2 for analysis
2.3.8 Procedure for the preparation of the first dilution of samples
obtained by surface swabbing or surface washing
The diluent retaining the contamination collected with swabs, sponges or surface washing is, in itself, already the first dilution of the sample. The subsequent treat-ment of serial decimal dilution is performed using this suspension as point of departure. Since the initial dilu-tion is not the standard 1:10 diludilu-tion, this difference
must be taken into account when doing the final calcu-lations of the results, as described in Chapters 3 and 4.
2.4 Serial decimal dilution of the sample
The preparation and inoculation of serial dilutions of the sample are required for quantitative tests, to reduce the number of microorganisms per unit of volume, and make it possible to count them. This series of dilutions is generally decimal or ten-fold for ease of calculation of final results.
The number of dilutions necessary depends on the expected level of contamination and should be such as to allow for, in plate counts, obtaining plates with num-bers of colonies varying between 25–30 and 250–300 (see Chapter 3) or between 15 and 150 in yeast and mold counts. In counts by the Most Probable Number Method (MPN) the number of dilutions must allow for obtaining positive tubes at the lowest dilutions and neg-atives tubes at the highest dilutions (see Chapter 4).
According to the general procedure described by the Compendium (Swanson et al., 2001), the second dilution is to be initiated immediately upon completion of the first dilution. The duration of the complete procedure, from the preparation of the first dilution until inocula-tion of all culture media, should not exceed 15 minutes (except when described in case-specific chapters).
According to the general procedure described by ISO 6887-1:1999, the duration of the complete procedure should not exceed 45 minutes and the time interval between the end of the preparation of the first dilution and the beginning of the second and subsequent dilu-tions should not exceed 30 minutes (except when speci-fied in specific procedures).
For dehydrated or dried foods (except for milk pow-der, egg powpow-der, and live yeast powder) ISO 6887-4:2003/Cor.1:2004 recommends a resuscitation step before preparing the second dilution. In general, leave the sample to rest for about 30 ± 5 min at laboratory temperature. Do not exceed a temperature of 25°C before preparation of further dilutions.
In all cases in which volumes are transferred, the uncertainty of the measurement must not exceed 5%
(ISO 6887-1:1999).
How to prepare the second dilution (10−2): Transfer aseptically 1 ml of the first dilution (10−1) to 9 ml dilu-ent. The diluents are the same as those recommended for the first dilution. In the second dilution there are
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no special cases in which a different diluent is required from the one used to prepare the first dilution.
Do not dip the tip of the pipette to a depth of more than 1 cm when pipetting the volume from the first to the second dilution (ISO 6887-1:1999). If the first dilution does not contain suspended particles, the mate-rial may be agitated before transferring the volume from the first to the second dilution. If there are suspended particles, ISO 6887-1:1999 recommends not to agitate and wait until the suspended particles settle to the bot-tom before transferring the volume. In the case of vis-cous samples, which adhere to the internal wall of the pipette, ISO 6887-5:2010 recommends dispensing the volume and subsequently wash the pipette with diluent (by aspirating several times) to ensure that all the mate-rial be transferred to the second dilution.
How to prepare subsequent dilutions: Transfer 1 ml of the previous dilution to 9 ml diluent. Before withdrawing the volume to be transferred, agitate the tube vigorously, inverting it 25 times in a 30-cm arc (within 7 s) or using a laboratory vortex mixer (15 s).
2.5 References
Davis, G.L. & Hickey, P.J. (2004) Media and dilution water prepa-ration. In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association. Chapter 4, pp. 93–101.
Downes, F.P. & Ito, K. (eds) (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association.
Duncan, S.E., Yaun, B.R. & Sumner, S.S. (2004) Microbiological Methods for Dairy Products. In: Wehr, H.M. & Frank, J.F (eds).
Standard Methods for the Examination of Dairy Products. 17th edi-tion. Washington, American Public Health Associaedi-tion. Chapter 9, pp. 249–268.
Frank, J.F. & Yousef, A.E. (2004) Tests for groups of microrganisms.
In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association. Chapter 8, pp. 227–248.
Gray, R.J.H. & Pinkas, J.M. (2001) Gums and spices. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbio-logical Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 52, pp. 533–540.
Hall, P., Ledenbach, L. & Flowers, R. (2001) Acid producing micro-organisms. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition.
Washington, American Public Health Association. Chapter 19, pp. 201–207.
Hunt, M.E. & Rice, E.W. (2005) Microbiological examination. In:
Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds).
Standard Methods for the Examination of Water & Wastewater.
21st edition. Washington, American Public Health Association
(APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF). Part 9000, pp. 9.1–9.169.
