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Overview: manual cell counting and automated cell counters

To count or not to count ? Often, that's the question. However, you should realize that counting cells is a necessary and potent measure to culture stock and experimental cell cultures in a standardized and reproducible fashion. Counting may give you much more insight than just the number of  cells . Setting up a growth curve and just adding your values on a weekly basis may make your understanding of what is going on in the dish -e.g. before  an experiment fails - a crucial asset to save time, money and frustration. Besides the classical manual coutinng using a hemocytomerter (e.g. Neubauer chamber), there are now several automated systems available. 

Comparison of cell counting systems

Type of count Advantages Disadvantages

Manual

Neubauerkammer

Trypanblaufärbung in Neubauerkammer
  • low costs for investment
  • no maintenance costs
  • lowest costs per count (only consumables = trypan blue etc.)
  • cells visible while counting
  • live/dead count possible
  • hard to standardize, especially between personnel
  • statistically the worst result, especially if limited numbers of squares are counted (usually 0,1 to 0,4 μl samples are counted)
  • "inconvenient" counts, especially when several samples have to be counted
  • training necessary (once) but often omitted or incomplete
  • high variability of results between personnel
  • no measurement of cell diameter = size

Chamber system
  • easier to standardize than manual counts
  • statistically in the middle range, usually about 0,4 μl samples are counted
  • variability between personnel only derives from pipetting and measurement imprecision
  • cells visible while counting
  • live/dead count possible
  • cell diameter measurable
  • investment middle to high
  • none  to medium costs for maintenance
  • highest costs per count (0,8 to 2,4 Euro per count)
  • training necessary as counting has to be adjusted for all new cell lines / types
  • settings for new cell types more complex than with capillary systems

Capillary system
  • easier to standardize than manual counts
  • middle range costs per count (~20ct)
  • statistically the best results, usually about 2 μl samples are counted
  • variability between personnel only derives from pipetting and measurement imprecision
  • no toxic waste (trypan blue or DNA-intercalating agents are toxic and have to be disposed of according to national regulations)
  • live /dead count possible
  • cell size, volume, and aggregation measureable
  • Settings for new cell types relatively easily adjustable compared to chamber systems
  • investment middle to high
  • medium to high costs for maintenance
  • training necessary as counter settings have to be adjusted for all new cell lines
  • cells not visible while counting

 

Manual cell counting in a hemocytometer (e.g. Neubauer or Thoma chamber)

Manual counting in a hemocytometer is the cheapest variant of cell counting. There are several types of counting chambers depending on the intended use. In cell culture, most often the Neubauer improved or Thoma improved chambers are used. Chambers may be purchased from different distributers. A good overview over the counting patterns (with German explanations) is given here: Optik Labor.

However, this type of counting is the hardest to standardize. Especially, results between personnel differ greatly as the counting is based on subjective decisions (inside/outside, dead/alive, cell/debris). In addition, in many cases not enough squares are counted and the result is accordingly ,  less statistically  reliable.

 

Automated counting

 A large array of counters is available at the moment. The types range from counter "only" to more sophisticated machines that may perform additional functions. Below, you can see a comparison of machines divided into measurement types. Please note, that we have decided to compare automated microscopes and impedance machines under the point "cell analysis".

Trypanblaufärbung in Neubauerkammer