12 Quality Control In broad terms, the quality control staff, under the supervision of a qualified food chemist, should be responsible for the following: (1) The evaluation of quality of all incoming raw material, supplementary ingredients used in processing, chemicals, packaging material, cellulose and other film, paper and fibreboard containers, and certain consumable stores: (2) All routine laboratory tests on raw material, finished products, lye and blancher liquor, boiler and processing water; (3) Sampling procedures; (4) Recording processing data, ie, drying temperatures, peeling and blanching procedures, cooking records for precooked products, such as meats, etc; (5) Research and development work on new products, packaging and processing methods; (6) Logging of complaints from consumers, and pursuing these to origin and eliminating the cause; (7) Supervision of methods of plant cleaning and sanitation, and establishing firm principles of hygiene for personnel and plant maintenance; (8) Arrangements for and supervision of pest and rodent control by contractors primarily concerned in these duties; (9) Liaison at all times with production management. 233 LABORATORY ANALYTICAL TESTS Having received samples representative of a batch of finished product, or incoming raw material, the staff will carry out the following tests: Moisture For moisture tests, either a vacuum oven, set at 70°C, or a fan assisted air oven, set at 10Oo-103"C, is used. The former is more accurate, albeit slower, on account of the low temperature used, as this prevents the charring and burning of the sample when it is completely dried out, and allows a more exact result to be calculated. Samples of raw vegetable should be grated finely before testing, and dry products should be ground in a small coffee grinder. The sample material is dried down to zero moisture, and the loss in weight expressed as a percentage of the original to the nearest 0.1 percent. Two hours is the average time for drying down most dehydrated vegetables, from 8 percent to zero moisture; raw vegetable samples will take 16 hr. These times apply when using an air oven. A more rapid calculation of moisture in dry products can be made by using an infrared moisture tester. The sample must be finely ground so that it passes through a 10 mesh sieve; a given weight is dried out under an infra red lamp, the loss in weight being recorded by a scale on the tester. Results can be obtained in up to 7min, and in as short a period as 4min for some vegetables. These moisture testers, however, are not as accurate as the oven-type, and results can vary about 0.5 percent when compared with the latter. It is, nevertheless, an indispensable instrument for the shift chargehand to use for rapid tests on material in course of process, particularly for testing products drying in finishing bins, where it is important for the operator to know the drying end-point accurately, before discharging the material to the packing department. Sulphur Dioxide Tests for SO, concentration are carried out by the Monier Williams method, or what was described in the early days of dehydration as the 'Committee' method. The former method is rather more accurate but the latter is more generally used with vegetables, for the sake of speed. Basically, the calculation is made by boiling a 20g sample of the ground product in dilute hydrochloric acid and titrating iodine into the distillate as follows: 250ml of distilled water are placed in a 500ml flask connected by a splash-head to a vertical condenser terminating in a bubbler feeding into a 600ml beaker. 