms such
as Neisseria gonorrhoeae. It is constructed to maintain a temperature of 35°C – 37°C
(95°F – 98.6°F).

A. Function Checks: See “Annual Temperature Chart and Incubator Preventive

Maintenance”
1. Read and record internal temperature
a. A suspended thermometer is to be placed inside the incubator in a location where
it is protected and easily read without disturbing.
b. A temperature chart will be posted on the incubator.
c. Temperatures will be checked and recorded on the temperature chart each day
that the clinic is open.
d. If the temperature is not within the range of 35°C to 37°C, actions will be taken
to remedy the problem (e.g., adjust the thermostat).
i. If the thermostat is adjusted, recheck the temperature.
ii. After allowing time for the temperature to change, read the temperature
again.
iii. Continue adjusting the thermostat and monitoring the temperature until an
acceptable temperature is obtained and maintained.
iv. If this cannot be accomplished, contact a repairperson.
v. Document all corrective actions on the temperature chart.
vi. If necessary, relocate the contents of the incubator to protect them from
extremely high temperatures.
2. Check door gasket – Ensure that the door gasket seals properly when the door is
closed.

B. Maintenance
Clean interior of incubator – The interior of the unit will be cleaned at least monthly.
1. Empty the incubator, and clean the interior with a warm solution of water and
appropriate disinfectant.
2. Rinse the interior with clean water and dry thoroughly.
3. Removable parts may be washed in warm, sudsy water. Avoid soap-filled pads or
metal scouring pads.
4. Return parts and contents to the incubator.

VI. References

A. College of American Pathologist. Laboratory Instrument Evaluation Verification and

Maintenance Manual, 4th edition (ISBN 0-930304-35-7), 1989.

B. National Committee for Clinic Laboratory Standards. Temperature calibration of water

baths, instruments, and temperature sensors – second edition; Approved Standard.
NCCLS Document 12-A2. Villanova, Pa.: NCCLS, 1990.

ACHDLS - Miscellaneous Equipment 24 Revised February 2012

 Centrifuge Preventive Maintenance
County Year
Site
Monthly maintenance consists of unplugging the centrifuge, cleaning the exterior and interior of the centrifuge with a suitable
disinfectant, and drying thoroughly. After cleaning, plug the centrifuge back into the power source. If the centrifuge is not in use,
monthly cleaning is not necessary. Centrifuge must have a cover in use during operation. Document on the chart when the centrifuge
is not in use.
MONTHLY JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC
Clean Exterior and Interior
Date
Initials

Function Checks below will be performed by the Technical Consultant annually.

RPM’s Acceptable Unacceptable
Time on centrifuge
Time on stopwatch Acceptable Unacceptable
Date
Technical Consultant Signature

CORRECTION ACTION LOG:

Date Problem Resolution Initials

ADPH/BCL/QM REV 10-09 Write NIU when not in use.

 Annual Temperature Chart
Check (F) __ or (C) __ Scale used.
Circle equipment and acceptable range used: Incubator 95°F -99°F (35°C - 37°C) Refrigerator 35°F - 46°F (2 - 8°C) Freezer 5F or colder (-15°C or colder) or Room 64°F - 86°F (18°C - 30°C)

Date Jan Intls Feb Intls Mar Intls Apr Intls May Intls June Intls July Intls Aug Intls Sept Intls Oct Intls Nov Intls Dec Intls

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

Correction Action Log on Back County _______________ Year ____________

FOOD OR BEVERAGE IS PROHIBITED FROM STORAGE IN CLINIC REFRIGERATORS/FREEZERS AT ALL TIMES!
ADPH/BCL//QM 10-09
 Temperature Corrective Action Log

Date Temp Adj Intls Recheck Temperature (C) DATE COMMENT

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ADPH/BCL/QM 10-09
 Incubator Preventive Maintenance
County Year
Site

MONTHLY JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC
Clean Exterior and Interior
Check Door Gasket for Proper Seal
Insure Latch Secures Properly
Date
Initials

Climate control bags must be properly sealed when used in the incubator. Please note on the
“Corrective Action Log” below if problems occur. No response recorded indicates that there
have been no climate control bag issues.

CORRECTION ACTION LOG:

Date Problem Resolution Initials

ADPH/BCL/QM REV 10-09 Write NIU when not in use.

Waived Tests
 WAIVED TESTS
PERFORMED BY ALABAMA COUNTY HEALTH DEPARTMENT
LABORATORY SYSTEMS
I. Hemoglobin detection with the HemoCue Hb 201+ Analyzer

A. Provides determination of hemoglobin quickly, easily and with quality results.

Capillary, venous, or arterial whole blood may be used. Refer to your HemoCue 201+
Manual for the basic instructions as well as technical specifications. Manuals are
provided by HemoCue, Inc.

B. Specimen Collection
1. To perform a test using capillary blood, the cuvette holder should be in its loading
position. The display will show three flashing dashes and the HemoCue symbol.
2. Make sure the patient’s hand is warm and relaxed. Use only the middle or ring
finger for sampling. Avoid fingers on which rings are present.
3. Clean the finger with alcohol or a suitable disinfectant and allow it to dry or wipe it
off with a dry, lint-free wipe.
4. Using your thumb, lightly press the finger from the top of the knuckle towards the
tip. This stimulates the blood flow towards the sampling point.

5. For best blood flow and least pain, sample at the side of the fingertip, not in the
center.
6. While applying light pressure towards the fingertip, puncture the finger using a
lancet.
7. Wipe away the first 2 or 3 drops of blood.
8. Re-apply light pressure towards the fingertip until another drop of blood appears.

ACHDLS –Waived Tests 25 Revised February 2012

 9. When the blood drop is large enough, fill the microcuvette in 1 continuous process.
Do NOT refill!
10. Wipe off excess blood from the outside of the microcuvette with a clean, lint-free
wipe being careful not to touch the open end of the microcuvette, which could result
in blood being drawn out of it.
11. Look for air bubbles in the filled microcuvette. If present, discard the microcuvette,
and fill a new one from a new drop of blood. Small bubbles around the edge can be
ignored.

12. Place the filled microcuvette in the cuvette holder. This must be performed within
10 minutes after filling the microcuvette!
13. Gently slide the cuvette holder to the measuring position.
14. During the measurement, an hourglass and 3 fixed dashes will be shown on the
display.
15. After 15-60 seconds, the hemoglobin value of the sample is displayed. The result
will remain on the display as long as the cuvette holder is in the measuring position.
When operating on battery power, the analyzer will automatically turn off after
approximately 5 minutes.
16. Although the reagents are present in the microcuvette in extremely low quantities,
consult local environment authorities for proper disposal. Always handle blood
specimens with care; they might be infectious.

ACHDLS –Waived Tests 26 Revised February 2012

 C. Report results on CHR-11.

See “HemoCue Maintenance Chart”

See “Annual Temperature Chart”
See “HemoCue Trouble Shooting Chart” (also found in HemoCue 201+ Manual)

II. Occult Blood Test

A. This product is a guaiac slide test for the qualitative detection of fecal occult blood. It
is useful in the diagnosis of a number of gastrointestinal disorders and is recommended
for use in:
1. Routine physical examinations.
2. Routine hospital testing.
3. Mass screening for colorectal cancer.

B. Please refer to the package inserts of the specific kit you are using.

C. Report results on CHR-11.

Note: A package insert of the test kit should be placed in a sheet protector and placed in
this manual immediately after this section.

III. Strep A (Rapid Test)

ACHDLS –Waived Tests 27 Revised February 2012

 A. An Enzyme Immunoassay for the Rapid Detection and Confirmation of Group A
Streptococci.

B. Please refer to the package inserts of the specific kit you are using.

C. Report results on CHR-11.

Note: A package insert of the test kit should be placed in a sheet protector and placed in
this manual immediately after this section.

IV. Urinalysis - Dipstick Method

A. The chemical analysis of urine is performed using one of many different urine reagent
strips (dipsticks) available from a variety of manufacturers. Reagent strips for
urinalysis are firm plastic strips to which are affixed several separate reagent pads. A
color reaction develops upon contact of the urine with the reagent pads. The following
are commonly available on urine dipsticks:
1. Protein.
2. Glucose.
3. Ketone bodies.
4. Bilirubin.
5. Leukocyte esterase.
6. pH.
7. Occult blood/hemoglobin.
8. Urobilinogen.
9. Nitrite.
10. Specific gravity.

B. Refer to the package inserts for the specific reagent areas on the product you are using.

C. Report results on CHR-11.

Note: A package insert of the test kit should be placed in a sheet protector and placed in
this manual immediately after this section.

V. Urine Pregnancy Test

A. Urine pregnancy test measures the presence of human chorionic gonadotrophin (HCG)
in urine for the early detection of pregnancy.

B. Refer to the package inserts of the specific kit you are using.

C. Report results on CHR-11 or CHR-12c.

Note: A package insert of the test kit should be placed in a sheet protector and placed in
this manual immediately after this section.

ACHDLS –Waived Tests 28 Revised February 2012

County HemoCue Maintenance Chart Year
Instrument SS# Write P (pass) or F (fail) for internal control function.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
January
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
February
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
March
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
April
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
May
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
June
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
July
Clean Cuvette Holder
Check Internal Control
Initials

Monthly Maintenance January February March April May June July

Optical Lens Monthly
Cleaning
Date/Initials

“NIU” indicates “not in use”. ADPH/BCL/QM Rev 10/11

County HemoCue Maintenance Chart Year
Instrument SS# Write P (pass) or F (fail) for internal control function.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
August
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
September
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
October
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
November
Clean Cuvette Holder
Check Internal Control
Initials
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
December
Clean Cuvette Holder
Check Internal Control
Initials

Monthly Maintenance August September October November December

Optical Lens Monthly
Cleaning
Date/Initials

CORRECTIVE ACTION LOG: Date / Problem / Corrective Action / Initials

HemoCue Controls (Lot number, range and values will be documented by Technical Consultant):
Level 1 -
Level 2 -
Level 3 -

“NIU” indicates “not in use”. ADPH/BCL/QM Rev 10/11

 Annual Temperature Chart
Check (F) __ or (C) __ Scale used.
Circle equipment and acceptable range used: Incubator 95°F -99°F (35°C - 37°C) Refrigerator 35°F - 46°F (2 - 8°C) Freezer 5F or colder (-15°C or colder) or Room 64°F - 86°F (18°C - 30°C)

Date Jan Intls Feb Intls Mar Intls Apr Intls May Intls June Intls July Intls Aug Intls Sept Intls Oct Intls Nov Intls Dec Intls

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Correction Action Log on Back County _______________ Year ____________
FOOD OR BEVERAGE IS PROHIBITED FROM STORAGE IN CLINIC REFRIGERATORS/FREEZERS AT ALL TIMES!
ADPH/BCL//QM 10-09
 Temperature Corrective Action Log

Date Temp Adj Intls Recheck Temperature (C) DATE COMMENT

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ADPH/BCL//QM 10-09
 HemoCue Hb 201+ Trouble Shooting Guide

If you are unable to resolve the problem by following this Trouble Shooting Guide, please
contact HemoCue, Inc. The analyzer has no serviceable parts.

