From one cell or several thousand. Nately achieved with dilutions that have plate counts in the 150-250. Agar plate is between 25 and 250 fur most bacteria. ]OURNAL OF VALIDATION TECHNOLOGY (SUMMER 2011) 43. Mates increase with increasing serial dilutions, and errors in. What are the advantages of dilution plate method? Counts performed by methods other than the serial dilution-agar plate procedure? The advantages and disadvantages of spread plate method.

Abstract Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious and error-prone. We therefore implemented a colony counting system with a novel segmentation algorithm to discriminate bacterial colonies from blood and other agar plates. A colony counter hardware was designed and a novel segmentation algorithm was written in MATLAB. In brief, pre-processing with Top-Hat-filtering to obtain a uniform background was followed by the segmentation step, during which the colony images were extracted from the blood agar and individual colonies were separated.

A Bayes classifier was then applied to count the final number of bacterial colonies as some of the colonies could still be concatenated to form larger groups. To assess accuracy and performance of the colony counter, we tested automated colony counting of different agar plates with known CFU numbers of S.

Pneumoniae, P. Aeruginosa and M. Catarrhalis and showed excellent performance. Introduction Microbiological research techniques often rely on accurate determination of colony forming units (CFUs).

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Routinely, this is done by aliquoting a small amount of a liquid culture and plating out several serial dilutions onto culture plates (Petri dishes containing semisolid medium). After incubation in appropriate conditions for the microorganism of choice, the colonies are counted to determine the number of CFU. This is done by manually counting of colonies on plates illuminated by transmitted light.

The concentration of bacteria in the original culture can then be calculated based on the assumption that each colony has raised from one single bacterium (colony forming unit, CFU). This process is time-consuming, tedious and error prone. There is a tendency to analyse only high dilutions of the initial culture as these have fewer colonies to count. Unfortunately, in low count assays minor counting errors have significant effects on the calculated concentration in the primary liquid medium. On the other hand, accurate counting of plates with high numbers of CFUs is error prone since it requires a high level of attention by the performer. Therefore, often only parts of a plate are analyzed and used to estimate the whole plate count after extrapolation. Furthermore high numbers of CFUs on a plate can lead to false redults due to overcrowding of bacteria.

This study aimed to design an automated colony counter which reliably detects, bacterial counts and colony size on semisolid agar plates of 85 mm diameter. The system should be suitable for the study of important human pathogens, such as Streptococcus pneumoniae, Moraxella catarrhalis and Pseudomonas aeruginosa. These bacteria are grown on diverse agar plates including Columbia blood sheep agar (CSBA), chocolate agar and brain heart infusion (BHI) agar plates.

The system should also be user-friendly and cost-effective with an algorithm that is adaptable to other culture media and microorganisms. Bacterial strains and growth conditions A total of 7 clinical isolates of S. Pneumoniae were selected from two nationwide surveillance programs collecting nasopharyngeal and invasive isolates,.

Additionally, a clinical isolate of Pseudomonas aeruginosa and ATCC strain 25238 of Moraxella catarrhalis were used for validation of the automated colony counter (one isolate for each species). For liquid culture all isolates were grown in brain heart infusion (BHI) broth, supplemented with 5% fetal calf serum (FCS) for S. For culture on solid media, S. Pneumoniae and Pseudomonas aeruginosa were grown on CSBA plates and Moraxella catarrhalis on BHI plates. All agar plates were produced in house.

Strains were grown at 37°C in a 5% CO 2 atmosphere. For counting experiments strains were grown in liquid medium to an OD 600 nm 0.3–0.4. Ten-fold serial dilutions of culture were made in phosphate buffered saline (PBS, pH 7.4) and 100 µl of dilutions (usually of 10 −4 to 10 −7) were plated out on agar plates using glass inoculators and a small rotating disk.

Plates were incubated at 37°C overnight in a 5% CO 2 atmosphere before counting of colonies. Colony Counter Hardware Configuration The completely installed system is shown in. An aluminium rack with a drawer to insert and remove Petri dishes into the machine was constructed. This drawer is eloxadised to minimize light scattering. Attached on the back is a power supply unit combined with a dimmer. This bottom assembly is linked to the camera holding arm with a column. The circular dark field illumination is also attached to the column.