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BRITISH STANDARD BRITISH STANDARD BRITISH STANDARD BRITISH STANDARD BS 5763-0:1996 ISO 7218:1996 Incorporating Amendment No. 1 Methods for Methods for Microbiological examination of food and animal feeding stuffs — Part 0: General laboratory practices ICS 07.100.30; 65.120; 67.040
Amendments issued since publication Amendments issued since publication
Amd. No. Date Comments
13218 30 January 2002 Indicated by a sideline
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Contents Contents Contents Page Page Committees responsible Inside front cover National foreword ii Introduction 1 Scope 2 Normative reference 3 Premises 4 Installations and equipment 5 Personnel 6 Preparation of the equipment 7 Preparation and sterilization of culture media and�reagents 8 Laboratory samples 9 Examination techniques and expression of results Annex A (normative) Confidence interval limits for estimated�counts Annex B (normative) MPN tables Annex C (informative) Bibliography Figure 1 — Example of plating out: Direct method Table 1 — Extreme cases Table 2 — Examples of the selection of positive results for�calculating�MPN�values Table A.1 — Counting from one Petri dish Table A.2 — Counting from two Petri dishes Table B.1 — MPN table for 3 ° 1 g (ml), 3 ° 0,1 g (ml) and 3 ° 0,01 g (ml) Table B.2 — Explanation of the results
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National foreword National foreword This Part of BS 5763 has been prepared by Technical Committee AW/9 and is identical with ISO 7218… Cross reference Cross reference The British Standards which implement international or European publications referred to in this … A British Standard does not purport to include all the necessary provisions of a contract. Users … Compliance with a British Standard does not of itself confer immunity from legal obligations. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages Summary of pages Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, pages�1 to 35 and a… The BSI copyright notice displayed in this document indicates when the document was last issued. The sidelining in this document indicates the most recent changes by amendment.
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Introduction Introduction When conducting microbiological examinations, it is especially important — that only those microorganisms which are present in the samples are isolated or enumerated, and — that only those microorganisms which are present in the samples are isolated or enumerated, and — that the microorganisms do not contaminate the environment. In order to achieve this, it is necessary to pay attention to personal hygiene and to use working… Since, in this International Standard, it is possible to give only a few examples of the precauti… Ultimately, it is the responsibility of the head of the laboratory to judge whether the manipulat… A large number of manipulations can, for example, unintentionally lead to cross-contamination and… In order to conduct the examinations correctly, it is necessary to take certain precautions when … Certain precautions must be taken, not only for reasons of hygiene, but also to ensure good repro… If the guidance in this International Standard is followed, this will also contribute towards the… 1 Scope 1 Scope 1 Scope This International Standard gives general instructions for carrying out microbiological examinati… The purpose of this International Standard is to help to ensure the validity of the examinations,… This International Standard may be used wholly or partly for the accreditation of a laboratory by… 2 Normative reference 2 Normative reference The following standard contains provisions which, through reference in this text, constitute prov… ISO 6887:1983, 3 Premises 3 Premises 3.1 Test areas 3.1 Test areas The areas required for the specific operation of a microbiology laboratory are as follows:
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3.2 Additional areas 3.2 Additional areas The areas included in this category are, for example: 3.3 Location of the premises 3.3 Location of the premises The environment within which the microbiological analyses are carried out shall not affect the re… Care shall be taken to locate the premises so as to avoid risk of cross-contamination. Applicatio… Care shall be taken to ensure protection against extreme conditions such as excess temperature, d… The surface area shall be sufficiently large to keep the work areas clean and orderly. For all te… During the course of the tests, care shall be taken to limit access to the test areas to only tho… Separate rooms and/or separate areas and/or specific enclosures shall be provided for the following: Incubators, refrigerators and freezers can be placed in specific, specially adapted rooms. 3.4 Equipping the premises 3.4 Equipping the premises 3.4.1 The test premises shall be fitted out in the following ways in order to reduce the risks of… 3.4.1 The test premises shall be fitted out in the following ways in order to reduce the risks of… 3.4.2 The ambient temperature and air quality (microorganism content, humidity, dust spreading ra… 3.4.2 The ambient temperature and air quality (microorganism content, humidity, dust spreading ra… When tests are to be conducted in a low-contamination atmosphere, the room shall be specially equ… This equipment shall comply with the relevant regulations.
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3.4.3 The laboratory bench tops and furniture shall be manufactured in smooth, impermeable materi… 3.4.3 The laboratory bench tops and furniture shall be manufactured in smooth, impermeable materi… Laboratory furniture shall be designed so as to facilitate cleaning the floors (e.g. movable furn… Enclosed storage facilities shall be available for storing documents used when manipulating the s… NOTE�It is desirable that documents or books which are not frequently used be placed outside the … 3.4.4 The premises shall be well lit with avoidance of interfering reflections. It is advisable t… 3.4.4 The premises shall be well lit with avoidance of interfering reflections. It is advisable t… 3.5 Maintenance and inspection 3.5 Maintenance and inspection The floors, walls, ceilings, laboratory bench tops and furniture shall be subjected to regular ma… Regular cleaning and disinfection shall be carried out in order to keep the premises in a conditi… The ventilation systems and their filters shall be regularly maintained and filters changed when … The microbiological quality of surfaces and air shall be monitored regularly. Surface contamination may be estimated by directly applying to the surface a contact plate contai… NOTE�Other methods can also be used in order to estimate surface and air contamination. 4 Installations and equipment 4 Installations and equipment In general, all installations and equipment shall be kept clean and in proper working condition. … In general, all installations and equipment shall be kept clean and in proper working condition. … 4.1 Microbiological cabinets 4.1 Microbiological cabinets 4.1.1 Description 4.1.1 Description A cabinet is a dust-removed work station equipped with horizontal or vertical laminar air-flow. I… Conventionally, the maximum tolerable number of particles per cubic metre with a size greater tha… Cabinets are of two types: a) clean-air cabinets, which are intended to protect the product from extraneous contamination, a… a) clean-air cabinets, which are intended to protect the product from extraneous contamination, a… b) safety cabinets, which are intended to protect the product from extraneous contamination, and … Safety cabinets should be used for all work involving pathogens. 4.1.2 Maintenance and inspection 4.1.2 Maintenance and inspection The efficiency of a safety cabinet shall be checked on receipt and thereafter at regular interval… Cabinets should be cleaned and disinfected after use. Periodic verification of any microbial cont… A periodic verification of the proportion of microorganisms present shall be carried out using th…