20g of the dried product for analysis are put into the distilled water in 234 the flask, with 10ml of concentrated hydrochloric acid. Heat is applied to bring about boiling in 2-3min. The distillate is collected in 200ml of water in the beaker at the bottom of the condenser, and N/100 iodine is titrated from a burette to maintain a slight excess of iodine. A little starch solution indicator is added to the beaker. A marked reduction in the rate of evolution of iodine combining substances is taken as the end-point, or when more than one minute is required to decolorise 0.2ml N/100 iodine. The distillation should be complete in 5-l0min. Calculations: lml N/100 iodine titrated = 0.32mgm sulphur dioxide. Peroxidase Test for Vegetables During blanching, the enzymes present in active form become progressively inactivated by heat. Of these enzymes, the peroxidase complex is readily perceived because of the colour reaction it promotes with a reagent. A suitable reagent is a solution of Guaiacol and hydrogen peroxide made as follows: (1) 1 per cent w/v Guaiacol dissolved in distilled water: (2) Mix 25ml of 20 volume hydrogen peroxide in 75ml of water, and add 100ml of 1 per cent Guaiacol solution. The sample for analysis is liberally wetted by the above solution, and the development of a brown colour indicates active, or positive, peroxidase. If the colour is not apparent in one minute, the result is negative, and it can be assumed that blanching is adequate. If the result is positive, it is necessary to adjust the blanching conditions, either by increase of temperature or product immersion time. Blemish Count A limited number of minor blemishes is often permitted, commercially, in dehydrated vegetables but the tolerance must be rigorously controlled. Blemish may arise from disease in the fresh vegetable, skin, root or growth defect, or can stem from a processing fault, such as scorching, under or overblanching, or inefficient peeling. Obvious blemish should not be present, and the only tolerated blemish is what is described as ‘minor’, ie, slight blemish in the dry state, which mainly disappears on reconstitution. The standard will obviously vary from processor to processor but, on average, the permitted tolerance is from 5-7 minor blemishes in a 50g sample of dehydrated vegetable. A similar standard applies to meats. 235 Reconstitution Ratio The dried product must be regularly checked for the reconstitution value, in order that correct cooking instructions can be supplied to the ultimate user. A 50g sample is rehydrated and cooked in the prescribed manner and time. The cooking water is drained off, and the drained weight is calculated against the original dry weight, to give a reconstitution ratio. Culinary Report This test is combined with the previous ratio test, and a system of quality marking is implemented and recorded for flavour, texture and colour. The criterion of quality, overall, is that of the freshly cooked fresh vegetable. Bacteriological Tests Dried vegetables must not contain bacteria likely to be harmful to man, and the conditions of manufacture must be such that bacterial toxins cannot be formed. General bacterial counts must be low, and it is the microbiologist’s duty to ensure that factory and personnel hygiene are of a particularly high standard. The use of bactericidal hand creams by operators who handle the raw material - particularly meat and poultry products - is a very essential precaution. Bacteriological equipment required for elementary tests will comprise two incubators (maintained at 37” and 55”C), a good microscope and an adequate supply of Petri dishes for culturing. Meat Product Tests In addition to bacteriological tests, for meat products, there are special tests required to establish free fatty acids, fat content, peroxide value and salt content. Potato Product Tests In addition to the specific tests noted above, for vegetables, tests are made for reducing sugars in raw potatoes and in the dried product in its various forms, and also ‘blue value’ tests to evaluate free starch. Residual Oxygen Test When products are gas packed, tests are made to establish the percentage of residual oxygen in the pack. An Orsat apparatus is required for this, and the commercially accepted standard for residual oxygen in a nitrogen-flushed pack is 2 percent or less. 236 Periodical Tests It is necessary that checks should be made at regular intervals for the presence of arsenic, lead and iron in dehydrated products. Metal abrasion is often a source of contamination in a product. This can arise from various items of plant and equipment, hence the necessity for using stainless steel in food handling machinery as far as possible, particularly at the 