Method and apparatus for collecting representative microbiological water and liquid samples

With a method and equipment according to this invention the greatest possible expansion launch of hygiene indicators and other bacteria and other germs in water samples is ensured in microbe cultivations for example on Colilert.TM. Media using both static cultivations and bubbled cultures like at a PMEU (Portable Microbe Enrichment Unit) type cultivation. The aim is the most effective possible microbiological detection value of a water sample or process sample regardless of whether the stated cultivation is executed without bubbling or other mixing or with it. The equipment and method according to the invention applies to and can be connected with manual or automatic sampling.


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The microbiological contamination control of waterworks and water consumption functions is in most cases depending on the detection of particular bacterial species and breeds, which indicate the state of hygiene. The hygiene control norms from severaldifferent fields list the Escherichia coli bacterium, and bacteria of similar kind, the so called coliform bacteria, as well as the faecal enterococci as such bacterial species and breeds. The monitoring of corresponding bacteria is often central inthe control processes of the cleanliness of water supply networks. Additionally, there are risks in, for example, the spreading of strains that are resistant to antibiotics through waste water treatment methods (Hakalehto, 2006). By way of instance, the adhering ofhygiene criterion collapsed in 2007 in the town of Nokia, in Finland. Waste water, that was to some extent treated but nevertheless contained considerable amounts of different pathogens and assorted hygiene signs, escaped to the wash water network.At least 10 000 persons had been subjected to the contaminated water before water use was banned, which caused the spread of severe bacteria, viruses and protozoa originated autoimmune disorders in Nokia. Furthermore, various business segments sufferedfinancial losses due to lack of clean water. These health hazards and fiscal detriment could have been averted, if the microbiological monitoring was more sophisticated and extensive in the water consumption functions and waterworks.

The intensified observation in question could have based, by way of instance, on the fast elucidation and detection of the same coliform and enterococci indicators that have been presented from the standards and norms of cleanliness of water. The speciesof germs in concern originate specifically from the component of the human alimentary tract in which the secretion of bile limits the growth of many species of microbes (Hakalehto et al., 2010). This component of the intestines is from the duodenum, in thearea in which the most important part of the nutrient uptake of the human organism occurs. The secretion of bile acids from the gut through the retina passage through the Papilla Vateri happens in this part of their intestines. As per a largepatient database, two thirds of the bacteria occurring in this area important for the functions of the body are coliform and enterococci bacteria. It is also likely that there exists a delicate condition of equilibrium, a balanced conditionbetween the E-coli and other coliform bacteria, which produce organic acids and the conforms belonging to the Klebsiella/Enterobacter group, which balance the production of acids, due to that, in healthy persons the pH value of the duodenum is about 6(Hakalehto et al., 2008). This”dualistic” condition of equilibrium is simulated with the Portable Microbe Enrichment Unit (PMEU) and its importance to the human system is an important reason to anticipate the bacteria, which participate in maintaining up of thisbalance, available in the intestinal tract of almost all persons. The keeping of this balance, consequently, has a substantial significance to, the well-being of the intestine and, at precisely the exact same time, the entire human system (Hakalehto, 2011).

When studying the flora and specifically the coliform bacteria in the intestines of a new born baby, it was detected that the massive use of antibiotics slowed down, but failed to stop, bacteria belonging to this Enterobaktericeaefamily from settling to the intestinal canal (Pesola & Hakalehto, 2011). In addition to this, the coliform bacteria formed an important element of their microbial flora in the intestines of new born infants who’d been awarded nourishment of different types (Pesola et al. 2009). According to results of different studies, it is justifiable to use cleanliness indicators at the discovery of intestine-derived contamination. After an enrichment cultivation of the water samples, it is possible to analyse in moredetail the origin of this microbial burden. For instance, at Lake Kallavesi (Kuopio, Finland) it has been possible to typify enterococci indicators, which have been enriched in the PMEU, with a biochemical typification method, and to demonstrate clearlythat the kinds of bacterial strains near industrial plants vary from those breed types isolated from below the treated effluent discharge of a municipal sewage treatment plant (Heitto et al., 2009; Hakalehto, 2010). To be able to employ safe detectionof indicator bacteria and other micro-organisms in the respective stages of the production and distribution of tap water, one recommendable choice is to construct an automatic microbe management system. It is advantageous to base that the microbe management systemon the discovery of hygiene indicators from samples whose automated or manually driven sampling frequency can be raised if the quality of water is suspected to be compromised. The decision to improve the sampling frequency of this contaminationcontrol could be based on, for instance, sensory observations or effects of physiochemical measurements. The procedure can be connected to the management system which changes the frequency, regional distribution and quality of sampling or otherparameters. By means of an automated system (or rather a manual system) samples could be collected in the PMEU or corresponding equipment which, on the contrary to the PMEU does not possess bubbling caused by sterile air or gas. In both cases,microbiological culture media is added into the water. It can be inserted as dry powder that’s made mixable into the sample, by way of instance, by putting it beforehand into the sampling dish or sampling syringe. When at all possible, the stream of sterile gas can also beused for the mixing of the sample.

