Cells in Bovine Milk: Differential Staining in Suspension: Collecting, Counting and Examining on Millipore Membrane

Abstract

Differential staining of somatic cells in bovine milk preserved with 12 mg of HgCl₂ and 52 mg of K₂Cr₂O₇ in 50 ml was performed as follows. (1) To a silicone-coated serological test tube are added 2 ml of 0.1% Triton X-100 solution, pH 7.0 (prepared with distilled water and filtered through a membrane filter), and 0.16 ml of benzidine-H₂O₂ solution (benzidine 0.2 gr/200 ml distilled water, filtered, to which are added 0.2 ml of 3% H₂O₂). (2) A 0.2 ml sample of milk is added, mixed well and the tube allowed to stand for 3 min at room-temperature. (3) 0.025 ml of May-Grünwald stain (British Drug Houses standard stain solution, No. 16934/3097) and 0.08 ml of Giemsa stain (improved Giemsa stain G. T. Gurr R66, in solution; BDH Cat. No. 17401) are added, mixed well and allowed to stand for 20 min in a water bath at 48-50 C. The contents and two rinsings of the tube with Triton solution at 48-50 C are poured into a siliconized Pyrex microanalysis filter holder (Millipore XX10 02500) with a Millipore (25 mm dia. type DA 0.65 μ) plain white filter in place. (4) The suspension is filtered immediately at a reduced pressure of about 125 mm Hg. (5) The membrane is washed by flushing about 7 ml of the Triton solution at 48-50 C through it, removed and dried on a glass slide at 35 C for about 10 min. (6) the membrane is saturated with immersion oil until it becomes transparent and is then overlaid with a coverslip for microscopic examination. This technique contrasts structural characteristics of the various cells formed in milk and shows also the peroxidase activity of certain leucocytes. Thus it facilitates differentiation between kinds of leucocytes, and distinguishes leucocytes from epithelial cells. This often enables the observer to assign an origin to otherwise unidentifiable cellular debris.