'wet' end of the process. Galvanized iron is sometimes resorted to for equipment in contact with the dry material, mainly on grounds of cost, but this should be kept to a minimum, in view of the contamination risk. Arsenic contamination can arise from flue gases, where a direct system of heat is used, but, if proper attention is paid to the use of correct fuels, and the hot air supply is kept clean, this should not give rise to serious problems. SPECIFICATIONS Finally, a set of specifications must be established by the technical staff, to cover every product to be handled. It is the duty of the laboratory staff to acquaint production personnel with these specifications, and then evaluate the production samples for compliance. Obviously, it is of paramount importance that there is no undue delay in carrying out quality control tests, and it is vital that these run continuously and concurrently with the 24 hour production schedule. Failure to maintain a round-the-clock check can mean considerable quantities of material being turned down as substandard because they do not meet the specification. This can be very costly to the dehydrator, as a salvage operation is not always easy or practicable. Liaison between quality control and production is, therefore, of paramount importance and this cannot be overstressed. Bacteriological specifications may well vary, according to the demands of the customer, but the following specification would be generally accepted as the norm. Meat Products: Total Count 5000 per g. Escherichia coli - absent from 0.lg. Salmonella species - absent in 25g Total Count - 50,000 per g. Maximum Yeast and mould count: 200 per g. Bacillus coli - absent from 0.lg. Salmonella species: absent in 25g Vegetables: The complete specification, therefore, will appear under the following headings, against which the Laboratory will report on every batch of material: 237 SPECIFICATION Culinary- Colour: Flavour: Texture: Blemish per 50g: S02ppm- Target: Maximum: Minimum: Maximum: Minimum: Moisture YO- Target: Peroxidase: Screening: Fat YO (Meats) Peroxide Value of extracted fat (Meats): Free Fatty Acids (Meats): Bacteria per g: Total Count: Escherichia coli: Bacillus Coli-Salmonella species Moulds/Yeasts per g: Arsenic ppm: Lead ppm: Iron ppm: Packing - Residual Oxygen YO: Some major buyers of potato granules for use in snack foods require additional tests as under as well as those listed above: (1) Level of Glycerol monostearate (2) Tetrasodium pyrophosphate (3) Anti-oxidants (BHA and/or BHT) Maximum 1%-typically 0.6% Maximum 0.5Y0-typically 0.3% Maximum 25mg/kg-typically 15-20mg/ kg 0.9 - 1.0 g./cc (uncompacted) Brabender Amylograph units (4) Reducing sugar 2.0% (5) Bulk density (6) Rehydration Characteristics 550-1000 Packaging Specification for potato granules intended for use in value added products. 238 Packaging (Factory) All product packed in drums under nitrogen pending call- off. Drums: Gas tight construction with septum allowing head space analysis. Packed under a nitrogen atmosphere containing a maximum of 2% oxygen. Drum to be internally lacquered (Shipping) Sacks: 25kg per sack effectively sealed to prevent spillage. Shelf-life: Drums 12 months. Sacks 4 months. Holding temperature: Maximum 7°C. Laboratory methods for determining moisture, rehydration viscosity, free fatty acids, and reducing sugars are appended in Tables 12.1,12.2,12.3 and 12.4 and are extracted from buyers’ specifications for bought-in material. Other tests are already outlined in this chapter. 239 METHODS OF ANALYSIS MOISTURE IN POTATO GRANULES BY VACUUM OVEN I Apparatus 1. 2. Vacuum oven. 3. 4. 5. 6. Balance accurate to f 0.001g. Vacuum pump capable of maintaining vacuum equivalent to 25mm Hg or less. Aluminium moisture dishes, diameter 2-1 /2in., height 5/8in., with slip-over cover. Gas drying bottle containing concentrated H,SO,. Desiccator containing drying agent. (Silica Gel self-indicating). I1 Method 1. Weigh approximately 2g of potato granules into an aluminium dish previously dried at 98"-100"C to constant weight, and cooled to room temperature in a desiccator. Duplicate test. Place dishes with cocked lids (do not remove lids) in vacuum oven for 6hr at 70°C under vacuum equivalent to 25mm of mercury. When the correct vacuum pressure is obtained, the time interval of 6hr is then begun. At this time, air at the rate of 2 bubbles per second is allowed to enter the vacuum oven through a sulphuric acid drying bottle. 