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Bacteriological analytical manual pdf download.
Bacteriological analytical manual pdf download
Open the lamp diaphragm until it is just past the field of view. This may not be possible with low power objectives of some microscopes until the auxiliary lens in the condenser is correctly adjusted. If the microscope has no lamp diaphragm, place a piece of paper with a small hole cut in it over the lamp opening and make the same adjustments to focus on its inner edge.
Microscopes with mirror and external light source are not discussed here, but the principles are similar. Follow the manufacturer's instructions for adjusting the lamp filament if possible. Set the substage aperture diaphragm next. This adjustment has a crucial effect on the resolution and contrast of the image. To approximate this setting without removing an eyepiece, open the substage diaphragm fully and gradually close it while looking through the microscope until the image gains a sudden increase in sharpness and detail.
Replace the eyepiece. If the lighting is too bright, use the rheostat, if provided, to turn it down, or add neutral density or other filters. Resolution will suffer if it is stopped down closed too far or opened too much. Although stopping down gives more contrast, it impairs resolution, and spurious details are formed by diffraction lines or fringes. Repeat the procedures for Koehlor illumination with each objective used. For phase-contrast microscopy, follow the same basic steps.
Do not use the substage diaphragm but make adjustments to bring the phase annulus and annular diaphragm into coincidence. Refer to the manufacturer's instructions. Use of a green filter is recommended. Standard Methods for the Examination of Dairy Products, 16th ed.
Chapter Chapter 2. Bryce and Paul L. Poelma Revised: November Top. The conventional plate count method for examining frozen, chilled, precooked, or prepared foods, outlined below, conforms to AOAC Official Methods of Analysis, sec.
The suitable colony counting range 10 is The automated spiral plate count method for the examination of foods and cosmetics 5 , outlined below, conforms to AOAC Official Methods of Analysis, sec. For procedural details of the standard plate count, see ref.
Guidelines for calculating and reporting plate counts have been changed to conform with the anticipated changes in the 16th edition of Standard Methods for the Examination of Dairy Products 2 and the International Dairy Federation IDF.
Conventional Plate Count MethodA. Work area, level table with ample surface in room that is clean, well-lighted foot-candles at working surface and well-ventilated, and reasonably free of dust and drafts.
Storage space, free of dust and insects and adequate for protection of equipment and supplies 3. Petri dishes, glass or plastic at least 15 x 90 mm 4. Pipets with pipet aids no mouth pipetting or pipettors, 1, 5, and 10 ml, graduated in 0. Dilution bottles, 6 oz ml , borosilicate-resistant glass, with rubber stoppers or plastic screw caps 6. Pipet and petri dish containers, adequate for protection 7. Circulating water bath, for tempering agar, thermostatically controlled to 45 1C 8.
Incubator, 35 1C; milk, 32 1C 9. Colony counter, dark-field, Quebec, or equivalent, with suitable light source and grid plate Tally register Dilution blanks, 90 1 ml Butterfield's phosphate-buffered dilution water R11 ; milk, 99 2 ml Plate count agar standard methods M Refrigerator, to cool and maintain samples at C; milk, Freezer, to maintain frozen samples from to C Procedure for analysis of frozen, chilled, precooked, or prepared foods Using separate sterile pipets, prepare decimal dilutions of , , , and others as appropriate, of food homogenate see Chapter 1 for sample preparation by transferring 10 ml of previous dilution to 90 ml of diluent.
Avoid sampling foam. Shake all dilutions 25 times in 30 cm 1 ft arc within 7 s. Pipet 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes. Reshake dilution bottle 25 times in 30 cm arc within 7 s if it stands more than 3 min before it is pipetted into petri dish.
Add ml plate count agar cooled to 45 1C to each plate within 15 min of original dilution. For milk samples, pour an agar control, pour a dilution water control and pipet water for a pipet control.
Add agar to the latter two for each series of samples. Add agar immediately to petri dishes when sample diluent contains hygroscopic materials, e. Pour agar and dilution water control plates for each series of samples. Immediately mix sample dilutions and agar medium thoroughly and uniformly by alternate rotation and back-and-forth motion of plates on flat level surface.
Let agar solidify. Invert solidified petri dishes, and incubate promptly for 48 2 h at 35C. Do not stack plates when pouring agar or when agar is solidifying.
Guidelines for calculating and reporting APCs in uncommon cases Official Methods of Analysis 3 does not provide guidelines for counting and reporting plate counts, whereas Standard Methods for the Examination of Dairy Products, 16th ed. Report all aerobic plate counts 2 computed from duplicate plates. For milk samples, report all aerobic plate 2 counts computed from duplicate plates containing less than 25 colonies as less than 25 estimated count.
Report all aerobic plate counts 2 computed from duplicate plates containing more than colonies as estimated counts. Counts outside the normal range may give erroneous indications of the actual bacterial composition of the sample.