In many cases, it has been found out that the use of the PMEU in the enrichment cultivation of germs helps intestinal germs (Pesola & Hakalehto, 2011) and germs of environmental samples (Hakalehto, 2010) to begin their growth process(in microbe cultivation,”growth” is referred to as the accumulation of the number of cells or individuals ). The beginning of growth has been proven to be in connection with the growth confirmation of pathogens, such as salmonella (Hakalehto et al., 2007)and campylobacteria (Pitkanen in al., 2009) as well as of indicator species (Hakalehto, 2011), and this has been shown to be in connection with the gas phase utilized during the cultivation. This can be applied to the use of microbiologic general substrateand selective substrate, for example Colilert.TM. Liquid-broth (Idexx Inc., USA) in water microbiology and other hygiene research (Hakalehto, 2010). This Colilert.TM. Method could have been used effectively also in Finland at the case of Nokia in 2007,especially collectively with PMEU and automatic sampling attached to it.

If the automatic sampling system is, however, attached to a semi automatic system would be the gas bubbling of the substrates isn’t being used or it hasn’t yet been taken under account, it’s possible to equip these procedures with the possibility ofextended cultivation or even the division of the sample for studies using many different farming methods. It’s common for all microbiological diagnoses that substrate is added to the samples so as to raise growth. The top of gas to thecultivation is a valuable way to activate and accelerate growth (Hakalehto et al., 2007; Pitkanen et al., 2009; Hakalehto, 2011). In reality a liquid sample, like a water sample, that can cause the sample mix comes to contact thegas volume it displaces. To be able to ensure the correctness of this cultivation, the gas must be entirely sterile and, as presented above, a suitable gas leak would be advantageous for the reviving of microbes in the sample. According toprevious experimental effects, this would ensure a real to life impression of coliforms, enterobacteria and assorted types of pathogens in the sample. The gas that is emitted from the cultivations was successfully utilized in the detection ofindicators and pathogens (Hakalehto et al., 2009). Additionally, on the basis of unpublished project results the monitoring of heterotroph microbe flora of waterworks and water consumption works ought to be appreciated because hygiene indicators alone do not revealall contaminations (Hakalehto, 2010). Similarly, on the grounds of partially unpublished effects of Finnoflag Oy, Using this Colilert.TM. Method operates, at least just as well in the two gasified and static cultivations also from the perspective ofgrowth rate. The outcomes of the national Polaris-project which support this monitoring have been published for the first time at the home page of Samplion Oy in the lecture material of Elias Hakalehto. The presentation was held at the Veden Vaarat(Dangers at Waters) Seminar on Aug.. 26, 2010, in Tampere, where police and waterworks were represented and the substance is published on the Samplion Oy home page

The importance of the detection of coliform bacteria, and especially the Escherichia coli bacterium is central from the microbiological hygiene management of water, and using gases in the use of the Colilert.TM. Method and othercorresponding methods will have an important significance for this. The implementation of a microbiological testing method resembling the one cited above for discovering coliform and E. coli has been confirmed in spring 2010 by Valtion TeknillinenTutkimuskeskus (the Technical Research Centre of Finland; VTT Expert Services) at Espoo (Wirtanen & Salo, 2010). The Colilert.TM. Method as well as the liquid-broth media utilized with it are based in part on using antibiotics as a selective feature insubstrate fluids and cultivation. On the flip side, the gasifying of the culture media that occurs in PMEU farming has a substantial influence on the effectiveness of the selective effect. In joint projects of THL (the National Institute forHealth and Welfare, Finland) and Finnoflag Oy it had been noted that, such as in the detection of campylobacteria from water samples, the selectivity of antibiotics improved due to gas bubbles (Pitkanen et. al., 2009). Because of this, it might beadvantageous to decrease the level of antibiotics at the guidelines of the usage of Colilert.TM. Culture media to acquire an optimal result in terms of development speed. These concentration levels are, however, the manufacturer’s proprietary information and areincluded in many detection methods based on existing standards. On the other hand, the reduction of antibiotic concentration can be regarded as potentially beneficial particularly, as an example, in the instance of Colilert.TM. Media along with other similar discerning culturemedia. Relating to this, the usage of gases in and/or top of gases into farming containers is beneficial to ensure the start of microbe growth and also to optimize the speed of it. Both these factors continue to have significance in theprevention of sterile catastrophes like the one that happened in Nokia at 2007 and at similar smaller water support related cases. For example, in The Netherlands, legislator has required that water authorities and parties involved in producingwater must use the best available technologies to ensure water quality. Thus, if farming is executed or has to be implemented without a gas leak and bubbling by gasoline, there is good reason to assume and require the available knowledge of theeffect of gases on different bacteria and most notably to E. coli and other coliforms should carefully be considered and exploited at the use of the civilization, civilization media or sample. 1 potential in practical field work would be to diluteselective culture broths containing antibiotics, i.e. culture media, with other culture media for PMEU cultivations, in the event the benefits of the gas flows may maximally be taken into use. Another possibility, as explained previously, is an assembly forexample with the company Idexx or other producers of culture media in order to modify the concentration of the culture broth.

As distinct studies have regularly come to the conclusion that both the top of gasoline into a cultivation and the gas condition in the farming at the beginning of expansion are meaningful in the detection of various bacteria and other microbesin lab studies (Hakalehto, 2010; Hakalehto, 2011), it may be considered that the consideration of the matter is advantageous in water management where many bacterial strains or single cells are subject to significant”environmental stress”. Theflowing rate, pressure, composition and temperature of the gas are meaningful for microbial growth and the beginning of it. As an example, it’s been mentioned that the carbon dioxide level of the gas or sample condition has a role concerning the maximumcorrectness of the outcomes of the analysis regardless of if or not it is automatic or manual. Thus, in order to be successful in receiving results, it is very important to prepare for the arrangement of gasoline leak or gasifying during sampling and/or beginning andexecution of this cultivation.

IP reviewed by Plant-Grow agriculture technology news