MILK QUALITY & SAFETY TESTING MANUAL For Afghanistan

Transcription

1 MILK QUALITY & SAFETY TESTING MANUAL For Afghanistan AUTHORS PAUL EBNER, PhD Associate Professor, Purdue University, College of Agriculture, Animal Sciences MOHAMMAD ALAM GHORYAR, PhD Associate Professor, Herat University, Faculty of Agriculture AS-631 Afghan Milk Manual.indd 1 SHOAIB AHMAD SHAKHES, DVM, MVSc. Associate Professor, Herat University, Faculty of Veterinary Science AHMAD ZIA GHANIZADAH, MSc. Associate Professor, Herat University, Faculty of Science

2 AUTHORS Paul Ebner, PhD Associate Professor, Purdue University, College of Agriculture, Animal Sciences Mohammad Alam Ghoryar, PhD Associate Professor, Herat University, Faculty of Agriculture Shoaib Ahmad Shakhes, DVM, MVSc., Associate Professor, Herat University, Faculty of Veterinary Science Ahmad Zia Ghanizadah, MSc., Associate Professor, Herat University, Faculty of Science Prepared for the USWDP Food Technology Bridge Program at Herat University Front cover photo credit: Zahra Rahimi AS-631 It is the policy of the Purdue University Cooperative Extension Service that all persons have equal opportunity and access to its educational programs, services, activities, and facilities without regard to race, religion, color, sex, age, national origin or ancestry, marital status, parental status, sexual orientation, disability or status as a veteran. Purdue University is an Affirmative Action institution. This material may be available in alternative formats. January EXT-INFO Order or download materials from Purdue Extension The Education Store 2 AS-631 Afghan Milk Manual.indd 2

3 TABLE OF CONTENTS INTRODUCTION 4 MILK QUALITY TESTS 8 Physical Appearance 8 Clot on Boiling (COB) Test 9 Ethanol/Alcohol Test 10 Alcohol/Alizarin (AA) Test 11 Acidity Test 12 Resazurin Test 13 Protein Content Test 14 Milk Fat Content Test 15 Using Milk Analysis Machines 15 MICROBIAL TESTS 16 Total Plate Count (TPC) 16 Total Coliform Test 20 TESTS FOR ADULTERATION 21 Using a Lactometer 21 Using a ph meter 23 General Test for Inhibitors 24 Soap Test 25 Starch Test 26 Table Sugar Test 27 Acid-based Preservatives Test 28 Test for Sodium (bi-) Carbonate 29 Formaldehyde Test 30 Antibiotic Residue Tests 31 POWDERED MILK 32 Processing and Technology 32 Preliminary Physical Test of Powdered Milk 32 Measurement of Scorched Particles 33 Solubility Test 34 REFERENCES 35 TABLE OF CONTENTS 3 AS-631 Afghan Milk Manual.indd 3

4 INTRODUCTION There are many factors that affect milk quality and safety. At the same time, there are numerous quality and safety tests that can easily be done at the farm, in the market, or in the lab based on both microbiology and chemistry. Many quality and safety issues are associated with microbial growth in the milking environment and the milk itself. Most of the microbes are bacteria, but milk can also be contaminated with molds or yeast. Microbes can contaminate milk at almost any point of the production process. The types and concentrations of microbes found in milk are significantly influenced by: 1. animal husbandry and care; 2. animal health; 3. milk handling and storage; and 4. processing. Assuring milk quality is a team effort requiring farmers, animal scientists, veterinarians, chemists, microbiologists, and food technicians. Microbes: a general term for microorganisms including viruses, bacteria, yeasts, molds, and protozoa. Collection Microbes are regularly introduced to milk during collection. That means milk quality and safety starts at the farm. The biggest contributors to contamination during collection are animal health and unsanitary milking conditions. ANIMAL HEALTH Milking animals are often affected by mastitis. Mastitis is inflammation most often caused by bacterial infections of the udder. When bacterial concentrations are high, bacteria can be transferred to milk during the milking process. Mastitis can be controlled, even prevented, by proper sanitation and, in severe cases, the use of antibiotics. It is important to note that milk collected from cows being treated with antibiotics should be discarded because the antibiotics and antibiotic residues can enter the milk. Milk containing antibiotics or antibiotic residues is considered adulterated. 4 INTRODUCTION AS-631 Afghan Milk Manual.indd 4

5 SANITATION Unsanitary milking conditions regularly lead to contamination of milk. Milking areas should be kept as clean as possible. Accumulation of feces/urine greatly increases the likelihood of microbial contamination of milk. Similarly, milk collectors can easily introduce bacteria to both the milking animal and the collected milk through unwashed hands and clothing. Improper or insufficient cleaning of milk collection containers often leads to contamination of milk. Milk is high in nutrients and is actually often used to grow bacteria in the lab. As such, milk left in a collection container even trace amounts will support the growth of many types of bacteria. If new milk is added to the contaminated container, it too becomes contaminated. Therefore, it is important to use containers that can be easily cleaned. However, if the wrong chemicals are used to clean milk collection containers, or if containers are not properly rinsed after cleaning, new milk can become contaminated with these chemicals. Such contaminated milk is not suitable for consumption in any form, even when processed into cheese, yogurt or other dairy products. Mastitis: Inflammation of the udder and udder tissue. In some cases, the udder and tissue can be visibly damaged. Other cases are subclinical in that the udder is infected, but may not show any symptoms. Mastitis is often transferred from cow to cow through milk equipment, milkers (unwashed hands), housing, and bedding. Holding or Transportation It should always be assumed that fresh milk contains some level of bacterial contamination. Therefore, controlling or limiting microbial growth in milk is key to ensuring quality and safety. Milk is considered a TCS Time and Temperature Control for Safety food whose safety and quality is dependent on controlling time and temperature. Lower temperatures (e.g., refrigeration) limit microbial growth. Keeping fresh milk under unrefrigerated conditions (e.g., room temperature or higher) for long periods allows bacteria to rapidly grow to very high concentrations. In many countries, fresh milk must be refrigerated within 45 minutes of collection. Where that is not feasible, alternative measures to limit microbial growth must be used. Impact of Contamination Milk with high levels of bacterial contamination is problematic for several reasons. Foremost is safety. Dairy environments (e.g., animals, feces, barns, milking equipment, etc.) are often contaminated with common bacterial pathogens such as Salmonella, E. coli, and Campylobacter. These bacteria can cause diarrhea, renal failure, and, in severe cases, death. They are especially serious for infants, children and the elderly. The second issue is quality. Milk with higher levels of contamination spoils very quickly. When bacteria are allowed to grow to high levels, milk can become very acidic, producing sour flavors. With some time, sour milk will coagulate and, in general, is not usable. This can significantly reduce shelf life and profitability. Finally, high levels of contamination can impact further processing of milk into products such as cheese, cream, ice cream, and yogurt, as we will see later. Shelf life: How long a product can remain on a store shelf before it becomes spoiled, rotten, or otherwise unusable. The shelf life of high-quality, pasteurized, and refrigerated milk is generally around 7 to 10 days. INTRODUCTION 5 AS-631 Afghan Milk Manual.indd 5