25mm of mercury means that the internal pressure in the vacuum oven will be 5mm of mercury above perfect vacuum. Remove the dishes after tightening their covers. Transfer to the desiccator, and allow to cool to ambient temperature (approximately 10-15min) before weighing. 2. 3. 4. Calculate the moisture. I11 Calculations Loss in weight x 100 sample weight 1. % Moisture = 2. Duplicate samples should be within 5 0.1% moisture - *^ EVALUATION OF POTATO GRANULE REHYDRATION CHARACTERISTICS USING THE BRABENDER VISCOGRAPH (VARIABLE SPEED) (COLD METHOD) I Apparatus 1. Brabender Viscograph, A. Set at 75rpm B. Fitted with a 2000cm per g sensitivity cartridge. 2. 500ml graduated cylinder. 3. Balance capable of weighing f 0.lgram. I1 Calibration and Checkout Procedures 1. Once per day the stirrer and the bowl should be checked with a gauge. A. Insert gauge into bowl. The pins should be so aligned as to go through the holes in the gauge. Note: Mis-alignment is noted by a clicking sound. B. The pins on the stirrer should also be aligned with the gauge. 2. If the sensitivity is changed, zero the instrument as follows: A. Place 500ml cold distilled water into bowl. B. Turn on the machine as noted in 111.1 and 111.7. C. Loosen sensitivity cartridge lock nuts and turn the head until the pen reads zero without using any added weights. D. Tighten lock nuts while holding the sensitivity cartridge so that the pen is on zero. E. Run for approximately 5min to ensure the pen is on zero. 111 Procedure 1. Turn water and power on. 2. Set thermoregulator transport in centre position. (Leave in this position). 3. Put the toggle switch under coding heading into the (Fast Uncontrolled) position. 4. Check to make sure pen is working and set at zero. 5. Place the cleaned mixing bowl into position and add 400ml of distilled water at 4°C. 6. Add lOOg of potato granules and stir vigorously to wet all granules. 7. Within 30sec lower stirrer and lock pin in place. Then press red button to start. 8. Timer should be set for 10min. 9. Lower cooling probe. 10. The answer is reported by taking the chart reading after l0min. 24 I DETERMINATION OF CHLOROFORM EXTRACTABLE FATTY SUBSTANCES IN POTATO GRANULES Apparatus Whatman Extraction Thimble 30mm by 100mm (41 by 123mm) Analytical Balance Cotton Wool Quickfit Round Bottom Flask 250ml. (500ml). Soxhlet Extractor Soxhlet Condenser Isomantle Drying Oven 100°C Desiccator & Desiccant Reagents Petroleum Spirit (40" - 60°C). Method 1. Weigh accurately, approximately 20g (50g) sample into an extraction thimble 30 by lOOmm (41 by 123mm). 2. Plug with wad of cotton wool (fat-free). 3. Weigh accurately a clean dry 250ml(550ml) Round Bottom Flask. 4. Pour 150ml (300ml) Petroleum Spirit into the flask. 5. Place the thimble containing the sample into a Soxhlet extractor. 6. Connect the Soxhlet extractor to the flask. and a condenser. 7. Place in an Isomantle. 8. Extract for at least 4hr. 9. Distil off Petroleum Spirit. 10. Place the flask in the drying oven for lhr. 11. Remove and place in the desiccator to cool. 12. Reweigh the flask. Calculation Increase in weight of flask x 100 Weight of Sample % = Extractable Fat = 242 REDUCING SUGARS IN POTATO GRANULES USING DINITROSALICYLIC ACID REAGENT Purpose To enable reducing sugar contents of potato granules to be determined. Modifications in weights and dilutions allow its application to other food products: Principle Sugar is extracted from the sample using cold water (thus minimising interference from protein and fat). A filtered aliquot is then heated with dinitrosalicylic acid reagent. A coupling reaction occurs and produces an orange red colour, which can be measured at a wavelength of 580mm using a simple filter colorimeter. Apparatus Boiling water bath Test tubes approx 25 by 150mm 5ml Pipettes Measuring cylinder Magnetic stirrer Colorimeter with appropriate filters