Tài liệu Practical Food Microbiology 3rd Edition - Part 9 pptx

Live bivalve molluscs and other shellfish Council Directive 91/492/EEC [1] sets out the designation of production areas of bivalve molluscs based on levels of Escherichia coli or faecal coliforms/100 g of flesh and intravalvular liquid. A three-dilution, five-tube most probable number (MPN) method is specified for testing without precise details, but other bacteri- ological methods of equivalent accuracy are permitted. In the UK testing for E. coli is performed because it is considered to be a more specific indicator of faecal pollution than faecal coliforms. The microbiological criteria for the classification of shellfish harvesting areas are shown in Table 9.1. In addition to the classification criteria, diarrhoetic shellfish poison (DSP) must be absent from the shellfish flesh and levels of paralytic shellfish poison (PSP) must be below 80mg/100 g of flesh. If these levels are exceeded fishing is prohibited in that harvesting area until compliance is achieved. Since the publication of the Directive another shellfish poison known as amnesic shellfish poison (ASP) has been identified; levels of ASP should be below 20mg/g of flesh [2]. 9 Live bivalve molluscs and other shellfish 229 Testing for DSP, PSP and ASP is normally performed by reference laboratories. Routine monitoring of shellfish harvesting areas in the UK for marine biotoxins is a statutory responsibility of the Food Standards Agency who can advise on the specialist laboratories currently contracted to undertake this task. In England the reference facility for outbreak related samples is the Food Safety Microbiology Laboratory, Central Public Health Laboratory. Tel: 020 82004400, ext. 3521/4113. The UK National Reference Laboratory for biotoxins is the Fisheries Research Services (FRS) Marine Laboratory, Aberdeen AB11 9DB. Tel: 01224 876544. An end-product standard is defined for shellfish intended for immediate human consumption. For faecal coliforms and E. coli this is given as category A in Table 9.1. In addition, the shellfish should meet the standards defined above for biotoxins and Salmonella should be absent in 25 g of shellfish flesh. The Di- rective recognized the absence of routine virus testing procedures and this is still true today. However consumption of molluscs containing viruses, in particular Norwalk-like virus (NLV; also known as small round structured virus or SRSV) is the most common cause of illness from this type of food. At present, methods for the direct detection of viral pathogens (NLV and hepatitis A virus or HAV) in shellfish are all based on the polymerase chain reaction (PCR). However, processing of shellfish extracts to recover low levels of contaminating virus and to remove PCR inhibitors is difficult. Currently these methods are complex, poorly standardized and restricted to specialist facilities [3]. The relationship between the levels of E. coli and the presence of virus particles in depurated shell- fish is poor, but studies have shown a much better relationship between the levels of certain types of phage, in particular F-specific RNA bacteriophage [4], and the risk of viral contamination. Phage detection methods are much simpler to perform than virus detection methods and might be incorporated more easily into routine examination of live bivalve molluscs. 230 Section nine Table 9.1 Classification of harvesting areas. Category Escherichia coli/100 g Faecal coliforms/100 g Interpretation A <230 <300 May go for direct consumption B 90% of samples not 90% of samples not Must be depurated or relayed to to exceed 4600 to exceed 6000 meet Category A (may also be heat treated by approved method) C Must not exceed Must not exceed Must be relayed for long period 46 000* 60 000 (>2 months) to meet Category A or B (may also be heat treated by approved method) D >46 000* >60 000 Prohibited (may also be prohibited on health grounds rather than monitoring results) *Figures not included in EEC regulations. Testing for viral contamination (NLV and HAV) is also currently performed by reference facilities. The Enteric Virus Unit at the Central Public Health Laboratory, Tel. 0208 200 4400, and other peripheral Public Health Laboratory Service (PHLS) laboratories can advise on analysis of clinical