Dilution factors may exaggerate low counts less than 25 , and crowded plates greater than may be difficult to count or may inhibit the growth of some bacteria, resulting in a low count. Report counts less than 25 or more than colonies as estimated aerobic plate counts EAPC. Use the following guide:. Normal plates Select spreader-free plate s. Count all colony forming units CFU , including those of pinpoint size, on selected plate s.
Record dilution s used and total number of colonies counted. Plates with more than colonies. When number of CFU per plate exceeds , for all dilutions, record the counts as too numerous to count TNTC for all but the plate closest to , and count CFU in those portions of plate that are representative of colony distribution. See ref. Spreading colonies are usually of 3 distinct types: 1 a chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump; 2 one that develops in film of water between agar and bottom of dish; and 3 one that forms in film of water at edge or on surface of agar.
When it is necessary to count plates containing spreaders not eliminated by a or b above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one colony.
Do not count each individual growth in such chains as a separate colony. Types 2 and 3 usually result in distinct colonies and are counted as such. Combine the spreader count and the colony count to compute the APC.
Plates with no CFU. When plates from all dilutions have no colonies, report APC as less than 1 times the corresponding lowest dilution used. Mark calculated APC with asterisk to denote that it was estimated from counts outside the per plate range.
When plate s from a sample are known to be contaminated or otherwise unsatisfactory, record the result s as laboratory accident LA.
Computing and recording counts see refs 6, 8 To avoid creating a fictitious impression of precision and accuracy when computing APC, report only the first two significant digits. Round off to two significant figures only at the time of conversion to SPC.
For milk samples, when plates for all dilutions have no colonies, report APC as less than 25 colonies estimated count. Round by raising the second digit to the next highest number when the third digit is 6, 7, 8, or 9 and use zeros for each successive digit toward the right from the second digit. Round down when the third digit is 1, 2, 3, or 4. When the third digit is 5, round up when the second digit is.
When counts of duplicate plates fall within and without the colony range, use only those counts that fall within this range. All plates with fewer than 25 CFU. In this method, a mechanical plater inoculates a rotating agar plate with liquid sample. The sample volume dispensed decreases as the dispensing stylus moves from the center to the edge of the rotating plate.
The microbial concentration is determined by counting the colonies on a part of the petri dish where they are easily countable and dividing this count by the appropriate volume. Additional dilutions may be made for suspected high microbial concentrations. Spiral plater Spiral Systems Instruments, Inc. Spiral colony counter Spiral Systems with special grid for relating deposited sample volumes to specific portions of petri dishes 3. Vacuum trap for disposal of liquids liter vacuum bottle to act as vacuum reservoir and vacuum source of cm Hg 4.
Disposable micro beakers, 5 ml 5. Petri dishes, plastic or glass, x 15 mm or x 15 mm 6. Plate count agar standard methods M 7.
Calculator optional , inexpensive electronic hand calculator is recommended 8. Polyethylene bags for storing prepared plates 9. Sterile dilution water Syringe, with Luer tip for obstructions in stylus; capacity not critical Work area, storage space, refrigerator, thermometers, tally, incubator, as described for Conventional Plate Count Method, above.
Sodium hypochlorite solution 5. Available commercially. Preparation of agar plates. Automatic dispenser with sterile delivery system is recommended to prepare agar plates. Agar volume dispensed into plates is reproducible and contamination rate is low compared to hand-pouring of agar in open laboratory. When possible, use laminar air flow hood along with automated dispenser.
Pour same quantity of agar into all plates so that same height of agar will be presented to spiral plater stylus tip to maintain contact angle. Agar plates should be level during cooling. Let agar solidify on level surface with poured plates stacked no higher than 10 dishes.
Place solidified agar plates in polyethylene bags, close with ties or heat-sealer, and store inverted at Bring prepoured plates to room temperature before inoculation. Preparation of samples. As described in Chapter 1, select that part of sample with smallest amount of connective tissues or fat globules. Description of spiral plater. Spiral plater inoculates surface of prepared agar plate to permit enumeration of microorganisms in solutions containing between and , microorganisms per ml.
Operator with minimum training can inoculate 50 plates per h. Within range stated, dilution bottles or pipets and other auxiliary equipment are not required. Required bench space is minimal, and time to check instrument alignment is less than 2 min. Plater deposits decreasing amount of sample in Archimedean spiral on surface of prepoured agar plate.
Volume of sample on any portion of plate is known. After incubation, colonies appear along line of spiral. If colonies on a portion of plate are sufficiently spaced from each other, count them on special grid which associates a calibrated volume with each area.
Estimate number of microorganisms in sample by dividing number of colonies in a defined area by volume contained in same area. Studies have shown the method to be proficient not only with milk 4 but also with other foods 7, Plating procedure Check stylus tip angle daily and adjust if necessary.
