Using a Counting ChamberFor microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.

A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.

To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.

It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not not careful. One entire grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed.

Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly distributed. To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling.

Here is a way to determine a particle count using a Neubauer hemocytometer. Suppose that you conduct a count as described above, and count 187 particles in the five small squares described.

Each square has an area of 1/25 mm-squared (that is, 0.04 mm-squared) and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004 mm-cubed. You have five squares with combined volume of 5x(0.004) = 0.02 mm-cubed. Thus you counted 187 particles in a volume of 0.02 mm-cubed, giving you 187/(0.02) = 9350 particles per mm-cubed. There are 1000 cubic millimeters in one cubic centimeter (same as a milliliter), so your particle count is 9,350,000 per ml.

Cells are often large enough to require counting over a larger surface area. For example, you might count the total number of cells in the four large corner squares plus the middle combined. Each square has surface area of 1 mm-squared and a depth of 0.1 mm, giving it a volume of 0.1 mm-cubed. Suppose that you counted 125 cells (total) in the five squares. You then have 125 cells per 0.5 mm-cubed, which is 250 cells/mm-cubed. Again, multiply by 1000 to determine cell count per ml (250,000).

Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. For example, suppose that for counting you had to dilute a suspension of Chlamydomonas 10 fold. Suppose you obtained a final count of 250,000 cells/ml as described above. Then the count in the original (undiluted) suspension is 10 x 250,000 which is 2,500,000 cells/ml.

 

Counting Chambers

Counting Chambers from Hausser

These chambers are the finest quality, optically ground, and polished milled glass chambers available. The chamber is diamond etched and has a double improved Neubauer Ruling, which has a worldwide reputation in hospitals and laboratories for unmatched reliability, meeting the most demanding of standards. The standard Hausser blood counting chambers are one piece construction (measuring 75mmx32mmx4.5mm) ensuring long term durability and absolute accuracy in measurement and count.

We offer both the standard (incline) and the new “V-Load” counting hambers for different charging methods. The tolerances are ( 2% of the volume). Cell Depth: 0.100 mm ( +/-2%); Volume: 0.1 Microliter Ruling; Pattern: Improved Neubauer, 1/400 Square mm Rulings cover 9 square millimeters. Boundary lines of the Neubauer rulings are the center lines of the groups of three. (These are indicated in the illustration ). The central square millimeter is ruled into 25 groups of 16 small squares, each group separated by triple lines, the middle one of

which is the boundary. The ruled surface is 0.10 mm below the cover glass, so that the volume over each of 16 small squares is 0.00025 cubic mm.

Made to Last

In addition to the standard ruling patterns which are etched permanently onto the polished surface, we offer the popular “Bright-Line” counting chamber. This bright-line counting chamber allows the ruling to appear brightly illuminated providing sharp contrast to the bright lines and cells, helping the user with accurate counts and less eyestrain. The triple lines show clearly which cells lie within the counting area and eliminate guesswork.We guarantee that all marks are permanent, and can not be removed even after repeated washings.

Other Chambers and Accessories

In addition to the blood counting chambers, we also offer a variety of other counting chambers and accessories which compliment our discipline.

The Phase Hemacytometer: used for phase microscopy; it is a one piece brightlined hemacytometer with a reduced thickness.

The Petroff Hausser Counting Chamber: used for bacteria counting. The Sperm Counting Chambers: used primarily in fertility testing; it is offered in a series of

depths (10, 20, 40 microns). The Howard Mold Counter and the Sedgewick Rafter Counting Chamber are also available as

well as a series of hemacytometer and counting chamber cover glasses.

Double Neubauer Ruled Metallized Counting Chamber

An “H” shape moat which forms 2 counting areas are metal plated and double Neubauer ruled providing sharp bright line contrast for consistent cell counts. A “V” cut on each area is used to

control the possibility of overflow. Triple dividing lines clearly show cells within the counting area. The ruled surface is 0.1mm below the cover glass; liquid capacity is 0.1mm3/mm2 and 0.00025mm3/grid square.

Ordering: The complete set includes one counting chamber, two 0.5mm cover glasses, one red cell pipette, one white blood cell pipette, tubing, mouthpieces and carrying case.

