The SPERM STAIN can clearly differentiate the morphological structures of the spermatozoa for a perfect functional assessment. The SPERM STAIN constitutes a system to differentiate the blood cell like components and the spermatozoa as well. The system combines the great polychromic results of classical methods such as May – Grunwald or Giemsa but with a really fast method executed in only 15 seconds.
The SPERM STAIN system is based on the original Romanowsky staining method for differential staining of several cellular structures.
Kit 3 x 100 ml. (Ref.99 03 85 ). Content:
1. SPERM STAIN NR. 1 1 x 10 ml. (Ref. 99 03 95).
Hexamethyl-p-roseniline methanolic solution. Handle with care.
2. SPERM STAIN NR. 2 1 x 100 ml. (Ref. 99 03 96).
Xanthene buffered solution.
3. SPERM STAIN NR. 3 1 x 100 ml. (Ref. 99 03 97.
Thiazine buffered solution
1- Fix the smear by immersion in the Working Reagent Nr. 1 ; 5 times, 1 second each. Let drain.
2- Stain the smear by immersion in the Working Reagent Nr. 2 ; 5 times, 1 second each. Let drain again.
3- Stain the smear by immersion in the working Reagent Nr. 3 ; 5 times, 1 second each.
Working reagent Nr. 1 Dilute the vial of SPERM STAIN NR 1 to 1000 ml. with Methanol (we recommend a minimum purity of 99.5% and a water content lower than 0.1%)
Working reagent Nr. 2 Dilute the content of SPERM STAIN NR 2 with 900 ml of deionised water. Please, use high quality deionized water, no tap water.
Working reagent Nr. 3 Dilute the content of SPERM STAIN NR 3 with 900 ml of deionised water. Please, use high quality deionized water, no tap water.
Storage and stability
The components of the kit, stored at room temperature (15 - 25ºC) will remain stable until the expiration date stated on the label. The working reagents are stable at room temperature (15 – 25 ºC) for a minimum of 3 months when reagents contamination or excessive evaporation are avoided.
Reagent nº 1, as a methanolic solution is flammable and toxic by contact, inhalation and ingestion. Handle with care. Waste products must be handled attending your local regulations.
Sperm samples: prepare the smear with 15 µl of fresh sperm on a standard glass slide. Leave it for air drying at least 10 minutes. It is recommended to make a thin and homogeneous spreading of the sample for the best fixation of the dyes and avoiding blurring hypercolorations.
Rinse the smears gently with deionized water and leave them for air-drying.
In order to prevent deterioration of the smears by the immersion oil coversliping is recommend with an appropriate mounting media such as DPX, Eukitt ®. Use two drops and a standard glass coverslip ( 50 x 22 mm ).
Staining intensity can be modified by varying the number of the immersions in solutions 2 and 3, depending on what color is preferred to be emphasized. Curettes with stain shall always be stored capped, specially 1 in order to avoid undesirable evaporations that could promote color deviations from the usual stainings.
Sperm head: Homogeneous dark violet Sperm acrosome: Pale violet, clearer then the head colour Midpiece and tail: dark violet Background: Pale pink