Use vacuum to hold microscope. Liquids are moved through system by vacuum. Clean stylus tip by rinsing for 1 s with sodium hypochlorite solution followed by sterile dilution water for 1 s before sample introduction.
This rinse procedure between processing of each sample minimizes cross-contamination. After rinsing, draw sample into tip of Teflon tubing by vacuum applied to 2-way valve. When tubing and syringe are filled with sample, close valve attached to syringe. Place agar plate on platform, place stylus tip on agar surface, and start motor. During inoculation, label petri plate lid. After agar has been inoculated, stylus lifts from agar surface and spiral plater automatically stops. Remove inoculated plate from platform and cover it.
Move stylus back to starting position. Vacuum-rinse system with hypochlorite and water, and then introduce new sample.
Invert plates and promptly place them in incubator for 48 3 h at 35 1C. Sterility controls Check sterility of spiral plater for each series of samples by plating sterile dilution water. They should not be excessively dry, as indicated by large wrinkles or glazed appearance. They should not have water droplets on surface of agar or differences greater than 2 mm in agar depth, and they should not be stored at Reduced flow rate through tubing indicates obstructions or material in system.
To clear obstructions, remove valve from syringe, insert hand-held syringe with Luer fitting containing water, and apply pressure. Use alcohol rinse to remove residual material adhering to walls of system. Dissolve accumulated residue with chromic acid. Rinse well after cleaning. Counting grid 1. Use same counting grid for both and mm petri dishes.
A mask is supplied for use with mm dishes. Counting grid is divided into 8 equal wedges; each wedge is divided by 4 arcs labeled l, 2, 3, and 4 from outside grid edge. Other lines within these arcs are added for ease of counting. A segment is the area between 2 arc lines within a wedge. Number of areas counted e. Spiral plater deposits sample on agar plate in the same way each time.
The grid relates colonies on spiral plate to the volume in which they were contained. When colonies are counted with grid, sample volume becomes greater as counting starts at outside edge of plate and proceeds toward center of plate.
The volume of sample represented by various parts of the counting grid is shown in operator's manual that accompanies spiral plater. Grid area. Plate all Incubate both sets of plates for 48 3 h at 35 1C.
Calculate concentrations for each dilution. Count spiral plates over grid surface, using counting rule of 20 described in H, below , and record number of colonies counted and grid area over which they were counted. Use the following formula:.
To check total volume dispensed by spiral plater, weigh amount dispensed from stylus tip. Collect in tared 5 ml plastic beaker and weigh on analytical balance 0. Examination and reporting of spiral plate counts. Counting rule of After incubation, center spiral plate over grid by adjusting holding arms on viewer.
Choose any wedge and begin counting colonies from outer edge of first segment toward center until 20 colonies have been counted. Complete by counting remaining colonies in segment where 20th colony occurs.
In this counting procedure, numbers such as 3b, 4c Fig. Any count irregularities in sample composition are controlled by counting the same segments in the opposite wedge and recording results.
Example of spirally inoculated plate Fig. Two segments of each wedge were counted on opposite sides of plate with 31 and 30 colonies, respectively. The sample volume contained in the darkened segments is 0. To estimate number of microorganisms, divide count by volume contained in all segments counted.
See example under Fig. If the number of colonies exceeds 75 in second, third, or fourth segment, which also contains the 20th colony, the estimated number of microorganisms will generally be low because of coincidence error associated with crowding of colonies.
In this case, count each circumferentially adjacent segment in all 8 wedges, counting at least 50 colonies, e. Calculate contained volume in counted segments of wedges and divide into number of colonies. When fewer than 20 colonies are counted on the total plate, report results as "less than estimated SPLC per ml. Report results of such plates as laboratory accident LA. If spreader covers entire plate, discard plate. If spreader covers half of plate area, count only those colonies that are well distributed in spreader-free areas.
Compute SPLC unless restricted by detection of inhibitory substances in sample, excessive spreader growth, or laboratory accidents. Round off counts as described in ID, above. Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. Association of Official Analytical Chemists. Official Methods of Analysis, 15th ed. Donnelly, C. Gilchrist, J. Peeler, and J.
Spiral plate count method for the examination of raw and pasteurized milk. Donnelly, J. Collaborative study comparing the spiral plate and aerobic plate count methods. International Dairy Federation. IDF, Brussels, Belgium. Jarvis, B. Lach, and J. Evaluation of the spiral plate maker for the enumeration of microorganisms in foods. Niemela, S. Statistical evaluation of Results from Quantitative Microbiological Examinations.