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4.2 Balance 4.2 Balance 4.2.1 Use 4.2.1 Use A food microbiology laboratory shall be equipped with balances of the required range and accuracy… These balances are mainly used for weighing the test portion of the sample to be analysed and the… 4.2.2 Maintenance and inspection 4.2.2 Maintenance and inspection The balance shall be placed on a stable horizontal support and shall be protected from vibrations. It shall be checked regularly by calibration with working standards (preferably each working day)… The balance pan shall be cleaned, if necessary, after each use and at least once a day. The mecha… 4.3 Homogenizer 4.3 Homogenizer 4.3.1 Description 4.3.1 Description This equipment is used to prepare the initial suspension from the test sample of non-liquid produ… The following apparatus may be used: In certain special cases, the homogenization can be carried out with sterilizable glass beads hav… 4.3.2 Use 4.3.2 Use The usual operating time of a peristaltic homogenizer is 1�min to 2�min. This type of apparatus s… — products which risk puncturing the bag (presence of sharp, hard or dry particles), or — products which risk puncturing the bag (presence of sharp, hard or dry particles), or — products which are difficult to homogenize because of their texture (e.g. salami-type sausage). The rotary homogenizer shall operate for a duration such that the number of revolutions is betwee… Glass beads can be used for the preparation, by shaking, of the initial suspensions of certain vi… 4.3.3 Maintenance and inspection 4.3.3 Maintenance and inspection The different appliances shall be inspected and maintained in accordance with the manufacturers’ … 4.4 pH-meter 4.4 pH-meter 4.4.1 Description 4.4.1 Description A pH-meter is used to measure the potential difference, at a determined temperature, between a me… NOTE�The measuring electrode and the reference electrode are usually grouped together in a combin…
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4.4.2 Use 4.4.2 Use A pH-meter is used to measure the pH of each batch of culture media and reagents ( 4.4.3 Maintenance and inspection 4.4.3 Maintenance and inspection The pH-meter shall be calibrated, in accordance with the manufacturer’s instructions, using at le… The electrodes shall be checked and maintained in accordance with the manufacturer’s instructions… Prior to each use, check that the measuring bulb of the electrodes is completely immersed in dist… Clean the electrodes after each use. In order to take into account the soiling and ageing of the … Store the electrodes in accordance with the manufacturer’s instructions. 4.5 Autoclave 4.5 Autoclave 4.5.1 Description 4.5.1 Description An autoclave is an appliance which enables a saturated steam temperature of at least 121 °C to be… 4.5.2 Use 4.5.2 Use During the same sterilization cycle, the autoclave shall not be used to sterilize clean equipment… The autoclave shall be equipped with: — at least one safety valve; — at least one safety valve; — a pressure gauge; — a drain cock; — a regulation device allowing the temperature to be maintained to within ±1 °C of the scheduled … — a thermometer or a recording thermocouple. It should preferably also be equipped with a duration indicator or a programmer/timer. With steam sterilization all air must be expelled prior to the pressure build-up. If the autoclav… 4.5.3 Maintenance and inspection 4.5.3 Maintenance and inspection The autoclave shall be kept in perfect operating condition and shall be regularly inspected by th… All the monitoring instruments shall be kept in perfect working order and shall be verified regul… Descaling, if necessary, and draining operations shall be carried out regularly.
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4.6 Incubator 4.6 Incubator 4.6.1 Description 4.6.1 Description An incubator consists of a chamber which enables a temperature to be kept stable and evenly distr… 4.6.2 Use 4.6.2 Use Incubators shall be equipped with a regulation system which allows the temperature to be kept eve… If the ambient temperature is close to or higher than that of the incubator, it is necessary to f… Incubators walls should be protected from direct sunlight. If possible, incubators should not be completely filled in one single operation because the cultu… When loading incubators, attention should be paid to air circulation; under no circumstances shal… 4.6.3 Maintenance and inspection 4.6.3 Maintenance and inspection The homogeneous temperature within the working volume shall be checked using several thermometers… The measurement accuracy should be four times better than the requested accuracy (e.g. for a requ… The temperature stability shall be checked, for example, with one or more maximum and minimum the… The incubator temperature shall be checked at least every working day. For this purpose, each inc… The inner and outer walls of the incubator shall be regularly cleaned and disinfected and, if app… 4.7 Refrigerator or cold-storage room 4.7 Refrigerator or cold-storage room 4.7.1 Description 4.7.1 Description These are chambers which allow cold storage to be guaranteed. The temperature, unless otherwise s… 4.7.2 Use 4.7.2 Use Different chambers shall be available for the storage of: The refrigerators and cold-storage rooms shall be loaded in such a way that appropriate air circu…
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4.7.3 Maintenance and inspection 4.7.3 Maintenance and inspection The temperature of each chamber shall be checked each working day using a thermometer or a perman… The following maintenance operations shall be carried out regularly: — removal of dust from the blades or from the external heat-exchange plates; — removal of dust from the blades or from the external heat-exchange plates; — defrosting; — cleaning and disinfection of the inside of the chambers. 4.8 Freezer 4.8 Freezer 4.8.1 Description 4.8.1 Description A freezer has chambers which allow frozen storage to be guaranteed. The temperature, unless other… 4.8.2 Use 4.8.2 Use Different chambers shall be available for the storage of: — some non-inoculated culture media and reagents; — some non-inoculated culture media and reagents; — samples for analysis; — microorganism strains. The freezer shall be loaded in such a way that a sufficiently low temperature is maintained, in p… 4.8.3 Maintenance and inspection 4.8.3 Maintenance and inspection The temperature of each chamber shall be checked each working day using a thermometer or a perman… The following maintenance operations shall be carried out regularly: 4.9 Thermostatically controlled bath 4.9 Thermostatically controlled bath 4.9.1 Description 4.9.1 Description This is a bath which allows a specified temperature to be maintained. Unless otherwise stated, th… 4.9.2 Use 4.9.2 Use The main uses are the following: For precise temperature control, the thermostatically controlled bath shall be equipped with a ci…