6 Pasteurization the use of heat to kill microbes significantly increases the quality and safety of milk. For milk, minimum pasteurization standards require heating milk to 63ºC (143ºF) for 30 minutes. Pasteurization is designed to kill pathogenic bacteria, but not all bacteria. Milk pasteurized under standard conditions should not be considered sterile. Milk that has high amounts of microbial growth before pasteurization will still spoil much more quickly after pasteurization than milk with low amounts of microbial growth. Assessing Milk Quality There are numerous easy and rapid tests to determine milk quality. These tests rely on well-defined properties of milk, including its basic composition and the presence or absence of certain compounds. MILK COMPOSITION Milk is approximately 87% water. The remaining constituents are referred to as solids and are what separates many of the physical properties of milk from other liquids. Total solids (TS) refers to all non water components of milk, including fats, proteins, sugars, vitamins, and minerals. Fats make up about 4% of fresh milk; that varies slightly depending on animal species and breed. The remaining solids are often called solids non fat (SNF). Of the SNF portion, the majority is protein, which makes up about 5% of milk, and sugars, which make up about 3.5% of milk. The remainder is vitamins and minerals (0.5%). The total solids give milk distinct qualities from other liquids. One of the first milk quality tests is to measure density. The proteins, sugars, vitamins, and minerals add weight to milk. Because the composition of milk varies only slightly from animal to animal, the density of fresh milk should always be within an established range (see table below). When water is added to milk, or fat is removed, or adulterants are added, the density of the milk often changes. The density of milk is easily measured with a lactometer; that process is described later in the Using a Lactometer section. MICROBIAL CONTAMINATION The total plate count test and the coliform test each is described later are two of several methods that use standard microbiology to measure microbial growth and concentrations in milk. When bacteria grow, they often produce high amounts of acid. In milk, high amounts of acid produce sour flavors that significantly reduce shelf life and usability. Many milk quality tests are designed to measure contamination based on the ph, or acidity. There are tests, described below, that can give a preliminary indication that the acidity of the milk is too high (qualitative test) as well as tests that can more closely measure the percentage or amount of acidity (quantitative tests). 6 INTRODUCTION AS-631 Afghan Milk Manual.indd 6

7 ABNORMAL MILK/ADULTERATION. Milk is easily and often adulterated, not always with the consumer s best interests in mind. For example, collected milk can be combined with colostrum, the milk produced by a mother immediately after giving birth. It is generally high in antibodies and other proteins that can affect taste, color, usability, and quality of milk. More often, however, milk is adulterated with additives that mask or hide problems, make milk appear to be of higher quality than it is, change its color, or increase its volume. When buffers, such as sodium carbonate, are added to old milk or milk with high bacterial growth, it prevents acidity while making the milk seem to be fresh. Water is added to milk to increase volume and, in turn, payment. Detecting water in milk is rather easy; diluted milk is less dense. To increase density, other adulterants, such as sugar, salt, and other solids, are added. Why would milk producers add chemicals that are high in nitrogen? Because when tested, such milk is judged to be higher in protein, and in systems where perceived quality, not quantity, determines the wholesale price, the producer s profit rises. Other chemicals may be added to milk to prevent growth of microbes. Some of the most common chemical preservatives, such as formaldehyde, are extremely toxic. These additions are always violations, and there are numerous cases where people were poisoned when the adulterant was a toxic substance. Table 1. General Properties of Milk PROPERTY WHOLE MILK, NON-HOMOGENIZED ph Acidity % Density Kg/L Acidity % Freezing point to C Water Total solids Fat Nonfat solids Protein Sugars Vitamins/mineral TPC Coliforms 87% (whole milk) 13% 4% (whole milk, nonhomogenized) 9% 3.5% 5.0% <0.5% <100,000 CFU/mL (raw milk)* <200 CFU/mL (pasteurized milk)* <100 CFU/mL (raw milk)* Undetectable (pasteurized milk)* *these standards differ from country to country. 7 AS-631 Afghan Milk Manual.indd 7

8 GENERAL MILK QUALITY TESTS Physical Appearance Clot on Boiling (COB) Test Ethanol/Alcohol Test Alcohol/Alizarin (AA) Test Acidity Test Resazurin Test Protein Content Test Milk Fat Content Test Using Milk Analysis Machines Physical Appearance To conduct a preliminary examination of a milk sample, first look at the container and assess its overall cleanliness. Examine seals or lids to see if they are working properly. Next, open the container and immediately smell the milk. The milk should not contain any off odors. Off odors can be produced by the animal s diet, chemical adulteration, natural hormones in the milk, the milk collection environment, and, most commonly, bacterial growth. Milk with high bacterial counts will have a characteristic sour odor. Fresh milk should appear white. Chemical adulteration and colostrum can produce off colors. If a small amount of milk is allowed to run down a polished surface, it should leave a streak of milk. Finally, the milk should be free of clumping. Milk that is spoiled or otherwise unusable due to microbial growth is often easy to identify based simply on appearance and odor. Most milk quality parameters, however, require more advanced testing for assessment. Several are described in the following pages. 8 GENERAL MILK QUALITY TESTS GENERAL MILK QUALITY TESTS AS-631 Afghan Milk Manual.indd 8

