Etiket Arşivleri: Milk

Laboratory‎ > ‎Milk


The purpose of this experiment was to investigate the coliform in milk and learn methylene blue reduction test and direct microscopic count method for milk.


The Standard Plate Count (SPC) procedure is used to determine the number of bacteria in a sample. In most cases the initial day SPC represents those bacteria that survive pasteurization (thermoduric), although gross contamination after pasteurization can cause high counts. The regulatory standard of < 20,000/ml is generally easily achieved. Most initial day bacteria counts are <500/ml while counts higher than 1000/ml suggest a potential contamination problem, either in the raw milk supply or within the processing equipment.

The ideal milk shows no increase in bacteria counts during refrigerated storage. When milk is held under refrigeration, only bacteria capable of growth under these conditions will grow. While most bacteria prefer warmer temperatures for growth, some bacteria, referred to as psychrotrophs (“cold-loving”), are capable of growth at 45oF or less. The most common types of psychrotrophic bacteria that rapidly spoil milk do not survive pasteurization; thus their presence in milk is the result of post-pasteurization contaminants due to less than adequate sanitation practices. The initial day SPC of fresh pasteurized milk is not a good indicator of the numbers of psychrotrophs present since most bacteria that survive pasteurization are not psychrotrophic (a few types of thermoduric bacteria will grow slowly under refrigeration conditions). A significant increase in the SPC after 7-10 days of refrigeration storage is evidence of psychrotrophic growth and suggests that post-pasteurization contamination has occurred and that shelf-life will be shortened. Generally, when the SPC exceeds 1 – 100 million, the product will become unacceptable due to flavor defects related to bacterial growth. The key to preventing spoilage and extending the shelf-life of a product is to prevent post-pasteurization contamination through a well-designed quality assurance program. It only takes one psychrotrophic bacteria per container of milk to cause spoilage.

The coliform bacteria (coli) count is used as an index of sanitation during the handling and processing of milk products. Coliforms are killed by pasteurization, thus when present in milk, they are regarded as post-pasteurization contaminants resulting from poor sanitation.  Though the standard is “not to exceed 10/ml,” detection of any coliform bacteria suggests that there is some point in processing that has been neglected in regard to effective cleaning and sanitation procedures. As a rule, the detection of coliforms in milk will indicate the potential for a shortened shelf-life due to concurrent contamination with psychrotrophic bacteria. Milks with coliform counts exceeding 10/ml are not tasted on subsequent days in this program.


  • Tcyptone glucose yeast agar

  •  Milk

  •  Methylene blue solution

  •  Tubes

  •  Pipette

  •  Spreader

  •  Bunsen burner

  •  Test tube rack

  •  Etuv

  •  Ethyle alcohol


1 – Total Count:

Firstly; milk was diluted from non dilution milk to 10-5 dilution in the test tube. In order to make 1 ml non-dilution milk was taken and it was added to 9 ml distilled water and thus 10-1 milk dilution was occurred and this process was continued to 10-5 dilution. After that; 0, 2 ml dilution was taken with pipette from each dilution (non-dilution, 10-1, 10-2, 10-3, 10-4, 10-5) these were inoculated to tcyptone glucose yeast agar with spread plate method. Then; these were incubated at 37 oC for 24 hours. After incubation, between 300 – 30 being microorganisms were counted and in ml numbers of microorganisms were calculated.

2 – Methylene Blue Reduction Test:

Secondly; raw milk and pasteurized milk were studied separately. 10 ml milk was put in the test tube and 3 – 4 drops methylene blue solution was added to milk. Then; milk and methylene blue solution were mixed vigorously, then; these tubes were incubated at 37 oC for 30 minutes. Milk was examined and observed whether decolorization was occurred or not.


Total Count:








A – raw milk







B – paste. Milk







C – raw milk







Group A)         For 10-2 dilution: (284*102) / 0, 2 ml = 142000

For 10-3 dilution: (65*103) / 0, 2 ml      = 325000

For 10-4 dilution: (38*104) / 0, 2 ml      = 1900000

(142000+325000+1900000) / 3 = 789000

Group B)         For 10-1 dilution: (47*101) / 0, 2 ml      = 2350

Group C)         For 10-3 dilution: (198*103) / 0, 2 ml = 990000

                        For 10-4 dilution: (33*104) / 0, 2 ml      =1650000

(990000+1650000) / 2 = 1320000

Methylene Blue Reduction à this method depends on the ability of microorganisms to change oxidation-reduction potential of medium. Bacteria consume dissolved oxygen in medium and produce some enzyme. These enzymes oxidize substrate and hydrogen removed from substrate and hydrogen was held with methylene blue solution and light blue of milk converted to white or colorless.


3 – Coliform Test:

Firstly; milk was diluted from non-dilution to 10-5 dilution, then 0, 2 ml diluted and milks were spread out on violet red bile agar with spread plate method then plates were incubated at 37 oC for 48 hours. After 48 hours number of colonies was counted and in ml number of microorganisms was calculated.

4 – Direct Microscopic Count Method:

In here; firstly; slide was taken and on slide 1 cm2 area was determined and one loopful raw milk was put on this area and raw milk was spread out with distilled water in this area. Then; on slide milk was waited to get dry in air. After that; one loopful xylol was added on each square and waited for 1 min then slide was washed with water. Afterwards; 1 – 2 drops methylene blue was added onto each square and waited for 2 min and again slide was washed with water. Next; slide was got dry in air. Finally; on each square, one drop iol immersion was added and milk was examined under 100X objective.