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4.9.3 Maintenance and inspection 4.9.3 Maintenance and inspection Each bath shall be equipped with a thermometer or a thermocouple independent of the automatic reg… The bath temperature shall be checked each time it is used, preferably daily. The level of the liquid in the bath (water, ethylene glycol, etc.) shall be checked regularly. In order to avoid any microbial proliferation, the liquid shall be changed frequently. 4.10 Sterilizing oven 4.10 Sterilizing oven 4.10.1 Description 4.10.1 Description A sterilizing oven is a chamber which allows a temperature of 170�°C to 180�°C to be attained for… 4.10.2 Use 4.10.2 Use Only metal or glass equipment is sterilized in the sterilizing oven. When the correct temperature… WARNING — Volumetric glassware shall not be sterilized in a sterilizing oven. WARNING — Volumetric glassware shall not be sterilized in a sterilizing oven. The temperature shall be evenly distributed within the chamber. The oven shall be equipped with: — a thermostat; — a thermostat; — a thermometer or a recording thermocouple. It should preferably also be equipped with a duration indicator or a programmer/timer. 4.10.3 Maintenance and inspection 4.10.3 Maintenance and inspection Check that the temperature is homogeneous over the working volume. The oven shall be kept in perfect operating condition and the monitoring instruments shall be ver… Regular cleaning is recommended. 4.11 Microwave oven 4.11 Microwave oven 4.11.1 Description 4.11.1 Description This is an apparatus which allows a product to be heated using microwaves. 4.11.2 Use 4.11.2 Use In the present state of the art, only one use is possible: melting of the agar culture media. The apparatus used operates at atmospheric pressure. It shall be able to heat the culture media i… WARNING — Handle with care! Heating up agar culture media in a microwave oven can cause delay in … WARNING — Handle with care! Heating up agar culture media in a microwave oven can cause delay in … NOTE�In the absence of sufficient assessment of the efficiency of microwaves for sterilizing cult… 4.12 Optical microscope 4.12 Optical microscope 4.12.1 Use 4.12.1 Use An optical microscope contains objectives with different magnifications. Using a high-magnificati… NOTE�Instruments (plate microscopes) with low-power magnification and ideally stereoscopic focusi…
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4.12.2 Maintenance and inspection 4.12.2 Maintenance and inspection After use involving immersion, the lens shall be cleaned, in such a way as not to affect the opti… At least once a year, an authorized member of the personnel shall carry out a general cleaning op… 4.13 Gas burner or wire incinerator 4.13 Gas burner or wire incinerator A gas burner is used for creating and maintaining a protection zone around a hot spot. It is used… The wire incinerator is preferable when sterilizing metal wires or loops after manipulating patho… 4.14 Dispenser for culture media and reagents 4.14 Dispenser for culture media and reagents 4.14.1 Description 4.14.1 Description This is an instrument or apparatus used to distribute culture media and reagents into tubes, bott… 4.14.2 Use 4.14.2 Use In the case of the aseptic distribution of sterile culture media and reagents, all parts of the i… The accuracy of the instrument or apparatus shall be appropriate to the accuracy of the volume to… 4.14.3 Maintenance and inspection 4.14.3 Maintenance and inspection These instruments shall be kept in perfect condition in accordance with the manufacturer’s instru… The volumes delivered shall be monitored regularly. 4.15 Mechanical stirrer 4.15 Mechanical stirrer This allows the homogeneous mixing of diverse liquid media (e.g. decimal dilutions and samples of… Its principle is based on an eccentric rotational movement of the contents of test tubes (vortex). 4.16 Colony-counting device 4.16 Colony-counting device This should preferably be equipped with a lighting system with a dark background, a magnifying gl… 4.17 Equipment for culture in a modified atmosphere 4.17 Equipment for culture in a modified atmosphere 4.17.1 Description 4.17.1 Description This is a jar that can be hermetically sealed or any other appropriate apparatus which enables mo… 4.17.2 Use 4.17.2 Use The composition of the atmosphere obtained by a gas mixture (e.g. from a gas cylinder) or by any …
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4.17.3 Maintenance and inspection 4.17.3 Maintenance and inspection An indicator for monitoring the nature of the atmosphere shall be placed in each chamber during e… If a catalyst is fitted, this shall be regularly regenerated according to the manufacturer’s inst… This equipment shall be regularly cleaned and disinfected. 4.18 Other equipment 4.18 Other equipment Other equipment and apparatus are in everyday use, including the following: filtration apparatus,… 5 Personnel 5 Personnel 5.1 Competence 5.1 Competence All personnel working in a microbiology laboratory shall have received adequate training to enabl… The personnel in charge of performing the tests shall have a good knowledge of and sufficient pra… All personnel shall receive relevant updated information, as necessary, in hygiene and laboratory… 5.2 Hygiene 5.2 Hygiene In the field of personal hygiene, the following precautions shall be taken in order to avoid cont… 6 Preparation of the equipment 6 Preparation of the equipment 6.1 Preparation 6.1 Preparation The equipment used in microbiology shall be prepared in such a manner as to guarantee its cleanli… The equipment used shall be washed prior to use, even if new. Stopper tubes and cap bottles prior to sterilization by appropriate means (combed cotton, metal, … Stopper the pipettes with combed cotton or any other appropriate material.
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If necessary, the equipment to be sterilized should be placed in special containers or wrapped in… 6.2 Sterilization 6.2 Sterilization 6.2.1 Sterilization by dry heat 6.2.1 Sterilization by dry heat Heat in a sterilizing oven ( 6.2.2 Sterilization by moist heat 6.2.2 Sterilization by moist heat Heat for at least 15�min at a minimum of 121�°C in an autoclave ( 6.3 Disposable apparatus 6.3 Disposable apparatus Disposable apparatus may be used in the same way as the re-usable glassware (Petri dishes, pipett… It is then advisable to make certain, by asking the manufacturer, that the proposed equipment is … Disposable apparatus shall be decontaminated prior to its disposal. Besides the methods described in 6.4 Management of clean equipment 6.4 Management of clean equipment Clean equipment shall be protected against dust during storage, under conditions which maintain i… 6.5 Management of sterile equipment 6.5 Management of sterile equipment Prior to use, the equipment shall be stored under conditions which allow it to remain sterile. Si… When sterilizing equipment intended for microbiology, an expiry date (or a manufacturing date) sh… 6.6 Decontamination 6.6 Decontamination After use (culture of microorganisms or contact with microorganisms), the equipment and its conte… Pasteur pipettes shall only be used once. Plastic equipment may be directly incinerated, if permitted by the laws of the country. 6.7 Washing 6.7 Washing Wash equipment only after it has been decontaminated. Empty the containers of their contents. Prior to washing, separate seals from stoppers or caps, if appropriate.