Symptom Explanation Action

The analyzer shows May be a temporary fault Turn off the analyzer and turn it on
an error code. again after 30 seconds. Take a new
microcuvette and repeat the
measurement. If the problem
continues, see specific error code.
E00 No stable endpoint is found 1a. Check the expiration date of the
within the time range. microcuvettes.
1. The cuvette is faulty. 1b. Use a new microcuvette and
2. The circuit board is out of repeat the measurement.
order. 2. The analyzer needs service.
Contact HemoCue, Inc.
E01-E05 1. Dirty optronic unit, faulty 1a. Turn off the analyzer and clean
electronic or optronic unit. the optronic unit as described in
the maintenance section.
1b. The analyzer needs service.
Contact HemoCue, Inc.
E06 1. Unstable blank value. The 1. Turn off the analyzer and allow it
analyzer might be cold. to reach room temperature. If the
problem continues, the analyzer
needs service.
Contact HemoCue, Inc.
E07 1. The battery power is too 1a. The batteries need to be replaced.
low. Turn off the analyzer and replace
the batteries (four type AA).
1b. Use the power adapter.
E08 The absorbance is too high. 1a. Check that the analyzer and
1. An item is blocking the microcuvetts are being used
light in the cuvette holder. according to the HemoCue Hb
201+ operating manual and
instructions for use.
1b. The analyzer needs service.
Contact HemoCue, Inc.
E09-E30 1. Dirty optronic unit, faulty 1a. Turn off the analyzer and clean
electronic or optronic unit. the optronic unit as described in
the maintenance section.
1b. The analyzer needs service.
Contact HemoCue, Inc.
HHH 1. Measured value 1. Consult Nursing Protocol for
exceeds 25.6 g/dl (256 action.
g/L, 15.9 mmol/L)
Symptom Explanation Action
No characters on 1. The analyzer is not 1a. Check that the power adaptor is
the display receiving power. connected to the AC power
2. If on battery power, the supply.
batteries need to be 1b. Check that the power adaptor is
replaced. securely connected to the
3. The display is out of analyzer.
order. 1c. Check that the cable is not
damaged.
2. Turn off the analyzer and replace
the batteries (four type AA).
3. The analyzer needs service.
Contact HemoCue, Inc.
The display gives 1. The display is out of 1. The analyzer needs service.
erroneous order. Contact HemoCue, Inc.
characters. 2. The microprocessor is out 2. The analyzer needs service.
of order. Contact HemoCue, Inc.
The display shows a 1. The batteries need to be 1. Turn off the analyzer and replace
“partially shaded” replaced. the batteries (four type AA).
battery. 2. If on AC power, the power 2a. Check that the power adaptor is
adaptor or the circuit properly connected and working.
board is out of order. 2b. The analyzer needs service.
Contact HemoCue, Inc.
The display does 1. The magnet in the cuvette 1. The analyzer needs service.
not switch to the holder may be missing. Contact HemoCue, Inc.
three flashing 2. The magnetic sensor is out 2. The analyzer needs service.
dashes (ready for of order. Contact HemoCue, Inc.
measuring).
Measurements on 1. The microcuvettes are 1. Check the expiration date and the
control materials beyond their expiration storage conditions of the
are out of range - date, damaged or have microcuvettes.
too high or too low. been improperly stored. 2. Measure the sample with a new
2. The optical eye of the microcuvette.
microcuvette is 3. Make sure that the control is
contaminated. mixed properly and at room
3. The control has not been temperature.
mixed properly and/or is 4. Check the microcuvette for air
not at room temperature. bubbles. Measure the sample with
4. Air bubbles in the a new microcuvette.
microcuvette. 5. Clean the optronic unit as
5. The optronic unit is dirty. described in the maintenance
6. The control is not suitable section.
for use with the HemoCue 6. Only use controls intended for the
Hb 201+ system. HemoCue Hb 201+ system.
7. The calibration of the Contact HemoCue, Inc. for
analyzer has been control information.
Symptom Explanation Action
changed. 7. The analyzer needs service.
8. The controls are beyond Contact HemoCue, Inc.
their expiration dates or 8. Check the expiration dates and
have been improperly storage conditions of the controls.
stored. Take a new microcuvette and
repeat the measurement from a
new vial/bottle of control.
Measurements on 1. Improper sampling 1. See pages 8-17 in the HemoCue
patient samples are technique. Hb 201+ manual.
higher or lower than 2. The microcuvettes are 2. Check the expiration dates and
anticipated. beyond their expiration storage conditions of the
date, damaged or have microcuvettes. Check the entire
been improperly stored. system with a commercial control.
3. The optical eye of the 3. Measure the sample with a new
microcuvette is microcuvette.
contaminated. 4. Check the microcuvette for air
4. Air bubbles in the bubbles. Measure the sample
microcuvette. with a new microcuvette.
5. The optronic unit is dirty. 5. Clean the optronic unit as
6. The calibration of the described in the maintenance
analyzer has changed. section.
6. The analyzer needs service.
Contact HemoCue, Inc.
Brightfield Microscopy
 BRIGHTFIELD MICROSCOPY
I. Principle

A. The microscope is perhaps the piece of equipment that receives the most use and,
unfortunately, misuse in the clinical laboratory. Microscopy is necessary to see
microbial cells and to determine morphological characteristics. It is a basic part of
work in many areas of the laboratory such as hematology, urinalysis, and microbiology.
Because the microscope is such an important piece of equipment and a precise
instrument, it must be kept in excellent condition, optically and mechanically. It must
be kept clean and aligned.

B. In simple terms, a microscope is a magnifying glass. The compound light, or

brightfield microscope (used in most clinical laboratories), consists of two magnifying
lenses: the objective and the eyepiece (ocular). It is used to magnify an object so it can
be seen with the human eye.

C. The total magnification observed is the product of the magnifications of these two
lenses. In other words, the magnification of the objective times the magnification of
the ocular equals the total magnification. The magnitude of magnification is inscribed
on each objective as a number. These magnification units are in terms of diameters
(10x, 40x, and 100x). Thus, 10x means that the diameter of an object is magnified 10
times its original size.

D. Because of the manner in which light travels through the brightfield microscope, the
image that is seen is upside down and reversed. The right side appears as the left, the
top as the bottom, and vice versa. This should be kept in mind when moving the slide
(or object) being observed.

II. Glossary of Terms

A. Aperture diaphragm - either a rotating disk or an iris diaphragm on the condenser used
to direct a cone of light to the specimen and objective. It should never be used to
regulate brightness. Resolution, control, and depth of field depend on the correct
setting of the aperture diaphragm.

B. Arm - for holding the microscope while carrying it.

C. Coarse adjustment knob - for rapid focusing of the specimen.

D. Compound microscope - a microscope made up of two lens systems: eyepiece and

objective.

E. Condenser - the lens system beneath the microscope stage positioned to concentrate
light correctly on the specimen and direct light rays into the objective. When the
condenser is used at a lowered position, the resolving power is reduced.

ACHDLS - Brightfield Microscopy 29 Revised February 2012

 F. Depth of field - distance just above and below the focal plane (area being examined)
that can be focused clearly.

G. Eyepiece – lens system of the microscope nearest to the eyes.

H. Field diaphragm - an aperture diaphragm which restricts area of illumination.

I. Fine adjustment knob - focuses the lens in small increments.

J. Immersion oil – oil with the same refractive index as glass, 1.515; used between the
cover glass and an oil immersion objective to prevent scattering of light in air.

K. Interpupillary distance - the distance between the eyes. The eyepieces of a binocular
scope must be adjusted so that left and right images merge into one.

L. Koehler illumination - optical illumination providing bright, evenly dispersed, glare-

free light with good contrast and resolution.

M. Nosepiece - a revolving plate that holds the objectives.

N. Ocular – eyepiece (lens system of the microscope nearest to the eyes).

O. Parcentric - the ability to center a specimen in the field of view for one objective and
have almost the same field in place when rotating to another objective.

P. Parfocal - the objectives are constructed so that only slight refocusing with the fine
adjustment knob is needed after rotating to another objective.

Q. Resolution - the ability of a microscope to reveal fine detail in a specimen. The better
the resolving power of a microscope, the closer two objects can be and still be
distinguished as two objects.

R. Stage - the platform on which the microscope slide is placed.

S. Working distance - distance between the coverslip of a slide and the tip of an objective.
The low power objective has the greatest working distance. The oil immersion
objective has a very small working distance.

III. Parts of the Microscope

A. The basic structures of compound microscopes are categorized into 4 groups (see
“Microscope Diagram”): the framework, the illumination system, the magnification
system and the adjustment system.

1. Framework - consists of several units

a. The base - a firm, horseshoe-shaped foot on which the microscope rests.

ACHDLS - Brightfield Microscopy 30 Revised February 2012

 b. The arm - the structure that supports the magnifying and adjusting systems. It is
also the handle by which the microscope can be carried without damaging the
delicate parts.
c. The stage - the horizontal platform, or shelf, on which the object being observed
is placed. Most microscopes have a mechanical stage, which makes it easier to
manipulate the object being observed.

2. Illumination - Good microscope work cannot be accomplished without proper

illumination.
a. Light source (or bulb) – The illumination system begins with a source of light.
Is often built in the microscope.
This illumination system has a control to regulate light intensity, ensuring
adequate illumination and comfort for the employee.
Is located at the base of the microscope, and the light is directed up through
the condenser system.
It is important that the bulb is positioned correctly for proper alignment
throughout the microscope.
Modern microscopes are designed so that the light bulb filament will be
centered if the bulb is installed properly.
b. Condenser
Microscopes generally use a substage “Abbe” condenser.
It directs and focuses the beam of light from the bulb onto the material under
examination.
Its position is adjustable; it can be raised or lowered beneath the stage by an
adjustment knob.
The iris in the condenser can also be opened or closed by an adjustment
knob.
The condenser must be adjusted with each objective used in order to
maximize the light focus and the resolving power of the microscope. This
adjustment is often necessary in the clinical laboratory when observing wet,
unstained preparations such as vaginal preps.
c. Field diaphragm
It controls the amount of light passing through the material under
observation.
It is located at the bottom of the microscope.
It contains an iris that can be opened or closed to adjust the intensity of the
light by a lever.
Proper illumination techniques involve a combination of proper light
intensity regulation, light source position, condenser position, and field size
regulation.

d. Koehler Illumination
For optimum results in light microscopy, it is crucial that the light path be set
properly before the light reaches the specimen. In 1893, German scientist

ACHDLS - Brightfield Microscopy 31 Revised February 2012

 August Koehler introduced this technique for applying exact control of the light
path in the illuminating beam.
i. Turn the lamp to the lowest setting.
ii. Raise the condenser to the highest setting.
iii. Open the condenser completely and close the field diaphragm completely.
iv. Adjust eyepieces so only one circle of light is observed.
v. Place prepared slide in slide brackets, centering over the light well.
vi. Starting with 10x objective and stage closest together, focus on the
specimen. This can be done without using a slide.
vii. A small circle of light should be visible in the eyepieces.
viii. While viewing, lower the condenser slightly until the image is in focus (a
polygonal outline with a pink-reddish tinge). You may need to close the
condenser diaphragm.
ix. Center the polygonal image in the field of view using the condenser
centering screws.
x. Open the field diaphragm to the edge of the field of view. Note: If needed,
re-center the image by adjusting the condenser centering screws.
xi. After it appears centered, open the field diaphragm until the image just
clears the field of view.
Note: According to which manufacturer’s microscope is in the county health
department, it may not be possible to set Koehler Illumination. Some
manufacturers align the optics during manufacturing so that it does not have to
be done on a daily basis. The microscope must have a field diaphragm in order
to set Koehler Illumination. In addition, if one person uses the microscope, it is
not necessary to set Koehler Illumination daily. If there are questions
concerning optical alignment, perform Koehler Illumination if possible.

3. Magnification System – contains important parts and plays a vital role in the use of
the microscope.
The ocular (eyepiece) is a lens that magnifies the image formed by the
objective.
The usual magnification of the ocular is 10x.
Most microscopes have two oculars and are called binocular microscopes.
Some microscopes only have one ocular and are called monocular microscopes.
The magnification produced by the ocular, when multiplied by the
magnification produced by the objective, gives the total magnification of the
object being viewed.
There are usually three objectives on each microscope, with magnifying powers
of 10x, 40x, and 100x. They are mounted on the nosepiece - a pivot enabling a
quick change of the objectives. They are described or rated according to focal
length (inscribed on the outside of the objective). The focal length of a lens is
very close to the working distance. The greater the magnifying power of a lens,
the smaller the focal length or the working distance.
a. Low-Power Objective:
i. Is usually a 10x magnification lens.

ACHDLS - Brightfield Microscopy 32 Revised February 2012

 ii. Is used for the initial scanning and observation in most microscope
work.
iii. Is employed for the initial focusing and light adjustment of the
microscope.
iv. There are two terms often associated with a microscope: parfocal and
parcentric.
o Parfocal means if one objective is in focus and a switch is made to
another objective, the focus will not be lost. Thus, the microscope
can be focused under low power and then switched to the high power
or oil immersion objective, and it will still be in focus except for fine
adjustment.
o Parcentric means that when an object is centered in the field of view
using low power and then switched to high power or oil immersion,
the object will still be in view and centered with only minor
adjustments needed.
b. High-Power Objective (High-Dry Objective):
i. Is usually a 40x magnification lens.
ii. Is used for more detailed study, as the total magnification with a 10x
eyepiece is 400x rather than the 100x of the low-power system.
iii. Is used to study wet preparations in more detail.
c. Oil-Immersion Objective:
i. Is generally a 100x lens. The objective almost rests on the microscope
slide when in use.
ii. An oil-immersion lens requires a special grade of oil (immersion oil –
used to increase the resolving power of the objective), to be placed
between the objective and the slide or coverglass.
iii. The oil-immersion lens, with a total magnification of 1000x when used
with a 10x eyepiece, is generally the limit of magnification with the
brightfield microscope. It is routinely used for morphologic
examination of microbes. The short working distance requires dry films,
so wet preparations cannot be examined under an oil-immersion lens.

