8/24/2016 Learning Objectives Food Microbiology 1.Discuss methods available for microbiological analysis of food 2.Compare methods of analysis and indicate Chapter4 AN INTRODUCTION advantages and disadvantages of each method Detection and 3.Detail procedures for collection and Enumeration of processing of food samples Microbes in Food 4.Calculate the microbial load of a sample 5.Recognize the difference between conventional and rapid microbiological methods The Good,the Bad,and the Ugly Determining Types and Numbers of Microbes Microbes are ubiquitous Important aspect of food microbiology Good microbes-used in Aerobic plate count (APC) the production of food estimates the number of microbes in a food such as yogurt,wine,beer sample cheese strict anaerobes are not included in the count Bad microbes cause food changes during processing,handling,and storage of food spoilage Increases for raw meats during storage ·Ugly microbes cause SET STo May decrease for dry or frozen foods human illness Depending on the food type,APC can be as low as 10 or as high as a million cells per gram
8/24/2016 1 Chapter 4 Detection and Enumeration of Microbes in Food 1 Learning Objectives 1. Discuss methods available for microbiological analysis of food 2. Compare methods of analysis and indicate advantages and disadvantages of each method 3. Detail procedures for collection and processing of food samples 4. Calculate the microbial load of a sample 5. Recognize the difference between conventional and rapid microbiological methods 2 The Good, the Bad, and the Ugly • Microbes are ubiquitous • Good microbes – used in the production of food such as yogurt, wine, beer, cheese • Bad microbes cause food spoilage • Ugly microbes cause human illness 3 Determining Types and Numbers of Microbes • Important aspect of food microbiology • Aerobic plate count (APC) - estimates the number of microbes in a food sample - strict anaerobes are not included in the count - changes during processing, handling, and storage of food * Increases for raw meats during storage * May decrease for dry or frozen foods - Depending on the food type, APC can be as low as 10 or as high as a million cells per gram 4
8/24/2016 Black Pepper Microbial Load Aerobic Plate Count Differs in products from the same origin -Ground meat greater microbial load thar cuts of meat Bacteria multiply during processing or on the equipment used to process it Starting number impacts the number in the final product (even heat treated food) High numbers occur even in foods properly heat treated due to 10-4 10-5 1.Poor-quality ingredients 10-6 2.Poor sanitation 3.Recontamination 4.Improper handling Determining a Microbial Load Sample Collection and Processing Methods used vary from . Estimation of the number of microbes in or food to food and for specific on food is needed to determine if a food microorganisms product meets specifications,standards, or quidelines FDA outlines its methods in Bacteriological Analytical Rule of thumb,as the microbial load Manua/BAM increases,the food quality decreases USDA publishes(meat and -Exceptions are fermented foods poultry)Microbiology Microbiological analysis of food refers to Laboratory Guidebook determining the levels of bacteria Online pdf book formats
8/24/2016 2 Black Pepper Aerobic Plate Count 10-4 10-5 10-6 5 Microbial Load • Differs in products from the same origin - Ground meat greater microbial load than cuts of meat - Bacteria multiply during processing or on the equipment used to process it • Starting number impacts the number in the final product (even heat treated food) • High numbers occur even in foods properly heat treated due to 1. Poor-quality ingredients 2. Poor sanitation 3. Recontamination 4. Improper handling 6 Determining a Microbial Load • Estimation of the number of microbes in or on food is needed to determine if a food product meets specifications, standards, or guidelines • Rule of thumb, as the microbial load increases, the food quality decreases - Exceptions are fermented foods • Microbiological analysis of food refers to determining the levels of bacteria 7 Sample Collection and Processing • Methods used vary from food to food and for specific microorganisms • FDA outlines its methods in Bacteriological Analytical Manual BAM • USDA publishes (meat and poultry) Microbiology Laboratory Guidebook • Online pdf book formats 8
8/24/2016 Sample Collection and Processing Sample Collection and Processing Sample collection should not introduce microbes A sample will only yield significant and into the food sample meaningful results if Best,if individual containers are submitted for 1.It represents the mass of the analysis material sampled If not,use aseptic technique to collect a sample 2.The sample is handled in a manner Instruments used to sample should be sterile that prevents changes in the Containers(bags or wide-mouthed jars)should microbial numbers between collection be sterile and analysis Containers should be labeled with food name, date collected,and other information used to track or analyze the results Sample Collection and Processing Sample Preparation ·Sample should be Samples should be analyzed as quickly as possible to prevent any change in the microbial population of ba If this is not possible: -Samples should be refrigerated ender for large volume food Frozen samples should be kept frozen testing procedures -Products normally refrigerated should such as FDA Bacteriological never be frozen as this may cause Analytical Manual (BAM) microbial death 3
