Automated counting of bacterial colony forming units on agar plates

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.     

ENUMERATING 3MTM PETRIFILMTM AEROBIC COUNT PLATES USING THE PETRISCAN® AUTOMATED COLONY COUNTER

In the food and dairy industries, aerobic plate counts are determined by a time-consuming and laborious hand-counting method. The PetriScan ® automated colony counter was developed to improve efficiency in the microbiology laboratory. In this study, colony counts of food, dairy, and milk products plated on 3M^TM Petrifilm^TM Aerobic Count Plates were compared using both automated and manual count plate methods. For sample variation, 16 different food, dairy, and milk products were used. Samples were prepared and serially diluted using Butterfield's diluent according to approved AOAC methods and APHA's Standard Methods. Plates were inoculated, incubated, and counted according to AOAC methods. For data collection, plates with counts between 5 and 300 colonies were included. A total of 55 low (5–30), 29 medium (31–100), and 23 high (101–300) count plates were used. Duplicate results were recorded for both methods; hand counts were tallied by two scientists. The duplicates of the mean log values for manual counts varied by 0.0005 and 0.0007, and the duplicates for the automated counts varied by 0.0011. The mean log value difference between the automated and manual counts for pooled data was 0.035. The correlation coefficient for the regression line comparing the automated and manual count methods for pooled data was 0.98. The regression equation was y = 0.9257x + 0.0781. These results demonstrate that the PetriScan® automated colony counter is a comparable and practical alternative to the standard method of manually counting plates.     

Evaluation of the spiral plate and laser colony counting techniques for the enumeration of bacteria in foods

Summary (1) In a parallel study, samples of food and dairy products, bacterial cultures and spore suspensions were examined by two operators using both the spiral plate and surface drop techniques for counting bacteria. (2) Statistical analyses of the results showed no differences between the methods at the 5% level of probability; regression and correlation coefficients were highly significant. A variation between paired counts of less than 0.5 log₁₀ cycles was given by 95% of the samples. (3) The replicate variances of both methods were <0.006, indicating good agreement betweeen duplicate plates. (4) An electronic laser counter used in this study was found to give comparable results (r=0.966) to the grid-method of colony counting in a substantially shorter time. (5) Analysis of operation times and material requirements for each method showed that significant savings in cost, time, space and support labour could be achieved with the spiral plate method over conventional techniques.     

Encephalitozoon cuniculi: quantitation of parasites and evaluation of viability

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

Evaluation of an Automated Colony Counter

An automated colony counter was found to readily detect surface and subsurface bacterial colonies of 0.3-mm size or greater with a high degree of precision. On a logarithmic scale, counting efficiency consistently ranged from 89 to 95% of corresponding manual count determinations for plates containing up to 1,000 colonies. In routine application, however, automated plate counts up to approximately 400 colonies were selected as a more practical range for operation. The automated counter was easily interfaced with an automated data acquisition system.     

ATP生物荧光法快速检测化妆品中菌落总数的研究

本文从快速检测的必要性出发,介绍了ATP生物荧光法及其原理,并针对某款化妆品通过一系列实验建立了一种快速检测菌落总数的方法。将该方法用于CTFA(美国化妆品香料香精协会,Cosmetic Toiletry and Fragrance Association)实验中,并通过与平板计数法结果进行比对,发现该方法可以有效的运用于样品细菌总数的快速检测。     

Improved analysis of hemagglutination assays for quantitation of lectin activity

A method to quantitate lectin activity based on hemagglutination assay in microtiter plates is described. In addition to the normal method of visual titer evaluation an electronic particle counter is used for counting of nonagglutinated cells in the microtiter wells; this allows a rapid, quantitative determination of the amount of lectin required to agglutinate 50% of the countable single cells. It is also recommended that counting results should be related to a standard curve of concanavalin A to improve the reproducibility of the assay.     

The use of spiral plating and microscopic colony counting for the rapid quantitation of Mycobacterium paratuberculosis

AIMS: To evaluate a spiral plating and microscopic colony counting technique to hasten the quantitation of Mycobacterium paratuberculosis. METHODS AND RESULTS: Broth and milk cultures of M. paratuberculosis were spirally plated onto Middlebrook agar plates and microscopically counted at 8 and 14 days of incubation. The same plates were recounted at 27-28 days of incubation when grossly visible colonies were present. The results were statistically compared with no difference in CFU ml-1 derived from the shorter vs longer incubation times. Other mycobacteria isolates were also plated and microscopically examined and found to be easily distinguishable from M. paratuberculosis. CONCLUSIONS: Microscopic quantitation of spirally plated M. paratuberculosis cultures can be achieved within 8-14 days of plate incubation and compare favourably to counts derived after prolonged incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique could greatly hasten the quantitation of viable M. paratuberculosis.     

Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems

An environmental isolate (13- 1BB ) of Salmonella enteritidis serogroup C1 was inoculated into sterile Potomac River water microcosms to observe survival and culturability of the organism by employing acridine orange direct count, fluorescent antibody direct count, direct viable count, plate count on veal infusion agar and xylose lysine decarboxylase agar, and indirect enumeration by the most-probable-number method (MPN), using media selective for Salmonella. Loss of culturability on laboratory media was observed within 48 h. However, cultures could be "resuscitated" and cultured on solid media, following addition of nutrients to the microcosms . Cells, resuscitated 4 days after apparent "die-off" (0 colony-forming units (cfu)/mL) using plate count techniques, yielded numbers of cfu in the same order of magnitude as had been observed before the onset of nutrient limitation. Microscopic techniques for direct viable counting indicated that viability is maintained for as long as 60 days after depletion of nutrients, although attempts to culture these cells, by addition of nutrient, after 21 days yielded apparently sterile plates. Thus, longer periods of "dormancy" appear to require conditions other than simple nutrient addition for resumption of cell growth and division.     

Quantification of unscheduled DNA synthesis by a whole cell counting method

A procedure was developed for the quantification of the autoradiographic assay for unscheduled DNA synthesis. Relative to commonly used practices for grain counting, this procedure provides a more accurate net nuclear grain count by eliminating the subjectivity currently associated with selection of the areas to be counted for the cytoplasmic background count. Briefly, the object area and aperture area modes of an ARTEK 880 colony counter are used to collect values for the total number of silver grains over a particular cell (nuclear and cytoplasmic counts), as well as for the nuclear and cytoplasmic areas. These values are then employed in a short algorithm to determine the net nuclear grain count. This new method provides greater sensitivity for defining weak UDS responses and the data collected readily lends itself to statistical analysis.     

Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia

The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions Actinobacteria, Acidobacteria, Proteobacteria, and Verrucomicrobia.     

Improved Culturability of Soil Bacteria and Isolation in Pure Culture of Novel Members of the Divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia

The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions Actinobacteria, Acidobacteria, Proteobacteria, and Verrucomicrobia.     

Killing of Cells in Bacterial Colonies

Bacterial colonies grown on membrane filters and transferred to plates of inhibitor-containing medium decrease in number of total viable colony-forming cells. The decrease in viable cell count occurs in direct proportion to the concentration of inhibitor present and the length of time the colonies are exposed to the inhibitor, suggesting that the kinetics of cell kill approximate a first-order chemical reaction.     

Counting of p32 in plant tissues using the Cerenkov effect

Summary A procedure for counting p³² in plant tissues is presented. The method, based on the use of Cerenkov radiation, involves practically no sample preparation. Plant tissue are placed into vials containing water or hexane and counted with a liquid scintillation counter. Counts obtained, using this procedure were found to be linearily related to that obtained with a G.M. tube. The counting efficiency was, however, higher with the proposed method. The use of hexane is advantageous if leakage of p³² from the tissue is possible, or when higher counting efficiency is desireable. The use of different liquids may also enable a discriminative count of different beta emitters. As suggested recently⁸ use of wavelength shifter may further increase efficiency of counting Cerenkov radiation.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods.

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.     

Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system

The micro-colony method was used to enumerate viable bacteria in composts. Cells were vacuum-filtered onto polycarbonate filters and incubated for 18 h on LB medium at 37 degrees C. Bacteria on the filters were stained with SYBR Green II, and enumerated using a newly developed micro-colony auto counting system which can automatically count micro-colonies on half the area of the filter within 90 s. A large number of bacteria in samples retained physiological activity and formed micro-colonies within 18 h, whereas most could not form large colonies on conventional media within 1 week. The results showed that this convenient technique can enumerate viable bacteria in compost rapidly for its efficient quality control.     

The effect of sample composition and vial type on Ĉerenkov counting in a liquid scintillation counter

The effect of sample vial type and sample composition on the ?erenkov count rate detected from ³²P and ³⁶Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate.     

Encephalitozoon cuniculi: Quantitation of Parasites and Evaluation of Viability1

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.