BIOL1040 Lecture Notes - Lecture 11: Capillary Action, Pipette, Cell Culture
23 May 2018
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MICROENUMERATION
MICROENUMERATION
• Process of counting "small things"
• In many biological, pathological and medical fields – necessary to be able to count number of
cells in a preparation
• i.e. number of spermatazoa, red blood cells, bacteria
PROCEDURE FOR CARRYING OUT A CELL COUNT:
• Diluting a sample – so a manageable number of cells are present
• Placing diluted sample into a counting chamber
• Counting chamber placed under microscope and cells counted
• Number of cells in original sample estimated using dilution factors for counted sample
Haemocytometer most widely used
• Originally designed for performing blood cell counts
• In cell culture labs
• Used in brewing industry for counting yeast cells
• Counting chamber – heavy glass slide and
• Lands – central area of counting chamber – ground flat and marked with grid lines of accurate
dimensions
• Cover slip support ridge – raised area to support cover slip
• Depth – uniform gap (0.1 or 0.2mm) between underside of cover slip and the lands
• Moats – separate the ruled counting area from the rest of the slide
• Since we know the size of the ruled area and the depth (ie the distance between the lands and
the coverslip) chamber the volume of liquid in the space can be calculated.
• When a suspension of cells is added to the chamber the cells will settle out of suspension onto
the grid area and they can be counted. The result of the count is then the number of cells in a
known volume of fluid.
• This can then be extrapolated to the number of cells in the original sample using the dilution
factor.
• The Improved Neubauer has a depth of 0.1mm.
• It has a ruled area of 9 x 1mm2 primary squares
• The four large corner squares (1mm2 each) are each divided into 16 smaller squares with
areas of 0.25 x 0.25mm.
• The central 1mm2 area is divided into 25 squares each 0.04mm2.
• Each of these squares is further divided into 16 squares, 0.05 x 0.05mm in size.
Using a counting Chamber
• Chamber and cover slip must be thoroughly cleaned and dried
• Care with drying (pat dry to avoid scratching the grid markings)
• Place chamber in flat surface and slide cover slip into position
• Mix suspension to be counted
• Take sample using capillary tube or automatic pipette
• Wipe any excess liquid from outside of capillary tube
• Completely fill chamber by holding capillary tube at 45° angle and gently touch tip against the
edge of cover slip
• Area under cover slip fills by capillary action.
find more resources at oneclass.com
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Document Summary
In many biological, pathological and medical fields necessary to be able to count number of cells in a preparation i. e. number of spermatazoa, red blood cells, bacteria. Haemocytometer most widely used: originally designed for performing blood cell counts, used in brewing industry for counting yeast cells. In cell culture labs: counting chamber heavy glass slide and. Cell dilutions: when counting blood cells it is necessary to count red and white blood cells separately and to use different types of diluting fluids. Red cell diluting fluid: the fluid must retain cells normal shape and not agglutinate. Fluid composed of 1% formalin and 3% tri- sodium citrate (dissolve 3 grams tri-sodium in 50ml distilled water, add 1 ml formalin then make up to 100ml using volumetric flask. Filter and store in clean glass reagent bottle. 100ml with distilled water then add few drops 2% gentian violet solution to give pale violet colour.