Catalog # DescriptionHauser

No. Price

63510-10 Complete Chamber Set 3120 285.00

63510-11 Chamber with 2 Cover Glasses 3100 226.00

63510-13 Chamber only 3110 215.00

63510-20 Phase Counting Chamber 3200 243.00

Etched - Transparent Field with Opaque PatternLevy Double Neubauer

Ruled Hemacytometer

Double improved Neubauer rulings are diamond etched directly into a polished recess to reduce damage from scratches. Opaque surfaces above and below the counting area offer an easy focal reference. The chamber comes with a quick orientation mark for the microscope. 0.1mm cell depth.

Ordering: The complete set includes one counting chamber, two 0.5mm cover glasses, pipettes, and carrying case.

Catalog # Description Hauser No. Price

63511-10 Complete Set 3520 320.00

63511-11 Chamber with Two Cover Glasses 3500 225.00

63511-13 Chamber only 3510 215.00

Other Counting Chambers

Fuchs-Rosenthal Counting Chamber:

Designed for counting leukocytes, eosinophils and cells in cerebrospinal fluid. Grid pattern is etched directly onto the glass surface. The chamber is 0.2mm deep

Ordering: Chamber set includes: one chamber and 2 octagonal cover glasses.

Catalog # Description Hauser No. Price

63512-10 Fuchs Resenthal Counting Chamber 3720 315.00

Petroff-Hausser Counting Chamber:

New improved Neubauer ruling pattern with 1/400 sq. mm of ruling. Designed for the counting of bacteria, sperm, blood platelets, and the content of vaccines. Cell is 0.2mm deep with 1.5mm thick glass slide allows the use off dark field microscopy.

Catalog # Description Hauser No. Price

63512-20 Petroff Hausser Chamber 3900 465.00

63512-21 Petroff-Hausser Chamber w/ Case 3920 575.00

63512-22 Petroff-Hausser Chamber no grid, w/ case 3900SP 465.00

63512-23 Petroff-Hausser Chamber 10 microns w/ Grid 3901 620.00

63512-24 Petroff-Hausser Chamber 20 microns w/ Grid 3902 620.00

63512-25 Petroff-Hausser Chamber 10 microns w/o Grid 3951 620.00

63512-26 Petroff-Hausser Chamber 20 microns w/o Grid 3952 620.00

Nageotte Counting Chamber:

Bright Line double ruling, 0.5mm cell depth.

Catalog # Description Hauser No. Price

63512-30 Nageotte Chamber with Coverglasses 4000 475.00

Palmer, McMaster, Speirs-Levy and Howard ChambersMcMaster Egg Slide Chamber 63512-75

Speirs-Levy Eosinophil Counting Chamber 63512-56

Howard Mold Counter 63512-55

 

Catalog # Description Hauser No. Price

63512-50 Palmer Chamber (Nanoplankton) 3850 207.00

63512-75 McMaster Egg Slide Chamber 3875 330.00

63512-56 Speirs-Levy Eosinophil Counting Chamber 3556 725.00

63512-55 Howard Mold Counter with Two Cover Slips 3820 291.00

Leica Bright-Line Counting Chamber

Designed by Leica for accurate counting of blood cells platelets, dust/yeast cluster and specimen samples. Sharp contrast from rhodium plated on “V-Load”. Triple dividing lines clearly delineate counting area for accurate and precise results. Molded from a single piece of thermal and shock resistant glass with a polished chamber and cover glass assuring precise capillary loading and even cell distribution. Fluid volume is 0.1 mm3/mm2 and 0.00025mm2 per each square.

Ordering: The complete set includes: one counting chamber, two 0.4mm cover glass, one red cell pipette, one white cell pipette, tubing, mouthpieces, and storage case

