HETTICH-Methods
Slide Preparation of Native Urine for Cytological Examination
As is well known among experts satisfactory cyto preparations of native urine take more than just
sedimenting the specimen on commercial slides. Urine contains few cells, which, in addition, poorly
adhere to slides. The result is a cell-poor, insufficient preparation. To improve the cell harvest it is
recommendable to either coat the slides or apply an adequate fixative. Poly-L-Lysin (PLL from SIGMA,
ordering No. P8920) is a proven coating medium, Saccomanno`s fluid a likewise timetested fixative.
A) Sedimentation of native urine onto coated slides
assemble a cyto insert (see assembly diagram) consisting of a coated slide, a slide carrier, and an 8 ml
cyto chamber (Cat. No. 1666).
fill in the urine specimen
centrifuge at 1100 x g (3000 rpm) for 5 minutes
decant the supernatant
remove the cyto chamber
fixate the wet urine sediment in 99% ethanol
Coating slides with Poly-L-Lysin
1. By immersion
dilute PLL 1:10 with distilled water and fill it into a cuvette
immerse commercial slides without prior cleaning in this solution for 5 minutes, the PLL solution
should have room temperature
take the slides out and lay them into a drying oven, let them dry for about 15 minutes at 60°C, air-drying,
of course, takes longer, as soon as the PLL layer is dry the slide is ready for use
2. By centrifugation
dilute PLL 1:10
place the slide without prior cleaning on a slide carrier and mount an 8 ml cyto chamber (Cat. No. 1666)
on it
fill 100 µl diluted PLL into the cyto chamber
centrifuge at 3000 rpm for 1 hour, after centrifugation the slide is coated and ready for use, the urine
specimen can be filled right into the cyto chamber
Coating by immersion can be carried out fast and easily. Sometimes, the coating, however, does not
form an even layer. Although coating by centrifugation takes a little longer, it always yields very good
coating results.
B) Preparation of native urine with Saccomanno`s solution
centrifuge the urine specimen in a tube at 1700 x g for 10 minutes
decant the supernatant
Important: Always whirl up the sediment that concentrates at the bottom of the tube before adding
Saccomanno`s solution (we recommend to tap the tube carefully on a solid surface).
add Saccomanno`s solution and allow the specimen to rest for 30 minutes at room temperature
assemble a cyto insert and fill in the specimen
centrifuge at 1100 x g for 5 minutes
decant the supernatant
dismount the cyto chamber and allow the sediment to air-dry, the preparation is ready for further
processing or despatch, if required
before staining, remove the wax-like film on the sediment by immersing the slide in ethanol (50%) for
approx. 10 minutes
Production of Saccomanno`s solution
100 ml consist of:
43 ml distilled water
53 ml ethanol (95%)
4 ml polyethylene glycol stock solution
Polyethylene glycol stock solution:
Heat polyethylene glycol 1500 (from Merck Darmstadt, ordering No. 807489) and distilled water
separately to 60°C. Mix equal volumes of the heated ingredients (e. g. 50 ml polyethylene glycol and 50
ml distilled water). Before preparing Saccomanno`s fluid allow the stock solution to cool down to room
temperature.
Both methods described above provide preparations of high cell yield and excellent cell presentation.
Further, sediments treated with Saccomanno`s solution feature a perfectly clean background, and the
urothelial cells are presented on the same optical level.
PLL coating, on the other hand, preserves also smaller particles like bacteria.
Industrial Professional Engineering Services
|
