1、BRITISH STANDARD BS4285-2.5: 1989 Direct counts Microbiological examination for dairy purposes Part2: Methods of general application for enumeration of microorganisms Section2.5 Enumeration of bacteria by direct microscopic counts IMPORTANT NOTE.It is essential that Parts0 and1 of this standard, whi
2、ch are published separately, be read in conjunction with this Section. UDC637.1.055.07:579.67+637.1.055.075:579.672.083.16:579.8.087.23BS4285-2.5:1989 This British Standard, having been prepared under the directionof the Dairying Standards Policy Committee, waspublished under the authorityof the Boa
3、rd of BSI andcomes into effect on 29 September 1989 BSI04-1999 BS4285 first published March 1968 Supplement No.1 to BS4285:1968 first published April1970 First revision, Section2.5, December 1984 Second revision, The Committees responsible for this British Standard are shown inPart0. The following B
4、SI references relate to the work on this standard: Committee reference DAC/4 Draft for comment88/52307DC ISBN 0 580 17390 9 Foreword This Section of BS4285 has been prepared under the direction of the Dairying Standards Policy Committee. It supersedes BS4285-2.5:1984 which is withdrawn. This revisio
5、n of this Section of BS4285 incorporates a more detailed description of the direct epifluorescent filter technique (DEFT) (seeclause5), the direct microscopic count technique (seeclause4) being identical with that in the 1984 edition. A British Standard does not purport to include all the necessary
6、provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligation. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, pages1 to4,
7、an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date of issue CommentsBS4285-2.5:1989 BSI 04-1999
8、 i Contents Page Foreword Inside front cover 1 Scope 1 2 Definition 1 3 Principle 1 4 Method A. Direct microscopic count technique 1 5 Method B. Direct epifluorescent filter technique (DEFT) 2 6 Test report 4 Table 1 Microscope factors 2 Table 2 Quantities of sample, trypsin and surfactant solutions
9、 for test 4 Publications referred to Inside back coverii blankBS4285-2.5:1989 BSI 04-1999 1 1 Scope This Section of BS4285 describes two methods of general application for the direct enumeration of bacteria: a) method A: direct microscopic count technique for total counts in milk; b) method B: direc
10、t epifluorescent filter technique (DEFT) for total counts or counts of bacteria in milk, certain milk products and samples such as rinses. NOTE 1In samples (including plant rinses) that have received some degree of heat treatment (equivalent to pasteurisation or greater), certain organisms, particul
11、arly Gram-positive cocci, will fluoresce orange (i.e.as active) but will be non-viable by plate count methods. Therefore great care is needed in interpreting results using such samples. NOTE 2The titles of the publications referred to in this standard are listed on inside back cover. 2 Definition Fo
12、r the purposes of this Section of BS4285, the following definition applies. bacteria. those organisms which can be recognized from experience, under the microscope, as having the characteristic size and appearance of organisms known as bacteria which, in method A, are stained the appropriate colour
13、or, in method B, fluoresce green, yellow or orangered after staining with acridine orange 3 Principle 3.1 Method A A small test portion is fixed on a microscope slide4.2.4, defatted, and stained with methylene blue or Newmans stain4.1. The prepared film is examined, the bacteria counted and the numb
14、er per millilitre of sample calculated. 3.2 Method B A known quantity of sample or primary dilution is treated with trypsin and surfactant and then filtered so that bacteria are concentrated on the filter where they are stained with acridine orange. The bacteria are counted and the number per millil
15、itre of sample calculated. 4 Method A. Direct microscopic count technique 4.1 Reagents 4.1.1 Methylene blue stain Dissolve the methylene blue in the ethanol and add the water. 4.1.2 Newmans stain Add the ethanol to the tetrachloroethane and heat on a water bath in a fume cupboard to a temperature no
16、t exceeding70 C. Add the methylene blue and shake until the dye dissolves. Cool, add acetic acid slowly, mix and filter. WARNING. The vapour of tetrachloroethane is toxic and all operations involving Newmans stain shall be carried out in a fume cupboard. 4.1.3 Xylene, or other suitable fat solvent 4
17、.1.4 Ethanol,95%V/V 4.2 Apparatus NOTEFor details of apparatus including its preparation and sterilization, see BS4285-1.2. 4.2.1 Ordinary microbiological laboratory apparatus. 4.2.2 Pipettes or syringes, calibrated to deliver0.01mL. 4.2.3 Microscope, with2.1mm oil immersion objective and 10 eyepiec
