1、ICS 67.180.10Untersuchung von Honig Bestimmung der relativen PollenhufigkeitIn keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.Ref. No. DIN 10760 : 2002-05English price group 08 Sa
2、les No. 010812.02DEUTSCHE NORM May 200210760Continued on pages 2 to 5. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Norm
3、en).Determination of relative pollen content of honeyTranslation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.ForewordThis standard has been prepared by Technical Committee Honiguntersuchung of the NormenausschussLebensmittel und
4、landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee).1 ScopeThis standard specifies a method of determining the relative pollen content of honey.2 ConceptRelative pollen contentContent of the individual pollen species as a percentage of the total pollen content.3 P
5、rincipleA specimen is prepared for microscopic examination of the pollen grains suspended in the honey. A certainnumber of pollen grains is identified and the proportion of the individual pollen species is calculated as apercentage of the total content.4 Reagents4.1 GeneralSince the method described
6、 here is not an analysis, but a count of specific plant cells (pollen grains), noparticular purity requirements are specified for the reagents used, except for water, which shall be distilled.The term solution as used in this standard shall be understood to be an aqueous solution.4.2 Glycerol gelati
7、n, for KAISER microscopy.5 Apparatus5.1 In addition to standard laboratory equipment, the equipment specified in subclauses 5.2 to 5.12 shallbe used.5.2 Stainless-steel screen, of aperture size 0,50 mm.Page 2DIN 10760 : 2002-055.3 1000 g centrifuge, g being the relative centrifugal acceleration, whi
8、ch is given by25101181r, frg =(1)wherer is the distance of the sedimentation point (bottom of the centrifuge tube) from the axis of rotation, in cm;fris the rotational speed, in min1.5.4 Tapered centrifuge tubes, of nominal capacity at least 40 ml.5.5 Test tube shaker.5.6 Microscope, with G180 320 t
9、o G180 1000 magnification.5.7 Microscope slides, measuring 76 mm G180 26 mm.5.8 Cover glasses, measuring 22 mm G180 22 mm.5.9 Hotplate.5.10 Disposable plastic Pasteur pipettes, of nominal capacity 1 ml.5.11 Microspatula.5.12 Pollen counter.6 SamplingPending the publication of a standard on sampling,
10、 the sampling procedure shall be agreed upon.A representative laboratory sample of not less than 200 g shall be taken for the examination.7 Procedure7.1 Sample preparation7.1.1 Pure liquid or set honeyAdequately homogenize the laboratory sample by stirring it vigorously for not less than three minut
11、es, takingcare to minimize the amount of air included, especially if the sample is also to be used to determinehydroxymethylfurfural.7.1.2 Impure liquid or set honeyAfter removing coarse impurities, stir the honey at ambient temperature until smooth and pass it through a screen(as in subclause 5.2),
12、 using a spatula in the case of set honey.7.1.3 Comb honeyDe-cap the combs if still capped, then separate the honey completely from the combs without heating, usinga screen (as in subclause 5.2).7.2 Preparation of specimens7.2.1 Weigh 10 g into a centrifuge tube (as in subclause 5.4). To dissolve th
13、e honey, add 20 ml of cold distilledwater or distilled water at not more than 40 C.Centrifuge the solution at 1000 g for ten minutes, then pour off the supernatant and add a further 20 ml of distilledwater in order to dissolve the sugar crystals in the honey completely. Use a microspatula when doing
14、 this in orderto reach the tip of the centrifuge tube or use a Pasteur pipette to swirl up the mixture. Then centrifuge for fiveminutes at 1000 g.Decant the supernatant, keeping the centrifuge tube tilted downwards in order to allow as much as possible ofthe remaining liquid to drain on an absorbent
15、 paper.Heat a hotplate to 40 C and liquefy glycerol gelatin as embedding medium by heating either in a water bath atnot more than 40 C or on the hotplate. Preheat the microscope slides on the hotplate.7.2.2 Thoroughly swirl up the sediment with a Pasteur pipette. Use the pipette to transfer the sedi
