van der Horst G, Maree L
Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa.
Biotech Histochem. 2009 Dec;84(6):299-308. doi: 10.3109/10520290902984274.
Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue fixative and SpermBlue stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue fixative or by adding 1 ml SpermBlue fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue stain or adding four drops of SpermBlue stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue staining process. SpermBlue stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 x or 1000 x magnification for most of the species studied.
我们的研究旨在探索一种简单的方法，以对人类和动物精子的顶体、头部、中段和鞭毛进行差异染色。另一个前提条件是，染色样本的精子形态可以通过自动精子形态分析（ASMA）进行分析。我们开发了一种使用SpermBlue固定剂和SpermBlue染色剂的新染色方法，它们与精液等渗。整个固定和染色过程仅需25分钟。需要三个主要步骤。首先，使用精液或稀释介质中的精子制作常规精子涂片。涂片在室温下自然风干。其次，通过将带有干燥涂片的载玻片放入装有SpermBlue固定剂的染色盘中，或将1毫升SpermBlue固定剂添加到载玻片上，将涂片固定10分钟。第三，通过将载玻片浸入装有SpermBlue染色剂的染色盘中，或将四滴SpermBlue染色剂添加到固定的涂片上，将固定的涂片染色15分钟。将染色后的载玻片轻轻浸入蒸馏水中，然后自然风干并封固在DPX或等效介质中。该方法简单，适用于现场条件。使用SpermBlue染色方法成功地对人类、三种猴类、马、野猪、公牛、公羊、小鼠、大鼠、家鸡、鱼类和无脊椎动物的精子进行了染色。SpermBlue将人类和动物精子染成不同色调或强度的蓝色。可以清楚地区分顶体、精子头部、中段、尾部的主段，甚至短的末段。对于大多数研究的物种，Sperm Class Analyzer ASMA系统成功地用于在600倍或1000倍放大倍数下自动量化精子头部和中段的测量值。