4. Adjusting System
The body tube is the part of the microscope through which the light passes to
the ocular.
The tube length from the eyepiece to the objective lens is generally 160 mm.
This is the tube that actually conducts the image.
The adjustment system enables the body tube to move up or down for focusing
the objectives.
This system usually consists of two adjustments, one coarse and the other fine.
▪ Coarse adjustment - gives rapid movement over a wide range and is used to
obtain an approximate focus. This adjustment should only be used with the
10x objective.
▪ Fine adjustment - gives very slow movement over a limited range and is
used to obtain exact focus after prior coarse adjustment. This adjustment
should only be used with the 40x and 100x objectives.

ACHDLS - Brightfield Microscopy 33 Revised February 2012

IV. Maintenance
See “Microscope Maintenance Chart”

A. Daily Maintenance
1. Clean all optical surfaces with lens cleaner and cotton-tipped applicators and/or lens
paper.
a. Eyepieces, objectives, and condenser should be cleaned using a cotton-tipped
applicator.
b. The light source should be cleaned using lens paper.
2. Protect the microscope with a cover (or trash can liner if other cover is unavailable)
when not in use.
3. Initial that “1” and “2” have been completed.

B. Monthly Maintenance
1. Clean all nonoptical surfaces with mild detergent, and rinse with warm water.
2. Check KOH solution for contamination.
a. If contaminated, replace with in-date KOH.
b. If transferring KOH from a stock bottle to a working bottle, label the
working bottle with name of reagent, % strength, and expiration date.
3. Check saline solution for contamination.
a. If contaminated, replace with in-date saline.
b. If transferring saline from a stock bottle to a working bottle, label the working
bottle with name of reagent, % strength, and expiration date.
4. Check the appearance of the pH paper according to the pH paper package insert.
5. Initial that all duties listed above have been completed.

C. Annual Professional Microscope Maintenance

1. Microscopes should be serviced annually by a professional.
2. Document the company performing the annual cleaning.
3. Document the date the annual cleaning was performed.

V. Corrective Action Log

See “Microscope Maintenance Chart”

A. Document any problems encountered with the microscope.

B. Document date of the problem.

C. Document corrective action taken.

D. Document initials of employee performing corrective action.

VI. Additional Information

A. Turn lamp voltage down and then off if the microscope has a rheostat.

ACHDLS - Brightfield Microscopy 34 Revised February 2012

 B. If slideways and gears on mechanical stage become difficult to move, lubricate with
machine oil or light grease. Document in the corrective action section of the
maintenance chart.

C. Keep at least one extra bulb available in case the bulb in the microscope blows. Contact
your technical consultant for ordering information. Follow manufacturer’s instructions
for ordering and replacing bulbs, or check with your technical consultant.

D. If your microscope requires a halogen bulb, DO NOT TOUCH the new bulb with your
fingers or gloves. Install it with a lint-free tissue.

E. Avoid exposure to corrosive fumes, extreme heat or cold, or sudden drastic temperature
change. When moving from one temperature extreme to another, allow optical parts to
equilibrate until all moisture has evaporated.

F. If immersion oil is required, use only chemically inert, low fluorescent, PCB-free
immersion oil. Low viscosity is acceptable. DO NOT USE CEDAR WOOD OIL.
Different brands of immersion oil are incompatible and should not be mixed.

G. Use a small amount of oil in a frequently-cleaned application bottle to minimize growth

of fungi which may reflect erroneously on specimen content.

VII. Helpful Hints

A. Turn on the light source.

B. Be sure low objective is in position.

C. Rack down, then up until field is in focus.

D. When moving from one objective to another, use fine adjustment knob to refocus.

E. Never drag high-dry objective through oil.

F. The higher the magnification, the more light you will need.

G. Always clean oil from objective and/or condenser at the end of the day.

H. Be sure low power objective is in working position at the end of the day.

I. Cover the microscope when you have finished for the day.

J. Try not to place the microscope on the same work bench as a centrifuge to avoid the
microscope and specimen from vibrating. If they will be on the same bench, secure a
mat that absorbs vibrations to place under the microscope.

ACHDLS - Brightfield Microscopy 35 Revised February 2012

 Microscope Diagram

ACHDLS - Brightfield Microscopy 36 Revised February 2012

VIII.Troubleshooting

Several conditions can affect good function of the microscope.

1. The brightness of the viewing field is poor.

Problem Solution
The condenser is too low. Raise the condenser to correct its position.
The condenser iris diaphragm is closed. Open the diaphragm properly.

2. There are dark shadows in the field which move as the eyepiece is turned.
Problem Solution
The surface of the eyepiece has scratches. Replace the eyepiece.
The eyepiece is dirty. Clean the eyepiece.

3. The image with the high power objective is not clear.

Problem Solution
The slide is upside down. Turn the slide over.
There is an air bubble in the oil. Move 100x lens quickly from side to side.
There is dirt on the objective. Clean the lens.
The oil is too sticky. Use specified immersion oil.

4. The image with the low power objective is not clear.

Problem Solution
There is oil on the lens. Clean the lens.

5. Objective lens hits specimen before achieving focus.

Problem Solution
Objective not screwed in nosepiece securely. Securely tighten objective in nosepiece.
Stage mounted too high. Call Annual Maintenance Personnel

IX. References

A. Alabama Department of Public Health. Wet Prep Competency; Microscope Teaching

Video Series, 2000.

B. Olympus. BX41 System Microscope Instruction Manual, 2003.

C. Olympus Video Library. The Basic Microscope - Use & Care.

ACHDLS - Brightfield Microscopy 37 Revised February 2012

 Microscope Maintenance Chart
County Year Manufacturer

January 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
February 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
March 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
April 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
May 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
June 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
July 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials

Monthly Maintenance January February March April May June July

Clean nonoptical surfaces
Check KOH solution
Check Saline
Check pH paper exp. date
Date/Initials

Annual Professional Microscope Cleaning Company “NIU” indicates not in use.

Annual Professional Microscope Cleaning Service Date ADPH/BCL/QM/ Rev 12-11
 Microscope Maintenance Chart
County Year Manufacturer

August 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
September 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
October 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
November 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
December 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials

Monthly Maintenance August September October November December

Clean nonoptical surfaces
Check KOH solution
Check Saline
Check pH paper exp. date
Date/Initials

CORRECTIVE ACTION LOG: Date / Problem / Corrective Action / Initials

NIU” indicates not in use. ADPH/BCL/QM/ Rev 12-11

Vaginal Wet Mount
 VAGINAL WET MOUNT

I. Principle

The direct microscopic examination of a saline suspension of material taken from the area of
inflammation can provide a quick presumptive diagnosis for Gardnerella vaginalis and
Candida albicans and/or a definitive diagnosis for Trichomonas vaginalis. The addition of
KOH to a portion of the suspension helps clear debris, allowing better visualization of C.
albicans (yeast).

II. Specimen Collection and Handling

A. Specimens from the human body may be infectious. Use universal precautions.

B. Patient preparation and collection procedures should be performed only by qualified

personnel. Procedure for inserting speculum and performing vaginal examinations
are found in the ADPH Nurse Orientation Manual: Vaginal Examination and
Specimen Collection Education Module.

C. Collect the specimen by rolling a cotton or Dacron® swab in vaginal discharge

pooled in the posterior fornix or in excess discharge on the speculum blade.
COLLECTION ORDER: Wet smears are collected first followed by the pap smear,
Gonorrhea /Chlamydia specimen, then the pelvic bi-manual examination.

D. There is no way to preserve the specimen, so immediately examine the slide

preparation under the microscope since trophozoites rapidly lose their motility.

E. Process the specimen by preparing 1 or 2 slides as described in the test procedure.

III. Specimen Rejection

A. Do not test specimens that:

1. Exceed the time limit (10 minutes) for immediate examination.
2. Are not properly labeled or have questionable identity.
3. Are collected after the use of a lubricant.

B. If the specimen does not meet the criteria for acceptability, collect a new sample.

IV. Reagent, Supplies, and Equipment

A. Normal saline (0.9% sodium chloride)

B. Potassium hydroxide (KOH) - 10% solution in a dropper bottle

C. Speculum

ACHDLS - Vaginal Wet Mount 38 Revised February 2012

 D. Cotton swabs

E. Microscope slides - 1” x 3” - frosted end

F. Cover slips 22 x 22 mm

G. Microscope with mechanical stage, low power (10x) and high power (40x)
objectives

V. Reagent Storage and Usage

A. Store reagents at room temperature.

B. Solutions should be clear. Observe for turbidity, and discard if cloudiness is noted.

VI. Calibration

Calibration is not applicable.

VII. Quality Control

Quality control materials are not available.

VIII. Preliminary Procedures and Precautions

A. Set or verify Koehler Illumination on the microscope each day of use. This may not
be possible on some microscopes due to illumination being permanently set at the
factory.

B. Personnel who perform this procedure must be adequately trained as follows:

1. Know proper technique for preparing the specimen on a slide.
2. Have thorough knowledge of the proper use and care of a microscope.
3. Have thorough knowledge of the morphology of organisms and other
microscopic structures.
4. Have interpretive skills to determine the significance of the microscopic
elements observed.
5. Have the ability to distinguish medically significant organisms and structures
from background debris and artifacts.

IX. Procedure

A. Preparation of slides
1. Prepare slide by placing 1 drop of normal saline on 1 end of the slide and
1 drop of KOH on the other end of the slide.
2. On the slide, mix each solution separately using a swab. Dip swab in saline
first, then in KOH, or use 2 different swabs.

ACHDLS - Vaginal Wet Mount 39 Revised February 2012

 Note: Separate slides (1 for the saline and 1 for the KOH) may be used if
necessary. Remember to dip the swab in the saline first, then KOH.
3. Apply a cover slip to each smear by placing at a 45° angle at the edge of the
smear and easing down gently to avoid air bubbles. Blot excess liquid from the
edges.
Note: Cover slips often stick together. Carefully separate them so that only
1 is placed on the smear.
4. Label the frosted end of the slide with the patient’s name.

B. Detection of inadequate slides

If an inadequate slide is detected, prepare a new one. A separate slide may be used
for saline and KOH. Any of the following conditions would produce an inadequate
slide:
1. Too much saline or KOH on slide, causing material to move too rapidly.
2. Preparation too thick or too thin.
3. Drop of saline and drop of KOH mixed on the slide.
4. Swab mixed with KOH before saline.

C. Microscopic examination of the slide

1. Place the slide on the microscope stage.
2. Use low power to find and focus on the specimen using reduced light.
3. Turn to high power and scan 10 fields on the saline side examining for
Trichomonas, “clue cells,” yeast, white blood cells (WBCs), bacteria, and
normal epithelial cells.
4. Average the fields.
5. A systematic pattern of examining the specimen is to examine 3 sites on each
edge of the cover slip and 2 sites in the center.

Systematic Pattern of Exam

Examine @ 10X & 40X

X X X

X XX X

X X X

6. Move stage so the KOH specimen is under the high power objective. Examine
10 high power fields for yeast cells (other elements are usually destroyed by

ACHDLS - Vaginal Wet Mount 40 Revised February 2012

 KOH).
Note: Because there is little contrast between yeast or pseudohyphae and
background material, you may not observe these cells if the light is too bright.
7. A fishy odor (amine) may be noticed by performing the whiff test when KOH
is added. This is often noted when clue cells and/or Trichomonas are present
in the specimen. This odor is recorded as “Amine Test – Positive.”

