Colony-forming unit

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For the human hematopoietic cell, see Hematopoietic stem cell.

In microbiology, colony-forming unit (CFU) is a rough estimate of the number of viable bacteria or fungal cells in a sample. Viable is defined as the ability to multiply via binary fission under the controlled conditions. In contrast in a microscopic evaluation, all cells, dead and living are counted. The visual appearance of a colony in a cell culture requires significant growth - when counting colonies it is uncertain if the colony arose from one cell or 1,000 cells. Therefore results are reported as CFU/mL (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids to reflect this uncertainty (rather than cells/mL or cells/g).[citation needed]

Theory[edit]

A dilution made with bacteria and peptoned water is placed in an Agar plate (Agar plate count for food samples or Trypticase soy agar for clinic samples) and spread over the plate by tipping in the pattern shown.

The purpose of plate counting is to estimate the number of cells present based on their ability to give rise to colonies under specific conditions of nutrient medium, temperature and time. Theoretically, one viable cell can give rise to a colony through replication. However, solitary cells are the exception in nature, and most likely the progenitor of the colony was a mass of cells deposited together. In addition, many bacteria grow in chains (e.g. Streptococcus) or clumps (e.g. Staphylococcus). Estimation of microbial numbers by CFU will, in most cases, undercount the number of living cells present in a sample for these reasons.[citation needed]

The plate count is linear for E. coli over the range of 30 - 300 CFU on a standard sized petri dish.[1] Therefore, to ensure that a sample will yield CFU in this range requires dilution of the sample and plating of several dilutions. Typically ten-fold dilutions are used, and the dilution series is plated in replicates of 2 or 3 over the chosen range of dilutions. The CFU/plate is read from a plate in the linear range, and then the CFU/g (or CFU/mL) of the original is deduced mathematically, factoring in the amount plated and its dilution factor.[citation needed]

A solution of bacteria at an unknown concentration is often serially diluted in order to obtain at least one plate with a countable number of bacteria. In this figure, the "x10" plate is suitable for counting.

An advantage to this method is that different microbial species may give rise to colonies that are clearly different from each other, both microscopically and macroscopically. The colony morphology can be of great use in the identification of the microorganism present.[citation needed]

A prior understanding of the microscopic anatomy of the organism can give a better understanding of how the observed CFU/mL relates to the number of viable cells per milliliter. Alternatively it is possible to decrease the average number of cells per CFU in some cases by vortexing the sample before conducting the dilution. However many microorganisms are delicate and would suffer a decrease in the proportion of cells that are viable when placed in a vortex.[citation needed]

Uses[edit]

The plate count method is the standard method used in microbiology to estimate cell numbers.[citation needed] There are a variety of variations on this method which include:

  • The Pour Plate method wherein the sample is suspended in a petri dish using molten agar cooled to approximately 40-45°C (just above the point of solidification to minimize heat-induced cell death). After the nutrient agar solidifies the plate is incubated.[citation needed]
  • The Spread Plate method wherein the sample (in a small volume) is spread across the surface of a nutrient agar plate and allowed to dry before incubation for counting.[citation needed]
  • The Miles and Misra Methods or drop-plate method wherein a very small aliquot (usually about 10 microliters) of sample from each dilution in series is dropped onto a petri dish. The drop dish must be read while the colonies are very small to prevent the loss of CFU as they grow together.[citation needed]
  • The Membrane Filter method wherein the sample is filtered through a membrane filter, then the filter placed on the surface of a nutrient agar plate (bacteria side up). During incubation nutrients leach up through the filter to support the growing cells. As the surface area of most filters is less than that of a standard petri dish, the linear range of the plate count will be less.[citation needed]

A second method for estimating the number of cells in a sample is the Most probable number (MPN)[2] method.

Tools for counting colonies[edit]

The traditional way of enumerating CFUs with a "click-counter" and a pen. When the colonies are too numerous, it is frequent to count CFUs only on a fraction of the dish.

Counting colonies is traditionally performed manually using a pencil and a click-counter. This is generally a straightforward task, but can become very laborious and time consuming when many plates have to be enumerated. Alternatively semi-automatic (software) and automatic (hardware + software) solutions can be used.[citation needed]

Software for counting CFUs[edit]

Colonies can be enumerated from pictures of plates using software tools. The experimenters would generally take a picture of each plate they need to count and then analyse all the pictures (this can be done with a simple digital camera or even a webcam). Since it takes less than 10 seconds to take a single picture, as opposed to several minutes to count CFU manually, this approach generally saves a lot of time. In addition, it is more objective and allows extraction of other variables such as the size and colour of the colonies.

  • OpenCFU[1] is a free and open-source program designed to optimise user friendliness, speed and robustness. It offers a wide range of filters and control as well as a modern user interface. OpenCFU is written in C++ and uses OpenCV for image analysis.[3]
  • NICE is a program written in MATLAB providing an easy way to count colonies from images.[4][5]
Some ImageJ macros[6] and plugins and some CellProfiler pipelines[7] can be used to count colonies. This often requires the user to change the code in order to achieve an efficient work-flow, but can prove useful and flexible. One main issue is the absence of specific GUI which can make the interaction with the processing algorithms tedious.

Automated systems[edit]

An automated colony counter using image processing.

Completely automated systems are also available from some biotechnology manufacturers.[citation needed] They are generally expensive and not as flexible as standalone software since the hardware and software are designed to work together for a specific set-up.[citation needed] Alternatively, some automatic systems use the spiral plating paradigm.[citation needed]

Alternative parameters[edit]

Instead of CFU, the parameters MPN (most probable number) and MFU (modified Fishman units) can be used.[citation needed] MPN correlates with CFU.[citation needed] MFU also takes into account bacteria in VNBC (viable but non-culturable) mode.[citation needed]

See also[edit]

References[edit]

  1. ^ Breed RS, Dotterrer WD (May 1916). "The Number of Colonies Allowable on Satisfactory Agar Plates". Journal of Bacteriology 1 (3): 321–31. PMC 378655. PMID 16558698. 
  2. ^ http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm109656.htm[full citation needed]
  3. ^ Geissmann Q (2013). "OpenCFU, a new free and open-source software to count cell colonies and other circular objects". Plos One 8 (2): e54072. doi:10.1371/journal.pone.0054072. PMC 3574151. PMID 23457446. 
  4. ^ http://www.nist.gov/pml/div686/sources_detectors/nice.cfm[full citation needed]
  5. ^ Clarke ML, Burton RL, Hill AN, Litorja M, Nahm MH, Hwang J (August 2010). "Low-cost, high-throughput, automated counting of bacterial colonies". Cytometry Part A 77 (8): 790–7. doi:10.1002/cyto.a.20864. PMC 2909336. PMID 20140968. 
  6. ^ Cai Z, Chattopadhyay N, Liu WJ, Chan C, Pignol JP, Reilly RM (November 2011). "Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: comparison with manual counting". International Journal of Radiation Biology 87 (11): 1135–46. doi:10.3109/09553002.2011.622033. PMID 21913819. 
  7. ^ Vokes MS, Carpenter AE (April 2008). "Using CellProfiler for automatic identification and measurement of biological objects in images". Current Protocols in Molecular Biology. Chapter 14: Unit 14.17. doi:10.1002/0471142727.mb1417s82. PMID 18425761. 

Further reading[edit]