International Organization for Standardization (1999) ISO 6887-1:1999. Microbiology of food and animal feeding stuffs – Prepara-tion of test samples, initial suspension and decimal diluPrepara-tions for microbiological examination – Part 1: General rules for the prepa-ration of the initial suspension and decimal dilutions. Geneva, ISO.
International Organization for Standardization (2003) ISO 6887-2:2003. Microbiology of food and animal feeding stuffs – Prepa-ration of test samples, initial suspension and decimal dilutions for microbiological examination – Part 2: Specific rules for the prepara-tion of meat and meat products. Geneva, ISO.
International Organization for Standardization (2003) ISO 6887-3:2003. Microbiology of food and animal feeding stuffs – Prepa-ration of test samples, initial suspension and decimal dilutions for microbiological examination – Part 4: Specific rules for the prepara-tion of fish and fishery products. Geneva, ISO.
International Organization for Standardization (2004) ISO 6887-4:2003/Cor.1:2004. Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination – Part 4: Specific rules for the preparation of products other than milk and milk products, meat and meat products, and fish and fishery products. 1st edition:2003, Technical Corrigendum 1:2004. Geneva, ISO.
International Organization for Standardization (2010) ISO 6887-5:2010. Microbiology of food and animal feeding stuffs – Prepa-ration of test samples, initial suspension and decimal dilutions for microbiological examination – Part 5: Specific rules for the prepara-tion of milk and milk products. Geneva, ISO.
International Organization for Standardization (2007) ISO 7218:2007. Microbiology of food and animal stuffs – General requirements and guidance for microbiological examinations.
Geneva, ISO.
International Organization for Standardization (2009) ISO 17604:2003/Amd 1:2009. Microbiology of food and animal feeding stuffs – Carcass sampling for microbiological analysis. 1st edition:2003, Amendment 1:2009. Geneva, ISO.
Laird, D.T., Gambrel-Lenarz, S.A., Scher, F.M., Graham, T.E. &
Reddy, R. (2004) Microbiological count methods. In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association. Chapter 6, pp. 153–186.
Midura, T.F. & Bryant, R.G. (2001) Sampling plans, sample col-lection, shipment, and preparation for analysis. In: Downes, F.P.
& Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 2, pp. 13–23.
MLG/FSIS (2011) Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products. In: Micro-biology Laboratory Guidebook [Online] Washington, Food Safety and Inspection Service, United States Department of Agriculture.
Available from: http://www.fsis.usda.gov/PDF/MLG_4_05.pdf [Accessed 3rd November 2011].
Ricke, S.C., Birkhold, S.G. & Gast, R.K. (2001) Egg and egg prod-ucts. In: Downes, F.P. & Ito, K. (eds). Compendium of Meth-ods for the Microbiological Examination of FoMeth-ods. 4th edition.
Washington, American Public Health Association. Chapter 46, pp. 473–481.
Smittle, R.B. & Cirigliano, M.C. (2001) Salad dressings. In:
Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 53, pp. 541–544.
Swanson, K.M.J, Petran, R.L. & Hanlin, J.H. (2001) Culture methods for enumeration of microrganisms. In: Downes, F.P. &
Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 6, pp. 53–67.
Wehr, H. M. & Frank, J. F (eds) (2004) Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association.
Annex 2.1 Procedures for the homogenization of the content and withdrawal of the analytical unit of different types of foods Powdered products: Homogenize the sample by vigor-ously agitating and inverting the package with your hands until well mixed (ISO 6887-4:2003/Cor.1:2004 or stir the content with a sterile spatula or glass rod (Midura &
Bryant, 2001). If there is not enough free space inside the package to allow for appropriate homogenization, transfer the whole content to a larger flask and proceed in exactly the same way (ISO 6887-5:2010). Withdraw the analytical unit with a sterile spatula.
Pasty or ground products: Stir the content with a sterile spatula or glass rod until well homogenized.
Withdraw the analytical unit with a sterile spatula (Midura & Bryant, 2001).
Yogurts with fruit pieces: For yogurt containing fruit pieces, the Standard Methods for the Examination of Dairy Products (Duncan et al., 2004) recommends homogenizing the entire content of the sample unit in a blender for 1 min, before withdrawing the analytical unit.
Cheeses: The Standard Methods for the Examination of Dairy Products (Duncan et al., 2004) recommends macerating the whole content of the sample unit (with a sterile spatula) and withdrawing the analytical unit from the mixture.
Very hard food products: ISO 6887-4:2003/
Cor.1:2004 recommends placing the sample inside a sterile plastic bag and beat the material with a sterile hammer crumbling it into small bits and pieces. Mix well the fragmented sample material and withdraw the analytical unit with a sterile spatula. ISO 6887-5:2010, specific for dairy products, recommends: when using
a stomacher contain the sample and diluent in two or more sterile bags to prevent puncturing and possible sample spillage. When using rotary homogenizer do not homogenize for more than 2.5 min at a time. If necessary to mince or to grind the sample, do not exceed 1 min at time to avoid an excessive increasing in temperature.
