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.

Applications of PMEU method for biofilm research and testing of biocides against biofilm growth

Paper machine biofilms have been studied already several years with PMEU method by IM. Test coupons, made of steel brands used in paper machines, have been installed inside PMEU syringaes and the growth has been observed with UV Epifluorescence Microscopy after a short incubation period (see picture above).

This technique has now been modified for ordinary light microscopes, too. Steel coupons have been replaced by specified glass slides and the Gram-stained biofilms can be observed with Bright Field Microscopy - no expensive epifluorescence microscopes are needed in this application.

This method will detect all biofilm-producing microbes and testing of biofilm-preventing biocides is also possible simultaneously. Primary attachers typically appear on the slides in just hours and mature biofilms are available in 12...24 hours. This application is very suitable for all areas of industry where the hygiene of surfaces is important. It can also be applied in every environmental research projects where the formation of biofilms in natural water environments is the subject of the study. Hygiene control of public swimming pools etc. also benefit of this method.

Sulphate Reducing Bacteria in paper industry.

SRB is an interesting group of bacteria which can use sulphate as an electron acceptor for the respiration. Despite the chances of certain other bacteria to use sulphate as the sulphur source for their S-containing cell components, the "real" sulphate reducers transform SO4(2-) to S(-2) in their energy metabolism and oxygen actually inhibits their growth - they are therefore obligate anaerobes. Certain yield of energy may be achieved through fermentation by SRB's but this type of metabolism is regarded as relatively insignificant one for them.

These bacteria have first detected in waste waters of sulphite pulp mills but modern paper machine processes can also induce their growth if certain sulphur-containing compounds are available.

In addition to H2S production (which is a hazardous gas), colour problems can arise because the metal sulphides. FeS is an indicator compound in the analytical detection of SRB's but also a harmful agent of discolorization of paper and paperboard. Last but not least, SRB's have been shown to be conneceted to a certain type of iron corrosion and it is all possible to find those problems still today whenever technical structures with poor steel quality and certain types of organic deposits on their surfaces are combined.

SRB's have also other, peculiar features like the tendency to follow non-exponential growth curve. They have been the subject of firm microbiological research only since the middle of 20th century because their need of anaerobiosis was not understood earlier.

FINNOFLAG Ltd. is currently developing a novel method for the sensitive detection of SRB's with PMEU method - more about this topic in next posts.

The need of bacterial identification?

IM has discussed about alternative methods for the detection of hazardous or harmful bacteria with Dr. Elias Hakalehto.

It is most important to know the pathogens which will appear in patient samples. Clinical microbiologists shall know who are the enemies of the ill people: their metabolic capabilities, antibiotic resistence patterns etc. Their overall features are easy to find from literature or internet whenever the name of the species is known. This identification can be performed by selective cultivations on agar plates or in PMEU incubator, and further tests like microscopic examinations, API ID systems, immunological tests and/or PCR can be done to confirm the basic identification.

Paper mill is definitely another challenge for microbiologist. In some (relatively rare cases) the names of microorganisms are important to know: if the product shall have high hygiene quality (like LPB and other food-grade cartonboards) or questions about bioterrorism have been arisen (spore-forming Bacillus anthracis as an example). The occurrence of Legionella pneumophila is also a risk in the waste water treatment of paper industry today. Selective cultivations, either on plates or in PMEU, are the solid solutions for continuous microbiological control in those cases. PMEU is preferred because its speed (hours, compared to days with colony count analyses).

Papermakers shall focus more on the metabolic activities than the names of bacteria which they are living with in paper mills, however. Continuous inoculation of the paper production processes by contaminants, delivered with incoming lots of starches, mineral fillers, raw water, dry pulp etc. shall be controlled to avoid spoilage (amylolytic activity as an example), biofilm and slime growth, tastes and odours, spots and colours in the product etc. Because the wide range of bacterial species and their origin from the nature itself, clinical methods do not suit very well for this monitoring. There is no time to start labourous cultivations, pure cultures and identifications when the bacterial input continues day and night, "7/24". PMEU seems to be an excellent tool to check the basic features of process populations, their biocide resistence patterns included.

One important fact must also be taken into account. There are a lot of harmful microbes which actually cannot be cultivated on agar at all. One example are certain filamentous bacteria which may cause biofilm layers into the processes. They can be cultivated in some broths, however, but the usage of the original samples as the growth medium is the best way to detect them all. This can be done with ordinary mb laboratory equipment or with PMEU incubator.

Identification of bacterial species is still needed when the mapping of contamination routes into the processes is the subject of the study. IM will discuss about the microbiological mapping in his next posts.

Statistical methods in microbiology.

IM has discussed about the evaluation of novel microbiological methods with several professionals. His knowledge of statistical methods in microbiology bases on the lessons by Prof. Seppo Niemelä, who was (and still is) a well-known specialist in this not-so-well-known area of microbiology.