9 Clot on Boiling Test (COB) The COB test is an old but very easy test designed to determine the overall quality of milk. Though it should be considered a preliminary test, it can be easily done in the field or on the farm. In the COB test, the milk is boiled, and once the milk cools, physical characteristics are examined. When high-quality milk is boiled and cooled, it will remain a fluid with a general milk appearance. Milk that is acidic (an indication of bacterial growth) or milk that contains colostrum or other adulterations often cannot withstand boiling; once it cools, it will coagulate and form lumps or flakes. MATERIALS Milk sample Spoon Flame PROCEDURE 1. Start flame away from all flammable objects. 2. Place milk in spoon using a pipette or similar device. 3. Hold milk/spoon over flame until milk boils. 4. Allow milk to cool. 5. Check for coagulation. Add a small amount of milk sample to the spoon using a pipette or similar device. READING RESULTS 1. Milk with little to no microbial contamination/ growth. This milk has a neutral ph and withstands boiling and has a normal milk appearance once it cools. 2. Milk with lower ph due to bacterial growth. Such milk cannot withstand boiling and shows coagulation around the edges. 3. Milk with very low ph due to bacterial growth. Milk with a very low ph will show heavy coagulation and flakes when boiled. Be careful not to overfill the spoon, as the milk will easily boil over the sides once it is heated. NOTES ON THE COB TEST Hold the spoon/milk sample over a flame until milk thoroughly boils. GENERAL MILK QUALITY TESTS 9 AS-631 Afghan Milk Manual.indd 9

10 Ethanol/Alcohol Test The ethanol/alcohol test is a rapid way to determine overall quality of milk. It relies on how milk reacts with ethanol. High-quality or otherwise normal milk will not react with ethanol. Milk that is high in acidity (low ph), contains colostrum, or contains salt concentrates (indication of mastitis) often will coagulate or form flakes when exposed to ethanol. READING RESULTS MATERIALS Milk sample 68% ethanol (often abbreviated EtOH ) Mixing container PROCEDURE 1. Combine equal parts 68% ethanol and milk sample (example: 10 ml of milk sample + 10 ml of 68% ethanol). 2. Mix thoroughly. 3. Incubate at room temperature for 10 minutes. 4. Check samples for coagulation. READING RESULTS The samples pictured to the right are milk samples after the addition of ethanol and a 10-minute incubation at room temperature. Sample A is fresh milk with little to no microbial growth. It does not react with alcohol and retains a normal milk appearance. Sample B has a low ph (high acid) due to microbial growth. This milk reacts with alcohol, and heavy coagulation and flaking can be seen on the sides of the flask. Sample A Sample B NOTES ON ALCOHOL TEST 10 GENERAL MILK QUALITY TESTS AS-631 Afghan Milk Manual.indd 10

11 Alcohol/Alizarin (AA) Test The AA test is very similar to the basic alcohol/ethanol test. The only difference is the addition of alizarin to the alcohol. Alizarin is a color indicator that changes based on the ph of the milk. Because it can provide an estimate of the level of acidity, the AA test is an example of a semi-quantitative test. It is conducted similarly to the alcohol/ethanol test, but the analysis takes color changes into account. NOTES ON THE ALCOHOL/ALIZARIN TEST MATERIALS Milk sample Alizarin solution (4.0g alizarin dissolved in 1L 61% ethanol) Mixing container PROCEDURE 1. Combine equal parts alizarin solution and milk sample (example: 10 ml of milk sample + 10 ml of alizarin solution). 2. Mix thoroughly. 3. Incubate at room temperature for 10 minutes. 4. Check samples for coagulation, and record the color. READING RESULTS The normal ph of fresh milk is about High levels of microbial growth will produce acid, which lowers the ph of the milk. An estimation of the acidity of the milk samples can be made using the table below: MEASUREMENTS NORMAL SLIGHTLY ACIDIC ACIDIC ALKALINE ph or lower 6.8 or higher color red/brown yellow/brown yellow pink/purple physical characteristics *may have flakes. no coagulation no coagulation coagulation no coagulation A B C D E The picture above shows several milk samples combined with alizarin solution after a 10-minute incubation. Sample A is fresh milk with little to no bacterial growth. Once alizarin solution is added, there is no color change and it remains a red/brown color. Also, there is no coagulation. The samples increase in acidity from B to E due to higher levels of bacterial growth. With higher acidity, the samples increase in coagulation and become more yellow. GENERAL MILK QUALITY TESTS 11 AS-631 Afghan Milk Manual.indd 11

12 Acidity Test The acidity test, although only a little more involved than the alcohol based tests, provides more quantitative information. The test is used to determine the percentage of acidity. Like the alizarin test, the acidity test uses a color indicator (phenophtalein). The percentage acidity for normal-quality milk is about 0.16% to 0.18%. MATERIALS Milk sample Flask for mixing 10 ml pipette Burette (0.1 ml graduations) Mixing utensil (e.g., glass rod) Phenophtalein solution (0.5% phenophtalein in 50% ethanol) Sodium hydroxide (NaOH; base, 0.1M) PROCEDURE 1. Add 9 ml of the milk sample to a flask. 2. Add 1 ml of the phenophtalein solution to the milk sample. 3. Slowly and gradually, add sodium hydroxide from the burette to the milk sample. 4. Stop the assay when the milk sample reaches a pink color. Record the amount of sodium hydroxide needed to obtain the pink color. RECORDING RESULTS Add 0.1M NaOH to the milk sample until it reaches the color of the picture to the right. Record the volume (ml) of sodium hydroxide needed to develop the pink color. Divide this number by 10 to get the percentage acid of the milk sample. USING A BURETTE 1. Fill the bottom burette container with 0.1M NaOH. Squeeze the container to push liquid into the burette. 2. Fill the burette until the bottom of the meniscus is at zero. EXAMPLE 1.8 ml of NaOH were required to change the milk sample from normal milk color to pink. Therefore, the percentage acidity is 1.8 / 10 = 0.18%. This would be a normal acidity percentage for fresh milk without much contamination or microbial growth. Milk that is acidic will require higher volumes of NaOH to become pink and will therefore have a higher acidity percentage. The addition of sodium hydroxide is stopped when milk reaches a pink color similar to the sample here. 3. Open the valve slowly to adjust the flow rate of 0.1M NaOH. 12 GENERAL MILK QUALITY TESTS AS-631 Afghan Milk Manual.indd 12

