134:. 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. Often 100 μL are plated but also larger amounts up to 1 mL are used. Higher plating volumes increase drying times but often do not result in higher accuracy, since additional dilution steps may be needed. 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.
138:
319:
231:
347:(MPN) and Modified Fishman Units (MFU) can be used. The Most Probable Number method counts viable cells and is useful when enumerating low concentrations of cells or enumerating microbes in products where particulates make plate counting impractical. Modified Fishman Units take into account bacteria which are viable, but non-culturable.
334:
Some of the automated systems such as the systems from MATLAB allow the cells to be counted without having to stain them. This lets the colonies to be reused for other experiments without the risk of killing the microorganisms with stains. However, a disadvantage to these automated systems is that it
301:
In addition to software based on traditional desktop computers, apps for both
Android and iOS devices are available for semi-automated and automated colony counting. The integrated camera is used to take pictures of the agar plate and either an internal or an external algorithm is used to process the
314:
as many of the research techniques done by humans counting individual cells have a high chance of error involved. Due to the fact that researchers regularly manually count the cells with the assistance of a transmitted light, this error prone technique can have a significant effect on the calculated
247:
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
238:
Counting colonies is traditionally performed manually using a pen 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)
208:
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. 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
326:
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
164:
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
1223:
Cai, Zhongli; Chattopadhyay, Niladri; Liu, Wenchao
Jessica; Chan, Conrad; Pignol, Jean-Philippe; Reilly, Raymond M. (November 2011). "Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: Comparison with manual counting".
292:
Some ImageJ macros and plugins and some CellProfiler pipelines 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
80:
123:). Estimation of microbial numbers by CFU will, in most cases, undercount the number of living cells present in a sample for these reasons. This is because the counting of CFU assumes that every colony is separate and founded by a single viable microbial cell.
201:
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
216:
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
62:
under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a
105:
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 temperature, time, and nutrient medium. Theoretically, one viable cell can give rise to a
248:
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.
221:
However, with the techniques that require the use of an agar plate, no fluid solution can be used because the purity of the specimen cannot be unidentified and it is not possible to count the cells one by one in the liquid.
335:
is extremely difficult to differentiate between the microorganisms with dust or scratches on blood agar plates because both the dust and scratches can create a highly diverse combination of shapes and appearances.
110:
through replication. However, solitary cells are the exception in nature, and in most cases the progenitor of a colony is a mass of cells deposited together. In addition, many bacteria grow in chains (e.g.
633:
Badieyan, Saeedesadat; Dilmaghani-Marand, Arezou; Hajipour, Mohammad Javad; Ameri, Ali; Razzaghi, Mohammad Reza; Rafii-Tabar, Hashem; Mahmoudi, Morteza; Sasanpour, Pezhman (17 July 2018).
169:
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.
586:
Schug, Angela R.; Bartel, Alexander; Meurer, Marita; Scholtzek, Anissa D.; Brombach, Julian; Hensel, Vivian; Fanning, Séamus; Schwarz, Stefan; Feßler, Andrea T. (1 December 2020).
234:
The traditional way of enumerating CFUs with a "click-counter" and a pen. When the colonies are too numerous, it is common practice to count CFUs only on a fraction of the dish.
259:
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
886:
205:
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.
692:"Effects of sonication on bacteria viability in wastewater treatment plants evaluated by flow cytometry—Fecal indicators, wastewater and activated sludge"
1672:"Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium"
1376:
Austerjost, Jonas; Marquard, Daniel; Raddatz, Lukas; Geier, Dominik; Becker, Thomas; Scheper, Thomas; Lindner, Patrick; Beutel, Sascha (August 2017).
71:, it is uncertain if the colony arose from a single cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.
985:"AutoCellSeg: robust automatic colony forming unit (CFU)/cell analysis using adaptive image segmentation and easy-to-use post-editing techniques"
1335:
Moucka, Michael; Muigg, Veronika; Schlotterbeck, Ann-Kathrin; Stöger, Laurent; Gensch, Alexander; Heller, Stefanie; Egli, Adrian (August 2022).
916:
Brugger, Silvio D.; Baumberger, Christian; Jost, Marcel; Jenni, Werner; Brugger, Urs; Mühlemann, Kathrin (2012-03-20). Bereswill, Stefan (ed.).
1774:
1456:
855:
515:
505:
149:
An advantage to this method is that different microbial species may give rise to colonies that are clearly different from each other, both
145:
in order to obtain at least one plate with a countable number of bacteria. In this figure, the "x10" plate is suitable for counting.
894:
775:
1317:
1603:
Brugger, Silvio D.; Baumberger, Christian; Jost, Marcel; Jenni, Werner; Brugger, Urs; Mühlemann, Kathrin (20 March 2012).
256:
1732:
812:
1174:
Clarke, Matthew L.; Burton, Robert L.; Hill, A. Nayo; Litorja, Maritoni; Nahm, Moon H.; Hwang, Jeeseong (August 2010).
