Competition between tube and colony count methods

All began with beef broth.

Dr. Louis Pasteur invented this method for the cultivation of diverse microbes. The famous "Swan Neck" trial was also performed with beef broth.

One of the first solid media for microbiological cultivations was the surface of a potato, presented by Dr. Robert Koch.

Colony counts began to be more and more favored by microbiologist because the colonies gave a chance to the immediate isolations of strains. The visual appearance of colonies on solid agars also help to identify the actors of the play, the species of a sample. Membrane filtration method also rise the popularity of colony count method, as well as the relatively good accuracy of colony count analyses, compared to the broth methods.

The role of the tube methods, however, has turned to be more important today. The limitations of the colony count method, correlated with the features of the samples (turbidity, toxic compounds etc.) and the slow growth (compared to the broth cultivation), are obvious. Testing of growth-affecting compounds like biocides are also easier and more reliable to perform in a solid media. When testing of those agents shall be done in the real environment (like the process water of a paper machine), the only alternative is the tube test. Detection of the response of stimulating and inhibiting agents can be done with various methods (photometry, colorimetry, turbidity, ATP Assay etc.) easily. Quantitative analyses of microbial counts can also be performed much faster with a (MPN) tube method than with the colony count method.

As a conclusion: colony count methods suit very well for purposes like the counting of CFU values as well as the selective cultivations of the total population to detect certain microbial groups. Testing of the effects of diverse growth factors (temperature, pH, biocidic and biostatic compounds etc.) should be performed with the tube methods, however. Growth on/in a solid medium does not correlate with the growth of the population in its original environment. Biofilm trials shall always be performed in liquids, never on solid media.

Various analytical tools have been developed for the measurement of the growth responses (pH, turbidity, impedance) automatically from the tubes and the most novel method, PMEU "mini-fermentor", gives the chance to perform all tests with the highest speed and - if needed - in the original samples to simulate the real growth environment of the microbial population. This method will be presented in PIRA Paper Conference, Barcelona, in next October.

A new PMEU application: quantitative MPN analyses of microbial counts

PMEU method is based on the rapid cultivations of several samples. The old idea to apply it in MPN (Most Probable Number) analyses has now proven to be correct: referring the Finnish Standard Book "SFS-KÄSIKIRJA 94: Mikrobiologiset vesitutkimusmenetelmät" (Methods for Microbiological Water Analysis) and discussions with specialists, PMEU can be used as an alternative, rapid method instead of the traditional technique, tube series in water bath or in an incubator. PMEU itself works as an incubator with a temperature deviation of < 0.1 oC.

A combination of 4 (levels of dilutions) * 5 (repeats) allows to follow the Finnish standard SFS 4447 (The Tube Method in Microbiological Water Analysis) as well as standards derived of it like SFS-EN ISO 9308-3 (for and coliforms) and SFS-EN ISO 7899-1 (for enterococci). Standards usually give MPN tables in the framework of 3*5 tubes (eg. for dilutions from 0 to 0.01) but PMEU gives an extra level (eg. 0 to 0.001) which covers a wider range of microbial counts. Samples with unknown levels of microbial densities are therefore easier to analyse correctly.

It seems that the leading status of membrane filtration has revised today. There are types of samples which are difficult or impossible to analyse with them (too much suspended solids etc.) and tube tests like MPN should be chosen. PMEU Tube Tests should be preferred also in situations where fast results (in hours, compared with days with colony count analyses) are needed.

The microbiological control of certain paper industry samples (pulp slurries,starches, minerals) are better to perform with tube methods. An example of the priority of the tube methods can be seen when samples with polymers should be analyzed: polymers tend to stuck membranes immiadely but do not prevent any analyses performed with tube methods. Rapid detection of harmful or hazardous bacteria can also be done faster with selective broths than in/on selective agars.