X. Interpretation of Results

A. Trichomonas vaginalis
1. Characteristics of the discharge:
a. Yellowish to gray-green
b. Frothy
c. Copious
d. Foul odor
2. Microscopic description using 10x objective:
a. Tiny
b. Round
c. Transparent
d. The size of white cells
e. Rapid movements (jerks and loops)
3. Microscopic description using 40x objective:
a. Approximately 15 mm (10-20 mm)
b. Round
c. Ovoid
d. Globular
e. Oval nucleus
f. Motility: Whirls, jerks, turns, seeming to vibrate
g. Undulating membrane on 1 side only, like the fin of a fish, the main
impression is movement.
4. Flagella:
a. Four flagella at the anterior end
b. Whip-like
c. Very motile
d. Slightly larger than white cells
e. Can be easily identified by their undulating swimming motion
f. Distinguish the trichomonad from the white cell by using the 40x
objective to detect the beating flagella.
g. These organisms are called “friendly white cells” because the flagella
appear to wave.

B. Candida albicans (yeast) - (pseudohyphae and hyphae)

1. Characteristics of the discharge:
a. Thick
b. White (occasionally yellow or colorless)
c. Curd-like

ACHDLS - Vaginal Wet Mount 41 Revised February 2012

 d. Cheesy
e. Musty odor
2. Microscopic description using 40x objective:
a. Candida albicans
Vary in size (2-6µm)
Round or oval
Non-motile
Some show budding
b. Pseudohyphae, hyphae, and Mycelia filaments
Vary in length (20-100µm) & breadth (2-4µm)
Filaments with rounded ends
Distinguishing from other elements:
▪ Debris from epithelial cells or mucus may be mistaken for or may
obscure yeast cells.
▪ Examine for yeast on the KOH specimen since other elements are
usually destroyed by the KOH.

C. Gardnerella vaginalis
1. Characteristics of the discharge:
a. Thin
b. Homogenous
c. Grayish
d. Adherent
e. Foul odor
2. Microscopic description:
a. Small
b. Thin
c. Gram-negative rod
d. Bacterium
e. Parasitizes the surface of vaginal epithelial cells
3. Microscopic description of “clue cells":
a. Squamous, vaginal epithelial cells covered with many bacteria, giving
them a stippled or granular appearance.
b. Borders are obscured or fuzzy because of the adherence of the bacteria.
c. So many organisms may attach to a vaginal epithelial cell that its entire
border is obscured.
d. Absence of a distinct border, rather than the granular appearance of the
cytoplasmic or nuclear portion, identifies the clue cell.
e. Look for sheets of epithelial cells that are studded with bacteria.
4. Other elements seen in the presence of clue cells:
a. On the saline preparation trichomonads may be seen with clue cells, but
mycelia and clue cells are almost never found together.
5. Diagnosis of Gardnerella:
a. At least 20% of the epithelial cells present must be clue cells to establish
the diagnosis of Gardnerella.

ACHDLS - Vaginal Wet Mount 42 Revised February 2012

 b. A fishy odor (amine) may be noticed by performing the whiff test when
KOH is added if clue cells are present in the specimen. Record this fishy
odor as "Amine Test - Positive”.

XI. Reporting of Results

Note: Report all results on CHR-11 or CHR-12c with the date and time procedure was
performed along with initials and signature of person performing test.

A. Reporting of Organisms
1. Trichomonas vaginalis: Observation of a single motile trichomonad
establishes a diagnosis.
a. When Trichomonas are observed, report as “Present”.
b. When no Trichomonas are observed, report as “Absent”.
Note: The use of signs (e.g. “-“, “+”, “0”) and words (e.g. “positive”,
“negative”, “zero”, “none”) must be avoided as they can be misinterpreted.
No documentation means test is not performed.
2. Yeast: Observation of yeast, pseudohyphae, hyphae or buds establishes a
diagnosis.
a. When yeast, pseudohyphae, hyphae or buds are observed, report as
“Present”.
b. When no yeast, pseudohyphae, hyphae or buds are observed, report as
“Absent”.
Note: The use of signs (e.g. “-“, “+”, “0”) and words (e.g. “positive”,
“negative”, “zero”, “none”) must be avoided as they can be misinterpreted.
No documentation means test is not performed.
3. Clue Cells: Epithelial cells obliterated with bacteria.
a. Clue cells are significant only when symptoms are present.
b. Twenty percent of total epithelial cells must be clued to establish bacterial
vaginosis.
c. When clue cells are observed, report as “Present”.
d. When no clue cells are observed, report as “Absent”.
Note: The use of signs (e.g. “-“, “+”, “0”) and words (e.g. “positive”,
“negative”, “zero”, “none”) must be avoided as they can be misinterpreted.
No documentation means test is not performed.
4. White Blood Cells (WBCs):
a. Must be enumerated and reported by indicating low and high numbers
determined per high power field (hpf).
b. WBCs ≥ 10/hpf is significant in vaginal wet mount observation.
c. WBC quantity is determined by the following method:
i. Ten high powered fields are viewed microscopically using the
systematic pattern of exam (See Section IX.C.5).
ii. The average total of the 10 high powered fields is determined (See
Section XII) and reported using the following range:
Rare = 0-1/hpf
Few = 2-5/hpf
Moderate = 6-20/hpf

ACHDLS - Vaginal Wet Mount 43 Revised February 2012

 Many = 21-35/hpf
Numerous = > 35/hpf

Note: The range wordage (Rare, Few, Moderate, Many, Numerous) may
be used instead of the numbers. Do not use abbreviations. No
documentation means test is not performed.

5. "Amine Test” or “whiff test”- a tool (along with other test observations and
patient symptoms) to assist the clinician in establishing bacterial vaginosis.
a. The test is performed by placing KOH on the wet mount slide and noting
the presence or absence of a “fishy odor”.
b. When a fishy odor is detected, report as “Positive”.
c. When no fishy odor is detected, report as “Negative”.
Note: The use of signs (e.g. “-“, “+”, “0”) must be avoided as they can be
misinterpreted. No documentation means test is not performed.
6. Other findings that may be documented:
1. Bacteria: Quantitate as 1+, 2+, 3+ or 4+.
2. Red Blood Cells (RBCs)
a. When RBCs are observed, report as “RBCs”.

XII. Calculations

C/10 = A where
C = (Cellular total of 10 high power fields)
A = (Average cellular total /high power field)
Example: Craig uses the systematic pattern of exam to observe a wet mount slide. After
10 high power field observations, he notes the following:
HPF One = 2, HPF Two = 3, HPF Three = 1, HPF Four = 6, HPF Five = 1,
HPF Six = 1, HPF Seven = 2, HPF Eight = 1, HPF Nine = 3, HPF Ten = 3

C= 2+3+1+6+1+1+2+1+3+3
C = 23 Craig determines the total cellular elements.
A = 23/10 Craig determines the average total by dividing by 10 hpfs.
A = 2.3/hpf Craig determines the average to be 2.3/hpf.
Based on the range chart (See Section XI.4.c.ii), Craig’s average (2.3/hpf) is in the
“Few” “2-5/hpf” range. He may report his observation as “2-5/hpf” or “Few”.

XIII. Troubleshooting

A. If patient results do not agree with clinical symptoms, an error may have occurred.

B. Review the "Sources of Error" for specific problems which may affect testing and
attempt to determine the cause of the problem.

ACHDLS - Vaginal Wet Mount 44 Revised February 2012

 C. If the cause of the problem is not easily determined, follow a systematic approach to
troubleshoot the situation.

D. Double check the specimen identification to ensure that the testing is being
performed on a properly collected and labeled specimen on the correct patient.

E. Compare the procedure as you performed it with each step in the written procedure.
Make sure the written procedure was followed exactly as it is written.

F. Examine reagents, solutions, and/or materials used for:

1. The right color.
2. Turbidity or precipitation.
3. Expiration dates.

G. Check the equipment.

1. Have function verification checks been performed?
2. Are function check results within acceptable limits?
3. Has preventive maintenance been performed at proper intervals?

H. Does the testing personnel have adequate training and experience to properly
perform the procedure?

I. An abrupt change in patient results that coincides with a change in reagents,

materials, equipment, or testing personnel can easily indicate the source of the
problem.

XIV. Sources of Error

A. Specimen errors

1. Specimen not collected prior to use of lubricant.

2. Specimen not examined immediately, causing a loss of motility.

3. Preparation too thick or thin.

B. Reagent errors

1. Reagents not properly stored.

2. Reagents are contaminated.

3. Reagents are not the correct strength.

4. KOH accidentally mixed with saline.

ACHDLS - Vaginal Wet Mount 45 Revised February 2012

 5. Too much saline on slide, causing material to move rapidly across the field.

C. Equipment errors

1. Microscope condenser, objectives, eyepieces, etc., not clean.

2. Microscope not set at optimum Koehler Illumination.

3. Microscope not properly focused.

D. Procedure Errors

1. Exam not performed using a thorough and systematic review of the slides.

2. Testing personnel not able to correctly recognize and/or differentiate

microscopic findings.

3. Testing personnel not able to correctly use the microscope.

4. Failure to read slide using reduced light.

XV. Remedial Action

A. Once the potential source of the problem is discovered, take proper actions such as:
1. Collect new specimen.
2. Use new reagents/materials.
3. Correct the equipment problems or replace the equipment.
4. Follow written procedure exactly.
5. Change testing personnel until additional training can be conducted.

B. Retest the patient specimens and reevaluate results to determine if they are
acceptable.

C. If results are still unacceptable, continue to troubleshoot to determine the problem.

1. Do not report any patient results.

2. Call an outside source to help troubleshoot.

D. If the patient results are now acceptable, the problem is resolved.

E. Once the problem has been resolved, report patient results on CHR-11 or CHR-12c,
including time, date, and initials of analyst.

ACHDLS - Vaginal Wet Mount 46 Revised February 2012

 F. Document the problem and the corrective action taken on the appropriate laboratory
record form.

XVI. Normal Values

A. A normal patient value is negative for Trichomonas vaginalis, Candida albicans

and Gardnerella vaginalis.

B. A normal patient may have 1-3 WBCs and/or epithelial cells per high power field.

C. Few, if any, WBCs are usually seen with Gardnerella vaginalis. If increased
numbers of WBCs are seen with clue cells, consider other infections.

XVII. Reportable Range

Not applicable.

XVIII. Test Limitations

Take care in interpreting apparent results: artifacts are common in KOH preps as a result
of cell degeneration, air bubbles, crystallization, and glycerol.

XIX. Course of Action if Test System Becomes Inoperable

A. If the test cannot be performed due to the absence of materials or inadequate

materials, if possible, borrow from an appropriate source the materials, reagents,
equipment, etc. needed, and run the test.

B. Since the microscopic examination must be performed immediately after the

specimen is collected, do not store the specimen or send it to another laboratory for
examination.

C. If the problem cannot be resolved, reschedule the patient to return at a later date or
send the patient to another location equipped to perform the examination.

XX. References

A. Alabama Department of Public Health. Wet Prep Competency; Microscope

Teaching Video Series, 2000.

B. Olympus. BX41 System Microscope Instruction Manual, 2003.

C. Olympus Video Library. The Basic Microscope - Use & Care.

ACHDLS - Vaginal Wet Mount 47 Revised February 2012

 Cellular Components in Vaginal Specimens

ACHDLS - Vaginal Wet Mount 48 Revised February 2012

 Cellular Components in Vaginal Specimens

ACHDLS - Vaginal Wet Mount 49 Revised February 2012

 Cellular Components in Vaginal Specimens

ACHDLS - Vaginal Wet Mount 50 Revised February 2012

 Cellular Components in Vaginal Specimens

ACHDLS - Vaginal Wet Mount 51 Revised February 2012

 Rapid Plasma Reagin
(RPR)
18-MM Circle Card Test
 RAPID PLASMA REAGIN (RPR)
18-MM CIRCLE CARD TEST
I. Principle

The rapid plasma reagin (RPR) 18-mm circle card test is a macroscopic, nontreponemal
flocculation card test used to screen for syphilis. The antigen is prepared from a
modified Venereal Disease Research Laboratory (VDRL) antigen suspension
containing choline chloride to eliminate the need to heat inactivate serum,
ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension, and
finely divided charcoal products as a visualizing agent. In the test, the RPR antigen is
mixed with unheated or heated serum or with unheated plasma on a plastic-coated card.
The RPR test measures IgM and IgG antibodies to lipoidal material released from
damaged host cells as well as to lipoproteinlike material and possibly cardiolipin
released from the treponemes. The antilipoidal antibodies are produced not only as a
consequence of syphilis and other treponemal diseases but also in response to
nontreponemal diseases of an acute and chronic nature in which tissue damage occurs.
If antibodies are present, they combine with the lipid particles of the antigen causing
them to agglutinate. The charcoal particles coagglutinate with the antibodies and
appear as black clumps against the white card. If antibodies are not present, the test
mixture is uniformly gray. The test can be purchased in kit form or in component parts
from many commercial sources. Without some other evidence for the diagnosis of
syphilis, a reactive nontreponemal test does not confirm Treponema pallidum infection.