Testing of microbiological data is more complicated than similar analyses in chemistry. The main reason is the model of repeat distribution: chemistry follows the ordinary normal distribution but the colony counts of microbiological analyses are featured by the Poisson distribution. The reason for this difference is easy to understand: the count of molecules is overwhelming when compared to the limited count of colonies in microbiological cultivations. The dependence of variance on the mean of the data is another problem of colony count analyses, preventing the usage of parametric methods.

Luck enough, there are some non-parametric statistical analyses for Poisson-distributed data, helping the comparisons of means and trends of colony count results.

A novel problem seems to have arisen in microbiological evaluations since 70's. Many modern, automatized instruments of microbiology are not based on the measurements of chemical concentrations or counts of colonies but on specified metabolic activities of microbes. Examples of these analytical procedures are eg. measurements of impedance, turbidity, pH or CO2 production. Because these parameters are in a close connection to the growth rates - and to a new parameter, time - , their evaluations are very challenging procedures.

- More in next posts...

Identification of bacterial species.

Questions about the identification of certain bacterial species are rising time after time. Paper mills - and their customers - are interested to know if the product, paper or board, contains harmful bacteria like food-poisoning bacteria, or even hazardous ones. Could terrorists inoculate a paper machine with Bacillus anthracis? Could Listeria live in wet end processes? Bird-flu?

Another reason for the question is the safety of working environment. Are high densities of bacteria in the wet end air dangerous?

Fortunately all information of the environmental needs, growth factors and capability to survive dry end treatment (="pasteurization") show that there is no need to worry if those real pathogens could be encountered in paper industry products.

There are still some areas inside paper mill where the identifications are needed, like

* food-poisoning, sporeforming bacteria (most important: Bacillus cereus)
* bacteria causing raw material breakdown (amylolytic ones etc.)
* potential primary attachers, causing biofilm formation (several genus)
* bacteria which can cause health symptoms and diseases(Legionella pneumophila, certain coliform bacteria)

Identification of specified bacteria from wet end population isn't easy task. Mixed population, sample matrix, low number of target organisms, their distribution inside the machine all lead to very challenging task.

I will refer the traditional and novel methods for the detection of specified bacteria in next posts. Coming back...

Connections of paper industry microbiology to other sectors of microbiology: what is actually needed?

To make any definitions of paper industry microbiology, it makes sense to compare it with elder sectors of microbiology. Despite the microbiological problems of the paper processes and the paper products have been obvious since the beginning of machine-scale production of paper and board on 19th century, their effects have get worser when the scale, speed and raw material repertoir have increased during last decades. The tradition of the microbiological control, as well as the history of biocide research, intended in the "healthcare" of paper and board machines is therefore much shorter than in related areas like in dairy or food microbiology.

Paper manufacturing processes could be seen as ecosystems where several, complicated microbiological processes are continuing day and night. Microbial communities perform their important role as the actors of chemical transformations which shall modify most living and very many non-living substances into forms which will support the growth of other living creatures. Many species of immigrant bacteria, coming into the processes with the raw water, mechanical fibres and several additives, will feel fine: favourable temperature, pH level and nutrient concentrations, as well as good aeration and a huge supply of contact surfaces to build up biofilms, are available for them. They really do not make any difference between their lives outside and inside of the paper mill walls.

Unfortunately (not for the microbes but for the paper production) there are some features of paper machines which are similar with fermentor and bioreactor processes of biotechnological industry. So many growth factors (some of them were mentioned above) will be kept on so controlled levels that the adaptation of certain microbiological population cannot be avoided. It shall also be kept in mind that the long running periods will increase the microbiological risks by allowing long growing periods of microbes inside the machines.

How to control these problems?

Measures to dose biocidic compounds into the processes cannot be avoided because the conditions of paper and board machines cannot be adjusted on biocidic levels: the rise of the overall temperature over +80 oC is impossible, like the rise of pH value over 12. Before significant technical improvements to prevent the microbial growth in the paper machine processes could be done (if ever), the biocidic treatments and their rapid control methods like ON LINE biofilm measurements and frequent (at least once per 8 hours) AT LINE microbiological control of the main contaminating routes, wet end processes and towers containing white waters, pulps and brokes are the most important tools to secure the runnability of the machines and the quality of the products.

With the price of only 2-3 jumbo rolls can reliable instruments for the AT LINE microbiological control of the wet end processes be bought today. Alternative methods, many of them representing molecular biology methods, are available, but those which can show not only the counts of certain species but also the overall metabolic activities of the waterborne microbes and their potential to produce biofilms should be preferred. A combination of PMEU incubations and ATP Assays, with the addition of PCR if needed, is the most recommended procedure to show the effects of biocides on the microbial activity. PMEU method can be applied to biofilm testing, too.

The most important thing is that not only the counts of microbes (how high they may ever been) but also their overall metabolic activity and certain actions like breakdown of starches by amylase enzymes or production of H2S and H2 in anaerobic conditions shall be controlled all the time when the machines are running. All the laws of microbial ecology are present both in the nature and inside the machines - and they can lead to severe problems if counteracting does not work.