- Email: [email protected]
Vital Staining of Spermatozoa Performed by the Patient
Vol. 35, No.1, January 1981 Prinl€d in U.S A.
FERTILITY AND STERILITY Copyright < 1981 The American Fertility Society
VITAL STAINING OF SPERMATOZOA PERFORMED BY THE PATIENT
J0RGEN G. BERTHELSEN, M.D.* Laboratory of Reproductive Biology, Department of Obstetrics and Gynaecology YA, Rigshospitalet, University of Copenhagen, and Department of Clinical Chemistry, Aalborg Hospital North, Aalborg, Denmark
Differential staining of spermatozoa with eosin (vital staining) is used as a measure offunctional capability of these cells. 1 - 3 The spermatozoa should be stained soon after liquefaction in order to avoid the progressive deterioration of the spermatozoa after ejaculation. Vital staining does not replace motility determination, as there is no constant relationship between the results from these two analyses. When for practical reasons the semen sample cannot reach the laboratory within a few hours after ejaculation, the motility determination and vital staining of the spermatozoa may give incorrect results. In these cases vital staining performed by the patient soon after liquefaction should be of value as a complement to the semen analysis. Such a staining method is presented.
utes of gentle mixing with a wooden stick, the semen is made to ascend into a glass capillary tube. The capillary tube is held vertical, and one drop of semen is deposited on the eosin spot on a microscope slide. The eosin is dissolved completely in the semen by quick but gentle stirring with a wooden stick. When the mixture is homogeneous it is left undisturbed for 1 minute. An unprepared microscope slide is then placed on top ofthe semenstain mixture and the mixture is allowed to spread between the two slides. The slides are then drawn apart in a longitudinal direction and waved in the air until dry. The slides are mailed in a plastic container together with a tablet of desiccating agent in an airtight plastic bag. As long as the slides are kept this way, the proportion of stained spermatozoa will remain unchanged. Examination of the Returned Slides in the Laboratory. The slides are fitted with mounting media and cover glasses. Examination for stained (infertile) and unstained (potentially fertile) spermatozoa is performed using a microscope equipped with either negative 3 or positive phase-contrast at a magnification of x 1000.
METHOD
Preparation of Microscope Slides by the Laboratory. The staining is performed on microscope slides, prepared in advance by the laboratory. Eosin BA (Searle), 2.5 gm, is dissolved in 1 liter of 0.125M sodium phosphate buffer, pH 7.4. Using a microsyringe, 20 f.LI ofthis solution are spread in a circular area with a diameter of 15 mm on a microscope slide and air-dried. The prepared slides are stable for more than 2 years. Staining and Slide Preparation Performed by the Patient. The ejaculate is deposited directly into a glass vessel and allowed to liquefy. After 2 min-
EVALUATION OF THE METHOD
With the capillary tubes used, the mean concentration of eosin in the semen-stain mixture was 2.3 gmIliter (range 1.8 to 4.5 gm/liter) in 100 specimens from 10 different semen samples. Varying the staining duration from 30 seconds to 3 minutes and the stain concentration in the semen from 1.5 to 5 gm/liter did not affect the proportion of unstained spermatozoa in semen samples from 10 men (P > > 0.05, Friedman analysis of variance). Thus deviation from the recommended staining time and the variation in eosin concentration caused by the varying sizes of semen
Received May 1, 1980; revised August 15, 1980; accepted August 21, 1980. '*Reprint requests: Jl1lrgen G. Berthelsen, M.D, Laboratory of Reproductive Biology, Afsn. 4052, Rigshospitalet, DK 2100 Copenhagen 0, Denmark.
86
Vol. 35, No.1
COMMUNICATlONS·IN-BRlEF
drops released by one capillary tube were without importance. Among 600 slides prepared by 69 patients, 3.0% (95% confidence limits 1.8% to 4.7%) were not suitable because of faulty preparation. This small number indicates that the staining procedure is sufficiently simple for use by untrained persons. When slides are mounted with cover glasses, staining of the spermatozoa is stable. Reexamination of slides from 10 patients at intervals during 6 months showed no differences in the proportions of unstained spermatozoa (P > > 0.05, Friedman analysis of variance).