Report No. Tomasiewicz, D. Hotchkiss, G. Reinbold, R. Read, Jr. The most suitable number of colonies on plates for counting. Food Prot. Zipkes, M. Gilchrist, and J. Comparison of yeast and mold counts by spiral, pour, and streak plate methods. If the length or width of the object changes while doing this with either lens, then there is an astigmatism correction and wearing the glasses during microscope work may be recommended. Special high-point eyepieces are made with a longer exit pupil distance to easily accommodate the extra distance needed when eyeglasses are worn.
These eyepieces are identified in some way by the manufacturer. To take advantage of the optimal resolution and illumination of the microscope, a technique known as Koehlor illumination is used. NOTE: Some settings may have been preset by the manufacturer and will not be adjustable. With a slide specimen on the stage, use a low power X objective, and focus with the coarse and fine adjustments. Low magnification provides a larger field of view for easier searching of the specimen.
It also provides a greater working distance than higher power objectives, offering more safety against focusing too low and breaking the slide. The microscope may have an auxiliary swing-in or swing-out lens in the condenser. Use the coarse and fine adjustments to focus the specimen. To obtain Koehlor illumination, close the lamp field iris diaphragm, if present, at the base of the microscope, and bring into focus by vertical adjustment of the condenser. Use the centering screws or knobs on the condenser, if present, to center the focused circle in the field.
If not on the condenser, centering screws or knobs for this purpose may be on the base near the lamp diaphragm. Open the lamp diaphragm until it is just past the field of view. This may not be possible with low power objectives of some microscopes until the auxiliary lens in the condenser is correctly adjusted. If the microscope has no lamp diaphragm, place a piece of paper with a small hole cut in it over the lamp opening and make the same adjustments to focus on its inner edge.
Microscopes with mirror and external light source are not discussed here, but the principles are similar. Follow the manufacturer's instructions for adjusting the lamp filament if possible. Set the substage aperture diaphragm next.
This adjustment has a crucial effect on the resolution and contrast of the image. To approximate this setting without removing an eyepiece, open the substage diaphragm fully and gradually close it while looking through the microscope until the image gains a sudden increase in sharpness and detail. Replace the eyepiece.
If the lighting is too bright, use the rheostat, if provided, to turn it down, or add neutral density or other filters. Resolution will suffer if it is stopped down closed too far or opened too much. Although stopping down gives more contrast, it impairs resolution, and spurious details are formed by diffraction lines or fringes.
Repeat the procedures for Koehlor illumination with each objective used. For phase-contrast microscopy, follow the same basic steps. Do not use the substage diaphragm but make adjustments to bring the phase annulus and annular diaphragm into coincidence. Refer to the manufacturer's instructions. Use of a green filter is recommended. Standard Methods for the Examination of Dairy Products, 16th ed. Chapter Chapter 2. Bryce and Paul L.
The conventional plate count method for examining frozen, chilled, precooked, or prepared foods, outlined below, conforms to AOAC Official Methods of Analysis, sec. The suitable colony counting range 10 is The automated spiral plate count method for the examination of foods and cosmetics 5 , outlined below, conforms to AOAC Official Methods of Analysis, sec.
For procedural details of the standard plate count, see ref. Guidelines for calculating and reporting plate counts have been changed to conform with the anticipated changes in the 16th edition of Standard Methods for the Examination of Dairy Products 2 and the International Dairy Federation IDF procedures 6.
Conventional Plate Count Method A. Work area, level table with ample surface in room that is clean, well-lighted foot-candles at working surface and well-ventilated, and reasonably free of dust and drafts. Storage space, free of dust and insects and adequate for protection of equipment and supplies 3. Petri dishes, glass or plastic at least 15 x 90 mm 4. Pipets with pipet aids no mouth pipetting or pipettors, 1, 5, and 10 ml, graduated in 0. Dilution bottles, 6 oz ml , borosilicate-resistant glass, with rubber stoppers or plastic screw caps 6.
Pipet and petri dish containers, adequate for protection 7. Colony counter, dark-field, Quebec, or equivalent, with suitable light source and grid plate Tally register Plate count agar standard methods M Procedure for analysis of frozen, chilled, precooked, or prepared foods Using separate sterile pipets, prepare decimal dilutions of , , , and others as appropriate, of food homogenate see Chapter 1 for sample preparation by transferring 10 ml of previous dilution to 90 ml of diluent.
Avoid sampling foam. Shake all dilutions 25 times in 30 cm 1 ft arc within 7 s. Pipet 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes. Reshake dilution bottle 25 times in 30 cm arc within 7 s if it stands more than 3 min before it is pipetted into petri dish.
For milk samples, pour an agar control, pour a dilution water control and pipet water for a pipet control. Add agar to the latter two for each series of samples. Add agar immediately to petri dishes when sample diluent contains hygroscopic materials, e. Pour agar and dilution water control plates for each series of samples. Immediately mix sample dilutions and agar medium thoroughly and uniformly by alternate rotation and back-and-forth motion of plates on flat level surface.