8/24/2016 3 Sample Collection and Processing A sample will only yield significant and meaningful results if 1. It represents the mass of the material sampled 2. The sample is handled in a manner that prevents changes in the microbial numbers between collection and analysis 9 Sample Collection and Processing Sample collection should not introduce microbes into the food sample • Best, if individual containers are submitted for analysis • If not, use aseptic technique to collect a sample • Instruments used to sample should be sterile • Containers (bags or wide-mouthed jars) should be sterile • Containers should be labeled with food name, date collected, and other information used to track or analyze the results 10 Sample Collection and Processing Samples should be analyzed as quickly as possible to prevent any change in the microbial population If this is not possible: - Samples should be refrigerated - Frozen samples should be kept frozen - Products normally refrigerated should never be frozen as this may cause microbial death 11 Sample Preparation • Sample should be processed to produce a homogeneous suspension of bacteria that can be pipetted • Stomacher - a laboratory blender for large volume food testing procedures, such as FDA Bacteriological Analytical Manual (BAM) 12
8/24/2016 Sample Preparation Liquid sample To create a homogeneous sample,solid food I ml removed is mixed with sterile diluent,such as 0.1% and added to peptone water uffer diluent 1.Sample(25g)added to a sterile stomacher bag Sample spread in duplicate on plates using 2.Diluent (225 ml)added to the bag a sterile spreader ”lates 3.Processed in the stomacher incubated 4.Creates the first 1/10 dilution 中( 25250 ith Bacteria should not be allowed to multiply colonies are counted and work quickly and use appropriate expressed as buffers CFU mle A The Analysis Pour Plate Spread Plate Method Method Disadvantage of surface plating -two or more colonies grow together growth of spreaders Plate surfaces must be dry 9
8/24/2016 4 Sample Preparation To create a homogeneous sample, solid food is mixed with sterile diluent, such as 0.1% peptone water 1. Sample (25g) added to a sterile stomacher bag 2. Diluent (225 ml) added to the bag 3. Processed in the stomacher 4. Creates the first 1/10 dilution Bacteria should not be allowed to multiply • work quickly and use appropriate buffers 13 Sample spread in duplicate on plates using a sterile spreader Liquid sample 1 ml removed and added to buffer diluent Plates incubated Plates with 25-250 colonies are counted and expressed as CFU / ml14 Analysis Disadvantage of surface plating - two or more colonies grow together - growth of spreaders Plate surfaces must be dry 15 Pour Plate Method Spread Plate Method 16
8/24/2016 Pour Plate Method Analysis colonies embedded in the agar Colony Forming Units CFU In theory,one CFU arises from one cell may not be true -may arise from clusters/chains of cells -may give an underestimation of the number of bacteria in a sample Cells need to be separated Sample homogenization Sample dilution Countable plate Analysis contains 25 250 colonies Roll Tube Method ·Fewer or more ·Same as pour plate colonies affect but tubes are used accuracy Total number: d Bacterial dilution is multiply the added to 2 ml molten colomy count by agar in a tube and the dilution rolled around in cold factor water until solid ·Advantage-less 品 plate count agar is used 5
8/24/2016 5 Pour Plate Method colonies embedded in the agar 17 Analysis Colony Forming Units CFU • In theory, one CFU arises from one cell - may not be true - may arise from clusters / chains of cells - may give an underestimation of the number of bacteria in a sample • Cells need to be separated - Sample homogenization - Sample dilution 18 • Countable plate contains 25- 250 colonies • Fewer or more colonies affect accuracy • Total number: multiply the colony count by the dilution factor • Expressed as bacteria per gram or ml of food 19 Analysis Roll Tube Method • Same as pour plate but tubes are used • Bacterial dilution is added to 2 ml molten agar in a tube and rolled around in cold water until solid • Advantage – less plate count agar is used 20
8/24/2016 Spiral Plater Analysis Analysis ·.Robotic arm spreads Membrane Filter Method bacteria on an agar plate Fluids with low microbial Volume of the inoculum is counts are often analyzed decreased as it moves to the with this technique outer edge Direct microscopic count 1:10,000 dilution is achieved may be used Counting grid needed to count colonies ·Use fewer plates But limited to liquid samples Analysis Analysis Hydrophobic grid membrane Fluorescent dyes stain filter method bacteria Fluorescent bacteria are ·1,600 grid filters counted Restrict microbial growth ·Restrict colony size a microsope factor ·1 ml filtered Used with meat,poultry. ·Count grids with DAPI stained bacterial colonies cells from drinking water Live dead cell stains Most probable number determined 2出 6
8/24/2016 6 Spiral Plater Analysis • Robotic arm spreads bacteria on an agar plate • Volume of the inoculum is decreased as it moves to the outer edge • 1:10,000 dilution is achieved • Counting grid needed to count colonies • Use fewer plates • But limited to liquid samples 21 Analysis Membrane Filter Method • Fluids with low microbial counts are often analyzed with this technique • Direct microscopic count may be used 22 Analysis Direct Epifluorescent Filter Technique • Fluorescent dyes stain bacteria • Fluorescent bacteria are counted • Calculate: average number of cells per field is multipled by a microsope factor • Used with meat, poultry, milk, and food contact surfaces • Live / dead cell stains DAPI stained bacterial cells from drinking water 23 Analysis Hydrophobic grid membrane filter method • 1,600 grid filters • Restrict microbial growth • Restrict colony size • 1 ml filtered • Count grids with colonies • Most probable number determined 24