18、e. 4.2.4 Microscope slides, having a square with10mm sides either: a) outlined on the surface by etching or other suitable means; or b) using a template to outline this area on a plain slide. The slides shall be new and fat-free. 4.3 Determination of the microscope factor Measure the diameter of the
19、 microscope field with a stage micrometer with0.01mm divisions using the2.1mm objective, the10 eyepiece and a standard draw tube length. Take the mean of three measurements and calculate the radius in millimetres. Methylene blue(3,7-Bis(dimethylamino) phenothiazin-S-ylium chloride) 0.3g Ethanol,95%
20、V/V 30mL Water 100mL Methylene blue 1.0g Ethanol,95% V/V 54mL 1,1,2,2-tetrachloroethane 40mL Glacial acetic acid 6mLBS4285-2.5:1989 2 BSI 04-1999 If0.01mL of sample is spread over an area of100mm 2 , calculate the microscope factor from the formula: where r is the radius of the microscope field (inm
21、m). Table 1 gives the microscope factors corresponding to different field diameters. Table 1 Microscope factors 4.4 Sampling Take the laboratory sample in accordance with BS4285-1.1. 4.5 Preparation of test sample Prepare the test sample from the laboratory sample in accordance with BS4285-1.1. 4.6
22、Procedure 4.6.1 Preparation of film. Using the pipette(4.2.2) remove0.01mL of the well-mixed sample, wipe the exterior of the pipette except the tip with absorbent paper and spread the sample evenly over the area on a slide(4.2.4). Dry the preparation at a temperature of40C to45C on a level surface
23、protected from dust. The drying time shall not exceed5min, but shall not be too short or the film will not be fixed properly. 4.6.2 Staining. Carry out the procedure in eithera) orb). a) Procedure using methylene blue. Defat the fixed sample by flooding the slide with xylene or other suitable fat so
24、lvent for at least1min. Drain and allow to dry. Flood with ethanol for1min, drain and allow to dry. Flood the slide with methylene blue stain for10s to15s. Do not exceed this time or the slide will be overstained. Rinse carefully in water, drain and allow to dry slowly. When properly prepared, the b
25、ackground of the slide will be light blue. b) Procedure using Newmans stain NOTEThe use of Newmans stain permits defatting and staining at the same time. Observing the warning in4.1.2, flood the slide holding the fixed sample with Newmans stain for5min. Allow to drain until thoroughly dry. Wash gent
26、ly in water to remove surplus stain, drain and allow to dry, slowly and completely. 4.7 Examination of the stained film Examine the stained film under the2.1mm oil immersion objective using the 10 eyepiece and the standard draw tube length used in4.3. Count the number of bacteria in up to30fields ta
27、ken at random or until the count is50, recording clumps and chains of bacteria separated by less than one diameter as single organisms. From the average per field, calculate the number of organisms per millilitre as follows. Direct microscope count per millilitre=average count per field microscope f
28、actor. 5 Method B. Direct epifluorescent filter technique (DEFT) 5.1 Reagents 5.1.1 General. All reagents shall be of analytical grade and shall be sterilized by filtration through a0.224m pore size membrane filter into a clean sterile container and rinsed with particle free water before sterilizati
29、on, to remove bacteria and particulate matter. 5.1.2 Trypsin solutions, reconstituted according to the manufacturers instructions and sterilized by filtration (5.1.1). Transfer0.5mL quantities to10mL test tubes and use immediately or store at20C. NOTEFor information on the availability of trypsin, a
30、pply to BSI Enquiry Section, BSI, Linford Wood, Milton Keynes, MK146LE, enclosing a stamped addressed envelope for reply. 5.1.3 Surfactant solutions, two solutions, weak and strong, both sterilized by filtration are required. NOTE1g/L and5g/L solutions of Triton-X100 are recommended. In the case of
31、butter and cream samples only,1g/L and10g/L solutions of Tween80 may be used as an alternative. 5.1.4 Sodium citrate buffer solutions, at pH3.0 0.2 and pH6.6 0.2. These buffers are available commercially. Alternatively, they can be prepared by mixing solutions of0.1mol/L citric acid and0.1mol/L sodi
32、um hydroxide to give the required pH, followed by sterilization by filtration(5.1.1). 5.1.5 Acridine orange, zinc chloride solution (0.25% g/L) Acridine orange3,6-bis (dimethylamino) acridine (Colour Index no.46005), zinc chloride salt0.25g Sodium citrate buffer solution, pH6.6 0.2,1000mL Dissolve t
33、he acridine orange in the buffer solution and sterilize by filtration(5.1.1). Diameter Factor for the average number of bacteria per millilitre mm 0.206 300000 0.178 400000 0.160 500000 0.146 600000 10 000 r 2 -BS4285-2.5:1989 BSI 04-1999 3 WARNING. Wear face masks and protective gloves when handlin
34、g acridine orange powder as the compound is a suspected carcinogen. Use only bright red/orange powders as dull red or brown powders give poor staining properties. Prepare acridine orange, zinc chloride solutions freshly each week and protect them from light by storage in the dark. Use of commerciall