Let agar solidify. Do not stack plates when pouring agar or when agar is solidifying. Guidelines for calculating and reporting APCs in uncommon cases Official Methods of Analysis 3 does not provide guidelines for counting and reporting plate counts, whereas Standard Methods for the Examination of Dairy Products, 16th ed. Report all aerobic plate counts 2 computed from duplicate plates. For milk samples, report all aerobic plate 2 counts computed from duplicate plates containing less than 25 colonies as less than 25 estimated count.
Report all aerobic plate counts 2 computed from duplicate plates containing more than colonies as estimated counts. Counts outside the normal range may give erroneous indications of the actual bacterial composition of the sample. Dilution factors may exaggerate low counts less than 25 , and crowded plates greater than may be difficult to count or may inhibit the growth of some bacteria, resulting in a low count. Report counts less than 25 or more than colonies as estimated aerobic plate counts EAPC.
Use the following guide: 1. Normal plates Select spreader-free plate s. Count all colony forming units CFU , including those of pinpoint size, on selected plate s. Record dilution s used and total number of colonies counted.
Plates with more than colonies. When number of CFU per plate exceeds , for all dilutions, record the counts as too numerous to count TNTC for all but the plate closest to , and count CFU in those portions of plate that are representative of colony distribution. See ref. Spreading colonies are usually of 3 distinct types: 1 a chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump; 2 one that develops in film of water between agar and bottom of dish; and 3 one that forms in film of water at edge or on surface of agar.
When it is necessary to count plates containing spreaders not eliminated by a or b above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one colony. Do not count each individual growth in such chains as a separate colony.
Types 2 and 3 usually result in distinct colonies and are counted as such. Combine the spreader count and the colony count to compute the APC. Plates with no CFU. When plates from all dilutions have no colonies, report APC as less than 1 times the corresponding lowest dilution used.
Mark calculated APC with asterisk to denote that it was estimated from counts outside the per plate range. When plate s from a sample are known to be contaminated or otherwise unsatisfactory, record the result s as laboratory accident LA. Computing and recording counts see refs 6, 8 To avoid creating a fictitious impression of precision and accuracy when computing APC, report only the first two significant digits. Round off to two significant figures only at the time of conversion to SPC.
For milk samples, when plates for all dilutions have no colonies, report APC as less than 25 colonies estimated count. Round by raising the second digit to the next highest number when the third digit is 6, 7, 8, or 9 and use zeros for each successive digit toward the right from the second digit.
Round down when the third digit is 1, 2, 3, or 4. When the third digit is 5, round up when the second digit is odd and round down when the second digit is even. Plates with CFU. When counts of duplicate plates fall within and without the colony range, use only those counts that fall within this range. All plates with fewer than 25 CFU. In this method, a mechanical plater inoculates a rotating agar plate with liquid sample. The sample volume dispensed decreases as the dispensing stylus moves from the center to the edge of the rotating plate.
The microbial concentration is determined by counting the colonies on a part of the petri dish where they are easily countable and dividing this count by the appropriate volume.
Additional dilutions may be made for suspected high microbial concentrations. Spiral plater Spiral Systems Instruments, Inc. Spiral colony counter Spiral Systems with special grid for relating deposited sample volumes to specific portions of petri dishes 3. Vacuum trap for disposal of liquids liter vacuum bottle to act as vacuum reservoir and vacuum source of cm Hg 4. Disposable micro beakers, 5 ml 5. Petri dishes, plastic or glass, x 15 mm or x 15 mm 6. Plate count agar standard methods M 7.
Calculator optional , inexpensive electronic hand calculator is recommended 8. Polyethylene bags for storing prepared plates 9. Sterile dilution water Syringe, with Luer tip for obstructions in stylus; capacity not critical Work area, storage space, refrigerator, thermometers, tally, incubator, as described for Conventional Plate Count Method, above. Sodium hypochlorite solution 5.
Available commercially. Preparation of agar plates. Automatic dispenser with sterile delivery system is recommended to prepare agar plates. Agar volume dispensed into plates is reproducible and contamination rate is low compared to hand-pouring of agar in open laboratory. When possible, use laminar air flow hood along with automated dispenser. Pour same quantity of agar into all plates so that same height of agar will be presented to spiral plater stylus tip to maintain contact angle.
Agar plates should be level during cooling. Let agar solidify on level surface with poured plates stacked no higher than 10 dishes. Place solidified agar plates in polyethylene bags, close with ties or heat-sealer, and store inverted at Bring prepoured plates to room temperature before inoculation.
Preparation of samples. As described in Chapter 1, select that part of sample with smallest amount of connective tissues or fat globules. Description of spiral plater. Spiral plater inoculates surface of prepared agar plate to permit enumeration of microorganisms in solutions containing between and , microorganisms per ml.