II. Specimen Collection and Handling

A. Specimen
1. Avoid accidental infection when collecting and processing samples by
observing universal precautions.
2. Serum and plasma are suitable specimens for the qualitative test; however
serum is preferred for the quantitative test. Test plasma samples within 48
hours of collection.
3. An acceptable specimen does not contain particulate matter that would
interfere with reading test results.
Note: Hemolysis may be caused by transporting blood in freezing or
extremely hot weather without proper insulation.

B. Collection
1. Serum - Collect whole blood into a clean, dry tube without an anticoagulant.
2. Plasma - Collect blood in a tube containing EDTA as an anticoagulant.
Completely fill the tube or collect blood until the vacuum in the collection
tube has been exhausted.
3. Label each specimen with patient identifier and date.
C. Handling Serum

ACHDLS - Rapid Plasma Reagin 52 Revised February 2012

 1. Allow sufficient time (approximately 20 minutes) at room temperature for the
specimen to clot.
2. Centrifuge the specimen at room temperature at 1500 - 2000 rpms for at least
5 minutes to sediment cellular elements.
3. Keep serum specimens in the original collection tube if testing will be
performed within a few hours.
4. Remove serum from the clot and store at refrigerator temperature (2-8°C) if
testing is to be delayed. If a delay of more than 5 days is anticipated before
testing, freeze the specimen at -20°C or lower. Avoid repeated
freezing/thawing of specimens.
5. Although unheated serum specimens may be used, serum may be heated at
56°C for 30 minutes without affecting test outcome.
6. Specimens must be at room temperature (23-29°C or 73-85°F) at the time of
testing.

D. Handling Plasma
1. Centrifuge the specimen at room temperature at 1500 - 2000 rpms for at least
5 minutes to sediment cellular elements.
2. Plasma may need to be retained in the original collection tube if the test is to
be performed immediately. If not, plasma should be removed from cellular
elements.
3. Store plasma specimens at 2-8°C, and test within 48 hours. Plasma samples
must be at room temperature (23-29°C or 73-85°F) at the time of testing.
4. Do not heat plasma.
5. Do not use plasma specimens for confirmatory treponemal tests.

III. Specimen Rejection

A. Specimens exceed the acceptable time limit since collection.

B. Specimens are collected in the wrong tube.

C. Specimens are not properly labeled or the identification is questionable.

D. Specimens are not stored properly after collection.

E. Specimens are grossly contaminated, hemolyzed, or lipemic.

F. Core blood and spinal fluid are unsuitable for RPR testing.

IV. Reagent, Supplies, and Equipment (See “Health Department RPR Reagent
Requisition”)

A. RPR antigen suspension

ACHDLS - Rapid Plasma Reagin 53 Revised February 2012

 RPR antigen suspension is a stabilized combination of 0.003% cardiolipin,
0.020-0.022% lecithin, 0.09% cholesterol, 10% choline chloride, 0.0125M
EDTA, 0.01875% charcoal, 0.01M Na2HPO4, 0.01M KH2PO4, and 0.1%
thimerosal in distilled water.
The antigen suspension is packaged in ampules.

B. Control serum samples

They are lyophilized reactive (R), minimally reactive (MR), and nonreactive
(NR) control serum specimens on a card.

C. Disposable, calibrated 20-gauge needle without bevel, silicone treated.

D. Plastic antigen dispensing bottle, 1 dram

E. Plastic-coated RPR cards with 10 circles, each approximately 18 mm in diameter.

F. Dispenstirs®, a disposable, plastic dispensing/stirring device that delivers 50µl.

G. Mechanical rotator, fixed-speed or adjustable to 100 +/- 2 rpm, circumscribing a

circle ¾ inch or 2 cm in diameter on a horizontal plane.

H. Humidifying cover.

I. High-intensity incandescent lamp.

J. Discard containers and disinfectants.

K. Disposable gloves, safety glasses, and protective clothing.

V. Reagent Storage and Usage

A. RPR antigen suspension

Store unopened ampules at 2-8°C.
Do not store the antigen in bright sunlight or in temperatures above 29°C.
Do not freeze.
An unopened ampule of antigen is stable until the expiration date.
Once placed in the dispensing bottle and refrigerated at 2-8°C (35-46°F), the
antigen reactivity remains satisfactory for approximately 3 months.

B. Control serum samples

Store control cards according to the manufacturer’s directions.

C. Plastic-coated RPR cards

Store at room temperature.

ACHDLS - Rapid Plasma Reagin 54 Revised February 2012

VI. Calibration
See “STD Patient Log and QC Form”
See “RPR Rotator Preventive Maintenance”

A. Needles
1. Check the calibrated needle every day of use if the needle has been dropped,
wiped, or when control pattern is not met to ensure the delivery of the correct
volume of antigen suspension (60 drops +/-2 drops per ml; 17µl per drop).
2. Place the needle on a 1-ml syringe.
3. Fill the syringe or pipette with RPR antigen suspension.
4. Holding the syringe or pipette in a vertical position, count the number of
drops delivered in 0.5 ml.
5. The needle is correctly calibrated if 30 drops +/-1 drop is delivered in 0.5 ml.
6. Replace the needle if it does not meet this specification. Be sure to test the
calibration of the replacement needle.

B. Rotator
1. Speed - For rotators without a digital readout, the speed can be estimated by
counting the number of rotations per minute.
a. To count the rotations, place a pen or pencil next to the rotator and count
the number of times the rotator touches the pen or pencil in 60 seconds.
b. If the rotator is properly adjusted, the count should be 100 +/- 2.
c. The rotator’s speed should be calibrated each day of use.
2. Time - The rotator’s timer should be checked against another laboratory
timer or stopwatch quarterly. The rotator’s timer should be +/-15 seconds of
the set time.
3. Circumference - Check quarterly to make sure the rotator is producing a
circle with a diameter of 2 cm or ¾ inch.
a. Place a blank sheet of paper underneath the edge of the bottom of the
rotator.
b. With the rotator off, secure a pen or pencil next to the side of the
rotating platform, and position it so that the writing surface is touching
the piece of paper.
c. Turn the rotator on for several seconds, and let the pen or pencil “draw”
a circle on the piece of blank paper. This circle is the circumference of
the rotating action.
d. After stopping the machine from rotating, remove the paper and measure
with a ruler the diameter of the circle drawn on the paper. If the
diameter of the circle is not 2 cm or ¾ inches, call your technical
consultant.

VII. Quality Control

See “STD Patient Log and QC Form”
See “RPR Rotator Preventive Maintenance”

ACHDLS - Rapid Plasma Reagin 55 Revised February 2012

 It is the responsibility of the laboratorian to ensure that the reagents are of good quality
and standard reactivity. Chemicals and distilled water should be of high quality, and
solutions should be prepared according to the manufacturer’s directions.

A control card should be run each day patient specimens are tested. Controls will be
run before patient testing is performed. Control results must be recorded on the STD
Patient Log.

A. RPR Test Antigen

1. Test results on control serum specimens of graded reactivity must give
expected results.
2. The antigen suspension should show in tests with nonreactive serum
specimens the complete dispersion of antigen particles.

B. Daily Controls
1. Check room temperature. For accurate and reproducible test results, the RPR
card antigen suspension, controls, and test specimens must be at room
temperature (23-29°C or 73-85°F) when tests are performed.
2. At each routine test run, check the expiration date of the antigen.
3. Determine antigen suspension reactivity with control cards of graded
reactivity (reactive, minimally reactive, and nonreactive) which have been
reconstituted with distilled water.
4. Use only RPR antigen that reproduces the established reactivity pattern of
controls.
5. Date and initial.

C. Monthly Controls
1. Clean humidifier sponge.
2. Remove dust in and/or on the rotator.
3. Clean black rubber sheeting on top of oscillating plate.
4. Date and initial.

D. Quarterly Controls
1. Check rotator timer (8 min +/- 15 sec) and record data.
2. Check rotator circumference (2 cm or ¾ inch) and record data.
3. Date and initial.

VIII. Preliminary Procedures and Precautions

A. Check the temperature of the testing area.

B. Check the accuracy of the needle delivery.

C. Check the speed of the rotator.

ACHDLS - Rapid Plasma Reagin 56 Revised February 2012

 D. Check humidifying cover on rotator.

E. Run the Reactive, Minimal Reactive, and Nonreactive control samples.

IX. Procedure

A. To prepare antigen for testing:

1. Attach the hub of the dispensing needle to the fitting on the plastic
dispensing bottle.
2. Shake the antigen ampule to resuspend the particles.
3. Open the ampule.
4. Squeeze the dispensing bottle to collapse it.
5. Insert the needle into the ampule and withdraw all the antigen suspension
into the dispensing bottle.

B. Place a drop of serum or plasma onto an 18-mm circle of the RPR test card, using
a disposable Dispenstir.

C. Using the inverted Dispenstir (closed end), spread the serum or plasma to fill the
entire circle.

D. Gently shake the antigen dispensing bottle to resuspend the particles.

E. Holding the dispensing bottle and needle in a vertical position, dispense several
drops on the edge of the card to clear the needle of air.

F. Add exactly 1 free-falling drop of antigen suspension to each circle containing

serum or plasma. Do not mix.

G. Place the card on the mechanical rotator under a humidifying cover.

H. Rotate the card for 8 minutes at 100 rpms +/- 2 rpms.

I. Immediately remove the card from the rotator; briefly rotate and tilt the card by
hand (3 or 4 to-and-fro motions) to aid in differentiating nonreactive from
minimally reactive results.

J. Read the test reactions (without magnification) in the “wet” state under a high-
intensity incandescent lamp.

X. Interpretation of Results

A. The RPR card test is an aid in the diagnosis of syphilis.

ACHDLS - Rapid Plasma Reagin 57 Revised February 2012

 Clinicians combine the RPR card test with results of other serologic tests,
darkfield examinations, clinical signs and symptoms, and risk factors in
arriving at a syphilis diagnosis.
Without some other support for the diagnosis of syphilis, a reactive RPR card
test is commonly unrelated to T. pallidum infection.
The predictive value of a reactive RPR card test in a serologic diagnosis of
syphilis is increased when combined with a reactive treponemal test, such as
the fluorescent treponemal antibody absorption test or the
microhemagglutination assay for antibodies to T. Pallidum.

B. A reactive RPR card test may suggest past or present infection with a pathogenic
treponeme; however, it may also be a false-positive reaction.
False-positive reactions can result from laboratory error as well as serum
antibodies unrelated to syphilis infection.
Technical errors are detected by a nonreactive RPR card test with a second
serum specimen.
False-positive RPR card tests from infections with a nontreponemal disease or
other disease conditions are identified by an accompanying nonreactive
treponemal test.

C. A nonreactive RPR card test without clinical evidence of syphilis may suggest no
current infection or an effectively treated infection.
A nonreactive RPR card test without clinical evidence of syphilis can be seen
in early primary syphilis, in secondary syphilis, as a result of the prozone
reaction, and in some cases of late syphilis.
A nonreactive card test result does not rule out an incubating syphilis
infection.

XI. Reporting of Results

A. Report results as follows:

1. Reactive (R) or Nonreactive (NR)
a. See CHR-11 or CHR-12c and Instructions.
b. If specimen is a field blood, document as such on the “STD Patient Log
and QC Form”.

XII. Calculations

Not applicable.

XIII. Troubleshooting

A. If patient results do not agree with clinical symptoms, an error may have occurred.

ACHDLS - Rapid Plasma Reagin 58 Revised February 2012

 B. A testing problem is usually caused by one of the following:
1. Specimen error.
2. Reagent error.
3. Equipment error.
4. Human error in the proper use of the microscope or in the recognition and/or
differentiation of microscopic findings.

C. Review the "Sources of Error" for specific problems which may affect testing, and
attempt to determine the cause of the problem.

D. If the cause of the problem is not easily determined, follow a systematic approach to
troubleshoot the situation.

E. Double check the specimen identification to ensure that the testing is being
performed on a properly collected and labeled specimen on the correct patient.