13 Resazurin Test The Resazurin Test is a common way to determine overall bacterial growth in a milk sample. Like the alizarin and acidity tests, it uses a color indicator, which allows a qualitative assessment of milk quality. The test requires an incubator where milk samples can be incubated at 37ºC. The test is somewhat rapid (1 hour), but can be extended for up to three hours. MATERIALS Milk sample Sterile test tubes with stoppers Resazurin solution (~10 g resazurin dissolved in 200 ml distilled/sterile water) PROCEDURE 1. Add 1 ml of resazurin solution to the test tube. 2. Add 9 ml of milk sample to the test tube containing resazurin solution. 3. Place stopper on test tube. 4. Place tube in 37ºC incubator. 5. Once sample reaches approximately 37ºC, invert tube to mix sample. 6. Incubate 1 hour at 37ºC. 7. Observe and record results after 1 hour. The picture below shows several milk samples after a resazurin test. Sample A is fresh milk with little to no bacterial growth. After the resazurin solution is combined with the milk sample and incubated, goodquality milk will remain blue with no color change. Samples B through E increase in acidity (from left to right) due to higher amounts of bacterial growth. The color of the samples changes as well, becoming light purple to pink. Sample E is extremely acidic and shows heavy coagulation along with a color change. COLOR Blue (no color change) Blue to purple Purple to pink Pink to whitish pink White QUALITY Excellent Good Fair Poor Bad NOTES ON THE RESZURIN TEST GENERAL MILK QUALITY TESTS 13 AS-631 Afghan Milk Manual.indd 13

14 Protein Content Test (Formaldehyde Titration) This test will give an estimate of the percentage protein in a milk sample. The assay uses formaldehyde, which reacts with amino acids of proteins. When formaldehyde is mixed with protein, the solution becomes acidic. The protein percentage is estimated by measuring the amount of acid produced upon the addition of formaldehyde. This assay is conducted in the same way as the acidity test and uses the same equipment as well as the same color indicator. The pictures included in the section on the Acidity Test can be used as instructionals. MATERIALS Flask (>50mL) Burette (0.1 ml graduations) 10 ml pipette Stirring rod Potassium oxalate (saturated aqueous) saturated means the highest amount of the compound that will dissolve in water. For potassium oxalate, it is approximately 35g in 1L of water. Phenophtalein solution (0.5% phenophtalein in 50% ethanol) 40% formalin solution 0.1 M sodium hydroxide (NaOH) PROCEDURE 1. Add 10 ml of milk sample to flask. 2. Add 0.4 ml of potassium oxalate and 0.5 ml of phenophtalein solution to the milk sample. 3. Allow sample to stand for 2 minutes and add sodium hydroxide (with burette) until the sample becomes pink (see Acidity Test for pink color sample). 4. Add 2 ml of formalin to return the sample to normal color. 5. Continue to add sodium hydroxide until sample becomes pink again. 6. Record the amount of 0.1M NaOH needed to turn the milk sample pink AFTER the addition of formaldehyde. RECORDING RESULTS The percentage protein is the number of ml of sodium hydroxide added after the addition of formaldehyde, multiplied by EXAMPLE 2.1 ml of NaOH were required to make the milk sample turn pink AFTER formaldehyde was added. Therefore, the percentage protein of the milk sample is 2.1 X 1.74 = 3.65%. The protein of milk should be approximately 3.6% to 4.0%. Pasteurized milk often has a lower protein percentage (~3.6%). Sheep milk is in general is often higher in protein (~4.0%). While the percentage of protein can differ based on animal breed and diet, milk samples that have protein percentages much lower or much higher than these numbers may be adulterated. NOTES ON THE PROTEIN CONTENT TEST 14 GENERAL MILK QUALITY TESTS AS-631 Afghan Milk Manual.indd 14

15 Milk Fat Content Test There are several ways to measure the percentage of fat in a milk sample. Most of the tests use sulfuric acid in some manner. This is because all components of milk except fat are soluble in sulfuric acid. Therefore, adding sulfuric acid will separate the fat content from the rest of the milk. The test described below likely requires the least amount of reagents and equipment. MATERIALS Milk sample 90% Sulfuric acid Graduated container that can be centrifuged at low speeds PROCEDURE 1. Combine equal parts sulfuric acid and milk sample (e.g., 20mL sulfuric acid + 20mL milk sample). 2. Thoroughly mix sample. 3. Centrifuge at low speed (e.g., 1,000 rpm) for 1 minute. Fat has less mass than other milk constituents, including water, so it will rise to the top. 4. Measure fat fraction. RECORDING RESULTS The percentage of fat is calculated by dividing the fat fraction by the amount of milk in the sample. For example, if the fat fraction is 0.7mL then the fat percentage would be 0.7mL / 20mL = or 3.5%. Using Milk Analysis Machines Different types of equipment and machines are available for milk analysis. Such machines can estimate fat percentage, protein percentage and lactose percentage. Most will also estimate freezing point or density as an indication of whether water has been added to the milk. Milk analyzers usually use one of two types of technology ultrasound or infrared. Ultrasound analyzers have the benefit of often being less expensive; infrared analyzers are generally more accurate. 1. Example of a low-cost ultrasound milk analyzer. 2. Milk is drawn into the analyzer from one sample. The analyzer also contains a ph probe to simultaneously measure acidity from a second sample (in researcher s hand.) NOTES ON THE FAT CONTENT TEST NOTES ON MILK ANALYSIS MACHINES GENERAL MILK QUALITY TESTS 15 AS-631 Afghan Milk Manual.indd 15

16 MICROBIAL TESTS Total Plate Count (TPC) Pour-plate TPC Spread-plate TPC Total Coliform Test Total Plate Count (TPC) All fresh milk will contain bacteria. TPC tests measure overall bacterial levels. They measure total bacteria but do not distinguish between non-harmful bacteria and harmful or pathogenic bacteria. Other tests are used to detect specific pathogens, such as Salmonella, E. coli, or Campylobacter. Higher bacterial levels can significantly reduce the shelf life of milk and usability. Pasteurization will destroy many of these bacteria, but if levels are too high or milk is not refrigerated for long periods prior to pasteurization, high bacterial levels will produce acid and quickly spoil the milk. There are different ways to conduct a TPC assay. Described below are the pour plate method and the spread plate method. They are similar. The pour plate is generally more accepted, but the spread plate method is sufficient. Additionally, the terms APC (aerobic plate count), SPC (standard plate count), and TPC are often used interchangeably, but TPC is probably used most often. MATERIALS Medium (most simple media such as LB, nutrient, etc. are acceptable)» Enzymatic digestion of casein 5.0 g» Yeast extract 2.5g» Glucose, anhydrous 1.0g» Agar 17g» Bring up to 1 L with distilled water» Autoclave or otherwise sterilize Ringer s solution (other isotonic solutions such as PBS can also be used. Ringer s solution is isotonic relative to most animal fluids)» NaCl 6.5g» KCl 0.42g» CaCl g» Calcium bicarbonate 0.2g» Bring up to 1L Petri dishes Test tubes Pipettes Cell spreader Ethanol Incubator (37ºC) If possible, it s best to do these tests in duplicate. Taking an average of your results will give you a more accurate estimation of the bacterial concentration. 16 MICROBIAL TESTS AS-631 Afghan Milk Manual.indd 16