Operator with minimum training can inoculate 50 plates per h. Within range stated, dilution bottles or pipets and other auxiliary equipment are not required. Required bench space is minimal, and time to check instrument alignment is less than 2 min. Plater deposits decreasing amount of sample in Archimedean spiral on surface of prepoured agar plate.
Volume of sample on any portion of plate is known. After incubation, colonies appear along line of spiral. If colonies on a portion of plate are sufficiently spaced from each other, count them on special grid which associates a calibrated volume with each area. Estimate number of microorganisms in sample by dividing number of colonies in a defined area by volume contained in same area. Studies have shown the method to be proficient not only with milk 4 but also with other foods 7, Plating procedure Check stylus tip angle daily and adjust if necessary.
Use vacuum to hold microscope cover slip against face of stylus tip; if cover slip plane is parallel at about l mm from surface of platform, tip is properly oriented. Liquids are moved through system by vacuum.
Clean stylus tip by rinsing for 1 s with sodium hypochlorite solution followed by sterile dilution water for 1 s before sample introduction.
This rinse procedure between processing of each sample minimizes cross-contamination. After rinsing, draw sample into tip of Teflon tubing by vacuum applied to 2-way valve. When tubing and syringe are filled with sample, close valve attached to syringe. Place agar plate on platform, place stylus tip on agar surface, and start motor. During inoculation, label petri plate lid. After agar has been inoculated, stylus lifts from agar surface and spiral plater automatically stops.
Remove inoculated plate from platform and cover it. Move stylus back to starting position. Vacuum-rinse system with hypochlorite and water, and then introduce new sample. Sterility controls Check sterility of spiral plater for each series of samples by plating sterile dilution water. They should not be excessively dry, as indicated by large wrinkles or glazed appearance. They should not have water droplets on surface of agar or differences greater than 2 mm in agar depth, and they should not be stored at Reduced flow rate through tubing indicates obstructions or material in system.
To clear obstructions, remove valve from syringe, insert hand-held syringe with Luer fitting containing water, and apply pressure. Use alcohol rinse to remove residual material adhering to walls of system. Dissolve accumulated residue with chromic acid. Rinse well after cleaning. Counting grid 1. Use same counting grid for both and mm petri dishes. A mask is supplied for use with mm dishes. Counting grid is divided into 8 equal wedges; each wedge is divided by 4 arcs labeled l, 2, 3, and 4 from outside grid edge.
Other lines within these arcs are added for ease of counting. A segment is the area between 2 arc lines within a wedge. Number of areas counted e. Spiral plater deposits sample on agar plate in the same way each time. The grid relates colonies on spiral plate to the volume in which they were contained.
When colonies are counted with grid, sample volume becomes greater as counting starts at outside edge of plate and proceeds toward center of plate. The volume of sample represented by various parts of the counting grid is shown in operator's manual that accompanies spiral plater. Grid area constants have been checked by the manufacturer and are accurate.
Calculate concentrations for each dilution. Count spiral plates over grid surface, using counting rule of 20 described in H, below , and record number of colonies counted and grid area over which they were counted. Use the following formula: To check total volume dispensed by spiral plater, weigh amount dispensed from stylus tip. Figure 1. Examination and reporting of spiral plate counts. Counting rule of After incubation, center spiral plate over grid by adjusting holding arms on viewer.
Choose any wedge and begin counting colonies from outer edge of first segment toward center until 20 colonies have been counted. Complete by counting remaining colonies in segment where 20th colony occurs. In this counting procedure, numbers such as 3b, 4c Fig. Any count irregularities in sample composition are controlled by counting the same segments in the opposite wedge and recording results.
Example of spirally inoculated plate Fig. Two segments of each wedge were counted on opposite sides of plate with 31 and 30 colonies, respectively. The sample volume contained in the darkened segments is 0. To estimate number of microorganisms, divide count by volume contained in all segments counted. See example under Fig. If the number of colonies exceeds 75 in second, third, or fourth segment, which also contains the 20th colony, the estimated number of microorganisms will generally be low because of coincidence error associated with crowding of colonies.
In this case, count each circumferentially adjacent segment in all 8 wedges, counting at least 50 colonies, e.
Calculate contained volume in counted segments of wedges and divide into number of colonies. When fewer than 20 colonies are counted on the total plate, report results as "less than estimated SPLC per ml. Report results of such plates as laboratory accident LA.
If spreader covers entire plate, discard plate. If spreader covers half of plate area, count only those colonies that are well distributed in spreader-free areas. Compute SPLC unless restricted by detection of inhibitory substances in sample, excessive spreader growth, or laboratory accidents.
Round off counts as described in ID, above. Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. Association of Official Analytical Chemists. Official Methods of Analysis, 15th ed.