samples associated with shellfish outbreaks. The UK National Reference Laboratory for bacteriological and viral contamination of shellfish is the Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth DT4 8UB. Tel: 01305 206600. Method 1 Multiple tube method for Escherichia coli This method is the standard procedure used in the UK [5]. Minerals modified gluta- mate broth is used for the first stage of the test based on the detection of acid produc- tion followed by detection of b-glucuronidase activity at 44°C using a chromogenic agar for confirmation of the presence of Escherichia coli. continued Live bivalve molluscs and other shellfish 231 A pooled sample of at least six shellfish are required for testing to overcome the variability associated with individual shellfish. Additional shellfish should be sub- mitted by the sampling authority to allow for rejections. All samples should be stored dry at 4°C and examined preferably within 6 h of collection but no later than 24 h after collection. Samples should not be frozen. Sample size Oysters/clams 10–15 Mussels 15–30 Cockles 30–50. Equipment Stomacher (optional) Rotary blender (optional) Shucking knives Balance with resolution 0.1 g or greater Incubators at 37 ±1°C and 44±1°C. Reagents 0.1% peptone solution (in water), pH 7.2± 0.2 0.1% peptone/0.85% sodium chloride solution Minerals modified glutamate medium, double and single strength 5-bromo-4-chloro-3-indolyl b- D-glucuronide (BCIG) agar. Tryptone bile agar contain- ing 144 mmol/L 5-bromo-4-chloro-3-indolyl-b-D glucuronic acid (e.g. 0.075 g/L of cyclohexylammonium salt). Control cultures NCTC 9001 Escherichia coli Positive NCTC 13216 Escherichia coli b-glucuronidase weak positive NCTC 9528 Klebsiella aerogenes b-glucuronidase negative Sample preparation (a) Select at least 10 oysters and clams, 15 mussels or 30 cockles. Discard any gaping shellfish and those with obvious signs of damage. (b) Clean the molluscs by scraping, scrubbing and washing under cold running water of potable quality and allow to drain on clean paper towels. (c) Open the molluscs with a flamed and cooled shucking knife, as follows: Oysters/clams Insert the knife between the two shells towards the hinge end of the shellfish, push further into the shellfish and prise open the upper shell. Allow any liquor to drain into a sterile weighed bag or beaker. Push the blade through the shellfish and sever the muscle attachments by slicing across. Remove the upper shell and scrape the contents of the lower shell into the sterile bag or beaker. Repeat for at least 10 oysters/clams to obtain the required weight and add to the same bag or beaker. continued 232 Section nine Mussels/cockles Insert the knife between the shells through the byssal opening of the shellfish and separate the shells by twisting the knife. Collect any liquor in a weighed sterile bag or beaker. Cut the muscle between the two shells and scrape the contents into the sterile bag or beaker. Repeat for a minimum of 15 mussels or 30 cockles to obtain the required weight, adding the contents to the same bag or beaker. Preparation of homogenate Using stomacher (d) Place the bag containing the shellfish meat and liquor inside two more bags to prevent puncture from shell. (e) Place the bag in the stomacher and operate the machine for 2–3 min. (f) Transfer 50 g of the homogenate to another stomacher bag and add approxi- mately 100 mL from a measured 450mL volume of 0.1% peptone solution. (g) Place the bag in the stomacher and operate the machine for 2–3 min. Add the re- mainder of the 0.1% peptone solution and mix well. This gives the 10 -1 dilution. or: Using blender (d) Weigh the shellfish flesh and liquor and add two parts by mass of 0.1% peptone solution. (e) Homogenize mixture in a rotary blender for sufficient time to achieve 15 000– 20 000 revolutions. The duration should not exceed 2.5min. (f) Stand for 30s. (g) Swirl briefly, then transfer 30 mL of the homogenate to a measured 70 mL of 0.1% peptone solution and mix well. This gives the 10 -1 dilution. Preparation of dilutions (h) Prepare a 10 -2 dilution by transferring 1 mL of 10 -1 dilution to 9 mL of 0.1% peptone/0.85% sodium chloride solution. Further dilutions may also be required when raw molluscs are being examined for classification of shellfish harvesting