17 Pour-plate TPC SAMPLE PREPARATION 1. Add 9mL of Ringer s solution to 7 individual test tubes. Label the test tubes 1, 2, 3, etc. 2. Add 1mL of milk sample to the tube labeled 1 and mix thoroughly. 3. Take 1mL of the 1 mix sample and add it to the tube labeled Repeat for the remaining tubes. 1. Take care to thoroughly mix each tube before beginning the following dilution. 2. The final dilution should have 1mL more than the other dilutions as shown by the white arrow. PLATING 1. Add 1mL of the undiluted milk sample to an empty, sterile petri dish. 2. Add 9mL of medium (cooled to below 45C) to the petri dish and mix gently, but thoroughly. Label this plate 1 3. Repeat for all test tubes, labeling each plate with the corresponding test tube number ( 1, -2, -3, etc.) 4. Incubate at 37ºC for 24 hours. 1. Add 1mL of the diluted sample directly to the sterile, empty petri dish. 2. Add 9mL of cooled medium/ agar to the sample in the petri dish. 3. Gently mix the sample and the agar together by moving left to right and up and down. NOTES ON POUR-PLATE TPC MICROBIAL TESTS 17 AS-631 Afghan Milk Manual.indd 17

18 Spread-plate TPC With the spread plate method, the diluted milk samples are added directly to petri dishes that already contain agar medium. SAMPLE PREPARATION 1. Add 900uL Ringer s solution to 7 individual microcentrifuge tubes. Label the tubes starting with 2, and continue 2. Add 100uL of milk sample to the tube labeled 2 and mix thoroughly. 3. Take 100uL of the 2 mix sample and add it to the tube labeled Repeat for the remaining tubes. PLATING 1. Add 100uL from the undiluted milk sample to the first plate and spread the sample with a sterile spreader. Label this plate Repeat for samples from each test tube labeling each plate with the corresponding test tube number (-2, -3, -4, etc.) 3. Once plates have dried, turn them upside down before placing them in the incubator. This is to prevent condensation from dripping on to the agar. 4. Incubate at 37ºC for 24 hours. 1. Dip the spreader in ethanol. 2. Carefully hold spreader over flame to burn ethanol and sterilize spreader. 3. Apply 100 ul of the sample to the center of the plate. 4. Spin the plate and spread bacteria uniformly from the inside of the plate to the outside, being careful not to touch the edges of the plate. NOTES ON SPREAD-PLATE TPC 18 MICROBIAL TESTS AS-631 Afghan Milk Manual.indd 18

19 RECORDING RESULTS Plates with 25 to 250 colonies should be used to most accurately count colonies. When there are >250 colonies on a plate, do not try and count them. Just record that plate as TNTC (too numerous to count) and go to the next readable plate. Bacterial concentrations are reported in colony forming units (CFU) per ml. As such, some calculations have to be done to determine the true concentration. With the pour-plate method, 1mL of the milk sample was added to the first plate. If that plate had 100 colonies, the bacterial concentration of the milk sample would be 100 CFU/mL. This is an unlikely scenario with raw milk. The following example is more realistic. Example of the type of plate that is ideal for counting colonies. There are between 25 and 250 colonies that are well isolated from each other. It normally takes some practice and experience to produce accurate plate counts. Going slowly, thoroughly mixing samples, changing pipette tips whenever possible, and always plating the highest dilutions first can help in reducing error. PLATE Colonies TNTC TNTC TNTC TNTC TNTC TNTC In this scenario, taking into account the dilution and choosing the -3 plates for calculation they have between 25 and 250 colonies the estimated bacterial concentration would be: ((205 X 10 3 ) + (190 X 10 3 )) / 2 = X 10 3 CFU/mL Bacterial concentration are normally recorded in scientific notation, so the concentration would be 1.98 X 10 5 CFU/mL. Note that you will often see bacterial concentrations recorded in log10 as well. So, here the concentration could also be recorded as log10 (1.98 X 10 5 ) = 5.29 log10 CFU/mL. Calculating the bacterial concentration with the spread-plate method is similar, but there is an extra dilution: Only 100 ul was added to the plate instead of 1 ml, as in the pour-plate method. Therefore, there is no 1 plate. Directly plating only 100 ul of the milk sample is already diluted 1/10, so that is the -1 plate. PLATE Colonies TNTC TNTC TNTC TNTC As in the scenario above, you would choose the -3 plates, because they have colonies. The estimated bacterial concentration would be: ((185 X 10 4 ) + (196 X 10 4 )) / 2 = X 10 4 or 1.90 X 10 6 CFU/mL. In log10 it would be 6.27 log10 CFU/mL. MICROBIAL TESTS 19 AS-631 Afghan Milk Manual.indd 19

20 Total Coliform Test TPC tests measure total bacteria in a milk sample (good and bad). A coliform test is more specific. Coliforms are bacteria that are generally found in the intestinal systems of animals and, in turn, animal feces. So, the presence of coliforms in a milk sample (or water sample, etc.) is an indication of fecal contamination. The coliform test is done just like a TPC test but uses a selective medium. This medium allows coliforms to grow while inhibiting other types of bacteria, thereby selecting coliforms. In the protocol here, violet red bile agar (VRBA) is used; it s regularly recommended for testing milk and dairy products for coliforms. It is a standard medium to detect lactose fermenting coliforms. The medium contains crystal violet and bile salts, which inhibit the growth of most gram-positive bacteria. Neutral Red is a dye and is included as a ph indicator. Lactose fermenting colonies (likely coliforms) appear pink/red and may be surrounded by pink/red zones, indicating precipitation of bile salts. Note that VRBA does not get autoclaved. It is only boiled prior to pouring plates. The procedure is exactly the same as the TPC either the pour-plate or spread-plate method. Only the growth medium is different. 1. E. coli grown on VRBA agar. Typical colonies are pink. 2. Closer detail of E. coli on VRBA agar. Lighter pink zones can be seen surrounding the bacterial colonies, indicating precipitation of bile salts. NOTES ON THE COLIFORM TEST 20 MICROBIAL TESTS AS-631 Afghan Milk Manual.indd 20