1793:
1521:
197:
Colony-forming units are used to quantify results in many microbiological plating and counting methods, including:
177:
Concentrations of colony-forming units can be expressed using logarithmic notation, where the value shown is the
1728:
137:
836:"Successful Culture Techniques for Helicobacter Species: General Culture Techniques for Helicobacter pylori"
186:
20:
729:
459:"Measurement of In Situ Activities of Nonphotosynthetic Microorganisms in Aquatic and Terrestrial Habitats"
1798:
366:
213:
588:"Comparison of two methods for cell count determination in the course of biocide susceptibility testing"
458:
1616:
1125:
929:
402:"Phylogenetic identification and in situ detection of individual microbial cells without cultivation"
371:
344:
97:
1269:"Using CellProfiler for Automatic Identification and Measurement of Biological Objects in Images"
1249:
1115:
691:
615:
587:
1104:"OpenCFU, a new free and open-source software to count cell colonies and other circular objects"
1758:
1770:
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1205:
1153:
1084:
1066:
1022:
1004:
965:
947:
861:
851:
781:. International Commission on Microbiological Specifications for Foods (ICMSF). Archived from
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672:
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511:
486:
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439:
421:
158:
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558:
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429:
413:
178:
107:
68:
1546:
Gilchrist, J. E.; Campbell, J. E.; Donnelly, C. B.; Peeler, J. T.; Delaney, J. M. (1973).
376:
142:
842:, Methods in Molecular Biology, vol. 921, Totowa, NJ: Humana Press, pp. 17–27,
752:
1620:
1498:
1473:
1129:
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474:
318:
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119:
59:
39:
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563:
538:
434:
401:
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361:
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328:
182:
113:
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782:
79:
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230:
166:
64:
55:
27:
1284:
1629:
1563:
1237:
1138:
942:
707:
603:
417:
1353:
1336:
847:
635:"Detection and Discrimination of Bacterial Colonies with Mueller Matrix Imaging"
554:
311:
154:
150:
1061:
1000:
650:
1489:
983:
Khan, Arif ul Maula; Torelli, Angelo; Wolf, Ivo; Gretz, Norbert (8 May 2018).
131:
101:
for clinic samples) and spread over the plate by tipping in the pattern shown.
88:
1695:
1638:
1571:
1407:
1070:
1008:
951:
834:
Whitmire, Jeannette M.; Merrell, D. Scott (2012), Houghton, JeanMarie (ed.),
690:
Foladori, Paola; Laura, Bruni; Gianni, Andreottola; Giuliano, Ziglio (2007).
658:
482:
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572:
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concentration in the main liquid medium when the cells are in low numbers.
1713:
1589:
1474:"Evaluation of an Automated System for the Counting of Microbial Colonies"
510:(Second ed.). USA: CRC Press, Taylor and Francis Group. p. 864.
490:
443:
1729:"Bacterial Analytical Manual: Most Probable Number from Serial Dilutions"
1267:
Bray, Mark-Anthony; Vokes, Martha S.; Carpenter, Anne E. (January 2015).
1191:
43:
1439:
Jarvis, Basil (2016). "Errors associated with colony count procedures".
161:
can be of great use in the identification of the microorganism present.
1472:
Heuser, Elisa; Becker, Karsten; Idelevich, Evgeny A. (17 August 2023).
84:
776:"Principles of microbiological testing: Statistical basis of sampling"
297:
which can make the interaction with the processing algorithms tedious.
209:
standard Petri dish, the linear range of the plate count will be less.
1605:"Automated Counting of Bacterial Colony Forming Units on Agar Plates"
1176:"Low-cost, high-throughput, automated counting of bacterial colonies"
918:"Automated Counting of Bacterial Colony Forming Units on Agar Plates"
281:
274:
264:
260:
1337:"Performance of four bacterial cell counting apps for smartphones"
1120:
317:
229:
136:
78:
51:
47:
507:
Practical
Handbook of Microbiology, Second Edition (Google eBook)
1441:
Statistical
Aspects of the Microbiological Examination of Foods
1378:"A smart device application for the automated determination of
1045:"Machine learning for enumeration of cell colony forming units"
539:"The Number of Colonies Allowable on Satisfactory Agar Plates"
294:
141:
A solution of bacteria at an unknown concentration is often
1522:"Fully Automatic Colony Counter by AAA Lab Equipment Video"
1318:"Now Available for Purchase: Promega Colony Counter App"
277:
that provides an easy way to count colonies from images.
504:
Goldman, Emanuel; Green, Lorrence H (24 August 2008).
310:
Many of the automated systems are used to counteract
302:
picture data and to estimate the number of colonies.
130:
over the range of 30 to 300 CFU on a standard sized
1049:
Visual
Computing for Industry, Biomedicine, and Art
322:
An automated colony counter using image processing.
1750:
1670:Dehority, B A; Tirabasso, P A; Grifo, A P (1989).