16、ment to amicroscope slide and spread the sediment over an area of 22 mm G180 22 mm with a microspatula.NOTE: 22 mm is almost the width of the microscope slide and it is advisable to mark out the area over whichthe sediment is to be spread, using with a waterproof pen or a template.Page 3DIN 10760 :
17、2002-05Leave the microscope slide on the hotplate for up to one hour until the sediment is completely dry.After preheating a cover plate on the hotplate, transfer a drop of glycerol gelatin to it and form a large crossdiagonally with it. This holds the pollen in position while the cover glass is low
18、ered on the dried sediment, thisbeing done very slowly to prevent air bubbles forming.Do not place the drop of glycerol gelatin directly on the dried sediment.Leave the specimen on the hotplate for five minutes in order to ensure a good distribution of the glycerol gelatinand a perfect swelling of t
19、he pollen.Allow the gelatin to solidify before starting the microscope examination.7.2.3 As an alternative to the procedure described in subclause 7.2.2, suspend the sediment in 1 ml of distilledwater. Swirl up the sediment with a pipette, remove 0,5 ml and spread the latter on a microscope slide. S
20、preada further 0,3 ml and then 0,2 ml of the sediment on separate slides. Use the first specimen (0,5 ml) for normalpollen analysis, the second specimen (0,3 ml) for pollen analysis in the case of very pollen-rich honey and thethird specimen (0,2 ml) for yeast counting and pollen counting in the cas
21、e of extremely pollen-rich honey. Thisprocedure makes it possible to determine the absolute pollen content.The subsequent procedure shall be as described in subclause 7.2.2.7.3 Pollen countingChoose the magnification that results in a manageable number of pollen grains in each field of view. After a
22、preliminary check to identify the pollen species present, determine the quantities of important pollens bycounting at least 500 cumulatively in steps of 100. If the result fluctuates, increase the number of grains counted.Distribute fields of view and counting paths uniformly over the specimen.Count
23、 the pollen using the matrix in figure 1 to obtain a representative result.Figure 1: Pollen counting matrixDistribute counting stops as evenly as possible over the entire width, i.e. over a row of sediment. Using a counterof the type specified in subclause 5.12, count 100 pollen grains in each of fi
24、ve rows distributed evenly over thesediment, as shown in figure 1, to give a total count of 500. To obtain a total count of 1000, insert a further fiverows between the first five rows. Ignore any pollen concentrations due to bee bread.NOTE: Bee bread is pollen stored by bees in comb cells.The interv
25、als at which the counting stops have to be set will depend on the pollen content of the honey, i.e. thedensity of pollen in the specimen, and on the size of the microscope field of view. In the case of very low-pollenhoney it may be necessary to count an uninterrupted line.Provided the count stabili
26、zes, only 500 pollen grains need be counted. Report the result as a percentage.8 Evaluation8.1 CalculationCalculate the relative pollen content for the required plant species, Xp, as a percentage, using the followingequation:nAX100=p (2)where n is the total pollen content.Count 100 pollen in this ro
27、w600200 (counting a further 100 results in 200 pollen)700300 (and so on )800400900500100022 G180 22 mm cover glassa) One complete field of view.b) Example of how to position the counting stops.Page 4DIN 10760 : 2002-05For a more in-depth interpretation of the results and to classify the honey, it ma
28、y be advantageous to subtractthe number of pollen grains from nectar-free plants from the total pollen count, using the following equation:)(pnnAX=100(3)whereA is the number of pollen of the required plant species;n! is the number of pollen grains from nectar-free plants.Report the relative pollen c