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Carefully wash the stoppers or caps and the glassware in hot water with a commercial detergent so… Rinse all equipment in distilled water or water of equivalent quality. Specialized equipment may be used in order to facilitate cleaning operations (e.g. pipette washer… 7 Preparation and sterilization of culture media and reagents 7 Preparation and sterilization of culture media and reagents The accurate preparation of culture media is one of the fundamental steps in microbiological anal… The accurate preparation of culture media is one of the fundamental steps in microbiological anal… 7.1 Distilled water 7.1 Distilled water The water used shall be distilled water or water of equivalent quality; i.e. free from substances… The distilled water shall be stored in containers manufactured from inert materials (e.g. neutral… NOTE�In some cases, it is necessary to use freshly prepared water, without any dissolved carbon d… In order to be considered as being of good quality, the distilled water shall possess a resistivi… NOTE�Due to passing through an ion exchanger, demineralized water often has a high microorganism … 7.2 Preparation of culture media 7.2 Preparation of culture media Two types of preparation exist: Two types of preparation exist: The bottles containing the dehydrated basic ingredients or the dehydrated complete media shall be… Do not use beyond the stated shelf-life. As the dehydrated components and media are hygroscopic, it is essential to close the bottles quic… 7.2.1 Rehydration 7.2.1 Rehydration Follow the recommendations of the International Standard in question and/or those of the manufact… 7.2.2 Measurement of pH 7.2.2 Measurement of pH Measure the pH using a pH-meter ( The adjustment is normally carried out using a solution of approximately 40 g/l (about 1 mol/l) o… 7.2.3 Dispensing 7.2.3 Dispensing Dispense the medium into appropriate containers, either manually or using an automatic apparatus�( Use a container having a volume one, two or three times the volume to be dispensed in order to pr…
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7.3 Sterilization 7.3 Sterilization The sterilization of culture media and of reagents can be carried out using various techniques, i… The sterilization of culture media and of reagents can be carried out using various techniques, i… However, certain media and reagents can be used without any special sterilization procedures (ref… After sterilization, the media shall be monitored, in particular with respect to pH, colour, ster… 7.3.1 Sterilization by moist heat 7.3.1 Sterilization by moist heat This is performed in an independent autoclave ( Prior to autoclaving, a representative number of containers, in each part of the autoclave, shall… 7.3.2 Sterilization by filtration 7.3.2 Sterilization by filtration This can be performed under vacuum or pressurized conditions. Use membranes and filter elements with a pore diameter of 0,22 µm. They shall have been sterilize… Sterilize the different components of the filtration apparatus, assembled or not, in the autoclave ( 7.4 Storage 7.4 Storage Each package of bottles, tubes and Petri dishes shall be labelled and shall bear the following de… Each package of bottles, tubes and Petri dishes shall be labelled and shall bear the following de… 7.4.1 Laboratory-prepared culture media and reagents 7.4.1 Laboratory-prepared culture media and reagents Culture media dispensed in tubes or bottles and reagents which are not used immediately shall be … Unless specified in the International Standard in question, they shall be kept in the refrigerator ( Never use media which have become dehydrated. Prior to use, it is desirable that the temperature of the culture media be equilibrated to that o… 7.4.2 Ready-to-use culture media and reagents 7.4.2 Ready-to-use culture media and reagents It is necessary to comply with the manufacturer’s instructions: expiry date, storage temperature … 7.5 Melting of agar culture media 7.5 Melting of agar culture media Melt a culture medium by placing it in a boiling water bath or by any other process which gives i…
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In the case of particularly sensitive culture media, this melting duration shall be shortened, an… 7.6 De-aeration of culture media 7.6 De-aeration of culture media If necessary, just prior to use, heat the culture media in boiling water or under a flow of steam… 7.7 Preparation of Petri dishes 7.7 Preparation of Petri dishes Pour the molten agar culture medium into Petri dishes so as to obtain a thickness of at least 2�m… Use the thus-prepared Petri dishes immediately or store them under conditions which prevent their… Use them after drying. In general, for the surface inoculation of an agar culture medium, dry the dishes, preferably wit… Ready-prepared agar plates are available commercially. Store and use them according to the manufa… 8 Laboratory samples 8 Laboratory samples 8.1 Sampling 8.1 Sampling It is important that the laboratory receive a sample which is truly representative of the product… Sampling is not part of these general rules for microbiological examinations. See the specific In… 8.2 Transport 8.2 Transport The transportation of the samples to the laboratory shall ensure that they are kept under conditi… Particular attention shall be paid to observing the following storage temperatures for the follow… WARNING — Sensitive foodstuffs (e.g. innards, fresh fish) shall be stored at a temperature of bet… WARNING — Sensitive foodstuffs (e.g. innards, fresh fish) shall be stored at a temperature of bet… Spoiled stable units shall be transported in closed packaging for protection against possible lea… 8.3 Receipt and storage 8.3 Receipt and storage The laboratory personnel shall check the condition of the samples on receipt. If their condition …
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The samples admitted into the laboratory shall be documented in such a manner that their progress… The following information shall be noted: Samples awaiting examination shall be stored under conditions which prevent any alteration in the… Particular attention shall be paid to the storage temperature (see 8.4 Test portions 8.4 Test portions In order to avoid contamination of the environment and of the test portion, it is recommended to … The protection of the environment against contamination is of particular importance during the we… Handle the samples in such a manner as to avoid any and all risk of contamination. To achieve thi… — when not working in a safety cabinet, work in the vicinity of a flame; — when not working in a safety cabinet, work in the vicinity of a flame; — for a packaged product, clean the outside of the packing with 70 % ethanol where it will be ope… — any instrument which is used for opening the packaging (tin-opener, scissors, etc.) shall be st… — any instrument which is used for taking the sample (spoon, forceps, pipette, etc.) shall be ste… — carefully mark the reference of the laboratory sample on the containers, plastic bags, etc. (see� 8.5 Conservation and destruction of laboratory samples 8.5 Conservation and destruction of laboratory samples Except for special cases, keep the laboratory samples until all results have been obtained, or lo… Before discarding, decontaminate any deteriorated or dangerous laboratory samples. Discard undeteriorated laboratory samples directly.