1548:"Spiral Plate Method for Bacterial Determination"
343:Instead of colony-forming units, the parameters
1757:. Methods in Enzymology. Vol. 1. pp.
67:requires significant growth, and when counting
400:Amann, R I; Ludwig, W; Schleifer, K H (1995).
1749:Fishman, William H.; Bernfeld, Peter (1955).
8:
1038:
1036:
1226:International Journal of Radiation Biology
38:) is a unit which estimates the number of
1703:
1646:
1628:
1579:
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1415:
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1352:
1292:
1199:
1147:
1137:
1119:
1078:
1060:
1016:
959:
941:
666:
562:
457:Staley, James T.; Konopka, Allan (1985).
433:
189:to be computed as a simple subtraction.
1169:
1167:
392:
1676:Applied and Environmental Microbiology
1273:Current Protocols in Molecular Biology
181:of the concentration. This allows the
19:For the human hematopoietic cell, see
813:"USP 61: Microbial Enumeration Tests"
807:
805:
803:
730:"Log10 Colony Forming Units per Gram"
537:Breed, RS; Dotterrer, WD (May 1916).
7:
1316:Arduengo, Michele (29 March 2013).
475:10.1146/annurev.mi.39.100185.001541
1449:10.1016/b978-0-12-803973-1.00007-3
1341:Journal of Microbiological Methods
83:A dilution made with bacteria and
14:
1727:Blodgett, Robert (October 2010).
1688:10.1128/aem.55.11.2789-2792.1989
1043:Zhang, Louis (5 November 2022).
774:Cole, Martin (1 November 2005).
732:. Titi Tudorancea Encyclopedia
126:The plate count is linear for
1:
1285:10.1002/0471142727.mb1417s109
463:Annual Review of Microbiology
1767:10.1016/0076-6879(55)01035-5
1733:Food and Drug Administration
1630:10.1371/journal.pone.0033695
1564:10.1128/am.25.2.244-252.1973
1386:Engineering in Life Sciences
1238:10.3109/09553002.2011.622033
1139:10.1371/journal.pone.0054072
943:10.1371/journal.pone.0033695
815:. United States Pharmacopeia
708:10.1016/j.watres.2006.08.021
604:10.1016/j.vetmic.2020.108831
418:10.1128/mr.59.1.143-169.1995
1753:[31] Glucuronidases
1354:10.1016/j.mimet.2022.106508
1102:Geissmann, Quentin (2013).
887:"Serial Dilution Protocols"
848:10.1007/978-1-62703-005-2_4
751:Fung, Daniel Y. C. (2009).
555:10.1128/JB.1.3.321-331.1916
226:Tools for counting colonies
54:etc.) in a sample that are
1815:
1062:10.1186/s42492-022-00122-3
1001:10.1038/s41598-018-24916-9
755:. Bioscience International
651:10.1038/s41598-018-29059-5
243:Software for counting CFUs
18:
1490:10.1128/spectrum.00673-23
16:Parameter in microbiology
1382:colonies on agar plates"
273:is a program written in
1279:(1): 14.17.1–14.17.13.
592:Veterinary Microbiology
543:Journal of Bacteriology
406:Microbiological Reviews
239:solutions can be used.
214:Miles and Misra methods
187:decontamination process
58:, able to multiply via
21:Hematopoietic stem cell
1399:10.1002/elsc.201700056
891:www.microbelibrary.org
367:Miles and Misra method
323:
235:
146:
102:
87:water is placed in an
1478:Microbiology Spectrum
1443:. Elsevier: 119–140.
321:
233:
140:
82:
1552:Applied Microbiology
1192:10.1002/cyto.a.20864
840:Helicobacter Species
753:"Viable Cell Counts"
372:Most probable number
345:Most Probable Number
257:free and open-source
95:for food samples or
1621:2012PLoSO...733695B
1322:Promega Connections
1130:2013PLoSO...854072G
934:2012PLoSO...733695B
897:on 17 November 2015
267:for image analysis.
117:) or clumps (e.g.,
98:Trypticase soy agar
32:colony-forming unit
1794:Microbiology terms
989:Scientific Reports
885:Reynolds, Jackie.
788:on 31 October 2017
639:Scientific Reports
324:
236:
147:
103:
1776:978-0-12-181801-2
1682:(11): 2789–2792.
1458:978-0-12-803973-1
1232:(11): 1135–1146.
857:978-1-62703-004-5
517:978-0-8493-9365-5
339:Alternative units
306:Automated systems
179:base 10 logarithm
159:colony morphology
1806:
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1731:. United States
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1528:. August 7, 2015
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1180:Cytometry Part A
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893:. Archived from
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143:serially diluted
93:Agar plate count
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377:Replica plating
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155:macroscopically
151:microscopically
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40:microbial cells
36:CFU, cfu or Cfu
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1392:(8): 959–966.
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702:(1): 235–243.
696:Water Research
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