Beakers Graduated flasks Funnels Filter papers Test tubes Balance Reagents Dinitrosalicylic Acid Reagent lg of Dinitrosalicylic acid is dissolved in 20ml of Sodium Hydroxide solution (1.6g pellets in 20ml water). Reaction is complete when the dinitrosalicylic acid has all changed colour. Then add 50ml water to 20g Potassium Sodium (+) Tartrate. Dissolve by gentle warming if necessary then dilute to 100ml with water. Dilute DNSA Reagent Dilute 2 volumes of DNSA reagent with 3 volumes of water. Standard Glucose Solution Prepare fresh standard solutions containing lOOmgm of dried glucose per 100cm of water. This solution contains lmgm Glucose per ml. 243 Method (1) Weigh out 5g (approx.) of potato granules into a clean 400ml beaker. Record the weight of sample. (2) Add the sample to 250ml f 2mls of distilled water in a 400ml beaker. (3) Stir on a magnetic stirrer or a minimum of 15min. (4) Remove beaker from stirrer and allow contents to settle. (5) Decant the upper layer through a No.2 filter paper pouring back the first 10-20ml of filtrate into the filter. If filtrate is not clear pour back until a clear filtrate is obtained. (6) Pipette 5ml of filtrate into a boiling tube. (7) Add 5ml of dilute dinitrosalicylic acid solution. (8) To a second tube add 5ml distilled water and 5ml dilute dinitrosalicylic acid solution. This is the blank solution. (9) Place the tubes in a vigorously boiling water bath for lOmin, making sure the level of the contents in the tube is at least one inch below the surface of the boiling water. (10) Remove the test tubes and cool in cold water. (11) Transfer the cool blank solution to a clean cuvette and using a 580 filter, set (12) Replace the blank solution with the sample solution and read the optical (13) From a standard curve calculate the sugar content of the potato granules. Preparation of standard Curve Into tube (1)pipette 5ml water 2)pipette (3)pipette (4)pipette (5)pipette the colorimeter to zero. density (OD) on the absorbance scale. 4.5ml water and 0.5ml glucose solution 4.0ml water and 1.0ml glucose solution 3.0ml water and 2.0ml glucose solution 2.0ml water and 3.0ml glucose solution Draw a graph of OD against concentration of glucose solution. 244 METHODS FOR MICROBIOLOGICAL ASSESSMENTS Specifications require the following to be determined: Total Viable Count @ 30°C 100,000 per gr. ma (500,000 for onions) E. Coli 10 per gr. max. Coliforms 100 per gr. max. Yeast and Moulds 1000 per gr. max. B. Cereus 100 per gr. max. St. Aureus 100 per gr. max. Salmonella Absent in 25 gr. Cl. Perfringens 100 per gr. (not always) It is customary to use ready prepared selective Agars, and ’Oxide’ has been suggested throughout. The organisms under examination will have dis- tinctive characteristics and for routine work this should prove satisfactory. Over recent years a piece of equipment called a ‘Stomacher’ has been developed and its use has been widespread for resuscitation of bacteria. How- ever it relies on the use of pre-sterilized plastic bags which cannot be recycled. As these may not always be available overseas, the traditional method of preparation is submitted. It must be stressed that all glassware and media must be sterilized un- less stated otherwise in the method. Media to be autoclaved at 121°C (15 p.s.i.) for 15 minutes, and glassware (pipettes and petri dishes) to be dried at 121°C for a minimum of 2 hours. Disposable pre-sterilized petri dishes are available in the U.K normally. This reduces the likelihood of cross-contamination but cost and availability may preclude their use in some overseas locations. Thought will have to be given to sampling frequency - say, every 2 to 4 hours or on a weight basis, say, every 250 to 500kg batch, according to the overall production. Do not ‘bulk’ samples and, say, carry out daily or weekly assessments, since rogue high results may put the product out of specification and give a totally distorted view. More and more emphasis is now being placed on microbiology and regular structured testing will give valuable pointers as to the cleanliness of plant and soundness of raw material. Outbreaks of food contamination in recent years have highlighted the importance of microbiology in the factory. Separate methods for all the above assessments follow. 