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9 Examination techniques and expression of results 9 Examination techniques and expression of results 9.1 Hygienic precautions during the examination 9.1 Hygienic precautions during the examination Precautions shall be taken in order to conduct the work as far as possible under aseptic conditio… The manipulation of products likely to contain pathogenic bacteria ( Aerosols are a major cause of environmental contamination and of infection. Aerosols can be forme… It is therefore necessary to avoid their formation. 9.2 Preparation of the initial suspension and dilutions 9.2 Preparation of the initial suspension and dilutions See ISO 6887. In the case of the preparation of an initial suspension, the time which elapses between the end o…
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9.3 Counting using a solid medium 9.3 Counting using a solid medium 9.3.1 Inoculation for poured plates 9.3.1 Inoculation for poured plates Prepare the medium (by surfusion on a water bath at�47 °C), the Petri dishes, the dilution fluid … Dispense into the Petri dishes (labelled) the defined volumes of the dilutions to be examined. Po… If the presence of spreading colonies (e.g. 9.3.2 Surface inoculation 9.3.2 Surface inoculation Deposit the inoculum in the centre of the labelled Petri dish onto the agar culture medium (prepa… Glass balls may be used to assist in this spreading. In certain cases (stated in the relevant International Standard), the inoculum may be deposited o… 9.3.3 Incubation 9.3.3 Incubation Unless otherwise stated, immediately invert the inoculated dishes and place them quickly in the i… NOTE�In certain cases, it may be useful to make provision for duplicate inoculated dishes which w… After incubation, the dishes shall, if possible, be examined immediately. Otherwise, they may be … 9.3.4 Counting of colonies 9.3.4 Counting of colonies Following the period of incubation stated in the specific standard, count the colonies (total col… The different methods of calculation defined in When counting typical or presumed suspect colonies, the maximum number of all typical or atypical… NOTE 1�In certain cases, it may be difficult to count the colonies (for example where spreading m… NOTE 2�When counting typical or presumed suspect colonies, the description of the colonies will p…
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9.3.5 Expression of results 9.3.5 Expression of results 9.3.5.1 General 9.3.5.1 General 9.3.5.1.1 In this subclause, the cases dealt with correspond to the following general cases: 9.3.5.1.1 In this subclause, the cases dealt with correspond to the following general cases: — inoculation of two Petri dishes, 90 mm in diameter, per dilution; — inoculation of two Petri dishes, 90 mm in diameter, per dilution; — maximum number for the counting of total colonies is 300 per dish; — maximum number of all typical and atypical colonies present on a dish when counting typical or … — maximum number for the counting of typical or presumed suspect colonies is 150 per dish; — number of presumed suspect colonies ( — minimum number of colonies [total colonies, typical colonies or colonies complying with identif… These figures will be defined in the specific standards. When dishes with a diameter different from 90 mm are used, the maximum number of colonies shall b… 9.3.5.1.2 The methods of calculation defined below take account of the cases which occur most fre… 9.3.5.1.2 The methods of calculation defined below take account of the cases which occur most fre… 9.3.5.2 Method of calculation: General case (counting of total colonies or typical colonies) 9.3.5.2 Method of calculation: General case (counting of total colonies or typical colonies) For a result to be valid, it is generally considered necessary to count the colonies on at least … Calculate the number N N where
is the sum of the colonies counted on all the dishes retained from two successive dilutions, and …
V V is the volume of inoculum applied to each dish, in millilitres;
n1 n 1 is the number of dishes retained at the first dilution;
n2 n 2 is the number of dishes retained at the second dilution;
d d is the dilution factor corresponding to the first dilution retained [d = 1 when the undiluted liq… Round off the results calculated to two significant figures. In order to do this, if the third fi… Take as the result a number preferably between 1,0 and 9,9 multiplied by the appropriate power of… Express the result as follows: — number — number
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EXAMPLE Counting has produced the following results: — at the first dilution (10 — at the first dilution (10 — at the second dilution (10 By rounding off the result as recommended above, the number of microorganisms is 19 000 or 1,9 × 10 9.3.5.3 Method of calculation: Case after identification or confirmation 9.3.5.3 Method of calculation: Case after identification or confirmation When the method used requires identification or confirmation, a given number, a a where
b b is the number of colonies complying with identification or confirmation criteria among the inocul…
C C is the total number of presumed suspect colonies counted on the dish. Round off the results calculated to the nearest whole number. In order to do this, if the first f… Calculate the number Round off the result as recommended in Express the result as recommended in EXAMPLE� Counting has produced the following results: — at the first dilution (10 — at the first dilution (10 — at the second dilution (10 Testing of selected colonies was carried out: — for 66 colonies, 8 colonies, 6 of which complied with the criteria; hence — for 66 colonies, 8 colonies, 6 of which complied with the criteria; hence — for 80 colonies, 9 colonies, 6 of which complied with the criteria; hence — for 7 colonies, 5 colonies, 4 of which complied with the criteria; hence — for 4 colonies, all 4 of which have complied with the criteria; hence N N By rounding off the result as recommended in
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9.3.5.4 Method of calculation: Estimated counts 9.3.5.4 Method of calculation: Estimated counts 9.3.5.4.1 Case of two dishes (test sample or initial suspension or first dilution) containing les… 9.3.5.4.1 Case of two dishes (test sample or initial suspension or first dilution) containing les… If the two dishes from the test sample (liquid products), or from the initial suspension (other p… N N E where
is the sum of the colonies counted on the two dishes;
V V is the volume of inoculum applied to each dish, in millilitres;
n n is the number of dishes retained (in this case, n = 2);
d d is the dilution factor of the initial suspension or of the first dilution inoculated or retained … Round off the result as recommended in Express the result as follows: — estimated number — estimated number EXAMPLE� Counting has produced the following results: — at the first dilution (10 — at the first dilution (10 N N E By rounding off the result as recommended in 9.3.5.4.2 Case of two dishes (test sample or initial suspension or first dilution) containing no … 9.3.5.4.2 Case of two dishes (test sample or initial suspension or first dilution) containing no … If the two dishes from the test sample (liquid products), or from the initial suspension (other p… — less than 1/ — less than 1/ where 9.3.5.4.3 Special cases (counting of typical or presumed suspect colonies) 9.3.5.4.3 Special cases (counting of typical or presumed suspect colonies) 9.3.5.4.3.1 If the number of all typical and atypical colonies for the two dishes containing a fi… 9.3.5.4.3.1 If the number of all typical and atypical colonies for the two dishes containing a fi… — less than 1/ — less than 1/ where
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EXAMPLE� Counting has produced the following results: — at the first dilution (10 — at the first dilution (10 — at the second dilution (10 The result expressed in microorganisms is less than 1 000 and more than 100 per millilitre or per… 9.3.5.4.3.2 If the number of all typical and atypical colonies for the two dishes containing a fi… 9.3.5.4.3.2 If the number of all typical and atypical colonies for the two dishes containing a fi… — less than 1/ — less than 1/ where EXAMPLE� Counting has produced the following results: — at the first dilution (10 — at the first dilution (10 — at the second dilution (10 The result expressed in microorganisms is less than 100 per millilitre or per gram of product. 9.3.5.5 Method of calculation: Special cases 9.3.5.5 Method of calculation: Special cases 9.3.5.5.1 When the number of colonies counted (total colonies, typical colonies or presumed suspe… 9.3.5.5.1 When the number of colonies counted (total colonies, typical colonies or presumed suspe… — if the number of colonies for each of the two dishes containing dilution — if the number of colonies for each of the two dishes containing dilution — if the number of colonies for each of the two dishes containing the dilution NOTE�The figures corresponding to confidence intervals should be adapted to the maximum number st… EXAMPLE 1� Counting has produced the following results: — at the first dilution (10 — at the first dilution (10 — at the second dilution (10 Use the method of calculation for general cases using the dishes from the two dilutions retained. EXAMPLE 2� Counting has produced the following results: — at the first dilution (10–2) retained: more than 324 colonies on each one of the dishes; — at the first dilution (10–2) retained: more than 324 colonies on each one of the dishes; — at the second dilution (10–3) retained: 12 and 14 colonies. Begin estimated counting on the basis of the colonies counted on the two dishes from the 10–3 dil…