Donnelly, C. Gilchrist, J. Peeler, and J. Spiral plate count method for the examination of raw and pasteurized milk. Donnelly, J. Collaborative study comparing the spiral plate and aerobic plate count methods. International Dairy Federation. IDF, Brussels, Belgium. Jarvis, B. Lach, and J. Evaluation of the spiral plate maker for the enumeration of microorganisms in foods. Niemela, S. Statistical evaluation of Results from Quantitative Microbiological Examinations.
Report No. Tomasiewicz, D. Hotchkiss, G. Reinbold, R. Read, Jr. The most suitable number of colonies on plates for counting. Food Prot. Zipkes, M. Gilchrist, and J. Comparison of yeast and mold counts by spiral, pour, and streak plate methods. Chapter 3. Maturin and James T. It is always crucial to note the general condition of the food sample, such as its consistency, color, and odor. As much information as possible should be obtained about its pre- and post-collection history see Chapter l.
Microscopic examination and Gram staining must be carried out, as described in Chapter 2. To decide what treatments, enrichments, or other tests are needed, the microbiologist should evaluate the data in relation to two types of information: l the causes epidemiologically associated with the type and condition of the implicated food, and 2 the clinical signs and symptoms observed in afflicted individuals. If possible, clinical microbial isolates usually from stool specimens and blood serum samples for serological and biochemical testing should be obtained from patients by way of their physicians.
Table l lists the major microbial or chemical agents of foodborne disease and their commonly associated food sources. Recently reported causative agents of foodborne outbreaks, cases, and deaths are given in Table 2.
Clinical symptoms most often associated with specific microbial or chemical agents and their duration are listed in Table 3. Analysts should use these tables as an aid in deciding the most probable, less probable, and least likely associations.
The tables should not be used to assume a single cause or to eliminate possibilities entirely. This agency, which is the principal source of epidemiologic data on reported foodborne disease outbreaks in the United States, periodically publishes summary surveillance reports of foodborne diseases in the Morbidity and Mortality Weekly Report series.
Most reports of foodborne illness are submitted to CDC by state health departments. CDC defines a foodborne disease outbreak as an incident in which at least two or more persons experience a similar illness after ingestion of a common food, and epidemiologic analysis implicates the food as the source of the illness.
A few exceptions exist; for example, one case of botulism or chemical poisoning constitutes an outbreak. Although CDC's foodborne disease surveillance system has limitations i. With new pathogens there is an inevitable lag before methods are installed and reporting by clinical and food laboratories becomes routine.
Changes in food production or processing may make a food the vehicle or growth medium for microorganisms not previously associated with that product. For example, new varieties of tomatoes that are less acidic than the traditional types might support the growth and toxin production of Clostridium botulinum; freezing procedures improved to preserve taste may also preserve microorganisms that are killed in blast freezing.
The food microbiologist should be aware that the clinical symptoms and diagnosis of the patient's illness, available when analysis of the food sample must begin, may be preliminary or incomplete. To proceed from the generalities given in the tables to an analytical course of action, the microbiologist must use reason, imagination, and caution. Andrews, Reginald W. Bennett, Jeffrey W. Bier, Elisa L.
Hitchins, E. Jeffery Rhodehamel, and Tony T. Table 1. Number and percent of confirmed foodborne disease outbreaks cases, and deaths in the USA reported to CDC from through , listed by etiologic agent Outbreak Cases No. Griffin, J. Golding, and C. Weekly Rep. Special Supplement No. Table 3. Onset, duration, and symptoms of foodborne illnesses a1 Onset and duration of illness Predominant symptoms Associated organism or toxin Upper gastrointestinal tract symptoms nausea, vomiting occur first or predominate Less than 1 h h Nausea, vomiting, unusual taste,burning of mouth.
Metallic chemicals a Nausea, vomiting, cyanosis, headache, dizziness, dyspnea, trembling, weakness, loss of consciousness. Nitrites; b Paragonimus sp. Onset h, mean h, Nausea, vomiting, retching, diarrhea, abdominal pain, duration days prostration. Staphylococcus aureus and its enterotoxins; Sarcocystis hominis h h rarely h Vomiting, abdominal cramps,diarrhea, nausea. Bacillus cereus Nausea, vomiting, diarrhea, thirst, dilation of pupils, collapse, coma.
Amanita species mushrooms; c Sarcocystis suihominis Sore throat and respiratory symptoms occur h Sore throat, fever, nausea, vomiting, rhinorrhea, sometimes a rash. Streptococcus pyogenes Lancefield Group A days Inflamed throat and nose,spreading grayish exudate,fever, chills, sore throat,malaise, difficulty in swallowing,edema of cervical lymph node. Corynebacterium diphtheriae Lower gastrointestinal tract symptoms abdominal cramps, diarrhea occur first or predominate h, mean h Abdominal cramps, diarrhea, putrefactive diarrhea associated with C.
