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applica tion note

Colony Counting: Standard Operating Procedure for Colony Counter
Introduction A colony counter is an instrument used to count colonies of bacteria or other microorganisms growing on an agar plate.

The counting may be performed: 1. manually - were merely lighted surfaces on which the plate is placed, with the colonies marked off with a felt-tipped pen on the outer surface of the plate while the operator kept the count manually 2. automatically - to count the colonies electronically, by identifying individual areas of dark and light according to automatic or user-set thresholds, and counting the resulting contrasting spots Such counters are used to estimate the number of microorganisms within a liquid or product. An appropriate dilution, or several dilutions within the estimated appropriate range, is poured or spread using sterile technique on the agar plate, which is then incubated under the appropriate conditions for growth until individual colonies appear. Each colony marks the spot where a single organism was originally placed, thus the number of colonies on the plate equals the number of organisms within the volume of liquid spread on the plate. That concentration is then extrapolated by the known dilution from the original culture, to estimate the concentration of organisms within that original culture. The maximum number of colonies which may be effectively counted on a single plate is somewhere between 100 and 300, depending on the size of the colony and the type of organism. The colony counter should be able to count bacteria on spiral, spread, settle and pour plates methods.
Standard Operating Procedure - TITLE Colony counting by manual colony counter

- SCOPE To standardize the reading and counting of Colony Forming Unit (CFU) on agar plates - GLOSSARY Aerobic Plate Count, Agar, Agar plate, Bacteria , Colony Forming Unit, CFU, Colony, Dilution factor, Incubation, Medium, Petri dish, Pin-point, Poured method, Spread method, Spiral method, Sterilization - RESPONSIBILITY Microbiological Laboratory Manager - SAFETY Individual Protective Devices for biohazard safety - STANDARD ISO Standard MATERIAL Colony Counter Electronic Marking pen Petri dishes Autoclavable Plastic bag

Viewer Petri-Light

Pen electronic colony counter ColonyCount New

Petri plate Agar Petri

Autoclave bag Sterilbag Indicator

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- PROTOCOL 1. Turn on the electric power to light the LED and fluorescent lamp. 2. Place the updown Petri dish on the surface of the counter by adjusting the dish holder. 3. Adjust the focusing arm with the lens to the desired angle, if small colonies are to be counted. 4. Begin counting by touching the Petri dish with the marking pen. Every time a colony is marked, the electronic colony counter pen will register the count. 5. If the colonies are numerous and evenly distributed on the agar surface, it is possible to reduce the time and to count just to opposite segments of the plate and then calculate the total number of the cfu (multiplying for the number of segments). 6. When the counting of the Petri dish is completed, take note of the counted number in the report paper and replace the Petri dish with the next one. 7. At the end of activity, turn off the unit. 8. Guidelines for calculating and reporting. APCs: Official Methods of Analysis does not provide guidelines for counting and reporting plate counts, whereas Standard Methods for the Examination of Dairy Products, 16th ed. presents detailed guidelines; for uniformity, therefore, use APHA guidelines as modified (6,8). Report all aerobic plate counts computed from duplicate plates. For milk samples, report all aerobic plate counts computed from duplicate plates containing less than 25 colonies as less than 25 estimated count. Report all aerobic plate counts computed from duplicate plates containing more than 250 colonies as estimated counts. Counts outside the normal 25/250 range may give erroneous indications of the actual bacterial composition of the sample. Dilution factors may exaggerate low counts (less than 25), and crowded plates (greater than 250) may be difficult to count or may inhibit the growth of some bacteria, resulting in a low count. Report counts less than 25 or more than 250 colonies as estimated aerobic plate counts (EAPC). Use the following guide: 1. Normal plates (25/250). Select spreader-free plate(s). Count all colony forming units (CFU), including those of pinpoint size, on selected plate(s). Record dilution(s) used and total number of colonies counted. 2. Plates with more than 250 colonies. When number of CFU per plate exceeds 250, for all dilutions, record the counts as too numerous to count (TNTC) for all but the plate closest to 250, and count CFU in those portions of plate that are representative of colony distribution. See ref. 2 for detailed guidelines. Mark calculated APC with EAPC to denote that it was estimated from counts outside 25/250 per plate range (see D-3). 3. Spreaders. Spreading colonies are usually of 3 distinct types: 1) a chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump; 2) one that develops in film of water between agar and bottom of dish; and 3) one that forms in film of water at edge or on surface of agar. If plates prepared from sample have excessive spreader growth so that (a) area covered by spreaders, including total area of repressed growth, exceeds 50% of plate area, or (b) area of repressed growth exceeds 25% of plate area, report plates as spreaders. When it is necessary to count plates containing spreaders not eliminated by (a) or (b) above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one colony. Do not count each individual growth in such chains as a separate colony. Types 2 and 3 usually result in distinct colonies and are counted as such. Combine the spreader count and the colony count to compute the APC. 4. Plates with no CFU. When plates from all dilutions have no colonies, report APC as less than 1 times the corresponding lowest dilution used. Mark calculated APC with asterisk to denote that it was estimated from counts outside the 25/250 per plate range. When plate(s) from a sample are known to be contaminated or otherwise unsatisfactory, record the result(s) as laboratory accident (LA). Reference - Health Protection Agency UK Standard Methods for Aerobic Colony Count - U.S. FDA. Bacteriological Analytical Manual. Aerobic Plate Count

application note n.199/102 - microbiology

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