Eosin 0.67g, Nigrosin
5.0g Distilled water to 100 ml.
The eosin nigrosin
stained slide was used for the estimation of morphological defects under 100x oil emersion lens of light microscope.
Morphological observation was determined from a total count of 100 spermatozoa in smears obtained with nigrosin
and eosin for live-dead count.
Microscopic examination included 10% KOH mount, Gram stain, wet mount & Negative staining by Nigrosin
depending upon the type of sample.
Evaluation of the one step eosin nigrosin
staining technique for human sperm vitality assessment.
Following a 30 second incubation, 150 [micro]L of ten percent nigrosin
solution is added, and mixed.
Viability testing requires a very simple two-step staining procedure, using eosin-Y as the stain and nigrosin
as a counterstain.
The strips were stained with nigrosin
(Acid Black 2), dried, and made transparent with microscope immersion oil that had a refractive index of 1.474.
Determination of dead spermatozoa: For dead sperm ratio, Eosin and Nigrosin
stain was prepared, mixing 1% Eosin and 5% Nigrosin
in 3% sodium citrate dehydrate solution (Zemjanis, 1970).
KG, Karlsruhe, Germany) to four parts 10% nigrosin
aqueous solution [Sigma-Aldrich, USA]) using a glass rod to produce a smear on the slide.
Catalase negative colonies that showed the presence of capsule on nigrosin
stain and were optochin sensitive was further confirmed by bile solubility test.