Clostridium perfringens, Bacillus cereus, Streptococcus faecalis, S. Sometimes bloody or mucoid diarrhea, cutaneous lesions and hypotension associated with V. Salmonella species including S. Enteroviruses, rotavirus, enteric adenovirus, Norwalk-like viruses; anisakid nematodes, Nanophyetus salmincola; Cryptosporidium parvum weeks Mucoid diarrhea fatty stools , abdominal pain, weight loss. Giardia lamblia Nanophyetus salmincola Abdominal pain, diarrhea, constipation, headache, drowsiness, ulcers, variable; often asymptomatic.
Entamoeba histolytica Isospora belli Nervousness, insomnia, hunger pains, anorexia, weight loss, abdominal pain, sometimes gastroenteritis. Taenia saginata, T. Shellfish toxin d Gastroenteritis, nervousness,blurred vision, chest pain,cyanosis, twitching, convulsions. Organic phosphate e Excessive salivation, perspiration, gastroenteritis, irregular pulse,pupils constricted, asthmatic breathing.
Muscaria-type mushrooms f Tingling and numbness, dizziness, pallor, gastroenteritis, hemorrhage, and desquamation of skin, fixed eyes, loss of reflexes, twitching, paralysis. Tetraodon toxin g Tingling and numbness, gastroenteritis, dizziness, dry mouth, muscular aches, dilated eyes, blurred vision, paralysis. Ciguatera toxin h Nausea, vomiting, tingling, dizziness, weakness, anorexia,weight loss, confusion.
Chlorinated hydrocarbons i 2 h to 7 days, usually h Vertigo, double or blurred vision,loss of reflex to light, difficulty in swallowing, speaklng, and breathing, dry mouth, weakness, respiratory paralysis, death. Clostridium botulinum and its neurotoxins More than 72 h Numbness, weakness of legs, spastic paralysis, impairment of vision, blindness, coma. Organic mercury j Gastroenteritis, leg pain, ungainly high-stepping gait, foot and wrist drop.
Triorthocresyl phosphate k h Allergic symptoms facial flushing, itching occur Less than 1 h days Headache, dizziness, nausea, vomiting, peppery taste, burning of throat, facial swelling and flushing, stomach pain, itching of skin. Histamine 1 Numbness around mouth, tingling sensation, flushing, dizziness, headache, nausea, vomiting.
Monosodium glutamate m Flushing, sensation of warmth,itching, abdominal pain, puffing of face and knees. Nicotinic acid n Coughing, asthma. Ascaris lumbricoides General infection symptoms fever, chills, malaise, prostration, aches, swollen lymph nodes occur days, mean 9 days Gastroenteritis, fever, edema about eyes, perspiration, muscular pain, chills, prostration, labored breathing. Trichinella spiralis Gnathostoma sp. Paragonimus spp.
Alaria spp. Toxoplasma gondii days, mean days Fever, malaise, lassitude, anorexia, nausea, abdominal pain, jaundice. Etiological agent not yet isolated--probably viral especially Hepatitis A and E viruses Varying periods depends on specific illness Fever, chills, head- or joint ache, prostration, malaise, swollen lymph nodes, and other specific symptoms of disease in question.
Bacillus anthracis, Brucella melitensis, B. Examine foods for Proteus species or other organisms capable of decarboxylating histidine into histamine, and for histamine. Jackson, Joseph M. Madden, Walter E. Hill, and Karl C. The sanitary tests presented here include special tests for shellfish and a brief consideration of bottled water. Complete discussions of the definition and scope of the "coliforms" can be found elsewhere 5.
Identification criteria used are production of gas from glucose and other sugars and fermentation of lactose to acid and gas within 48 h at 35C coliforms and With all shellfish isolates, an incubation temperature of Some E. Some properties of enterovirulent E. Enteroadherent E.
The standard weight of analytical portions of food samples examined for the presence of sanitary and enteropathogenic E. If desired, 50 g portions may be used with appropriate scale-up of the suspending medium. Some properties of the enterovirulent E. Covered water bath, with circulating system to maintain temperature of To learn more, view our Privacy Policy. To browse Academia.
Innovations in methods for the microbiological analysis of food continue to appear at a rapid pace. The list of commercially available test kits and the discussion of rapid methods in Appendix 1 were thoroughly revised. Three chapters were added: the use of reverse transcription RT and the polymerase chain reaction PCR to detect and quantify contamination of shellfish with hepatitis A virus Chapter 26 ; new procedures for the alkaline phosphatase test to determine whether dairy foods were prepared with pasteurized milk Chapter 27 ; and the use of PCR to detect toxigenic Vibrio cholerae in foods.
In addition, there are updated Muhammad Akond. Kacem Mourad.
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