1、 TAPPI/ANSI T 449 om-14 SUGGESTED METHOD 1940 OFFICIAL STANDARD 1956 OFFICIAL TEST METHOD 1979 REVISED 1984 REVISED 1990 REVISED 2000 REVISED 2007 REVISED 2014 2014 TAPPI The information and data contained in this document were prepared by a technical committee of the Association. The committee and
2、the Association assume no liability or responsibility in connection with the use of such information or data, including but not limited to any liability under patent, copyright, or trade secret laws. The user is responsible for determining that this document is the most recent edition published. App
3、roved by the Standard Specific Interest Group for this Test Method TAPPI CAUTION: This Test Method may include safety precautions which are believed to be appropriate at the time of publication of the method. The intent of these is to alert the user of the method to safety issues related to such use
4、. The user is responsible for determining that the safety precautions are complete and are appropriate to their use of the method, and for ensuring that suitable safety practices have not changed since publication of the method. This method may require the use, disposal, or both, of chemicals which
5、may present serious health hazards to humans. Procedures for the handling of such substances are set forth on Material Safety Data Sheets which must be developed by all manufacturers and importers of potentially hazardous chemicals and maintained by all distributors of potentially hazardous chemical
6、s. Prior to the use of this method, the user must determine whether any of the chemicals to be used or disposed of are potentially hazardous and, if so, must follow strictly the procedures specified by both the manufacturer, as well as local, state, and federal authorities for safe use and disposal
7、of these chemicals. Bacteriological examination of paper and paperboard 1. Scope The following procedure is recommended for the bacteriological examination of paper and paperboard intended for use as single service containers and closures for dairy products. Because of the exacting technique require
8、d in bacteriological procedures, reproducible results can be obtained only by a trained technician. All tests should be performed under the appropriate laboratory conditions to ensure quality assurance and safety. 2. Significance This method is important because it provides a procedure for determini
9、ng the number of colony-forming units (CFUs) per gram of paper or paperboard in accordance with the requirements of the Dairymens Standard as published by the U. S. Department of Health and Human Services, Public Health Service, Food and Drug Administration. 3. Apparatus and materials 3.1 Alcohol, 7
10、0% ethyl alcohol for sterilizing instruments. Alternatively, methyl or isopropyl alcohol may be substituted. 3.2 Balance, sensitive to 0.1 g, with a pan large enough to hold the petri dishes. 3.3 Dilution blanks, commercially available, prefilled, presterilized phosphate buffered dilution blanks are
11、 recommended. Alternatively milk dilution bottles scribed at the 99 mL level (150 mL capacity) of borosilicate glass or other autoclavable plastic material, fitted with either screw caps or Escher rubber stoppers may be used. 3.4 Colony counter, any one of several types. A hand tally for recording t
12、he count is recommended. T 449 om-14 Bacteriological examination of paper and paperboard / 2 3.5 Envelopes, two pressure-sensitive sealing envelopes are needed per sample, one smaller 16.5 24.1 cm (6.5 9.5 in.) and one larger 22.9 30.5 cm (9 12 in). 3.6 Disintegrator, high speed electric blender wit
13、h a sterile metal (stainless steel preferred) cup or glass cup of 500 mL capacity. 3.7 Flaming equipment, an alcohol lamp, or a Bunsen burner, to flame tongs, scissors, knives, and the mouths of sterile containers. 3.8 Incubator, capable of maintaining a temperature of 32 1C. 3.9 Knife, preferably o
14、ne with an inserted blade of adjustable length to cut paper and board. 3.10 Petri dishes, commercially available, presterilized, disposable (90 15 mm) plates are recommended. 3.11 Pipets, commercially available presterilized 10 or 20 mL pipets with large or breakaway tips (3 mm opening) are recommen
15、ded. If dilution of the suspension is necessary, the presterilized, 1.1 mL dilution pipets will be needed. 3.12 Scissors, preferably with 10.2-cm (4-in.) cutting edges. 3.13 Nutrient substrate, Standard Methods Agar (SMA), commercially available as a premixed powder. Composition per liter: Agar 15.0