F. Compare the procedure as you performed it with each step in the written procedure.
Make sure the written procedure was followed exactly as it is written.

G. Examine reagents, solutions, and/or materials for:

1. The right color.
2. Turbidity or precipitation.
3. Expiration dates.

H. Check the equipment.

1. Have function verification checks been performed?
2. Are function check results within acceptable limits?
3. Has preventive maintenance been performed at proper intervals?

I. Does the testing personnel have adequate training and experience to properly
perform the procedure?

J. An abrupt change in patient results that coincides with a change in reagents,

materials, equipment, or testing personnel can easily indicate the source of the
problem.

XIV. Sources of Error

A. If the temperatures of the sera, reagents, or testing area are less than 23°C (73°F),
test reactivity decreases; if temperatures are greater than 29°C (85°F), test reactivity
increases.

B. If the speed of the mechanical rotator is too fast or slow, improper antigen-antibody
interaction will cause unpredictable results.

ACHDLS - Rapid Plasma Reagin 59 Revised February 2012

 C. If the time of rotation is too long, test reactivity may be increased. If the time of
rotation is too short, test reactivity may be decreased.

D. If the card is excessively rotated and tilted by hand after removal from the rotator, a
false reactive may occur.

E. If lighting produces a glare on the card, the reactions may be obscured.

F. If the antigen is outdated or not adequately tested for standard reactivity, the results
may be inaccurate.

G. If the serum is unevenly spread in the circle, the antigen and antibody may not mix
properly.

H. If hemolyzed, contaminated, or improperly collected serum or plasma specimens

are tested, the reactions may be masked.

I. If the moistened humidifying cover is not used to cover tests as they are being
rotated, proper humidity will not be maintained, and test components may dry on
the card causing false reactive results.

XV. Remedial Action

A. When you think the cause of the problem is discovered, take proper actions such as:
1. Collect new specimen.
2. Use new reagents/materials.
3. Correct equipment problems or replace the equipment.
4. Follow written procedure exactly.
5. Change testing personnel until additional training can be conducted.

B. Retest the patient specimens, and reevaluate to determine if results are acceptable.

C. If results are still unacceptable, continue to troubleshoot to determine the source of

the problem.

1. Do not report any patient results.

2. Call an outside source to help troubleshoot.

D. If the patient results are now acceptable, the problem is resolved.

E. Document the problem and the corrective action on the appropriate laboratory
record form.

XVI. Normal Values

ACHDLS - Rapid Plasma Reagin 60 Revised February 2012

 A. Nonreactive

XVII. Reportable Range

Not applicable.

XVIII. Test Limitations

A. The RPR card test cannot be used to test spinal fluids.

B. A prozone reaction may be encountered occasionally.

In a prozone reaction, complete or partial inhibition of reactivity occurs with
undiluted serum (maximum reactivity is obtained only with diluted serum).
A specimen should be tested for the prozone phenomenon when the clinician
suspects syphilis, but the qualitative RPR is nonreactive.

C. The RPR card test may be reactive in persons from areas where yaws, pinta or
nonvenereal syphilis is endemic. Generally, residual titer from these infections will
be < 1:8.

D. Biological false-positive reactions occur occasionally with cardiolipin antigens,

mainly in specimens from persons who abuse drugs, have diseases such as lupus
erythematosus, mononucleosis, malaria, leprosy, or viral pneumonia, or who have
recently been vaccinated.

E. Nontreponemal test titers of persons who have been treated in latent or late stages
of syphilis or who have become reinfected do not decrease as rapidly as those of
persons in the early stages of their first infection. In fact, these persons may remain
“serofast,” retaining a low-level reactive titer for life.

XIX. Course of Action if Test System Becomes Inoperable

A. If the test cannot be performed due to the absence of materials or inadequate

materials, if possible, borrow from an appropriate source the materials, reagents,
equipment, etc. needed, and run the test.

XX. Reference

A. Manual of Tests for Syphilis, 9th Edition, 1998.

ACHDLS - Rapid Plasma Reagin 61 Revised February 2012

 RPR REAGENT REQUISITION

Person Certified to
Perform RPR Testing:

Health Department::

Street Phone

City Zip Code

Date Requested:

RPR REAGENTS QUANT

TITY REQUESTED QUANTITY SHIPPED
(To be completed by Lab)

TESTING CARDS (50/PKG)

CONTROL CARDS (10/PKG)

ANTIGEN (3/BX)

NEEDLES (10/PKG or EACH)

DISPENSING BOTTLES (10/PKG)

DISPENSTIRS (500/BX)

ORDER FROM: Jane Duke, Charlene Thomas, or Craig Wilson

Bureau of Clinical Laboratories / Quality Management Section
8140 AUM Drive, 36117 (Physical address)
P.O. Box 244018, 36124-4018 (Mailing address)
Montgomery, Alabama
Phone: 334-213-2853
Fax: 334-274-9801

Please use this form when ordering RPR supplies. Individual components should be ordered as
needed. “Whole” kits are no longer in stock.

Please do not phone in orders unless absolutely necessary. Use of this order form will ensure
accuracy and expediency in filling of requisitions.

ADPH/BCL/QM 12-11
 STD Patient Log and QC Form
County Date / /
Site Page of

Patient Name/CHR RPR Darkfield Wet Prep Comments Initials

RPR Quality Control Data

Antigen Lot Number
Antigen Manufacturer Expiration Date CONTROL RESULTS
Antigen Open Date
Control Card Lot Number Reactive (R)
Control Card Expiration Date Minimal Reactive (MR)
Nonreactive (NR)
Test Area Temperature (23 - 29 C)
Rotator Speed (98 - 102) rpm’s RPR QC Performed By
Needle Check (58 - 62 drops/ml)
Note: Label the dispensing bottle with the antigen lot number, manufacturer’s expiration date, and date antigen was placed in the dispensing
bottle (Open Date). Ensure the antigen is used within three months of the antigen open date not to exceed the manufacturer’s date.

ADPH/BCL/QM/REV 04-10
 RPR Rotator Preventive Maintenance
CHD
Site
Instructions: Year
1. Write in actual value, if applicable
2. If no value, use a check mark.
3. Record date and initials when performed.
MONTHLY JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC
Clean Humidifier Sponge
Remove Dust in or on
the Rotator
Clean Black Sheeting on
Oscillating Plate
Date
Initials
QUARTERLY FIRST QUARTER SECOND QUARTER THIRD QUARTER FOURTH QUARTER
Check Rotator time
(8 min +/-- 15 sec)
Check Rotator Circumference
(2 cm or ¾ inch)
Date
Initials

Date Additional Comments or Notes Initials

ADPH/BCL/QM Rev 10-09 Write NIU when not in use.

Darkfield Microscopy
 DARKFIELD MICROSCOPY

I. Principle

A. The standard brightfield microscope may be equipped for darkfield examination by

replacing the brightfield condenser with either a double- or single- reflecting darkfield
condenser. Illumination for darkfield microscopy is obtained when light rays strike the
object in the field at an oblique angle so that no direct light rays enter the microscope
objective, only the rays reflected from the object. Therefore, the object appears self-
luminous against a dark background. When a fluid containing particles, including
bacteria or treponemes, is placed on a slide, the oblique rays are reflected from the
surfaces upward into the barrel of the microscope; these particles appear brightly
illuminated against a black background. This type of illumination can be obtained by
using a double-reflecting darkfield condenser or a single-reflecting darkfield condenser.

B. In the double-reflecting darkfield condenser, two reflecting surfaces produce intense

illumination; however, this type of condenser requires precise focusing and accurate
centering.

C. The single-reflecting condenser contains one reflecting surface which does not produce
a sharp focusing of the hollow cone of rays. This characteristic makes it easier to
manipulate, but less intense illumination is produced. Thus, the single-reflecting
condenser is less desirable when high intensity of illumination is required.

D. Most darkfield condensers require the numerical aperture (NA) of the oil immersion
objective be reduced below that of the condenser. This can be accomplished by
inserting a funnel stop in the objective or by using the oil immersion objective with a
built-in iris diaphragm.

II. Glossary of Terms

A. Aperture diaphragm - either a rotating disk or an iris diaphragm on the condenser used
to direct a cone of light to the specimen and enter the objective. It should never be used
to regulate brightness. Resolution, control, and depth of field depend on the correct
setting of the aperture diaphragm.

B. Arm - for holding the microscope while carrying it.

C. Coarse adjustment knob - for rapid focusing of the specimen.

D. Compound microscope - a microscope made of two lens systems: eyepiece and

objective.

E. Condenser - the lens system beneath the microscope stage positioned to concentrate
light correctly on the specimen and direct light rays into the objective. When the
condenser is used at a lowered position, the resolving power is reduced.

ACHDLS – Darkfield Microscopy 62 Revised February 2012

 F. Depth of field - distance just above and below the focal plane (area being examined)
that can be focused clearly.

G. Eyepiece – lens system of the microscope nearest to the eyes.

H. Field diaphragm - an aperture diaphragm which restricts area of illumination.

I. Fine adjustment knob - focuses the lens in small increments.

J. Immersion oil - oil with the same refractive index as glass, 1.515; used between the
cover glass and an oil immersion objective to prevent scattering of light in air.

K. Interpupillary distance - distance between the eyes. The eyepieces of a binocular scope
must be adjusted so that left and right images merge into one.

L. Koehler illumination - optical illumination providing bright, evenly dispersed, glare-

free light with good contrast and resolution.

M. Nosepiece - a revolving plate that holds the objectives.

N. Ocular – eyepiece (lens system of the microscope nearest to the eyes).

O. Parcentric - the ability to center a specimen in the field of view for one objective and
have almost the same field in place when rotating to another objective.

P. Parfocal - the objectives are constructed so that only slight refocusing with the fine
adjustment knob is needed after rotating to another objective.

Q. Resolution - the ability of a microscope to reveal fine detail in a specimen. The better
the resolving power of a microscope, the closer two objects can be and still be
distinguished as two objects.

R. Stage - the platform on which the microscope slide is placed.

S. Working distance - distance between the coverslip of a slide and the tip of an objective.
The low power objective has the greatest working distance. The oil immersion
objective has a very small working distance.

III. Adjusting the Microscope for Correct Alignment and Illumination

Always complete the microscope adjustment and have the microscope in satisfactory
working condition BEFORE collecting a darkfield specimen for examination.

Note: To complete the microscope adjustment and to verify that the microscope is in good
working order before examining patient material, prepare a suspension of gingival
scrapings in a drop of saline on a slide of proper thickness, mount with a coverslip, and use
this as a control slide.

ACHDLS – Darkfield Microscopy 63 Revised February 2012

 A. Place the control slide on the stage, and raise the substage containing the darkfield
condenser. The top of the darkfield condenser should be slightly below the level of the
stage, but as close to the control slide as possible without pushing it up.

B. Turn on the variable transformer to produce the maximum light intensity.

C. Lower the substage slightly and place 2-3 drops of immersion oil on the top of the
condenser.

D. Center the specimen over the condenser with the mechanical slide carrier.

E. Slowly raise the substage until complete oil contact between the top of the condenser
and the bottom of the slide occurs.

F. Rotate the nosepiece to center the 10x objective over the specimen.

G. Bring the specimen into focus by using the coarse adjustment knob.

H. At this point, center the light in the field by rotating the 2 centering screws at the base
of the darkfield condenser.

I. Rotate the nosepiece until the high-dry (40x) objective is in place over the specimen.

J. Bring the specimen into focus by using the fine adjustment knob only.

K. If a satisfactory image is obtained, place a small drop of immersion oil on the cover
glass.

L. Rotate the nosepiece until the oil immersion objective (100x) is in place over the
specimen and is in contact with the oil on the cover glass.

M. Bring the specimen into focus by using the fine adjustment knob only. (Use of the
coarse adjustment knob with the high-dry or oil immersion objectives may cause
damage to the objectives by allowing them to come in contact with the specimen slide).

IV. Maintenance
See “Microscope Maintenance Chart”

A. Daily Maintenance
1. Clean all optical surfaces using lens cleaner and lens paper.
2. Eyepieces and objectives
a. Dampen a cotton-tipped applicator with lens cleaner.
b. Begin in the middle of the eyepiece or objective, and work in a circular motion
toward the outer edges.
c. Lightly touch the surface with lens paper to dry it.
3. Condenser and illuminator
a. Dampen lens paper with lens cleaner and gently clean the surface.
b. Use a piece of dry lens paper to dry the surface.