Catalog # Description Hauser Price

63514-10 Bright-Line Chamber Set 1483 272.00

63514-11 Bright-Line Chamber w two 0.4mm Cover Glass 1492 206.00

63514-12 Bright-Line Chamber Only 1490 192.00

63514-20 Bright-Line Phase Chamber Set w/o Cover Glass 1475 220.00

63514-30 Hemacytometer Cover Glass, 0.4mm 1461 25.00

63514-31 Red Cell Pipette (Thoma) 1456 42.00

63514-32 White Cell Pipette (Thoma) 1458 42.00

63514-34 Plastic Case for Hemacytometer 1505 39.00

Accessories for Counting Chambers63516-10 Cover Glass 20x26x0.5mm 5000 12.00

63516-11 Cover Glass 25x28x0.5mm 5010 32.00

63516-12 Cover Glass 25x33x0.5mm 5020 32.00

63516-13 Cover Glass 27x37x0.5mm 5030 37.50

63516-20 Sedgewick Rafter Cover Glass 5040 32.00

63516-30 Petroff-Hausser Reinforced Cover Glass 5050 97.00

63516-31 Petroff-Hausser Cover Glass 20x26x0.2mm 5051 38.00

63516-40 Whipple Eyepiece Micrometer 5060 212.00

63516-42 Howard Eyepiece Micrometer 5070 176.00

63516-50 Nageotte Cover Glass, 30x33x0.5mm 5400 39.50

63516-61 Nageotte Cover Glass 30x33x1.5mm 5411 27.00

63516-45 Howard Thin Cover Glass 28 x 33 x 0.5mm Thick 5080 39.50

63516-46 Howard Thick Cover Glass 28 x 33 x 1mm Thick 5090 39.50

Cellometer™- The Disposable Cell Counting Chamber!!

Cell Counting Made Easy

These Disposable Cell Counters are an enclosed chamber with two ports for sample introduction. The chamber contains precisely spaced lines in a grid pattern. There are two counting chambers per slide for replicates The counting grid pattern is the Improved Neubauer, as in a common Hemacytometer. The consistent pattern design allows the standard cell counting procedure to be followed.

With a pipetor, 15 to 20 microlilter cell-containing sample is introduced into the counting chamber through one of the ports. Liquid spreads inside the chamber, replaces air, which escapes through the opposing port.

The cell counting chamber is placed under a phase contrast optical microscope with a typical magnification of 100. The microscope is focused both onto the grid pattern and the cell particles. Standard protocols are followed for cell counting.

1. Count the total number of cells found in 4 large corner squares (C_total) 2. Cell concentration = Dilution x C_total / Volume

Let the Cellometer™ replace your Hemacytometers, which are traditionally used for cell. Counting!!

Benefits of the Cellometer™:

1. No washing after counting. Extremely important for people whom work with infectious samples.2. No need to place the cover slide3. Easy sample introduction. By integrating the cover slide into the counting chamber, the

Cellometer™ eliminates the mistakes that may be caused by misplacement of the cover slide or misleading of the sample- two major sources for counting errors when using the Hemacytometer.

4. Made from plastic. Ideal for BioSafety level 3.

Technical Data

Comparisons of Counting ProtocolsStep Cellometer™ Hemacytometer

1 Mix cell suspension Mix cell suspension

2 Pipette 15 to 20 microliter into sample port Carefully load with 10 microliter solution

3 Count cells in 4 corner squares (C_total) Count cells in 4 corner squares (C_total)

4Cell Concentration = Dilution x (C_total / 4.0 ) x10 4

Cell Concentration = Dilution x (C_total / 4.0 ) x10 4

5 Dispose used Cellometer Wash and dry cover slide and base

Description of the experiment:

1. Cell Type: Human T lymphocyte 2. Counting Devices: Cellometer™ with glass base (CG2), all plastic Cellometer™ (CP2) and a

standard bright line Hemacytometer.3. Cell counting performed alternating between Cellometer™ and Hemacytometer.4. Statistical data analysis using Two-Sample T test.

Results: CP2-test 1 CP2-test 2 CG2

# of test (Cellometer™) 15 19 17

# of test (Hemacytometer) 8 17 17

Mean (Cellometer™) 1.78 x 10 5 1.83 x 10 5 1.87 x 10 5

Mean (Hemacytometer) 1.85 x 10 5 1.82 x 10 5 1.82 x 10 5

p-value 0.325 0.908 0.505

Conclusions:

1. Cell counts obtained using Cellometer™ and Hemacytometer are consistent.2. With Cellometer™, sample loading is simple and consistent.3. There is no washing and drying after using Cellometer™, which  Eliminates liquid waste and 

Saves time and effort

Ordering Information:

63561-25Cellometer™ Disposable Cell Counting Chamber Plastic Base

25/bx 80.00

63561-100Cellometer™ Disposable Cell Counting Chamber Plastic Base

100/bx 300.00