21 TESTS FOR ADULTERATION Using a Lactometer Using a ph meter General Test for Inhibitors Soap Test Starch Test Table Sugar Test Acid-based Preservatives Test Test for Sodium (bi-) Carbonate Formaldehyde Test Antibiotic Residue Tests Using a Lactometer A lactometer can be used for several purposes. It is basically a tool to measure the specific gravity or mass of milk. For example, milk has a higher specific gravity than water, so a lactometer can be used to test for added water. It can also be used to estimate total solids of milk. The total solids are basically the non-water portions of milk and make up about 13% of all milk (~4% fat and ~9% protein, lactate and other non-fat solids). The quality of milk for further processing, such as cheese or yogurt, is dependent in many ways upon the total solids concentration of the starting milk. MEASURING SPECIFIC GRAVITY OF MILK MATERIALS Cylinder (<1L) Lactometer PROCEDURE 1. Heat fresh milk sample to 40C and cool to room temperature. (Heating reduces the size of fat globules, which can impact the lactometer) 2. Mix cooled milk sample and add to cylinder. Add enough milk so that the lactometer will not touch the bottom of the cylinder. 3. Lower the lactometer into the milk sample and allow it to settle (float). This can take up to 1 minute. 4. Record the lactometer reading at the bottom of the meniscus. 5. Record the temperature. 1. Gently insert the lactometer into the milk sample. 2. Once the lactometer settles, read the density reading at the bottom of the meniscus. 3. Read the temperature on the thermometer portion of the lactometer. 21 AS-631 Afghan Milk Manual.indd 21

22 RECORDING RESULTS In the example above, the lactometer reads 120 and the temperature is 65ºF. If the milk is too warm or too cold, the lactometer reading has to be corrected. The acceptable range with the lactometer pictured above is 60ºF +/10 degrees. The lactometer reading must be adjusted for the temperature if it is outside of this range (+1 for every 10 degrees above 60ºF and 1 for every 10 degrees below 60ºF). The lactometer pictured above is New York style lactometer. The quevenne style is also common throughout the world. The only difference is the scale. With both lactometers, a reading of 0 translates to a specific gravity of 1 (water). To determine the specific gravity with the New York scale: Specific gravity = lactometer reading X ,000 From the sample above: READING THE MENISCUS 1. Here the lactometer is placed in water. Water is much less dense than milk, and the lactometer sinks to a much lower reading (0). Specific gravity = 120 X , = Kg/mL This is the normal specific gravity for fresh, unadulterated milk. The examples below illustrate how a lactometer can be used as a first step in determining if milk is adulterated. NOTES ON USING A LACTOMETER 2. This milk sample is diluted with water (about 50mL water was added to 450mL of milk). The milk appears normal, but its density is much lower as indicated by the lactometer reading (80). 22 TESTS FOR ADULTERATION AS-631 Afghan Milk Manual.indd 22

23 Using a ph Meter Used properly, a ph meter is a valuable tool for testing milk quality. Most ph meters require regular calibration for accurate measurement. A proper calibration prior to starting any experiments can save a lot of time and produce much more accurate results. Different ph meters have different protocols for calibration; calibration should follow the manufacturer s instructions. Below is a standard calibration protocol. NOTES ON USING A ph METER 1. Start by cleaning the probe. This is done by rinsing the probe with clean, distilled water. 2. Dry the probe gently with soft, clean tissue paper or a towel. 3. Be especially careful with the electrode end f the probe. It is easily damaged. To dry it, simply blot the end with the paper. 4. Press the CAL button and insert the probe into the ph 7.0 solution. 5. Once the screen stops blinking, remove the probe. The meter will adjust to ph After washing the probe, press the CAL button and insert the probe into the ph 4.0 solution. 7. Once the screen stops blinking, remove the probe. The meter will adjust to ph Repeat the procedure one last time with the ph 10.0 solution. The ph meter is now calibrated. 9. After calibration, insert the probe into the test sample. Record final ph reading. TESTS FOR ADULTERATION 23 AS-631 Afghan Milk Manual.indd 23

24 General Test for Inhibitors Many adulterants are added to milk to slow or stop bacterial growth. In doing so, these adulterants extend the shelf life of milk or give the appearance that the milk is fresh. Some of these adulterants are health hazards (e.g., high concentrations of antibiotics, formaldehyde, some acids, etc.). All adulterants that inhibit bacterial growth can significantly impact the further processing of milk because many of these processes rely on bacterial fermentation, such as in cheese and yogurt production. The General Test for Inhibitors can be used as a preliminary test to determine if milk contains any bacterial-inhibiting adulterants. MATERIALS Milk samples Bacterial starter culture (pure cultures can be used, but fresh yogurt or buttermilk can also be used) Test tubes Incubator 1. Two milk samples that appear identical, but one contains an inhibitor. PROCEDURE 1. Heat fresh milk to 90ºC for 5 minutes. 2. Cool milk to 37ºC or below. 3. Add 1mL of starter culture to each milk sample. 4. Incubate milk samples at 37ºC for 3 hours. 5. Measure acid production using the Acidity Test or by ph meter. RECORDING RESULTS When bacteria grow in milk, they produce acid. Many inhibitors (e.g., antibiotics, formaldehyde, preservatives, etc.) will reduce bacterial growth. Therefore, if inhibitors are present in the milk sample, there will be reduced bacterial growth of the starter culture, compared to milk without any inhibitors, and reduced acid production. The percentage of acidity in milk with inhibitors will then be lower than that of normal fresh milk. If using a ph meter, after incubation with the starter culture, milk with inhibitors will have a higher ph than that of milk without inhibitors. 2. After adding the starter culture and incubating for 3 hours, the ph of the milk without the inhibitor is 4.82, indicating the growth of bacteria and acid production. NOTES ON USING GENERAL TEST 3. After the same treatment, the ph of the milk sample that contains the inhibitor is only 5.63, indicating that the milk contains some substance that is inhibiting the growth of bacteria and the production of acid. 24 TESTS FOR ADULTERATION AS-631 Afghan Milk Manual.indd 24