8/24/2016 ·Advantages Direct Microscopic Count Grid with 25 large squares ·fast,simple no incubation Cover glass little sample processing Slide small sample size Disadvantages ·fatigue live and dead bacteria counted no value for foods with low numbers Sample spread on a microsope slide" Cover glass Fix,defat,and stain the cells Calibrated microscope used Location of squares know the diameter of the field c Number of cells or clumps per field divided by the area of re kh the field-number per square millimeter Metabolism-Based Methods of Analysis Methylene Blue Reduction Test Measurements of metabolism or production of Color imparted to milk by metabolic products can be used to estimate microbial methylene blue disappears numbers as oxygen is removed due Most are minaturized and the basis of rapid test to the formation of methods reduced substances ·Redox potential electron transfers measured using potentiometer Bacterial metabolism dye reduction tests-use redox indicators or dyes causes the color to to estimate bacterial numbers disappear larger the number-faster the color changes Some foods contain high levels of reductive Quicker the color changes substances the larger the number of Commonly used for raw milk bacteria present >
8/24/2016 7 Direct Microscopic Count • Advantages - fast, simple - no incubation - little sample processing - small sample size • Disadvantages - fatigue - live and dead bacteria counted - no value for foods with low numbers • Sample spread on a microsope slide • Fix, defat, and stain the cells • Calibrated microscope used - know the diameter of the field • Number of cells or clumps per field divided by the area of the field – number per square millimeter 25 26 Metabolism-Based Methods of Analysis • Measurements of metabolism or production of metabolic products can be used to estimate microbial numbers • Most are minaturized and the basis of rapid test methods • Redox potential - electron transfers measured using potentiometer - dye reduction tests – use redox indicators or dyes to estimate bacterial numbers larger the number – faster the color changes • Some foods contain high levels of reductive substances • Commonly used for raw milk 27 Methylene Blue Reduction Test • Color imparted to milk by methylene blue disappears as oxygen is removed due to the formation of reduced substances • Bacterial metabolism causes the color to disappear • Quicker the color changes the larger the number of bacteria present 28
8/24/2016 Surface Testing Swab Test Method ·Most common in food- Food contact surfaces should be tested processing facilities for their hygienic state Moistened cotton or calcium Inherent problem-consistent removal alginate swab (dissolves) of microbes from the surface Sterile templates define the area size Swab placed in a diluent Standard plate counts performed Ideal for rough,uneven surfaces Contact Agar Plates Other Methods a raised agar Simply incubate the plate These methods are all common Sterile sticky tape conventional methods ·Pressed against a Selective media may be used to premeasured surface Pressed to an agar plate enhance detection of specific microbes Sponge system May be combined with rapid methods to Moistened sponge wiped decrease the time required for across the test area identification of microbes found in food. ·Placed into diluent Standard plate count performed 8
8/24/2016 8 Surface Testing • Food contact surfaces should be tested for their hygienic state • Inherent problem – consistent removal of microbes from the surface 29 Swab Test Method • Most common in foodprocessing facilities • Moistened cotton or calcium alginate swab (dissolves) • Sterile templates define the area size • Swab placed in a diluent • Standard plate counts performed • Ideal for rough, uneven surfaces 30 Other Methods Contact Agar Plates • Petri plate has a raised agar surface • Simply incubate the plate Sterile sticky tape • Pressed against a premeasured surface • Pressed to an agar plate Sponge system • Moistened sponge wiped across the test area • Placed into diluent • Standard plate count performed 31 • These methods are all common conventional methods • Selective media may be used to enhance detection of specific microbes • May be combined with rapid methods to decrease the time required for identification of microbes found in food. 32
8/24/2016 Summary Summary Samples must be collected using aseptic Surface test methods are essential in techniques and held in such a way as to determining whether food contact prevent the growth of microoganisms. surfaces are being maintained in a Food samples should be blended or hygienic state homogenized prior to examination. Rapid methods based on conventional Standard plate count is used to provide biochemical methods are now available. an estimate of the number of reducing the time required to identify a microorganisms in a sample microbe
8/24/2016 9 Summary • Samples must be collected using aseptic techniques and held in such a way as to prevent the growth of microoganisms. • Food samples should be blended or homogenized prior to examination. • Standard plate count is used to provide an estimate of the number of microorganisms in a sample 33 Summary • Surface test methods are essential in determining whether food contact surfaces are being maintained in a hygienic state • Rapid methods based on conventional biochemical methods are now available, reducing the time required to identify a microbe 34