87 CONCLUSIONS
The presented method for vital staining of spermatozoa is so simple that it can be reliably employed by patients. The method may be equally applicable to other vital stains. REFERENCES 1. Morosow VA: An objective method for the evaluation of the activity of semen (in Russian). In Collected Papers from the Siberian Research Institute for Cattle Breeding, Novosibirsk, 1:91, 1938 2. Blom E: A one-minute live-dead sperm stain by means of eosin-nigrosin. Fertil Steril 1:176, 1950 3. Eliasson R, Treichl L: Supravital staining of human spermatozoa. Fertil Steril 22:134, 1971
FERTILITY AND STERILITY Copyright < 1981 The American Fertility Society
VITAL STAINING OF SPERMATOZOA PERFORMED BY THE PATIENT
J0RGEN G. BERTHELSEN, M.D.* Laboratory of Reproductive Biology, Department of Obstetrics and Gynaecology YA, Rigshospitalet, University of Copenhagen, and Department of Clinical Chemistry, Aalborg Hospital North, Aalborg, Denmark
Differential staining of spermatozoa with eosin (vital staining) is used as a measure offunctional capability of these cells. 1 - 3 The spermatozoa should be stained soon after liquefaction in order to avoid the progressive deterioration of the spermatozoa after ejaculation. Vital staining does not replace motility determination, as there is no constant relationship between the results from these two analyses. When for practical reasons the semen sample cannot reach the laboratory within a few hours after ejaculation, the motility determination and vital staining of the spermatozoa may give incorrect results. In these cases vital staining performed by the patient soon after liquefaction should be of value as a complement to the semen analysis. Such a staining method is presented.
utes of gentle mixing with a wooden stick, the semen is made to ascend into a glass capillary tube. The capillary tube is held vertical, and one drop of semen is deposited on the eosin spot on a microscope slide. The eosin is dissolved completely in the semen by quick but gentle stirring with a wooden stick. When the mixture is homogeneous it is left undisturbed for 1 minute. An unprepared microscope slide is then placed on top ofthe semenstain mixture and the mixture is allowed to spread between the two slides. The slides are then drawn apart in a longitudinal direction and waved in the air until dry. The slides are mailed in a plastic container together with a tablet of desiccating agent in an airtight plastic bag. As long as the slides are kept this way, the proportion of stained spermatozoa will remain unchanged. Examination of the Returned Slides in the Laboratory. The slides are fitted with mounting media and cover glasses. Examination for stained (infertile) and unstained (potentially fertile) spermatozoa is performed using a microscope equipped with either negative 3 or positive phase-contrast at a magnification of x 1000.
METHOD
Preparation of Microscope Slides by the Laboratory. The staining is performed on microscope slides, prepared in advance by the laboratory. Eosin BA (Searle), 2.5 gm, is dissolved in 1 liter of 0.125M sodium phosphate buffer, pH 7.4. Using a microsyringe, 20 f.LI ofthis solution are spread in a circular area with a diameter of 15 mm on a microscope slide and air-dried. The prepared slides are stable for more than 2 years. Staining and Slide Preparation Performed by the Patient. The ejaculate is deposited directly into a glass vessel and allowed to liquefy. After 2 min-
EVALUATION OF THE METHOD
With the capillary tubes used, the mean concentration of eosin in the semen-stain mixture was 2.3 gmIliter (range 1.8 to 4.5 gm/liter) in 100 specimens from 10 different semen samples. Varying the staining duration from 30 seconds to 3 minutes and the stain concentration in the semen from 1.5 to 5 gm/liter did not affect the proportion of unstained spermatozoa in semen samples from 10 men (P > > 0.05, Friedman analysis of variance). Thus deviation from the recommended staining time and the variation in eosin concentration caused by the varying sizes of semen
Received May 1, 1980; revised August 15, 1980; accepted August 21, 1980. '*Reprint requests: Jl1lrgen G. Berthelsen, M.D, Laboratory of Reproductive Biology, Afsn. 4052, Rigshospitalet, DK 2100 Copenhagen 0, Denmark.
86
Vol. 35, No.1
COMMUNICATlONS·IN-BRlEF
drops released by one capillary tube were without importance. Among 600 slides prepared by 69 patients, 3.0% (95% confidence limits 1.8% to 4.7%) were not suitable because of faulty preparation. This small number indicates that the staining procedure is sufficiently simple for use by untrained persons. When slides are mounted with cover glasses, staining of the spermatozoa is stable. Reexamination of slides from 10 patients at intervals during 6 months showed no differences in the proportions of unstained spermatozoa (P > > 0.05, Friedman analysis of variance).
87 CONCLUSIONS
The presented method for vital staining of spermatozoa is so simple that it can be reliably employed by patients. The method may be equally applicable to other vital stains. REFERENCES 1. Morosow VA: An objective method for the evaluation of the activity of semen (in Russian). In Collected Papers from the Siberian Research Institute for Cattle Breeding, Novosibirsk, 1:91, 1938 2. Blom E: A one-minute live-dead sperm stain by means of eosin-nigrosin. Fertil Steril 1:176, 1950 3. Eliasson R, Treichl L: Supravital staining of human spermatozoa. Fertil Steril 22:134, 1971