16、g Pancreatic digest of casein 5.0g Yeast extract 2.5g Glucose 1.0g pH 7.0 0.2 at 25C, sterilize by autoclaving for 20 minutes at a minimum of 121C (15 psi). 3.14 Sterilizing equipment, two types of suitable size: an autoclave for steam sterilization; a hot-air oven at 165C, with thermometer. 3.15 St
17、erile tongs or forceps. 3.16 Laminar flow hood, optional equipment. 4. Sterilization of equipment and media 4.1 Depending upon the nature of the equipment to be sterilized, use one of three methods as follows: 4.1.1 Steam sterilizer (autoclave). Sterilize the following by autoclaving for 20 min at a
18、 minimum of 121C, corresponding to 103 kPa (15 psi) steam pressure: (a) disintegration jars; (b) media (unless other conditions are specified by the manufacturer of the medium); (c) sample bottles; (d) corkborers; and (e) water blanks for dilution. Include a biological or chemical indicator strip to
19、 evaluate autoclave efficacy. 4.1.2 Dry heat. Sterilize the following by heating for at least 2 h at a minimum temperature of 165C: (a) heavy kraft envelopes and folders; (b) tongs; (c) knives; (d) pipets; and (e) scissors. Take care to dry glassware completely before heating and avoid scorching any
20、 paper containers or wrapper used for the instruments being sterilized. Since some kraft envelopes vary in their heat resistance, it is recommended that heat embrittlement be determined prior to selection. 4.1.3 Alcohol. Immerse the contact portion of scissors, tongs, knives, and similar instruments
21、 in alcohol. When needed for cutting or handling samples, remove; allow to drain for a few moments, then burn off the excess alcohol. Use extreme care when using an open flame around alcohol. 5. Sampling 5.1 Please refer to TAPPI T 400 “Sampling and Accepting a Single Lot of Paper, Paperboard, Conta
22、inerboard or Related Product.” 5.2 Cut each sample from the roll of paper with a clean, sharp knife placing each test unit in a different envelope. For each test unit cut into the roll two laps deep and discard these first two layers. Then cut three sides of a square approximately 10 10 inches (25 2
23、5 cm) into the roll six laps deep, forming a flap. Raise the flap and tape the three sides of the flap to prevent undue exposure and then cut the fourth (hinge) side of the flap. Place the sample into a clean paper envelope, seal and insert it into a manila or brown kraft envelope. Preferably the en
24、velopes should seal with a pressure-sensitive adhesive rather than one that must be moistened. 3 / Bacteriological examination of paper and paperboard T 449 om-14 6. Test specimens Place a closed, sterile petri dish on the balance and tare. Cut along the top of the envelope with sterile scissors and
25、 remove the paperboard with sterile tongs. Hold the sample along one edge and trim the remaining three edges with sterile scissors, discarding the outer edges. Make a series of parallel cuts about 1 cm wide along one edge of the sample. Remove the cover of the petri dish and cut squares of paper int
26、o the dish by making a series of cuts 1 cm wide perpendicular to those previously made. Cut enough paper to give a 3.0 g sample and replace petri dish cover. 7. Procedure NOTE 1: This procedure should be carried out in a room free of air currents and dust. The work area should be cleaned with a suit
27、able disinfectant before beginning and, if available, a laminar flow hood is recommended for plating. 7.1 Disintegration. Put 300 mL of sterile phosphate dilution water into the disintegration jar, add the 3 g of cut up paper or paperboard, and replace the cover (fiber consistency equals 1%). Disint
28、egrate at high speed for two (2) minutes. When many samples are run on the same disintegrator, cool the sterile dilution water to prevent the temperature from exceeding 45C (113F) in the disintegrator cup. The cover should remain on at all times except when removing sample. 7.2 Plating. 7.2.1 Immedi
29、ately after disintegration with a sterile 10- or 20-mL wide bore pipette, distribute 10 mL of the 1% suspension (representing 0.1 g of disintegrated stock) approximately equally among 5 petri dishes (i.e., 2.0 mL/plate). Pour 15 to 20 mL of standard methods agar (SMA), cooled to about 45C (113F) int
