Method and means for the determination of bacteria or somatic cells in milk.

0001The present invention relates to a method and means for determining microbial and somatic cells in raw milk samples, in particular a method for the simultaneous measurement of both cell types without expensive pretreatment.

0002In the known methods for determining microbial and somatic cells, these are each determined separately in two processes, the determination of bacteria in raw milk preferably being carried out by direct methods. These methods include microbiological colony counts and microscopic procedures. In the microcolony counting process, raw milk samples are either placed in or on an agar gel, incubated and the number of macrocolonies that form is counted. The number of bacteria in the raw milk is proportional to the number of macrocolonies found. In direct microscopic manual counting, a certain amount of raw milk is spread on a slide, left to dry and then stained with a staining solution. The colored bacteria are then counted manually. According to a modified method, the milk is pretreated with a proteolytic enzyme solution, sucked off through a filter and the bacteria remaining on the filter are stained; the stained bacteria are counted manually.

0003The microscopic count is automated according to the so-called BACTOSCAN method, in which the raw milk sample is first mixed with a lysing solution so that protein particles and somatic cells disintegrate. Gradient centrifugation between two media (sucrose and dextran) separates the bacteria from the rest of the milk. The bacterial solution is withdrawn from the centrifuge and freed of residual protein particles with a proteolytic solution. The remaining bacteria are then stained with acridine orange. An aliquot of the bacterial suspension is applied as a very thin film on the edge of a rotating disc. Irradiation of the film with xenon light causes the bacteria to fluoresce in red-orange. The fluorescent light generated in this way is received via a photodetector and converted into corresponding pulses, which are counted (EP-OS 8826).

0004Four methods are widely used for counting somatic cells in raw milk. This involves classic microscopic counting, counting using the Coulter method, counting using the Fossomatic method and the use of an autoanalyzer for cell counting. For the microscopic counting of somatic cells, a defined amount of milk is placed on a slide, the film is dried, stained and then counted. At least 2 films from a sample must be counted. According to the Coulter method, the sample is first fixed with formalin, heated and treated with various fat loss solutions. After appropriate dilution, the sample is fed to the measuring device and evaluated by conductometry.

0005The Fossomatic process requires a raw milk sample pre-diluted with buffer and stained with fluorescent dyes, which is sprayed in a fine jet onto a rotating disk that serves as the object plane for an automated fluorescence microscope. Stained cells generate fluorescent light, which is electronically recorded. The measurement impulses caused by this are counted and are a measure of the number of somatic cells in the sample. The principle used in the Autoanalyzer is based on the indirect method of stray light measurement. The fat in the milk is chemically dissolved and the scattered light, which is caused by the somatic cells, is measured.

0006In the known methods for the determination of bacterial and somatic cells, the determination is fundamentally only possible in separate analyzes; they are complicated and time consuming. The results of the colony counting process are only available after 48 hours. The determination according to the Bactoscan method has the disadvantage that the milk sample has to be subjected to a complex and unreproducible pretreatment and the bacterial numbers in the range below 50,000 / ml can no longer be determined exactly. The somatic cells must be determined in a separate measurement using the Coulter or Fossomatic method.

0007The invention has for its object to provide a method and means for the simultaneous determination of bacteria and somatic cells in milk, with which the above-mentioned disadvantages of the known methods can be avoided.

0008The invention relates to a method for determining bacteria and somatic cells in milk, which is characterized in that the milk is treated with a clarifying reagent and at least one fluorescent dye and the fluorescent particles are analyzed by flow cytometry or microscopy.

0009Another object of the invention is a means for performing this method, the one<img file="EP0342501A2_D0001.tif" /> In this way, 40 different raw milk samples with different levels of bacterial and somatic cells were measured by flow cytometry. The bacterial content of the same samples was also determined by the plate casting method and the content of somatic cells by the Fossomatic method. A comparison of the methods shows a very good agreement of the results.

Example 4

0010A clarifying reagent is made in a water bath<tables id="tabl0001" num="0001"><img file="EP0342501A2_D0002.tif" /></tables>and raw milk tempered to 30 ° C. In a test tube, 200 μl of milk are added to 1 ml of the clarifying reagent with vigorous stirring. After the solution has been clarified, 100 μl of a dye solution composed of 50 mg / l of thioflavin TCN in 50 mM Tris buffer are added to the solution.

0011Immediately after mixing the liquids, a Neubauer counting chamber is installed on each side loaded with a drop of the mixture. The cells are allowed to sediment and the fluorescent cells are then counted out under a microscope.

Example 5

0012A clarifying reagent<tables id="tabl0002" num="0002"><img file="EP0342501A2_D0003.tif" /></tables>is tempered to 50 C. 300 µl of raw milk (room temperature) are added to 1 ml of this reagent and shaken vigorously. After the solution has been clarified, 100 μl of a dye solution are removed<tables id="tabl0003" num="0003"><img file="EP0342501A2_D0004.tif" /></tables>added to the solution.

0013The fluorescence intensity of the solution obtained is measured in a flow cytometer in the emission range > 520 nm detected. A one-dimensional histogram is obtained in which the bacteria appear at low and the somatic cells at high fluorescence intensity. The summation of the relevant areas in the pulse height histogram gives the number of bacteria and somatic cells per ml of measured raw milk sample.