25 Soap Test When soap is added to milk, the milk often becomes thicker. Therefore, milk with soap as an adulterant can appear to have more cream. Soaps are bases, so testing for the presence of soap is a basic test of ph. This can be done using the same phenophtalein solution that is used for the Acidity test. MATERIALS Milk sample Phenophtalein solution (0.5% phenophtalein in 50% ethanol) PROCEDURE 1. Add 10mL of milk sample to test tube. 2. Add phenophtalein solution in drops to the milk sample. 3. Check for color changes. NOTES ON THE SOAP TEST RECORDING RESULTS 1. Two milk samples appear identical, but one has been adulterated with soap. 2. When phenophtalein solution is added to both samples, the adulterated sample becomes pink, indicating the presence of a basic chemical soap, in this case. TESTS FOR ADULTERATION 25 AS-631 Afghan Milk Manual.indd 25

26 Starch Test Like soap, starch can be added to milk as a thickener to give the appearance that the milk is high in cream. Starch can also be added to adulterated milk to increase its density; sugars are part of the total solids of milk and part of what makes milk heavier than water. In doing so it can mask the effects of diluting milk with water by giving the appearance that the adulterated milk has the same density as unadulterated milk. Starch is a complex carbohydrate (polysaccharide) that reacts with iodine, unlike the primary milk sugars lactose and galactose, which are single and disaccharides. Thus, the presence of starch can easily be detected by adding a small amount of iodine. MATERIALS Milk sample Test tubes Water bath (or other device that can hold boiling water) Iodine solution (1%) PROCEDURE 1. Add 5mL milk sample to a test tube that can be boiled. 2. Boil milk samples for 5 minutes. 3. Allow samples to cool to room temperature. 4. Add 2 to 3 drops of iodine solution to each sample. 5. Observe color changes. NOTES ON THE STARCH TEST RECORDING RESULTS 1. Two milk samples that appear identical. One sample has been adulterated with starch. 2. After boiling and the addition of iodine, the sample adulterated with starch becomes dark blue to black. 26 TESTS FOR ADULTERATION AS-631 Afghan Milk Manual.indd 26

27 Table Sugar Test Like starch, table sugar can be added to milk to increase the total solids. For instance, if milk is diluted with water to increase its volume, the milk density will decrease. Adding table sugar increases the concentration of solids and, in doing so, increases its density to appear like normal milk. Sucrose is also a component of most kinds of synthetic milks. There is a fundamental difference between table sugar (sucrose) and the sugars present in normal milk. Sucrose is ketose sugar that will react with resorcinol when it is reduced. To test for sucrose, an acid is added to reduce sucrose. Resorcinol is then added. The reduced sucrose will then react with resorcinol to form a red color. TESTS FOR ADULTERATION 27 AS-631 Afghan Milk Manual.indd 27

28 Acid-based Preservatives Test Acids are often used in food preservation, but they should not be added to milk that is sold as fresh milk. Preservatives are inhibitors and will reduce bacterial growth, which will extend the milk shelf life, but preservatives also interfere with any fermentation processes needed to make yogurt, cheese, or similar products. The test below is designed to identify acid-based preservatives, such as benzoic acid, but the General Test for Inhibitors can also be used to detect inhibitors in general. MATERIALS Milk samples Sulfuric acid Ferric chloride (5%) PROCEDURE 1. Place 10mL of milk sample in an appropriate test tube. 2. Add approximately 500uL of sulfuric acid to sample and shake thoroughly. 3. Add approximately 500uL of ferric chloride solution and shake well. 4. Observe any color changes. RECORDING RESULTS The presence of acid-based preservatives will result in a color change in the adulterated milk sample. These color changes can range from red to purple. Milk that has not been adulterated will remain white to slightly yellow due to sulfuric acid. RECORDING RESULTS 1. Two milk samples that appear identical, but one sample has been adulterated with an acidbased preservative. 2. The color change in the right sample indicates the presence of an acid based preservative. 28 TESTS FOR ADULTERATION AS-631 Afghan Milk Manual.indd 28

29 Test for Sodium (bi-) Carbonate Sodium carbonate and sodium bicarbonate can be added to milk to prevent acidity. As milk ages, bacteria will grow and produce acid. Sodium carbonate is a buffer and is used to neutralize that acid. Therefore, sodium carbonate is added to milk with high amounts of bacteria to raise the ph and give the appearance that the milk is fresh. Below are two basic tests for testing for the presence of sodium carbonate. RECORDING RESULTS Sodium Carbonate Test 1 MATERIALS Milk samples Rosalic acid (1%; also called aurin or corralin) Test tubes PROCEDURE 1. Add 10mL of milk sample to a test tube. 2. Add 100uL of rosalic acid solution. 3. Observe any color changes. 1. Three milk samples that appear identical, but two samples have different concentrations of sodium carbonate. Sodium Carbonate Test 2 MATERIALS Milk samples Beaker, test tube, or similar container Water bath, heat block, or similar heating device PROCEDURE 1. Add 2 to 3mL of milk sample to a test tube. 2. Heat milk samples at 90C until milk has evaporated. If other heat sources are used, be careful not to burn milk. 3. Observe color changes. 2. The A sample is unadulterated milk. Samples B and C contain increasing amounts of sodium carbonate. Upon heating, the milk begins to evaporate and the sodium carbonate leaves a yellow to orange residue. NOTES ON THE ACID-BASED PRESERVATIVES TEST TESTS FOR ADULTERATION 29 AS-631 Afghan Milk Manual.indd 29

30 Formaldehyde Test As mentioned previously, formaldehyde can be added to milk to cover up or mask contamination problems. This poses a clear human health hazard formaldehyde is highly toxic. Like in the general test for inhibitors, it is always a good idea to include a sample of highquality, fresh, unadulterated milk when conducting the formaldehyde test for comparison. MATERIALS Milk sample Test tubes 10% ferric chloride solution 90% sulfuric acid PROCEDURE 1. Add 5mL of milk sample and 5mL of sterile water to a test tube. 2. Add 1 drop of 10% ferric chloride solution to 10mL of sulfuric acid. 3. Add the ferric chloride/sulfuric acid solution to milk sample by pouring gently along the side of the tube. 4. Observe color changes. RECORDING RESULTS Milk that contains formaldehyde will have a purple/red color when the ferric chloride/sulfuric acid is added to the milk. Normal milk will be green and will change to brown if left to stand. This test will detect formaldehyde at 10ppm or about 1mL of formaldehyde in 100 liters of milk. NOTES ON THE FORMALDEHYDE TEST RECORDING RESULTS 1. Add the ferric chloride/sulfuric acid solution to the milk sample by applying the liquid to the side of the tube in drops. 2. The sample labeled A is fresh, unadulterated milk. After sitting, the bottom becomes dark brown. The sample labeled B is milk adulterated with formaldehyde. The bottom is a red/purple. 30 TESTS FOR ADULTERATION AS-631 Afghan Milk Manual.indd 30