areas, i.e. 10 -3 and 10 -4 . Procedure (i) Prepare 15 tubes of minerals modified glutamate medium, five containing 10 mL of double strength medium and 10 containing 10mL of single strength medium. (j) Add 10 mL of 10 -1 dilution to each of the five tubes containing double strength medium. (k) Add 1mL of 10 -1 dilution to each of five tubes of single strength medium. (l) Add 1 mL of 10 -2 dilution to each of five tubes of single strength medium. (m) Repeat step (l) with further dilutions if necessary. (n) Incubate all tubes at 37°C for 24±2h. (o) Examine all tubes for acid production, signified by a colour change to yellow. The presence of any acid, regardless of quantity, is regarded as a positive result. Absence of acid production after 24 ±2 h constitutes a negative result for E. coli. continued p. 237 Live bivalve molluscs and other shellfish 233 Table 9.2 Most probable number (MPN) of organisms [5]. Tables for multiple tube method using 5 ¥ 1g, 5 ¥0.1 g, 5¥0.01g. 1 g 0.1 g 0.01 g MPN/100 g Category A (<230 Escherichia coli) 00 0 <20 00 1 20 01 0 20 10 0 20 10 1 40 11 0 40 12 0 50 20 0 40 20 1 50 21 0 50 21 1 70 22 0 70 2 3 0 110 30 0 70 30 1 90 31 0 90 3 1 1 130 3 2 0 130 3 2 1 160 3 3 0 160 4 0 0 110 4 0 1 140 4 1 0 160 4 1 1 200 4 2 0 200 5 0 0 220 Category B (>230 E. coli, <4600 E. coli) 4 2 1 250 4 3 0 250 4 3 1 310 4 4 0 320 4 4 1 380 5 0 1 290 5 0 2 410 5 1 0 310 5 1 1 430 5 1 2 600 5 1 3 850 5 2 0 500 5 2 1 700 5 2 2 950 5 2 3 1200 5 3 0 750 Table 9.3 Most probable number (MPN) of organisms [5]. Tables for multiple tube method using 5 ¥ 0.1g, 5 ¥0.01 g, 5¥0.001g. 0.1 g 0.01 g 0.001 g MPN/100 g Category A (<230 Escherichia coli) 0 0 1 200 0 1 0 200 1 0 0 200 Category B (>230 E. coli, <4600 E. coli) 1 0 1 400 1 1 0 400 1 2 0 500 2 0 0 400 2 0 1 500 2 1 0 500 2 1 1 700 2 2 0 700 2 3 0 1100 3 0 0 700 3 0 1 900 3 1 0 900 3 1 1 1300 3 2 0 1300 234 Section nine Table 9.2 continued. 1 g 0.1 g 0.01 g MPN/100 g 5 3 1 1100 5 3 2 1400 5 3 3 1750 5 3 4 2100 5 4 0 1300 5 4 1 1700 5 4 2 2200 5 4 3 2800 5 4 4 3450 5 5 0 2400 5 5 1 3500 Category C (>4600 E. coli, <46 000 E. coli) 5 5 2 5400 5 5 3 9100 S 5 4 16 000 55 5 >18 000* *Needs further dilutions to clarify classification. Live bivalve molluscs and other shellfish 235 Table 9.3 continued. 0.1 g 0.01 g 0.001 g MPN/100 g 3 2 1 1600 3 3 0 1600 4 0 0 1100 4 0 1 1400 4 1 0 1600 4 1 1 2000 4 2 0 2000 4 2 1 2500 4 3 0 2500 4 3 1 3100 4 4 0 3200 4 4 1 3800 5 0 0 2200 5 0 1 2900 5 0 2 4100 5 1 0 3100 5 1 1 4300 Category C (>4600 E. coli, <46 000 E. coli) 5 1 2 6000 5 1 3 8500 5 2 0 5000 5 2 1 7000 5 2 2 9500 5 2 3 12 000 5 3 0 7500 5 3 1 11 000 5 3 2 14 000 5 2 3 17 500 5 3 4 21 000 5 4 0 13 000 5 4 1 17 000 5 4 2 22 000 5 4 3 28 000 5 4 4 34 500 5 5 0 24 000 5 5 1 35 000 Prohibited (>46 000 E. coli) 5 5 2 54 000 5 5 3 91 000 5 5 4 160 000 55 5 >180 000 236 Section nine Table 9.4 Most probably number (MPN) of organisms [5]. Tables for multiple tube method using 5 ¥ 0.01g, 5 ¥0.001 g, 5¥0.0001g. 0.01 g 0.001 g 0.0001 g MPN/100 g Category B (>230 Escherichia coli, <4600 E. coli) 0 0 1 2000 0 1 0 2000 1 0 0 2000 1 0 1 4000 1 1 0 4000 2 0 0 4000 Category C (>4600 E. coli, <46 000 E. coli) 1 2 0 5000 2 0 1 5000 2 1 0 5000 2 1 1 7000 2 2 0 7000 2 3 0 11 000 3 0 0 7000 3 0 1 9000 3 1 0 9000 3 1 1 13 000 3 2 0 13 000 3 2 1 16 000 3 3 0 16 000 4 0 0 11 000 4 0 1 14 000 4 1 0 16 000 4 1 1 20 000 4 2 0 20 000 4 2 1 25 000 4 3 0 25 000 4 3 1 31 000 4 4 0 32 000 4 4 1 38 000 5 0 0 22 000 5 0 1 29 000 5 0 2 41 000 5 1 0 31 000 5 1 1 43 000 Prohibited (>46 000 E. coli) 5 1 2 60 000 5 1 3 85 000 5 2 0 50 000 5 2 1 70 000 5 2 2 95 000 5 2 3 12 0000 Table 9.4 continued. 0.01 g 0.001 g 0.0001 g MPN/100 g 5 3 0 75 000 5 3 1 110 000 5 3 2 140 000 5 3 3 175 000 5 3 4 210 000 5 4 0 130 000 5 4 1 170 000 5 4 2 220 000 5 4 3 280 000 5 4 4 345 000 5 5 0 240 000 5 5 1 350 000 5 5 2 540 000 5 5 3 910 000 5 5 4 1 600000 Live bivalve molluscs and other shellfish 237 (p) Subculture each tube showing acid production to a section of BCIG agar, and streak to obtain isolated colonies. (q) Incubate the BCIG agar plates at 44°C for 20–24 h. (r) Examine the plates for the presence of blue colonies, typical of b-glucuronidase positive E. coli. (s) Consider tubes that yield growth of blue colonies on BCIG agar as positive for the presence of E. coli. Calculation (t) For each dilution, count the number of positive tubes. (u) If dilutions of 10 -3 or higher were used, select the highest dilution having five positive tubes and the next two higher dilutions. If no dilution contains five positive tubes, select the three highest dilutions amongst which at least one positive result was obtained. (v) Use the number of positive tubes at each dilution selected to determine the MPN by reference to the MPN table for the appropriate dilution range (Tables 9.2–9.4). 