245 SAMPLING AND SAMPLE PREPARATION Reagents Peptone Water Powder (CM9 - Oxoid) 0.1% Peptone Water 180ml in 200 ml medical flat bottles 0.1% Peptone Water 9ml in Universal media bottles Reagent Preparation Dissolve lgm of peptone water (CM9) in 1 litre distilled water and dispense either 180ml or 9ml per bottle as given above. Autoclave for 15 minutes at 15psi. Sampling Samples should be taken using clean equipment and cross contamination must be avoided. This is most easily effected by using a polythene bag which is inverted over the hand, the product is grasped by the ‘gloved’ hand and enclosed by re-inverting the bag. Sample Preparation Place part of sample into grinder and mill, reject, repeat operation twice, the third part is retained. Transfer 20gm into 180ml of sterilized peptone water using a flamed spoon/spatula, mix well, allow to stand for at least 20 minutes to resuscitate the bacteria. A 10% homogenate has been produced i.e. lml contains 0.lgm. Further dilutions are obtained by taking lml and adding to 9ml sterilized peptone water as shown below: 20gm + 180ml= 10% - lmllO% + 9ml= 1% lml1% + 9ml= 0.1% - lml 0.1% + 9ml= 0.01% - lml contains O.lgm or x 10 lml contains 0.01 gm or x 100 lml contains 0.001gm or x 1,000 1ml contains 0.0001gm or x io4 and so as may be required NOTE (1) 10% homogenates can be obtained by using lOgm + 90ml,25gm + 225gm (2) When moving from dilution to dilution a fresh clean sterilized pipette or 5gm + 45ml if desired. must be used. 246 TOTAL VIABLE COUNT (TVC) Media Plate Count Agar (CM463B - Oxoid) Peptone Water (CM9 - Oxoid) Media Preparation Add 23.5gm plate count agar to 1 litre of distilled water, dispense 9ml into universal bottles and sterilize for 15 minutes at 15psi. When cooled the agar will solidify and can be stored in this state for several months. For use the agar should be melted in a boiling waterbath and allowed to COO^ to 45-50°C. Method Take the 0.1% dilution of the sample as prepared under sample preparation and pipette 1.0ml via a sterile petri dish, add 9mls of cooled melted agar and mix by gently swirling the petri dish (or plate). Allow the mixture to set, place upside down in the incubator at 30°C and leave for 2 days. After this time the number of colonies are counted and multiplied by 1,000 to give a count per gm. If counts in excess of 300,000 per gm i.e. 300 per plate are expected then the 0.01% dilution should be used. The dilution should be increased so that the count does not exceed 300 per plate. Initially it may be necessary to carry out several dilutions in order to ascertain the range of results. E. (ESCHERICHIA) COLI Media Media Preparation Dissolve 51.5gm of agar in 1 litre distilled water, by bringing to the boil, dispense 10mls into universal bottles, sterilize for 15 minutes at 15psi. When cooled this media will set, prior to use melt in a boiling water bath and allow to cool to 45-50°C. Method Pipette lml of the 10% homogenate onto a sterile petri dish, add lOml of cooled, melted agar, swirl to mix and allow to set. Incubate upside down at 44°C for 24 hours. Count only the red/purple colonies and multiply by 10 to give the number of colonies per gm. MacConkey No 3 (Cm115 - Oxoid) 247 TOTAL COLIFORM BACTERIA Media Media Preparation Dissolve 38.5gm in 1 litre of distilled water by bringing to the boil. Allow to cool to 45-50°C. The media can be held at this temperature for a maximum of 3 hours. Violet Red Bile Agar (Cm107 - Oxoid) Do not autoclave this material. Method Pipette lml of the 10% homogenate onto a sterile petri dish, add lOmls of freshly prepared cooled agar, swirl to mix and allow to set. Incubate upside down at 37°C for 48 hours. After incubation count the red colonies and multiply by 10 to give count per gm. YEASTS AND MOULDS Media Rose Bengal Agar (CM49 - Oxoid) Chloramphenicol Supplement (SRT8 - Oxoid) Media Preparation Dissolve 16gm of agar in 500ml of distilled water by bringing to the boil. Sterilize at 121°C for 15 minutes and allow to cook to 50°C. Add the contents of 1 vial of supplement which has been dissolved in 2ml of acetone. Pour 10ml of agar onto sterile plates and store at 5°C. Method Pipette 0.2ml of the homogenate onto a prepared plate and spread over the surface of the agar using a sterile glass spreader. The spreader can be sterilized by flaming between use. The plate should be incubated upside down at 22°C -t 2°C for 4-5 days. Inspect the plate daily after 3 days as vigorous mould growth may make counting difficult. Count moulds (rough, hairy, fungal appearance) multiply by 50 to give number of moulds per gm. Count yeasts (smooth surfaced colonies) and similarly multiply by 50. 