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EXAMPLE 3� Counting (when a maximum number of 300 has been set for the counting of colonies) has produced th… — at the first dilution (10–2) retained: more than 324 colonies on each one of the two dishes; — at the first dilution (10–2) retained: more than 324 colonies on each one of the two dishes; — at the second dilution (10–3) retained: 8 and 6 colonies. The result of this counting is unacceptable. EXAMPLE 4� Counting (when a maximum number of 150 has been set for the counting of colonies) has produced th… — at the first dilution (10–2) retained: more than 167 colonies on each one of the two dishes (up… — at the first dilution (10–2) retained: more than 167 colonies on each one of the two dishes (up… — at the second dilution (10–3) retained: 8 and 6 colonies. Begin estimated counting on the basis of colonies counted on the two dishes from the 10–3 dilution. 9.3.5.5.2 Where the counting of colonies (total colonies or typical colonies or presumed suspect … 9.3.5.5.2 Where the counting of colonies (total colonies or typical colonies or presumed suspect … — more than 300/ — more than 300/ where
d d is the dilution factor of the last inoculated dilution;
b b is the number of colonies complying with identification or confirmation criteria among the inocul… 9.3.5.5.3 Where only the two dishes containing the last inoculated dilution contain less than 300… 9.3.5.5.3 Where only the two dishes containing the last inoculated dilution contain less than 300… N N where
is the sum of colonies counted on the two dishes, and where at least one contains a minimum of 15…
V V is the volume of the inoculum applied to each dish, in millilitres;
n n is the number of dishes retained (in this case, n = 2);
d d is the dilution factor corresponding to the dilution retained. Round off the result as recommended in Express the result as follows: — number — number EXAMPLE� Counting has produced the following results: — at the last dilution (10 — at the last dilution (10 — at the last dilution (10 N N By rounding off the result as recommended in
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9.3.6 Confidence limit 9.3.6 Confidence limit In order to estimate the validity of the result and to avoid too strict an interpretation, it is … In order to estimate the validity of the result and to avoid too strict an interpretation, it is … Other variations due to the technique itself intervene, particularly those connected with dilutio… 9.3.6.1 General case 9.3.6.1 General case With a probability of 95 %, the confidence interval ¸, which characterizes the microbial dispersi…
when
and where EXAMPLE 1: Extreme cases (see Extreme cases Extreme cases
Number of colonies counted Number of colonies counted Weighted mean Weighted mean Confidence interval Confidence interval
Dilution n Dilution Dilution n + 1 Dilution
300 30 278 to 324
300
300 30 – 7 % to + 8 %
�15 �1 10 to 20
�14
�15 �1 – 29 % to + 43 % EXAMPLE 2:�Confidence interval for the example given in With
¸ = (191,82 + 0,87 ± 18,30) × 10 The limits of the confidence interval are therefore
In this case, the limits of the confidence interval, expressed as a percentage and calculated on …
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9.3.6.2 Case after identification 9.3.6.2 Case after identification With a probability of 95 %, the confidence interval ¸, which characterizes the microbial dispersi… EXAMPLE:�For the case given in
¸ = (51,36 + 0,87 ± 9,47) × 10 The limits of the confidence interval are therefore
In this case, the limits of the confidence interval are between –16,7 % and +20,1 % inclusive. 9.3.6.3 Estimated counts 9.3.6.3 Estimated counts The confidence limits are given in 9.4 Counting using a liquid medium: Most probable number technique 9.4 Counting using a liquid medium: Most probable number technique NOTE�Two inoculation systems are possible. The most frequently used systems (called “symmetric” s… NOTE�Two inoculation systems are possible. The most frequently used systems (called “symmetric” s… 9.4.1 Inoculation 9.4.1 Inoculation Depending on the desired accuracy of the results, inoculate an appropriate number of flasks or tu… Introduce, by means of a pipette, the inoculum into the corresponding flasks or tubes. Use a new sterile pipette for each dilution. 9.4.2 Incubation 9.4.2 Incubation Place the inoculated flasks and/or tubes in an incubator or, preferably, in a thermostatically co… 9.4.3 Interpretation of results 9.4.3 Interpretation of results 9.4.3.1 Selection of dilutions 9.4.3.1 Selection of dilutions NOTE�The initial suspension and the test sample, if liquid, are considered as being “dilutions”. NOTE�The initial suspension and the test sample, if liquid, are considered as being “dilutions”. In the general case where three tubes or flasks per dilution have been inoculated, select, for ea… 9.4.3.1.1 Case 1: At least one dilution reveals three positive tubes (see 9.4.3.1.1 Case 1: At least one dilution reveals three positive tubes (see Select the highest dilution (i.e. the one having the smallest concentration of sample) which reve… If an insufficient number of dilutions has been prepared beyond the highest dilution revealing th…
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Examples of the selection of positive results for calculating MPN values Examples of the selection of positive results for calculating MPN values
Example Example Number of positive tubes obtained from three incubated tubes for the following inoculated quantit… Number of positive tubes obtained from three incubated tubes for the following inoculated quantit… a MPNb MPN
Liquid products Liquid products 10 ml 10 ml 1 ml 1 ml 10–1 ml 10 –1 10–2 ml 10 –2 ml 10–3 ml 10 –3 Liquid products Liquid products Other products Other products
Other products Other products 1 g 1 g 10–1 g 10 –1 g 10–2 g 10 –2 g 10–3 g 10 –3 g 10–4 g 10 –4 g ml–1 ml –1 g–1 g –1
1 3 3 3 2 2 1 1 0 1,5 ° 101 1,5 ° 102
2 3 3 3 3 3 0 0 — 2,4 ° 101 2,4 ° 102
3 2 2 1 1 1 1 0 0 7,4 7,4 ° 101
4 3 3 3 3 0 0 0 0 2,4 2,4 ° 101
5 2 2 2 2 0 0 1 0 2,1 ° 10–1 2,1
a Bold: combination selected. a Bold: combination selected. b Calculated from the MPN index for three tubes ( 9.4.3.1.2 Case 2: No dilution revealing three positive tubes 9.4.3.1.2 Case 2: No dilution revealing three positive tubes Case 1 cannot be applied. Select the three highest dilutions of the series (i.e. those having the… 9.4.3.1.3 Special cases 9.4.3.1.3 Special cases In all cases where more than one of the three dilutions selected according to 9.4.3.2 Calculation of the most probable number (MPN) 9.4.3.2 Calculation of the most probable number (MPN) 9.4.3.2.1 Check, according to the number of samples examined by batch in 9.4.3.2.1 Check, according to the number of samples examined by batch in Thus, for example, when only the results of category�1 are accepted, sequence 221 can only be tak… 9.4.3.2.2 For each sequence considered as being acceptable according to 9.4.3.2.2 For each sequence considered as being acceptable according to
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9.4.4 Expression of results 9.4.4 Expression of results From the MPN index (see
where In the case where the smallest dilution selected corresponds to the tubes prepared from a double … If the MPN is smaller than 0,3 microorganisms per millilitre (liquid products) or per gram (other… EXAMPLE In the case of a solid product, the MPN index is�24; the first tube selected corresponds to the i… C C s 9.4.5 Confidence limits 9.4.5 Confidence limits The confidence limits are given in the MPN table ( It is well known that large variations in results can be observed with the MPN technique. Consequ… 9.5 Detection method 9.5 Detection method A detection method is a method which determines the presence or absence of microorganisms defined… A detection method is a method which determines the presence or absence of microorganisms defined… 9.5.1 Principle 9.5.1 Principle Unless otherwise stated in the relevant International Standard, mix (liquid products) or homogeni… In certain cases, it is desirable to precede enrichment in the elective or selective broth by a p… 9.5.2 Interpretation of the result 9.5.2 Interpretation of the result If the target microorganism has been detected, give the result in the form: “present in the analysed If the target microorganism has not been detected, give the result in the form: “absent in the analysed Under no circumstances generalize to other higher quantities of product.