ACHDLS – Darkfield Microscopy 64 Revised February 2012

 4. Cover the microscope when not in use.
5. Document performance of maintenance and initials of the person performing
maintenance.

B. Monthly Maintenance
1. Clean all non-optical surfaces with warm water and mild detergent.
2. Please indicate N/A on the maintenance chart for the checking of KOH.
3. Check saline for cloudiness or contamination.
4. Document the maintenance on the chart.
5. Document the initials of the person performing maintenance.

C. Annual Professional Microscope Maintenance

1. Microscopes should be serviced annually by a professional.
2. Document the company performing the cleaning.
3. Document the date the annual cleaning was performed.

V. Corrective Action Log

See “Microscope Maintenance Chart”

A. Document any problems encountered with the microscope.

B. Document date of the problem.

C. Document corrective action(s) taken.

D. Document initials of employee performing corrective action(s).

VI. Additional Information

A. Turn lamp voltage down and then off if the microscope has a rheostat.

B. If slideways and gears on the mechanical stage become difficult to move, lubricate with
machine oil or light grease. Document in the corrective action section of the
maintenance chart.

C. Keep at least one extra bulb available in case the bulb in the microscope blows.
Contact your technical consultant for ordering information. Follow manufacturer’s
instructions for ordering and replacing bulbs, or check with your technical consultant.

D. If the microscope requires a halogen lamp, DO NOT TOUCH the new bulb with your
fingers or gloves. Hold the lamp with a lint-free tissue to install it.

E. Avoid exposure to corrosive fumes, extreme heat or cold, or sudden drastic temperature
change. When moving from one temperature extreme to another, allow optical parts to
equilibrate until all moisture is evaporated.

ACHDLS – Darkfield Microscopy 65 Revised February 2012

 F. If immersion oil is required, use only chemically inert, low fluorescent, PCB-free
immersion oil. Low viscosity is acceptable. DO NOT USE CEDAR WOOD OIL.
Different brands of immersion oil are incompatible and should not be mixed.

G. Use a small amount of oil in a frequently-cleaned application bottle to minimize growth

of fungi which may reflect erroneously on specimen content.

VII. Helpful Hints

A. Turn on the light source.

B. Be sure low objective is in position.

C. Rack down, then up until field is in focus.

D. When moving from one objective to another, use fine adjustment knob to refocus.

E. Never drag high-dry objective through the oil.

F. The higher the magnification, the more light will be needed.

G. Always clean the oil from the objective and/or condenser at the end of the day.

H. Be sure low power objective is in working position at the end of the day.

I. Cover the scope when you have finished for the day.

VIII. References

A. Alabama Department of Public Health. Wet Prep Competency; Microscope Teaching

Video Series, 2000.

B. Dark-Field Microscopy and Morphology of T. pallidum.

C. Manual of Tests for Syphilis, 9th Edition, 1998.

D. Olympus. BX41 System Microscope Instruction Manual, 2003.

E. Olympus Video Library. The Basic Microscope - Use & Care.

ACHDLS – Darkfield Microscopy 66 Revised February 2012

 Microscope Maintenance Chart
County Year Manufacturer

January 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
February 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
March 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
April 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
May 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
June 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
July 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials

Monthly Maintenance January February March April May June July

Clean nonoptical surfaces
Check KOH solution
Check Saline
Check pH paper exp. date
Date/Initials

Annual Professional Microscope Cleaning Company “NIU” indicates not in use.

Annual Professional Microscope Cleaning Service Date ADPH/BCL/QM/ Rev 12-11
 Microscope Maintenance Chart
County Year Manufacturer

August 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
September 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
October 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
November 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials
December 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Clean optical surfaces

Cover when not in use
Initials

Monthly Maintenance August September October November December

Clean nonoptical surfaces
Check KOH solution
Check Saline
Check pH paper exp. date
Date/Initials

CORRECTIVE ACTION LOG: Date / Problem / Corrective Action / Initials

“NIU” indicates “not in use". ADPH/BCL/QM/ Rev 12-11

Darkfield Microscopy for
the Detection and
Identification of
Treponema pallidum
 DARKFIELD MICROSCOPY FOR THE
DETECTION AND IDENTIFICATION OF TREPONEMA PALLIDUM
I. Principle

A. A clinical diagnosis of syphilis is confirmed by using darkfield microscopy to

demonstrate Treponema pallidum in material from suspected lesions or regional
lymph nodes. A positive darkfield result is an almost certain diagnosis of
primary, secondary, or early congenital syphilis. For patients with early primary
syphilis or for patients with syphilitic lesions and advanced acquired
immunodeficiency syndrome (AIDS), the darkfield examination may identify the
etiologic agent of syphilis and help diagnose the disease even when antibodies to
T. pallidum cannot be detected.

B. Proper equipment, adequately trained personnel, and the examination of several

slides may be required to demonstrate the presence of T. pallidum in lesion
material by darkfield microscopy.

II. Specimen Collection and Handling

To avoid infection when collecting specimens, observe universal precautions. Before

collecting specimens, ensure that the darkfield microscope is in good working order.

A. Specimen
1. The ideal specimen for darkfield examination is a serous fluid rich in T.
pallidum but contains few blood cells (treponemes may be obscured if many
cells are present).
2. Consider every genital lesion in sexually active patients as syphilis until
subjected to a darkfield examination and proven otherwise. Other lesions on
the skin or mucous membranes should be examined when syphilis is
suspected.
3. Darkfield examination of oral lesions is not recommended. All positive
darkfield tests from mouth specimens must be confirmed by a direct
fluorescent antibody test. The indigenous flora of the oral cavity frequently
contain a spiral organism, T. denticola, which is indistinguishable from T.
pallidum.
4. If topical antimicrobial therapy has been applied to a syphilitic lesion, it may
not be possible to demonstrate motile T. pallidum even if several specimens
are examined.

B. Collection
1. Lesions
a. Remove any scab or crust covering the lesion.
b. Secondary infection exudate, if any, should be removed with a sterile
gauze sponge.
c. If necessary, compress the base of the lesion or apply a suction cup to it

ACHDLS - Darkfield Microscopy for T. pallidum 67 Revised February 2012

 to promote the accumulation of tissue fluid on the ulcer surface.
d. Apply a glass slide to the opened lesion to transfer the fluid from the
lesion to the slide.
e. Place a cover glass on the specimen and flatten or depress it evenly on the
slide, using the blunt end of an applicator stick to remove air bubbles.
f. Examine the slide immediately.
g. To prevent drying, place additional slides containing specimens in a moist
chamber such as a large plastic petri dish containing a moist paper towel.
Note: The slide preparations should not contain a large volume of fluid
(large volumes cause a rapid liquid flow across the field), nor should the
preparation be so thin that it begins to dry before an adequate examination
can be made.
2. Dry papulosquamous lesions of the skin
a. Gently remove the superficial layer of skin with a scalpel, needle tip, or
mechanical abrader.
b. Try not to cause bleeding. If very little serous fluid appears, compress the
lesion.
c. Touch the corner of the surface of a microscope slide to the fluid to
transfer the material to the slide.
d. Material can also be collected by injecting a small drop of sterile saline
into the base of the lesion and aspirating the fluid with a small-gauge
needle and syringe.
3. Cervical/vaginal lesions
a. With visualization by a bivalve speculum, remove any cervical or vaginal
discharge.
b. Clean the area around the lesion with sterile gauze.
c. Obtain serous exudate from the lesion.
d. Serous exudate, if necessary, can be produced from the lesion by
compressing it with a Kelly clamp.
e. Prepare slides as described in number 1 of this section.
4. Mucous patches
a. Collect some of the mucous material and place it on a clean glass slide.
b. Place a cover glass on the specimen, and examine it immediately.

C. Handling
1. Label slide with patient identifier.
2. Examine the slide within 5-20 minutes of collection, either by bringing the
patient to the microscope or the microscope to the patient.
Note: Any appreciable delay in examining a specimen may result in questionable
findings because the motility of the treponemes may be reduced or completely
lost.

III. Specimen Rejection

A. Specimens not properly labeled or where the identification of the sample is

questionable should not be tested.

ACHDLS - Darkfield Microscopy for T. pallidum 68 Revised February 2012

IV. Reagent, Supplies, and Equipment

A. Reagents
1. Saline, physiological (0.85%), sterile
2. Disinfectant (70% alcohol or iodine solution on swabs)

B. Equipment
1. Microscope assembly containing:
a. Condenser – darkfield, oil immersion condenser; single- or double-
reflecting type
b. Substage - a rack-and-pinion focusing substage for holding a darkfield
condenser
c. Microscope stand with course and fine adjustment knobs and revolving
nosepiece for 3 objectives
d. Body - inclined monocular or binocular type
e. Stage - plain stage with an attachable graduated or ungraduated
mechanical slide carrier
f. Objectives - parfocal
10x - low-power used to focus on specimen and center condenser;
NOT used to search specimen
40x - 45x - high-power used to search the specimen
90x – 100x – oil immersion fitted with a funnel stop or built-in iris
diaphragm used for final identification of organisms
g. Oculars - 10x
h. Illuminator - should be built into the base of the microscope
It should not be attached to the darkfield condenser because the heat
generated may cause complete loss of a critical identifying criterion
such as motility.
The built-in base illuminator should consist of a 6.0- to 6.5-volt or
equivalent, high intensity lamp with a variable transformer for
regulating light intensity.
If a separate external illuminator is used for the microscope, it should
be equipped with an iris diaphragm and a 100-watt lamp which
requires the microscope to have a flat-surface mirror for reflecting the
light into the darkfield condenser.
i. Eye shields - can be obtained for the oculars of some binocular
microscope models, thus eliminating the necessity of having to work in a
darkened room.
2. Microscope slides - 1 x 3 inches (clean and free of scratches)
a. Using slides of the correct thickness is very important.
b. The thickness required by American-made microscopes is usually
engraved on the top of the darkfield condensers.
c. For foreign-made microscopes, refer to the manufacturer’s literature.
3. Cover glass - size number 1, 22 x 22 mm (clean and free of scratches)
4. Oil - immersion, nondrying, Cargille type A (Cargille code 1248, R. P.
Cargille, Inc., Cedar Grove, N.J.) or equivalent

ACHDLS - Darkfield Microscopy for T. pallidum 69 Revised February 2012

 5. Lens paper and lens cleaner
6. Applicator sticks
7. Surgical gloves (rubber or plastic)
8. Gauze, 2 x 2 inches, sterile
9. Scalpel
10. Pipette - sterile, disposable capillary with safety pipetting device
11. Speculum, bivalve
12. Clamp (Kelly or hemostat)
13. Syringe - 1 or 2 ml with 20-gauge needle, sterile
14. Petri dish, plastic, 150 x 15 mm
15. Containers and disinfectant for discards
16. Paper towels

V. Reagent Storage and Usage

A. Store reagents at room temperature.

VI. Calibration

Calibration is not applicable.

VII. Quality Control

A. To complete the microscope adjustment and verify that the microscope is in good
working order before examining the patient material, prepare a suspension of gingival
scrapings in a drop of saline on a slide of proper thickness, then mount with a cover
glass. Examine under oil immersion in a systematic manner.

B. Systematic Pattern of Exam.

Examine @ 10X & 40X

X X X

X XX X

X X X

C. Treponemes observed in the patient’s specimen must have characteristic morphology

and motility for T. pallidum described in this manual.

VIII. Preliminary Procedures and Precautions

ACHDLS - Darkfield Microscopy for T. pallidum 70 Revised February 2012

 A. Ensure that the darkfield microscope is in good working order, and employees are
properly trained in its use.

IX. Procedure

A. Examination of the specimen for T. pallidum

1. Place the slide to be examined on a previously adjusted darkfield microscope.
2. Search the entire specimen methodically with the high-dry objective for spiral
organisms that have the morphology and motility characteristics of T. pallidum.
Search carefully, systematically, and exhaustively before making a negative
report.
3. If a suspected treponeme is observed, center it in the field with the slide carrier
so it can be examined with the oil-immersion objective.
4. Rotate the nosepiece halfway so that a small drop of immersion oil can be placed
on the cover glass.
5. Continue to rotate the nosepiece until the oil-immersion objective is in place
over the specimen and in contact with the oil on the cover glass.
6. Examine the organism carefully, focusing the fine adjustment knob only.
7. When organisms are found that have the characteristic morphology and motility
of T. pallidum, report as positive.
8. After examining a slide, discard it into a container of 70% alcohol or other
suitable bactericidal solution such as 10% hypochlorite.
9. After examining all specimens for the day, remove the immersion oil from the
stage, the darkfield condenser, and the oil-immersion objective of the microscope.
Use only lens paper and lens cleaner to clean the oil-immersion objective to avoid
scratching the lens.
10. Keep the microscope free of oil and dust and in good working order at all times.