Pieces of solid foods: ISO 6887-4:2003/Cor.1:2004 recommends using an adequate instrument (sterile knife or pair of scissors) to break up or cut the material into smaller parts (taken from different points of the piece in its original shape), until obtaining the quantity required for analysis.
Eggs in the shell: For analysis of the internal content, Chapter 46 of the Compendium (Ricke et al., 2001) rec-ommends to wash the shell of the eggs with a brush, water and soap, drain off the excess liquid, immerse the eggs in ethanol 70% for 10 min and flame-sterilize.
Using sterile gloves open the eggs aseptically and place the internal content inside a sterile flask or bag, separat-ing the yolk from the egg white if the analysis requires so. Mix well and withdraw the analytical unit from the mixture.
ISO 6887-4:2003/Cor.1:2004 recommends three procedures, depending on the final purpose of the test:
• To analyze only external contamination, the surface washing method may be used. Alternatively, break the eggs, dispose of the internal content, place the egg shells in a sterile bag, crumble the egg shells, mix well and withdraw the analytical unit from the mixture.
• To analyze both external and internal contamina-tion, break the eggs, place the shells and the internal content in a sterile flask or bag, mix well and with-draw the analytical unit from the mixture.
• To analyze only the internal content, clean the shell with moistened gauze, dry with absorbent paper, immerse the eggs in Alcoholic Solution of Iodine and, using sterile gloves, remove aseptically the eggs from the solution, and let them stand to dry. Using gloves open the eggs aseptically and place the inter-nal content in a sterile flask or bag, separating the yolk from the egg white if the analysis requires so.
Mix well and withdraw the analytical unit of the mixture.
Meat cuts for analysis of non-surface contamina-tion: For the analysis of contamination of deep tissues of carcasses or meat cuts, ISO 6887-2:2003 recommends
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exposing an area of approximately 5 × 5 cm, using a sterile knife or pair of scissors to remove the skin, if present, and a surface layer of approximately 2 mm thickness.
Cauterize the exposed surface with a flame and, using another sterile knife or pair of scissors, remove a second layer of approximately 1 mm thick and 4 × 4 cm in size.
From this exposed area, withdraw the analytical unit(s) required for the analyses.
Bivalves: ISO 6887-3:2003 recommends rubbing the shells with a sterile, abrasive brush under running water (drinkable). Drain off any excess water, place onto a sterile surface and cover with sterile absorbent paper. Open at least six shells aseptically and subse-quently take out the organisms, along with the inter-valvular water and transfer everything to a sterile flask or plastic bag. Wash and disinfect the hands before ini-tiating the operation.
Gastropods: ISO 6887-3:2003 recommends rub-bing the shells with a sterile, abrasive brush under running water (drinkable or potable). Disinfect with alcohol 70%, place onto a sterile surface (if necessary between two sterile gauze layers). Break the shells with a sterile hammer and take out the meat parts aseptically and transfer them to a sterile bag or flask. Wash and disinfect the hands before initiating the operation.
Cephalopods: ISO 6887-3:2003 recommends removing the skin and withdrawing the analytical unit from the dorsal muscles and tentacles.
Whole crustaceans such as crabs: ISO 6887-3:2003 recommends breaking the shell and claws using a sterile hammer and removing the maximum amount of meat as possible when withdrawing the analytical unit.
Sea urchins: ISO 6887-3:2003 recommends wash-ing the organisms under runnwash-ing water (drinkable or potable), open the ventral side and remove all the flesh and internal fluids using a spatula.
Annex 2.2 Special cases in which there are variations in the analytical unit and/
or dilution and/or diluents recommended for the preparation of the first dilution of samples of different types of foods Liquids with low levels of contamination: In quanti-tative tests of liquid samples with low microbial counts,
it is common practice to inoculate an aliquot of the sample directly in or on the culture media, without pre-vious dilution. In this case, the first dilution may be done by inoculating 1 ml of the sample in 9 ml diluent.
Also common are tests using the membrane filtration technique, in which volumes of the sample are inocu-lated, without any dilution.
Fatty foods: For these foods, the Compendium (Midura & Bryant, 2001) recommends preparing the diluent with 1% (w/v) nonionic Tergitol 7 or an equiv-alent surfactant (Tween 80, for example) and homog-enize in a blender for 2 min, at low speed (8000 rpm).