24% BACILLUS CEREUS Media Bacillus Cereus Agar-(CM617 - Oxoid) B.C. Supplement-(SR99 - Oxoid) Egg Yolk Emulsion-(SR47 - Oxoid) Media Preparation Dissolve 20.5gm of agar in 475m1 distilled water by bringing to the boil. Sterilize at 121°C for 15 minutes. Cool to 50°C and asceptically add the contents of 1 vial of B. Cereus supplement which has been rehydrated with 2mls of sterile distilled water and 25ml of Egg Yolk Emulsion. Mix well and pour 100mls onto sterile petri dishes. Store plates at 4". Method Pipette O.lml of 10% homogenate onto a prepared place and spread using a sterile spreader. Incubate at 37°C for 24 hours. Examine plates for peacock blue colonies surrounded by egg precipitate of the same colour. Leave for a further 24 hours at room temperature (16°C) for all colonies to develop. Count colonies and multiply by 100 to give count per gm. STAPHYLOCOCCUS AUREUS Media Baird-Parker Medium (CM275 - Oxoid) Egg Yolk-Tellurite Emulsion (SR54 - Oxoid) Media Preparation Dissolve 63gm of Baird-Parker medium in 1 litre of distilled water, sterilize for 15 minutes at 121°C. Allow to cool to 50°C and asceptically add 50ml of Egg- tellurite emulsion, mix well. Pour approximately l.Omls onto sterilized petri dishes and allow to set, store prepared plates at 4°C. Method Pipette 0.1 ml of the 10% homogenate onto a prepared plate and spread over the surface using a glass spreader. Incubate upside down at 37°C for 24 hours. Count black shiny convex 1.0-10.5mm diameters st. Aureus colonies, If no colonies appear incubate for a further 24 hours. multiply by 100 to give number per gm. 249 SALMONELLA Media Brilliant Green Agar (CM 263 Oxoid) SS Agar (CM 99 Oxoid) Selenite Broth (CM 395 Oxoid) Sodium Biselenite (LIZ1 Oxoid) Media Preparation (a) Brilliant Green Agar: Dissolve 50gm of agar in 1 litre of distilled water by bringing to the boil, sterilize at 121°C for 15 minutes. (b) SS Agar: Dissolve 63gm of agar in 1 litre of distilled water by bringing to the boil. DO NOT STERILIZE. Allow to cool to 50°C and pour into sterile petri dishes. (c) Selenite Broth: Dissolve 4gm of sodium biselenite in 1 litre of distilled water, add 19gm of selenite broth powder, dissolve, fill into containers to 5cm and sterilize for 10 minutes in a boiling water bath. Method Prepare a 10% homogenate of 25gm of sample in 225m1 of peptone water and incubate at 37°C for 24 hours. Take lOmls of the homogenate after incubation and add to Selenite Broth, incubate for 48 hours at 42°C. Streak plates containing Brilliant Green Agar and SS Agar after 24 and 48 hours ex Selenite Broth. Incubate plates at 37°C for 24 hours. Salmonella will appear as pink colonies on BG agar and transparent colonies with black centres on SS agar. 250 CLOSTRIDIUM PERFRINGENS Media Perfringens Agar (O.P.S.P.)-(CM543 - Oxoid) Supplement A (SR76 - Oxoid) Supplement B (SR77 - Oxoid) Media Preparation Dissolve 22.8gm of agar in 500mls of distilled water by bringing to the boil. Sterilize for 15 minutes at 121"C, allow to cool to 50°C and asceptically add the contents of one vial each of supplements A and B which have been rehydrated by the addition of 2ml distilled water. Mix well and pour into sterile plates. Method Pipette lml of the 10% homogenate onto a prepared plate and spread with a sterile spread. Further dilutions may be used if the levels are high. Incubate anaerobically for 18-24 hours at 37°C (a suitable gas jar and gas generating kits are available from Oxoid - HPOllA, BRO38B, BRO42A and BRO55A). Count large black colonies and multiply x 10 to give a count per gm. 25 I