10 mL of the 10 -1 dilution is equivalent to 1 g of flesh, 1mL of the 10 -1 dilution is equivalent to 0.1 g of flesh, etc. Method 2 Salmonella spp. The sample should be prepared for examination as described in steps (a)–(c) in method 1. Homogenize the sample as described in steps (d)–(g) using either a stomacher or a blender, but use buffered peptone water instead of 0.1% peptone solution. Then proceed as described in Section 6.12, method 2. 238 Section nine Method 3 Phage detection F-specific RNA bacteriophages are bacterial viruses that have analogous morphology and genetic structure to human pathogenic viruses (NLV, enteroviruses and HAV) found in sewage. This, allied to their abundance in sewage and ease of enumeration, make them a good indicator of viral contamination in the marine environment. Their presence in shellfish is indicative of sewage pollution and potential contami- nation by human pathogenic viruses. They are particularly useful indicators of potential viral contamination in shellfish after treatment where traditional bacterial indicators are removed more readily than human viruses. F-specific RNA bacterio- phages are capable of infecting a specified F-pili producing bacterial host strain. Infection produces visible plaques on a confluent lawn grown under appropriate culture conditions with the infectious process being inhibited in the presence of ribonuclease (RNase) in the plating media Principle of method A culture of host strain is mixed with a small volume of molten nutrient medium. Shellfish homogenate is added and the mixture flooded on a solid nutrient agar base and allowed to set. This is then incubated at 37°C during which time the host multi- plies to produce a confluent lawn. Visible plaques form where bacteriophage is pres- ent. It is assumed that each plaque is derived from one bacteriophage. Where necessary, simultaneous examination of parallel plates with added RNase for confir- mation by differential counts is carried out. The results are expressed as the number of plaque forming units (pfu)/100 g of shellfish [6]. Sample size As for method 1. Equipment As for method 1, and in addition: Centrifuge Water bath at 45 ±2°C Spectrophotometer Colony counter Sterile glassware. Reagents 1.0% calcium-glucose solution 12.5% w/v nalidixic acid solution Tryptone yeast extract glucose broth (TYGB) Tryptone yeast extract glucose 2% agar (TYGA2) as plates Tryptone yeast extract glucose 1% agar (TYGA1) in 100 mL volumes 100% w/v RNase (store at –20°C) 0.1% peptone solution (negative control) MacConkey agar Glycerol Chloroform. continued [...]... counting Escherichia coli in live bivalve molluscs Comm Dis Public Health 199 8; 1: 188 96 6 ISO 1070 5-1 (BS 606 8-4 Part 11 199 6) Water Quality Microbiological Methods Detection and Enumeration of Bacteriophages Part 1: Enumeration of F-specific RNA Bacteriophages Geneva: International Organization for Standardization (ISO), 199 5 Live bivalve molluscs and other shellfish 241 ... European Communities Directive 97 /61/EC of 20 October 199 7 amending the annex to Directive 91 / 492 /EEC laying down the health conditions for the production and placing on the market of live bivalve molluscs Off J Eur Communities 199 7; L 295 : 35–6 Lees D Viruses and bivalve shellfish (review) Int J Food Microbiol 2000; 59: 81–116 Dore WJ, Henshilwood K, Lees DN Evaluation of F-specific RNA bacteriophage as... Shellfish containing levels of F-specific RNA bacteriophage of . single strength 5-bromo-4-chloro-3-indolyl b- D-glucuronide (BCIG) agar. Tryptone bile agar contain- ing 144 mmol/L 5-bromo-4-chloro-3-indolyl-b-D glucuronic. Health 199 8; 1: 188 96 . 6 ISO 1070 5-1 (BS 606 8-4 Part 11 199 6). Water Quality. Microbiological Methods. Detection and Enumeration of Bacteriophages. Part