ISO 6887-4:2003/Cor.1:2004 and ISO 6887-5:2010 recommend preparing the diluent with 1 g/l to 10 g/l Tween 80, depending on the fat level. For products containing 40% fat, for example, the diluent should be prepared with 4 g/l Tween 80. This procedure does not apply to margarine and spreads.
For margarine and spreads ISO 6887-4:2003/
Cor.1:2004 recommends an analytical unit of 40 g and the following procedure: add to the analytical unit a volume of diluent (without any supplements) propor-tional to the fat level of the margarine. For example, for margarines containing 82% fat and an analytical unit of 40 g, add 40 × 0.82 = 33 ml of diluent. Place the flask in a temperature-controlled hot water bath at 45°C, until the material is completely melted. This should not take more than 20 min. Mix with the aid of a magnetic agi-tator until a homogeneous emulsion is formed, which may take between 2 to 5 min, depending on the type of product. Allow to stand at room temperature until complete separation of the aqueous (lower) and fatty (upper) phases. Continue the analysis with the aque-ous phase, 1 ml of which corresponds to 1 g margarine.
Then prepare a 10−1 dilution by adding, for each m mil-liliters of aqueous phase, 9 m milmil-liliters of diluent.
The Standard Methods for the Examination of Dairy Products (Laird et al., 2004) recommends, for margarine and spreads the same procedures as the one described for butter.
Thickeners or products containing natural antimi-crobial compounds: For thickeners and other products the viscosity of which increases when added to or mixed with water (gums, pectin, cellulose, dried leafy herbs, such as oregano) the Compendium (Gray & Pinkas, 2001) and ISO 6887-1:1999 recommend working with an initial dilution greater than 1:10, such as, for instance, 1/20, 1/50, 1/100 or any other appropriate to the viscosity of the material.
For products containing natural antimicrobial com-pounds, such as spices and herbs (garlic, onion, clove, cinnamon, oregano, pepper) and certain teas and cof-fee, it is recommended that the first dilution be greater than 1:10 (1:100 for oregano and cinnamon, 1:1000 for cloves). Alternatively, it is possible to prepare the dilu-ent with 0.5% potassium sulfite (K2SO3) and analyze the sample with the normal initial dilution ratio.
The analytical unit may be 10g and the initial dilu-tion used must be taken into account when calculating the results. If the expected counts are low, one should, if possible, keep the inoculum at 0.1 g of the initial dilu-tion in the culture media. For more details on how to do this, follow the instructions and guidelines contained in Chapters 3 and 4.
Acid products: ISO 6887-4:2003/Cor.1:2004 and ISO 6887-5:2010 recommend that the pH value of the first dilution be neutralized with sterile NaOH. To facil-itate this step Saline Peptone Water with Bromocresol Purple (SPW-BCP) may be used. When adding NaOH the increase in pH may be accompanied by a change in color of the culture medium, which may change from yellow to purple when reaching pH 6.8 (neutral). The NaOH concentration (0.1 M or 1 M, for example) to be used will depend on the acidity of the sample and should be such that the amount added does not significantly alter the 1+9 proportion between sample and diluent.
A second option is to use buffered diluents but, even in this case, the addition of NaOH is often necessary to increase the buffering capacity of the medium.
Chapter 2 of the Compendium (Midura & Bryant, 2001) does not mention differentiated procedures for these samples, as far as general quantification tests are concerned. However, for samples of mayonnaise, and other salad dressings, Chapter 53 (Smittle & Crigliano, 2001) recommends that they be neutralized for colif-orm and S. aureus counts. See also the specific recom-mendations for fermented dairy products.
Fine flours or meals, cereal grains, animal feed:
ISO 6887-4:2003/Cor.1:2004 recommends adding the diluent to the analytical unit, leaving to stand for 20 to 30 min at room temperature and then homogenizing. If the viscosity of the suspension becomes too high, add a complementary quantity of diluent, until obtaining a 1:20 dilution ratio. This change in the initial dilution should be taken into account when calculating the results.
Chocolate in bars, bonbons: ISO 6887-4:2003/
Cor.1:2004 recommends pre-heating the diluent to 40°C before adding it to the analytical unit and subsequent mixing by hand stirring. Leave to stand for 20–30 min
at room temperature, until the material is completely melted. After that, homogenize in a stomacher.
Egg white: The Compendium (Midura & Bryant, 2001, Ricke et al., 2001) does not mention differenti-ated procedures for these samples. ISO 6887-4:2003/
Cor.1:2004 recommends the first dilution to be 1:40, in Buffered Peptone Water (BPW), to reduce the inhib-itory effect exerted by natural lysozyme on microorgan-isms in general. The initial dilution used must be taken into account when calculating the results.
Fermented products containing live
Fermented products containing live