35、y prepared concentrated solutions of acridine orange, zinc chloride is recommended for safety, convenience and reproducibility. 5.1.6 Propan-2-ol,95%(V/V), sterilized by filtration(5.1.1) through a solvent resistant filter. 5.2 Apparatus NOTE 1For details of apparatus including its preparation and s
36、terilization, see BS4285-1.2. NOTE 2For information on the availability of suitable apparatus and filters apply to BSI Enquiry Section, BSI, Linford Wood, Milton Keynes, MK146LE, enclosing a stamped addressed envelope for reply. 5.2.1 Ordinary microbiological laboratory apparatus. Because particles
37、of rubber and cotton fluoresce, stoppers and closures of these materials shall not be used. Plastics materials are acceptable. 5.2.2 Graduated pipettes (1.0mL and5.0mL), and test tubes (10mL) with metal caps or glass bottles (50mL) with tops of plastics material, all of which shall be well washed, r
38、insed in water sterilized by filtration, and then sterilized (seeBS4285-2.1). 5.2.3 Membrane filters, of25mm diameter and of0.64m uniform pore size made from polycarbonate. 5.2.4 Membrane filter holder assembly. NOTEThe filter support base should be of a type allowing minimum restriction to flow of
39、liquid through the support and has to offer sufficient support to the membrane to stop it deforming when the vacuum is applied. Stainless steel mesh supports coated with polytetrafluoroethylene (PTFE) are recommended. The filter towers shall be thoroughly cleaned and brushed before use using a suita
40、ble non-ionic, phosphate-free detergent and thoroughly rinsed with sterile, particle-free distilled water before use and between samples. Protect towers from atmospheric dust when not in use. 5.2.5 Vacuum pump, capable of establishing a vacuum of100kPa, connected via a vacuum trap fitted with a thre
41、e-way stopcock. 5.2.6 Water bath, capable of being maintained at50 0.5C. 5.2.7 Vortex mixer. 5.2.8 Epifluorescence microscope with 100 magnification oil immersion objective and 10 eyepieces with appropriate excitation and barrier filters to deliver incident excitation light of wavelength450nm to490n
42、m. 5.2.9 Immersion oil, non fluorescent and compatible with objective lense type. 5.3 Determination of the microscope factor Measure the diameter of the microscope field of view by use of a stage micrometer with0.01mm divisions using the 100 objective, the 10 eyepiece and a standard draw tube length
43、. Take the mean of three measurements and calculate the radius of the microscope field of view in millimetres. Calculate the microscope factor from the formula: where 5.4 Sampling Take the laboratory sample in accordance with BS4285-1.1. Do not use sample collecting vessels with rubber stoppers or c
44、losures (see5.2.1). NOTEUse of disposable plastics containers is recommended. However, reusable containers with wide mouths and no shoulders (in order that effective cleaning can be carried out) may be used. 5.5 Preparation of test sample Prepare the test sample from the laboratory sample in accorda
45、nce with BS4285-1.1. 5.6 Procedure 5.6.1 Treatment of test portion. Depending on the nature of the sample (see Table 2) pipette the appropriate volumes of the sample, trypsin solution and surfactant into a clean sterile test tube or bottle of sufficient volume. Ensure the sample and reagents are tho
46、roughly dispersed by vigorous mixing for at least30s using the vortex mixer(5.2.7). Incubate the mixture for10min in the water bath(5.2.6), controlled at50 0.5 C and mix again using the vortex mixer. A is the area of membrane through which the test portion is filtered (inmm 2 ); V is the volume of t
47、est portion (inmL); r is the radius (inmm) of the microscope field of view. A r 2 V -BS4285-2.5:1989 4 BSI 04-1999 5.6.2 Filtration. Place a membrane filter(5.2.3), with the shiny side uppermost, in the filter assembly(5.2.4) and attach the vacuum pump(5.2.5) to the assembly. At all stages of the su
48、bsequent procedure release the vacuum before adding liquid to the filter tower. Control the supply of vacuum to the filtration towers via the three-way stopcock of the vacuum trap to ensure an even and rapid distribution of the release of vacuum. Do not use vacuum pressures of greater than100kPa as
49、this can cause distortion of the membrane filter. Pre-warm the filter assembly by filtering5mL of the weak surfactant solution(5.1.3) pre-warmed to50C in the water bath. Filter the treated sample and rinse the sample treatment container with a further5mL of the weak surfactant solution pre-warmed to50C and filter this rinse. Finally, wash the filter with5mL of the weak surfactant solution. 5.6.3 Staining of bacteria on membrane. Carefully add1.0mL of the acridine orange solution(5.1.5) to the filter makin