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9.6 Basic identification techniques 9.6 Basic identification techniques 9.6.1 Preparation of a pure culture 9.6.1 Preparation of a pure culture 9.6.1.1 General 9.6.1.1 General Begin the preparation of a pure culture by the selection of a colony on or in an agar medium whic… Then inoculate the selected colony on a non-selective agar culture medium. After incubation, sele… Use the plating-out techniques described in 9.6.1.2 Plating out 9.6.1.2 Plating out Take a small quantity from the surface of a well-isolated colony using the tip of a sterile loop…. Take a small quantity from the surface of a well-isolated colony using the tip of a sterile loop…. 9.6.1.2.1 Direct method: Example 9.6.1.2.1 Direct method: Example Using the tip of the loop, inoculate, in close streaks, a portion of about one-third of the agar … 9.6.1.2.2 Method using dilution fluid 9.6.1.2.2 Method using dilution fluid Suspend the cells in 1 ml to 2 ml of the selected dilution fluid, rubbing the inoculated loop aga… Sterilize and cool down the loop. Using the loop, take a small portion of the microbial suspensio… 9.6.1.3 Incubation 9.6.1.3 Incubation Unless otherwise stated, invert the inoculated Petri dishes and place them in the incubator for t… 9.6.1.4 Selection 9.6.1.4 Selection After incubation, select a well-isolated colony from the dish, either for subsequent plating out,… If possible, the final tests should be carried out using cells stemming from one single colony. I…
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9.6.2 Gram’s stain (Modified Hucker technique) 9.6.2 Gram’s stain (Modified Hucker technique) This staining of bacterial cells allows description of the morphology of the bacteria and classif… This staining of bacterial cells allows description of the morphology of the bacteria and classif… 9.6.2.1 Solutions 9.6.2.1 Solutions Commercially available solutions may be used. In this case, follow the manufacturer’s recommendat… Commercially available solutions may be used. In this case, follow the manufacturer’s recommendat… 9.6.2.1.1 Crystal violet solution 9.6.2.1.1 Crystal violet solution 9.6.2.1.1.1 Composition 9.6.2.1.1.1 Composition
Crystal violet 2,0 g
Ethanol (95 %) 20 ml
Ammonium oxalate (C2H8N2O4) 0,8 g
Distilled water 80 ml 9.6.2.1.1.2 Preparation 9.6.2.1.1.2 Preparation Dissolve the Crystal violet in the ethanol and the ammonium oxalate in the distilled water. Mix t… 9.6.2.1.2 Iodine solution 9.6.2.1.2 Iodine solution 9.6.2.1.2.1 Composition 9.6.2.1.2.1 Composition
Iodine 1,0 g
Potassium iodide (KI) 2,0 g
Distilled water 100 ml 9.6.2.1.2.2 Preparation 9.6.2.1.2.2 Preparation Dissolve the potassium iodide in 10 ml of distilled water; add the iodine in fractions. After dis… 9.6.2.1.3 Safranine solution 9.6.2.1.3 Safranine solution 9.6.2.1.3.1 Composition 9.6.2.1.3.1 Composition
Safranine O 0,25 g
Ethanol (95 %) 10 ml
Distilled water 100 ml 9.6.2.1.3.2 Preparation 9.6.2.1.3.2 Preparation Dissolve the safranine in the ethanol then mix with the distilled water.
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9.6.2.2 Staining technique 9.6.2.2 Staining technique After fixing the bacterial film on the microscope slide prepared from a culture 18�h to 24�h old,… Gently rinse the inclined slide with water for a few seconds. Cover the slide with the iodine solution ( Gently rinse the inclined slide with water for a few seconds. Pour gently and continuously a film of ethanol�(95�%) onto the inclined slide over a period of no… Gently rinse the inclined slide with water in order to eliminate the ethanol. Cover the slide with the solution of safranine�( Gently rinse the inclined slide with water. Dry the slide. 9.6.2.3 Interpretation 9.6.2.3 Interpretation Examine the slide under the high-power oil objective of the microscope ( For a pure culture of certain bacterial types, both Gram-positive and Gram-negative cells can be … NOTE�Densely packed cells may give an uncharacteristic response. 9.6.3 Test for catalase 9.6.3 Test for catalase The detection of this enzyme, which decomposes hydrogen peroxide (H The detection of this enzyme, which decomposes hydrogen peroxide (H In all cases, unless otherwise stated in the relevant International Standard, the culture medium … NOTE 1�Certain lactic bacteria possess a “pseudo-catalase” which does not contain any haem group … NOTE 2�In the case of anaerobic bacteria, wait 30 s in the open air prior to adding the hydrogen … 9.6.3.1 From a broth culture 9.6.3.1 From a broth culture Add to 1 ml of the culture 0,5�ml of a 10 volume [3 % ( 9.6.3.2 From an agar medium culture 9.6.3.2 From an agar medium culture Cover the culture with 1 ml to 2 ml of a 10 volume hydrogen peroxide solution. Observe immediatel… 9.6.3.3 From a colony 9.6.3.3 From a colony Place separately two drops of a 10 volume hydrogen peroxide solution on a microscope slide. Pick off a colony with a sterile glass or plastic rod (especially not a metallic wire) and gently… The observations can be conducted macroscopically or using a low-magnification microscope.