31 Antiobiotic Residue Tests When animals are treated with antibiotics, antibiotic residues can easily pass to the milk. This can cause problems for further processing that relies on fermentation (e.g., yogurt, cheese, etc.) Therefore, it is important to separate milk from animals being treated with antibiotics from milk from untreated animals. Numerous tests are available to test for the presence of general and specific antibiotics in the milk. Basic tests simply combine milk samples with bacterial cultures and tests for bacterial growth. If antibiotics are present, bacterial growth will be inhibited. In these tests, a color indicator is usually included to measure ph. If bacteria grow and produce acid, the sample will change colors (negative). Samples that do not change color indicate that bacterial growth is inhibited (positive). In this way, the tests are identical in principle to the General Test for Inhibitors. Tests for specific antibiotics usually rely on immunochemistry. Milk samples are added to a paper strip that contains compounds that will bind to any antibiotics residues present. If bound, the strip changes colors, indicating a positive sample. 1. Examples of antibiotic residue test strips used in dairy industries around the globe. A small milk sample is applied to the strip, and the strip is incubated. If antibiotics are present, bands (red) appear in distinct patterns. TESTS FOR ADULTERATION 31 AS-631 Afghan Milk Manual.indd 31

32 POWDERED MILK Processing and Technology Preliminary Physical Test of Powdered Milk Measurement of Scorched Particles Solubility Test Processing and Technology Quality and safety testing of powdered milk is, in most cases, similar to tests used for fluid milk. Once the milk is reconstituted, all tests that are used for fluid milk can be used for powdered milk. It is important to note that powdered milk, once reconstituted, should retain all of the qualities and characteristics of fluid milk. Below is a table that describes some acceptable properties of powdered milk Whole milk powder Partially skimmed milk powder Skim milk powder MILKFAT (%) MAXIMUM WATER (%) TOTAL SOLIDS (%) Two basic methods are used to remove water from milk to produce powdered milk: roller drying and spraydrying. Of the two methods, roller-drying uses higher heat, which can damage milk proteins. Consequently, roller-dried powdered milk is often less soluble, has off flavors and is, generally, of lower quality than spray-dried powdered milk. Preliminary Physical Test of Powdered Milk Since powdered milk is simply milk with the water removed, it should retain all of the qualities of fluid milk. Upon initial inspection, the powder should be a uniform white to slightly off-white (cream color). It should be free of any brown or yellow colors. The powder itself should taste slightly sweet and have no off tastes or odors that are also found in fluid milk. Powdered milk should also be free of any burnt flavors or odors. Finally, the powder should appear dry, with uniform granules, and free of clumps or lumps that indicate improper moisture content or storage. 32 POWDERED MILK AS-631 Afghan Milk Manual.indd 32

33 Measurement of Scorched Particles As mentioned above, roller-drying uses high heat, which can damage powdered milk. Often, high heat will produce scorched or burnt milk. This can significantly lower the solubility and nutritive value of the powdered milk. Testing powdered milk to determine the level of scorched particles is easy, quick, and involves very few materials. RECORDING RESULTS Comparing the filters to the standard below will give the milk a grade (A to D) with A being highest quality and D being lowest quality. MATERIALS Milk sample Filter pad Flask Scorched particle standard PROCEDURE 1. Combine powdered milk (25.0g skim milk or 32.5g whole milk) with 250mL sterile water. 2. Mix at high speed for 1 minute. 3. Let milk sit for 10 minutes. 4. Pass milk sample through filter. 5. Collect filter and compare visually to the scorched particle standard. 1. These are the standards developed by the American Dry Milk Institute. Other standards are available as well. 2. This sample is from highquality powdered milk with little to no scorched particles. It would receive an A grade. 1. Place the filter in the top of a funnel or similar device. 2. Slowly pour the sample over the filter and collect waste milk in the container below. 3. This sample is from lowerquality powdered with a high concentration of scorched particles. It would receive a D grade. NOTES ON THE SCORCHED PARTICLES TEST POWDERED MILK 33 AS-631 Afghan Milk Manual.indd 33

34 Solubility Test If powdered milk is not soluble, it loses its nutritive value. The acceptable solubility standards, however, differ for roller-dried and spray-dried powdered milk. In general, roller- dried powdered milk is less soluble than spraydried powdered milk due to the process. Again, high heat tends to damage more milk proteins during roller drying, making it more difficult to completely dissolve roller-dried powdered milk. The solubility test is easy but requires a centrifuge. The protocol described here can be scaled up or down to match the type and size of centrifuge available. MATERIALS Milk sample Flask Centrifuge Centrifuge tubes PROCEDURE 1. Combine powdered milk (10g for skim milk, 13g for whole milk) with 100mL sterile water in a flask. 2. Shake vigorously for 1 minute. 3. Let milk sit for 10 minutes. 4. Shake again briefly. 5. Transfer reconstituted milk to a 50mL centrifuge tube. 6. Centrifuge samples at 5,000 rpm for 5 minutes to sediment solids. 7. Remove supernatant (liquid), being careful not to disturb the solid pellet at the bottom of the tube. 8. Re-suspend pellet in 50mL sterile water and repeat steps 6 and Measure the pellet in the bottom of the tube in ml. RECORDING RESULTS The size of the pellet left in the bottom of the centrifuge tube is the solubility index. Lowerquality milk powders will contain larger pellets because large portions of the powdered milk do not completely dissolve. Below is a table showing solubility standards for different types of powdered milk. 1. A pellet will form in the bottom of the tube after centrifugation. This example is a pellet from a high-quality (spray-dried) powdered milk sample. 2. This pellet is from a lowerquality powdered milk sample. The dark area is scorched milk. 3. Re-suspend the pellet with 1mL of water until it is completely dissolved. 4. The re-suspended pellet of the high-quality milk sample measures approximately 1.2mL. 5. The re-suspended pellet of the lower quality milk measures approximately 2.3mL. 34 POWDERED MILK AS-631 Afghan Milk Manual.indd 34