30、o each petri dish. When adding the agar, rotate the plate with gentile agitation so that the agar is thoroughly mixed with the disintegrated paper or paperboard. Make sure the pulp is evenly distributed and that no lumps remain. Replace the lid and allow the agar to solidify, 15 - 20 minutes on a fl
31、at surface. Pour agar into one control plate (without disintegrated paper or paperboard) from each container of nutrient medium to check its sterility. Pour a second control plate expose it to room atmosphere during plating operation not to exceed 30 minutes exposure. Finally, pour agar into one con
32、trol plate containing 1 mL of dilution water taken from a representative bottle from each lot of dilution blanks used. 7.2.2 If a higher dilution is required, add 10 mL of the 1% suspension to a sterile, 90-mL water blank and distribute 10 mL of this suspension equally among five petri dishes, which
33、 together will then contain 0.01 g of the original pulp specimen. 7.2.3 Adjust the dilution of pulp samples to provide a count of 30 to 300 colonies per plate. NOTE 2: The amount of fibrous material distributed over a given area of plate surface has been found to be an important factor in the number
34、 of colonies observed in a given sample of paper or board. It is therefore important to carefully follow directions regarding the fiber consistency, the volume of sample used for plating, and the number and size of plates used. 7.3 Incubation. 7.3.1 Once the plates have solidified, invert and incuba
35、te the plates at 32 1C for 48 3 hours. 7.4 Counting plate cultures. 7.4.1 After incubation, examine the plates for the presence and number of CFUs with a colony counter. 7.4.2 If any particles are observed in the culture which cannot be definitely identified as CFUs, examine them microscopically. Ex
36、amine control plates also and make a record of the number of CFUs found on each control plate. 7.4.3 Add the total number of colonies counted on each of the five plates and multiply by 10 (if the sample was further diluted, multiply by the appropriate dilution factor). This gives the number of CFUs
37、per gram of paper or paperboard. 8. Report Report the result as the number of colonies per gram of paper or paperboard. Indicate any deviation from the procedure. T 449 om-14 Bacteriological examination of paper and paperboard / 4 9. Precision 9.1 Repeatability (within a laboratory) = 5% 9.2 Reprodu
38、cibility (between laboratories) = 10% 9.3 These values are based on those reported in Standard Methods for Examination of Water and Wastewater. The user of these precision data is advised that it is based on actual mill testing, laboratory testing, or both. There is no knowledge of the exact degree
39、to which personnel skill or equipment were optimized during its generation. The precision quoted provides an estimate of typical variation in test results which may be encountered when this method is routinely used by two or more parties. 10. Keywords Paper, Paperboard, Bacteriology, Microbiology, B
40、iological control, Microorganism control, Dairymans standard 11. Additional information 11.1 Effective date of issue: November 4, 2014 11.2 The colony forming unit determination is done using the methods in T 449. Incorporating a dye, such as INT 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazoliu
41、m chloride, to enhance detection is an alteration of the test method, which could affect the historically determined plate count and rejection threshold. Redox dyes such as INT can both artificially increase the detection threshold, which could affect rejection of the board, or lower detection since
42、 certain bacteria do not react with the dye or are inhibited by it. Any alteration to the test method must be so stated. 11.3 Other than editorial, the only changes in the 2014 edition was the addition of the precaution concerning use of dyes in 11.2. References 1. American Public Health Association
43、, “Standard Methods for the Examination of Water and Waste Water,” 18th Edition, (1994). 2. American Public Health Association, “Standard Methods for the Examination of Dairy Products,” 16th Edition (1992). 3. Syracuse Research Corporation, “Manual of Recommended Laboratory Methods for Sampling and Microbiological Testing of Single-Service Food Packages and their Components.” Your comments and suggestions on this procedure are earnestly requested and should be sent to the TAPPI Standards Department.