29、ontent of the nectar-free plant species in the total pollen count.8.2 Precision8.2.1 Interlaboratory testingSee appendix for results of interlaboratory testing to determine the precision of the method.8.2.2 Repeatability limit(same operator, same equipment)The absolute difference between two success
30、ive results obtained under repeatability conditions will not exceedthe repeatability limit, r, in more than 5 % of cases.For a count of 500, the values are as follows.Crucifers:x= 31,9 % r = 7,80 %Roses:x= 24,2 % r = 5,82 %Maple:x=7,8% r = 5,11 %All clover species:x=5,1% r = 2,65 %For a count of 100
31、0, the values are as follows.Crucifersx= 32,6 % r = 5,19 %Roses:x= 24,6 % r = 8,14 %Maple:x=7,6% r = 4,21 %All clover species:x=5,5% r = 2,24 %8.2.3 Reproducibility limit(different operators, different equipment)The absolute difference between two individual results obtained under reproducibility co
32、nditions will not exceedthe reproducibility limit, R, in more than 5 % of cases.For a count of 500, the values are as follows.Crucifers:x= 31,9 % R = 10,42 %Roses:x= 24,2 % R = 7,30 %Maple:x=7,8% R = 9,11 %All clover species:x=5,1% R = 3,77 %For a count of 1000, the values are as follows.Crucifers:x
33、= 32,6 % R = 8,47 %Roses:x= 24,6 % R = 12,90 %Maple:x=7,6% R = 7,96 %All clover species:x=5,5% R = 4,62 %9 Test reportThe test report shall refer to this standard and include the following details as a minimum:a) type, origin and description of sample;b) type of sampling and date;c) dates of receipt
34、 and testing;d) relative pollen content for the particular plant species, Xp, as a percentage;e) reason for any deviation from this standard.Page 5DIN 10760 : 2002-05AppendixResults of interlaboratory testingAn interlaboratory test was performed by Technical Committee Honiguntersuchung and evaluated
35、 as specifiedin ISO 5725-1 and ISO 5725-2. The statistical results are summarized in table A.1.Table A.1: Results of interlaboratory testingAbsolute counts Relative counts3)Mean MeanRepeat-Repeat-Reproduci-Reproduci-Pollen typeability limitabilitybility limitbilityor group standard standarddeviation
36、 deviationn1)x xrsrRsRCruciferae 500 159 31,9 7,80 2,76 10,42 3,68Crucifers 1000 326 32,6 5,19 1,83 8,47 2,99Rosaceae 500 120 24,2 5,82 2,06 7,30 2,58Roses 1000 241 24,6 8,14 2,88 12,90 4,56Acer 500 39 7,8 5,11 1,81 9,11 3,22Maple 1000 76 7,6 4,21 1,49 7,96 2,81Trifolium, Lotus 500 25 5,1 2,65 0,94
37、3,77 1,33All clover species 1000 55 5,5 2,24 0,79 4,62 1,63Other pollen500 141 28,2types2)1000 280 28,01) Total number of pollen counted; the variation decreases as the count increases.2) The interlaboratory test distinguished between two further groups, namely nectar-free and otherpollen types. The
38、se complex groups were not included in the precision data.3) The greater the proportion of the particular pollen species or pollen group, the better are the values forthe repeatability and reproducibility limits.Normative referencesThis standard incorporates, by dated or undated reference, provision
39、s from other publications. These normativereferences are cited at the appropriate places in the text, and the titles of the publications are listed below. Fordated references, subsequent amendments to or revisions of any of these publications apply to this standardonly when incorporated in it by ame
40、ndment or revision. For undated references, the latest edition of thepublication referred to applies.ISO 5725-1 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 1: Generalprinciples and definitionsISO 5725-2 : 1994 Accuracy (trueness and precision) of measurement meth
41、ods and results Part 2: Basicmethod for the determination of repeatability and reproducibility of a standardmeasurement method.BibliographyLouveaux, J., Maurizio, A. and Vorwohl, G., Methods of melissopalynology. In: Bee World, 1978: 59 (4),pp. 139157.Behm, F., von der Ohe, K. and Henrich, W., Zuverlssigkeit der Pollenanalyse von Honig; Bestimmung derPollenhufigkeit (Reproducibility of results of the analysis of honey when determining the pollen content). In:Deut. Lebensm. Rundschau, 1996: 92 (6), pp. 183187.
