Colony-forming unit

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In microbiology, colony-forming unit (CFU) is an estimate of viable bacterial or fungal numbers. Unlike direct microscopic counts where all cells, dead and living, are counted, CFU estimates viable cells. The appearance of a visible colony requires significant growth of the initial cells plated - at the time of counting the colonies it is not possible to determine if the colony arose from one cell or 1,000 cells. Therefore, the results are given 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).

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 (viable defined as able to multiply via binary fission under the controlled conditions) can give rise to a colony through multiplication. 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.

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.

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 great use in the identification of the microorganism present.

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.

Uses[edit]

The plate count method is the standard method used in microbiology to estimate cell numbers. 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.
  • 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.
  • 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.
  • 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.

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.

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[2] is a program written in MATLAB providing an easy way to count colonies from images.[4]
Some ImageJ macros[5] and plugins and some CellProfiler pipelines[6] 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. 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. Alternatively, some automatic systems use the spiral plating paradigm.

Alternative parameters[edit]

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

Reference: Fishman W H and Bernfeld P 1955 Meth. Enzymol., pp 262-269

See also[edit]

Notes[edit]

  1. ^ Breed, Robert S.; Dotterrer, W. D. (May 1916). "The Number of Colonies Allowable on Satisfactory Agar Plates". Journal of Bacteriology 1 (3): 321–331. 
  2. ^ fda.gov
  3. ^ Geissmann, Quentin (2013-02-15). "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. Retrieved 2013-08-28. 
  4. ^ Clarke, Matthew L; Robert L Burton, A. Nayo Hill, Maritoni Litorja, Moon H Nahm, Jeeseong Hwang (2010-08-01). "Low‐cost, high‐throughput, automated counting of bacterial colonies". Cytometry Part A 77A (8): 790–797. doi:10.1002/cyto.a.20864. ISSN 1552-4930. Retrieved 2012-02-08. 
  5. ^ Cai, Zhongli; Niladri Chattopadhyay, Wenchao Jessica Liu, Conrad Chan, Jean-Philippe Pignol, Raymond M Reilly (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): 1135–1146. doi:10.3109/09553002.2011.622033. ISSN 1362-3095. Retrieved 2012-03-13. 
  6. ^ Vokes, Martha S; Anne E Carpenter (2008). "Using CellProfiler for automatic identification and measurement of biological objects in images.". Current Protocols in Molecular Biology. Chapter 14 (April): Unit 14.17. doi:10.1002/0471142727.mb1417s82.