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9.6.4 Test for oxidase 9.6.4 Test for oxidase The detection of oxidase is carried out by the change in colouring of a compound at the time of o… The detection of oxidase is carried out by the change in colouring of a compound at the time of o… 9.6.4.1 Reagent 9.6.4.1 Reagent 9.6.4.1.1 Composition 9.6.4.1.1 Composition
N, N, N½, N½-Tetramethyl-p-phenylenediamine dihydrochloride (C10H16N2·2HCl) N 1,0�g
Distilled water 100 ml 9.6.4.1.2 Preparation 9.6.4.1.2 Preparation Dissolve the reagent in the cold water. The reagent shall be prepared immediately prior to use. Commercially available disks or sticks may be used. In this case, follow the manufacturer’s recom… 9.6.4.2 Technique 9.6.4.2 Technique Moisten a piece of filter paper with the reagent. Take a sample of the bacterial culture obtained… 9.6.4.3 Interpretation of the result 9.6.4.3 Interpretation of the result In the case of the presence of oxidase, a violet to purple colour appears within a period of betw… 9.6.5 Use of biochemical galleries for identification 9.6.5 Use of biochemical galleries for identification Currently available biochemical galleries may be used for identification. However, all commercial…
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(normative) Confidence interval limits for estimated counts (normative) Confidence interval limits for estimated counts The values given in Counting from one Petri dish Counting from one Petri dish
Number of microorganismsa Number of microorganisms a Confidence limit at 95 % level Confidence limit at 95 % level Percent error for the limitb Percent error for the limit b
Lower Lower Upper Upper Lower Lower Upper Upper
�1 <1 �6 –97 +457
�2 <1 �7 –88 +261
�3 <1 �9 –79 +192
�4 �1 10 –73 +156
�5 �2 12 –68 +133
�6 �2 13 –63 +118
�7 �3 14 –60 +106
�8 �3 16 –57 �+97
�9 �4 17 –54 �+90
10 �5 18 –52 �+84
11 �6 20 –50 �+79
12 �6 21 –48 �+75
13 �7 22 –47 �+71
14 �8 24 –45 �+68
15 �8 25 –44 �+65
a Equal to the number of colonies. a Equal to the number of colonies. b Compared to the microorganism count (1
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Counting from two Petri dishes Counting from two Petri dishes
Number of coloniesa Number of colonies a Number of microorganisms Number of microorganisms Confidence limit at 95 % level Confidence limit at 95 % level Percent of error for the limitb Percent of error for the limit b
Lower Lower Upper Upper Lower Lower Upper Upper
�1 �1 <1 �3 –97 +457
�2 �1 <1 �4 –88 +261
�3 �2 <1 �4 –79 +192
�4 �2 �1 �5 –73 +156
�5 �2 �1 �6 –68 +133
�6 �3 �1 �6 –63 +118
�7 �4 �2 �7 –60 +106
�8 �4 �2 �8 –57 �+97
�9 �4 �2 �9 –54 �+90
10 �5 �2 �9 –52 �+84
11 �6 �3 10 –50 �+79
12 �6 �3 10 –48 �+75
13 �6 �3 11 –47 �+71
14 �7 �4 12 –45 �+68
15 �8 �4 12 –44 �+65
16 �8 �5 13 –43 �+62
17 �8 �5 14 –42 �+60
18 �9 �5 14 –41 �+58
19 10 �6 15 –40 �+56
20 10 �6 15 –39 �+54
21 10 �6 16 –38 �+53
22 11 �7 17 –37 �+51
23 12 �7 17 –36 �+50
24 12 �8 18 –36 �+49
25 12 �8 18 –35 �+48
26 13 �8 19 –35 �+47
27 14 �9 20 –34 �+46
28 14 �9 20 –34 �+45
29 14 �9 21 –33 �+44
30 15 10 21 –32 �+43
a Counted on two Petri dishes. a Counted on two Petri dishes. b Compared to the microorganism count (column 2).
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(normative) MPN tables (normative) MPN tables MPN table for 3 ° 1 g (ml), 3 ° 0,1 g (ml) and 3 ° 0,01 g (ml) MPN table for 3 ° 1 g (ml), 3 ° 0,1 g (ml) and 3 ° 0,01 g (ml)
Number of positive results Number of positive results MPN MPN Category when the number of tests is Category when the number of tests is Confidence limits Confidence limits
NOTE�The confidence limits given in Table B.1 are only intended to provide some idea about the in… NOTE�The confidence limits given in
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Explanation of the results Explanation of the results
Category Category Definition Definition
1 When the number of microorganisms in the product is equal to the MPN found, the result is one of …
2 When the number of microorganisms in the product is equal to the MPN found, the result is one of …
3 When the number of microorganisms in the product is equal to the MPN found, the result is one of …
0 When the number of microorganisms in the product is equal to the MPN found, the result is one of … Before starting testing, it shall be decided which category will be acceptable, i.e. only 1, 1 an…
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(informative) Bibliography (informative) Bibliography 1 Australian Standard AS 1766, 2 Australian Standard AS 2243, 3 Buttiaux, R., Beerens, H., and Tacquet, A. 4 5 Cruickshank et al. 6 Kenya Standard KS 05-176-1978, 7 Harrewijin, G.A. and Hartog, B.J. Guidelines to perform microbiological analyses of food and fo… 8 9 Jarvis, B. Statistical aspects of the microbiological analysis of foods. In: 10 Johnson, N.J and Kotz, S. 11 12 Harrigan W.F. and McCance M.E. 13 14 15 16 17 18 19 20 21 De Man, J.C. MPN tables (corrected).
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BS 5763-0:1996� ISO 7218:1996 BS 5763-0:1996� ISO 7218:1996 BSI BSI 389 Chiswick High Road London W4 4AL BSI�—�British�Standards�Institution BSI�—�British�Standards�Institution BSI is the independent national body responsible for preparing British�Standards. It presents the… Revisions British�Standards are updated by amendment or revision. Users of British�Standards should make su… It is the constant aim of BSI to improve the quality of our products and services. We would be gr… BSI offers members an individual updating service called PLUS which ensures that subscribers auto… Buying standards Orders for all BSI, international and foreign standards publications should be addressed to Custo… In response to orders for international standards, it is BSI policy to supply the BSI implementat… Information on standards BSI provides a wide range of information on national, European and international standards throug… Subscribing members of BSI are kept up to date with standards developments and receive substantia… Information regarding online access to British Standards via British Standards Online can be foun… Further information about BSI is available on the BSI website at Copyright Copyright subsists in all BSI publications. BSI also holds the copyright, in the UK, of the publi… This does not preclude the free use, in the course of implementing the standard, of necessary det… Details and advice can be obtained from the Copyright & Licensing Manager. Tel:�+44�(0)20�8996�70…