B. Differentiation of T. pallidum from other organisms and objects

1. Characteristics of T. pallidum
a. Morphology
i. Shape - delicate, corkscrew-shaped organism with rigid, uniform, tightly
wound, deep spirals
ii. Length - 6-20 micrometers (µm), average length = 10 µm. The average
organism is slightly longer than the diameter of a blood cell (8 µm).
iii. Width - approximately 0.13-0.15 µm
iv. Spiral wave length - approximately 1.0-1.5 µm
v. Spiral depth - approximately 0.5-0.7 µm
Note: Coil appearance is maintained despite active motility of the organism.
b. Motility
i. Translation (uniform movement in a straight line) - slow, may exhibit
deliberate forward and backward movement with occasional erratic
movement
ii. Rotation - slow to rapid rotation about the longitudinal axis (like a
corkscrew); may rotate without changing place

ACHDLS - Darkfield Microscopy for T. pallidum 71 Revised February 2012

 iii. Flexion - rotation is accompanied by soft bending, twisting, or undulation
from side to side, giving a shimmering, graceful effect. Bending of the
organism usually occurs in the middle and is stiffly executed, like the
bending of a coil spring which comes back into place when released.
iv. Distortion - may occur as a ring with ends seemingly attached or occur as
tortuous convolutions. When attached to or obstructed by heavier objects,
vigorous struggling distorts the coils.
2. Characteristics of T. refringens (formerly Borrelia refringens)
a. A member of the normal genital flora
b. Morphology
i. Shape - spiral organisms that may appear loosely coiled, thick, and coarse
ii. Length - approximately 5-16 µm
iii. Width - approximately 0.2-0.3 µm
c. Motility
i. Translation - rapid movement across or out of the field with a writhing
motion
ii. Rotation - active serpentine and rotating motion with marked flexion. The
organism may rotate so rapidly that it looks straight.
iii. Flexion - marked bending and frequent relaxation of coils
3. If the material on the slides contains numerous artifacts or refractile objects, the
untrained observer may be deceived by pieces of cellular debris, flagella, and
wavy fibrin filaments which can, because of Brownian movement, be quite
deceptive and must be interpreted cautiously.
4. In summary, T. pallidum is a thin, tightly wound, spiral organism capable of
extreme contortions from which it snaps back to its original form in a coiled
springlike manner. It may spin rapidly without translation, move slowly forward
and backward without obvious change in direction of rotation or pitch of coils, or
may move more slowly and thread its way corkscrew-fashion in viscous material.
A springlike rigidity is constant, and T. pallidum does not move rapidly from
place to place with a serpentine motion. Any coarsely wound spiral organism
exhibiting great flexion and rapid movement from place to place is NOT T.
pallidum.

ACHDLS - Darkfield Microscopy for T. pallidum 72 Revised February 2012

 5. Picture of T. refringens

ACHDLS - Darkfield Microscopy for T. pallidum 73 Revised February 2012

X. Interpretation of Results

A. The earliest and empirically most specific means of diagnosing syphilis (if yaws,
bejel, and pinta are excluded) is by darkfield microscopy. The demonstration of
treponemes with characteristic morphology and motility for T. pallidum
constitutes a positive diagnosis of syphilis in primary, secondary, or early
congenital stages, whatever the outcome of serologic testing. False-positive
darkfield tests may occur with oral specimens; therefore, such positive specimens
must be confirmed by direct fluorescent antibody tests specific for identification
of T. pallidum. When patients with untreated primary syphilis are positive by
darkfield microscopy but are serologically nonreactive, they usually become
serologically reactive within several days to several weeks. In other stages, the
patient should be seroreactive; if not, the darkfield interpretation and serologic
results should be analyzed to decide whether test results may be false positives or
false negatives, respectively.

B. Every genital lesion should be considered syphilitic until proven otherwise.

Extragenital lesions characterized by indolence, induration, and regional
lymphadenopathy should be regarded as possibly syphilitic. Failure to find the
organism does not exclude a diagnosis of syphilis.

C. Negative results may be reported for the following reasons:

1. The number of organisms was insufficient for detection.
2. The patient has received antitreponenal drugs, topically or systemically.
3. The lesion is “fading” or approaching natural resolution or disappearance.
4. The lesion is one of late syphilis.
5. The lesion is not syphilitic.

D. When the darkfield examination is negative in patients suspected of having

primary syphilis, repeated examination (on as many as 3 consecutive days) may
be indicated. Theoretically, serologic tests for syphilis should be repeated at 1
week, 1 month, and 3 months. If nonreactive, serologic results are obtained for
longer than 3 months in untreated patients, syphilis may be excluded as the cause
of such lesions. Among patients suspected of having syphilis in other stages, a
negative darkfield examination and nonreactive serologic tests suggest that
syphilis is extremely unlikely, and follow-up tests are unnecessary.

XI. Reporting of Results

A. Reporting Laboratory Findings: See CHD-11 (County Health Record) and

Instructions.

Results Report
Organisms found that have characteristic Darkfield positive
morphology and motility of T. pallidum

ACHDLS - Darkfield Microscopy for T. pallidum 74 Revised February 2012

 No treponemal organisms found, OR spiral Darkfield negative
organisms found but without characteristic
morphology and motility of T. pallidum

No T. pallidum found, but specimen had too Darkfield unsatisfactory

many refractile elements (blood cells, air
bubbles, tissue fragments) to be able to identify
any treponemes or the specimen is drying

XII. Calculations

Calculations are not applicable.

XIII. Troubleshooting

A. Review the "Sources of Error" for specific problems which may affect testing, and
attempt to determine the cause of the problem.

B. If the cause of the problem is not easily determined, follow a systematic approach
to troubleshoot the situation.

C. Double check the specimen identification to ensure that the testing is being
performed on a properly collected and labeled specimen on the correct patient.

D. Compare the procedure as you performed it with each step in the written
procedure. Make sure the written procedure was followed exactly as it is written.

E. Examine any reagents, solutions, and/or materials for:

1. The right color.
2. Turbidity or precipitation.
3. Expiration dates.

F. Check the equipment.

1. Have function verification checks been performed?
2. Are function check results within acceptable limits?
3. Has preventive maintenance been performed at proper intervals?

G. Do testing personnel have adequate training and experience to properly perform

the procedure?

H. An abrupt change in patient results that coincides with a change in reagents,

materials, equipment, or testing personnel can easily indicate the source of the
problem.

XIV. Sources of Error

ACHDLS - Darkfield Microscopy for T. pallidum 75 Revised February 2012

 A. Preparation errors
1. If the specimen contains too many blood cells, air bubbles, or tissue
fragments, these refractile elements can obscure the presence of T. pallidum.
2. If cover glasses and slides are dirty or scratched, obtaining a good darkfield
will be difficult.
3. If slides are too thick or thin, the apex of the cone of light will not coincide
with the object being studied.
4. If the cover glass is too thick, it is impossible to focus on the specimen
because of the short working distance of the oil immersion objective.
5. If there is excessive fluid between the glass slide and cover glass, the liquid
will flow rapidly across the field of vision, and it will be too deep to scan.
6. If there is too little fluid between the glass slide and cover glass, the specimen
will begin to dry, and the organisms will lose motility.

B. Microscopy errors
1. If immersion oil is not placed between the condenser and the slide or if
immersion oil with an incorrect refractory index is used, no light will reach
the specimen.
2. If slides of improper thickness are used, focusing on the lesion material and
illumination of the objects on the slide will be affected.
3. If, while using a paraboloid condenser, the concave side of the microscope
mirror is used with an external light source, the light intensity in the field of
view will decrease.
4. If the darkfield condenser is not properly centered, the object will be
illuminated poorly or not at all.
5. If the darkfield condenser is not properly focused, the most intense
illumination of the object will decrease.
6. If oil and dust are on the subsurface or reflecting area of the darkfield
condenser, the intensity of illumination of the object will decrease.
7. If the high numerical aperture of the oil-immersion objective is not
compensated with a funnel stop or an iris diaphragm, undiffracted direct light
will enter the oil objective.
8. If immersion oil is on the lens of the low-power (10x) or high-power (40x-
45x) objectives, the picture will be hazy without sharp definition.
9. If the specimen is inadequately illuminated, it will be impossible to
differentiate T. pallidum from other spiral organisms.
10. If the microscope is focused on the cover glass instead of on the specimen, a
false-negative report might be issued.
11. If the search of the specimen is inadequate or unmethodical, a false-negative
report might be issued.

C. Errors in differentiating T. pallidum from other organisms and objects

1. If one is unfamiliar with the morphology and motility characteristics of T.
pallidum, a false-positive or a false-negative report might be issued.

ACHDLS - Darkfield Microscopy for T. pallidum 76 Revised February 2012

 2. If one is unfamiliar with the characteristics of nonspecific spiral organisms,
tissue debris, fibrin strand, and other extraneous objects, a false-positive
report might be issued.
3. If one mistakes the effects of Brownian movement on spiral objects for
motility, a false-positive report might be issued.
4. If one sees occasional erratic movement of T. pallidum or no movement at all,
too much time may have elapsed between making and examining the slide.

XV. Remedial Action

A. When you think you have discovered the cause of the problem, take proper actions
such as:
1. Collect new specimen.
2. Use new reagents/materials.
3. Correct equipment problems or replace the equipment.
4. Follow written procedure exactly.
5. Change testing personnel until additional training can be conducted.

B. Retest the patient specimens and reevaluate to see if results are acceptable.

C. If results are still unacceptable, continue to troubleshoot to determine the source of

the problem.
1. Do not report any patient results.
2. Call an outside source to help troubleshoot.

D. If the patient results are now acceptable, the problem is resolved.

E. Document the problem and the corrective action on the appropriate laboratory
record form.

XVI. Normal Values

A. A normal patient is negative for Treponema pallidum.

XVII. Reportable Range

Not applicable.

XVIII. Test Limitations

A. Oral lesions at or near the gingival margin are unsatisfactory for darkfield
examination, as the indigenous flora in this area frequently contain a spiral
organism indistinguishable from T. pallidum.

B. The examination of lesion material from patients who have received

antitreponemal drugs topically or systemically may produce negative results.

ACHDLS - Darkfield Microscopy for T. pallidum 77 Revised February 2012

 C. Fading lesions of the skin are less likely to yield a positive darkfield because fewer
treponemes are present.

XIX. Course of Action if Test System Becomes Inoperable

A. If the test cannot be performed due to the absence of materials or inadequate

materials, if possible, borrow from an appropriate source the materials, reagents,
equipment, etc. needed and run the test.

XX. References

A. Alabama Department of Public Health. Wet Prep Competency; Microscope

Teaching Video Series, 2000.

B. Dark-Field Microscopy and Morphology of T. pallidum.

C. Manual of Tests for Syphilis, 9th Edition, 1998.

D. Olympus. BX41 System Microscope Instruction Manual, 2003.

E. Olympus Video Library. The Basic Microscope - Use & Care.

ACHDLS - Darkfield Microscopy for T. pallidum 78 Revised February 2012

 STD Patient Log and QC Form
County Date / /
Site Page of

Patient Name/CHR RPR Darkfield Wet Prep Comments Initials

RPR Quality Control Data

Antigen Lot Number
Antigen Manufacturer Expiration Date CONTROL RESULTS
Antigen Open Date
Control Card Lot Number Reactive (R)
Control Card Expiration Date Minimal Reactive (MR)
Nonreactive (NR)
Test Area Temperature (23 - 29 C)
Rotator Speed (98 - 102) rpm’s RPR QC Performed By
Needle Check (58 - 62 drops/ml)
Note: Label the dispensing bottle with the antigen lot number, manufacturer’s expiration date, and date antigen was placed in the dispensing
bottle (Open Date). Ensure the antigen is used within three months of the antigen open date not to exceed the manufacturer’s date.

ADPH/BCL/QM/REV 04-10