Answering your questions

Sept. 1, 2003
Edited by Daniel M. Baer, M.D.

Six-hour sperm motility exam,
enterococcus and group D streptococcus, and glucose tolerance test

Six-hour sperm motility exam

Our present procedure includes:
volume, viscosity, count (using Cell-Vu disposable counting chamber), initial motility (done within one hour of collection), morphology (Wright-Giemsa stain), vitality (if motility <40%) and another motility at six hours after collection.

As I attempt to update this procedure, I cannot document why the lab started doing a six-hour motility. Please direct me to resources, which would: 1) give information as to what a proper procedure should include, and 2) help me determine if a second motility at six or any other hour is appropriate.

Sperm motility is a complex process. Assessment of sperm motility both quantitatively, as well as qualitatively, is an important part of a semen analysis. Not only must the number of moving sperm be estimated, but the quality of their movement must also be assessed (a grading scale of 1 to 4 by most laboratories). The percentage motility must be performed soon after the collection of the semen sample (usually upon liquefaction, which can occur within 30 minutes). Depending upon the technique used to determine motility (e.g., slide technique, counting chamber or CASA), motility should be performed in duplicate and the mean results recorded on the report. It is true that some laboratories even determine the percentage of motility at intervals extending up to 24 hours; however, we find no justification for repeated determinations because sperm in the female reproductive tract can enter the cervical mucus within three minutes after they have left the semen. We, therefore, determine motility only once. We know that beginning several hours after collection of the semen sample, the motility will be somewhat reduced. In a clinical setting, it is important to determine how sperm reacts in cervical mucus. There is a test to determine sperm-cervical mucus interaction, Penetrak (Adaltis U.S. Inc, Allentown, PA), but this test is no longer manufactured, and there is no commercially available substitute.

John Sampson, PhD
Director, Andrology Northwest
Portland, OR


  1. WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 3rd ed. Cambridge University Press, 1987.
  2. Sampson J and Alexander N. Semen Analysis: A Laboratory Approach,
    Laboratory Medicine. Vol.13, No. 4: April 1982.

Enterococcus and group D

What is the difference between
Enterococcus and group D streptococcus?

Streptococci are gram-positive,
catalase-negative, facultatively anaerobic cocci. Traditionally, the streptococci were classified by phenotypic criteria, such as hemolytic reactions, biochemical tests and Lancefield serological groups. The Lancefield typing scheme was based on cell-wall polysaccharide antigens (beta-hemolytic groups A, B, C, F and G) or cell-wall lipoteichoic acid antigens (group D
streptococci). Based on these phenotypic criteria, the enterococci were classified as group D
streptococci. The nonenterococcal group D streptococci included
Streptococcus bovis, Streptococcus equinus, and Streptococcus

In the mid-1980s, molecular tests such as DNA-DNA and rRNA hybridization and 16S rRNA sequencing were used to investigate streptococcal taxonomy. As a consequence, the enterococci were placed in the genus
Enterococcus. The nonenterococcal group D streptococci remained classified as
streptococci. Enterococcal phenotypic traits include growth in 6.5% NaCl and hydrolysis of L-pyrrolidonyl-beta-naphthylamide (PYR). The nonenterococcal group D
streptococci do not grow in 6.5% NaCl or hydrolyzed PYR. Both the enterococci and S.
bovis inhabit the gastrointestinal tract of humans and animals. Clinically, S.
bovis bacteremia is often associated with malignancy of the colon, although it is not clear whether the organism is a marker for the disease or actually plays a role in gastrointestinal malignancy. The most clinically relevant difference between enterococci and the group D
streptococci is antimicrobial resistance. The enterococci are generally more resistant to the penicillins, cephalosporins, aminoglycosides and vancomycin, permitting them to survive in a hospital environment and cause nosocomial infections in patients receiving broad-spectrum antimicrobial agents.

David Sewell, PhD, ABMM
Director of Microbiology
Veterans Affairs Medical Center
Portland, OR


  1. Ruoff KL, Whiley RA, Beighton D.
    Streptococcus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH.
    Manual of Clinical Microbiology, 7th ed,. Washington, DC: American Society for Microbiology; 1999:283-297.
  2. Facklam RR, Sahm DF, Martins Teixeira L.
    Enterococcus. In: Manual of Clinical Microbiology, 7th ed. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Washington, DC: American Society for Microbiology; 1999:297-305.

Glucose tolerance test

What are the latest guidelines
for glucose tolerance testing, as far as glucose dosage and specimen draw times? What are the latest guidelines for gestational diabetes in regard to glucose amount, draw time, and patient fasting or

The glucose tolerance test is no
longer recommended as the standard for diabetes testing. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus suggests that fasting blood glucose levels greater than or equal to 126 mg/dL after an eight-hour fast on at least two occasions is diagnostic for diabetes.1 The literature currently favors a two-hour postprandial glucose testing after a loading dose of 75mg of glucose. The patient should be fasting prior to the loading dose. Other texts suggest a fasting blood sugar of >140 mg/dL on two occasions or a postprandial glucose of >200 mg/dL is indicative of diabetes mellitus and negates the need for a formal glucose tolerance test.2

If formal glucose tolerance testing is ordered, however, patients should ingest at least 150 grams/day of carbohydrates for the three days preceding the testing and should undergo an overnight fast prior to the administration of up to 75 grams of oral glucose based on weight (1.75grams of glucose/kg body weight).2 Specimens should be drawn prior to dosage and then at 30 minutes (optional), one-, two- and three-hours post-dose. The simultaneous testing of urine for glucose is considered optional. If requested, urine should be tested at one, two and three hours. Physicians must be aware, however, that an abnormal glucose tolerance test in the absence of fasting glucose levels >140 mg/dL or a two-hour postprandial glucose level of >200mg/dL indicates glucose intolerance, and not necessarily diabetes.2

For gestational diabetes testing, a formal oral glucose tolerance test is indicated if a blood sugar level of >140 mg is achieved following a 50-gram oral dose of glucose.1 In these cases, a three-hour tolerance test is conducted after the patient has completed a three-day diet of at least 150 grams of carbohydrates and an overnight fast. A 100-gram loading dose is administered followed by blood levels drawn at 30 minutes (optional) one, two and three hours. Diagnosis of gestational diabetes is then based on obtaining any two of the following four plasma glucose levels during the tolerance test: a fasting glucose of >105mg/dL; a one-hour glucose of >190 mg/dL; a two-hour glucose of >165 mg/dL and a three-hour glucose of >145mg/ dL. Simultaneous urine testing is optional. Hemoglobin A1C tests should not be used to diagnose diabetes.

Dennis Ernst, MT(ASCP)
The Center for Phlebotomy Education Inc.
Ramsey IN


  1. Henry JB., ed. Clinical Diagnosis and Management by Laboratory Methods. 20th ed. 2001. WB Saunders. Philadelphia, PA. 2001.
  2. Jacobs S., ed. Laboratory Test Handbook 4th ed. Lexi-Comp. Cleveland, OH. 1996.
Daniel M. Baer is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLOs editorial advisory board.
MLO’s Tips from the Clinical Experts department provides
practical, up-to-date solutions to readers’ technical and clinical
issues from a panel of experts in various fields. Readers may send
questions to Dan Baer by fax, (503) 636-7932; or e-mail, [email protected].

September 2003: Vol. 35, No. 9

© 2003 Nelson Publishing, Inc. All rights reserved.