Coliform Test:








A – raw milk







B – raw Milk







C – raw milk







Group A)         For 10-3 dilution: (165*103) / 0, 2 ml = 825000

Group C)         For 10-3 dilution: (50*103) / 0, 2 ml = 250000

Direct Microscopic Count Method:


1st region

2nd region

3rd region

4th region

5th region

1st half slide






2nd half slide






1st half slide à 3+6+8+14+21 = 52

2nd half slide à 2+4+5+10+15 = 36

Average number of microorganisms = (52+36) / 10 = 8, 8

Average number of m/o’s = 8, 8 * 15700 * 100 = 14130000

In here 100 is 0, 01 ml milk.


        In this experiment; we examined microbiological properties of milk. In milk number of microorganisms was calculated with total count method and coliform bacteria were investigated with coliform test. Also; methylene blue test was applied the milk. In this test one drop methylene blue solution was dropped into milk and colour of milk was slightly blue. After 30 minute slight blue colour of milk converted to white, this transformation was related with number of microorganisms in milk. Microorganisms were used the dissolved oxygen and microorganisms produced certain enzymes. These enzymes oxidize the substrate, thus hydrogen was removed from substrate. Hydrogen was held with methylene blue solution and color of milk was colourless this process was called decolourization. In addition; while raw milk decolorized for 30 minutes pasteurized milk decolorized for 4 – 5 hours. This showed that; in raw milk, number of microorganisms was more than in pasteurized milk.

        Secondly; number of microorganisms was determined with total count method. In each milk dilution, number of microorganisms was calculated with accordance to between 30 – 300 microorganisms and as a result; we observed that raw milk was to contain more number of microorganisms than pasteurized milk.

        Next; coliform test was applied the milk and in here number of microorganisms was calculated. Finally; in milk microorganisms was examined with microcopy and dark blue microorganisms were observed and at five regions average number of microorganisms were calculated, and these were determined at the result and calculation.

Milk Dairy Products


















Dairy Processes ( Prof.Dr. Sevim KAYA )


Collecting, cooling, transportation


Hand milking

Machine milking

Milk reception

Churn reception

Tanker reception

Measuring by volume

Measuring by weight

Chilling the incoming milk

Milk should be chilled to (and handled) <+4?C

Transportation using different ways:

By insulated tankers

In churns

All the ways, same rules

Kept chilled

Free from air

Treated as gently as possible

Milk ( Dr. Hüseyin BOZKURT )

Dr. Hüseyin Bozkurt

Milk and dairy products have made a major contribution to the diet. Composition of milk Component Whole Milk Skimmed Milk Butter Milk Cream (40% fat) Fat 3.82 0.06 0.50 40.0 Protein (Nx6.38) 3.25 3.35 3.35 2.00 Casein 2.50 2.60 2.60 1.56 Whey Protein 0.60 0.62 0.62 0.37 Lactose 4.80 4.95 4.95 2.90 Minerals 0.70 0.73 0.73 0.44 Water 87.4 91.0 90.5 54.6

Nutrients provided by pasteurized milk Nutrient Quantity in Milk Protein 34.0 g Fat 39.1 g Carbohydrate 49.4 g Calcium 1.2 g Iron 1.1 mg Vitamin A 471 mg Vitamin D 0.30 mg Thiamin 0.40 mg Riboflavin 1.6 mg Niacin 9.3 mg Vitamin C 10.4 mg

The total solids (TS) of milk are made up of butterfat (usually around 3.9 %) and solids-not-fat (SNF, around 8.6%). The principles components of the SNF of milk are proteins (casein and whey proteins, immunoglobulin’s, etc.) an lactose. Fat phase contain fat soluble vitamins and salts. Aqueous phase also contains antibiotic residue.

The fat in untreated milk floats freely and rises to the surface to form a creamline on the top. Centrifugal seperation of the milk is simply an accelereated form of the same phenomenon.

Thermisation Pasteurization Sterilization

Milk and dairy products are pasteurized to destroy pathogenic organisms, particularly Tuberculobacillus and to reduce the number of non-pathogenic organisms which might adversely affect product quality. • Batch pasteurization hold at least 30 min at temperature not less than 62.8C and not more than 65.6C. • Continuous pasteurization (HTST) hold for at least 15 s at a temperature of at least 71.7C

Prior to taking portions for each determination; ◦ The milk should be thoroughly mixed by continuous slow inversions of the sample bottle or by slowly pouring it into a beaker and back into another beaker and repeating the process many times. } In the routine examination of milk it is convenient to take ◦ the lactometer reading, ◦ determining the fat by the Gerber method, ◦ the total solids ◦ the non-fatty solids content can be calculated.

The development of sourness can be assessed by determining the acidity of the milk. ◦ The assessment of the degree of pasteurisation from the results of the phosphatase test is also a common routine procedure for milks which are sold as “Pasteurised”. } Other determinations are ◦ protein, ◦ lactose, ◦ ash, ◦ chloride and citric acid and ◦ examination for dirt, ◦ added dyes, ◦ preservatives, ◦ detergent and ◦ antibiotics. ◦ The methylene blue test and the bacteriological examination must be carried out on samples taken under aseptic precautions.

Cow milk Component Lowest Highest Total Acidity as Lactic acid (%) 0.135 0.202 Density 1.028 1.039 Fat Content (%) 3.0 — Solid Non-Fat Content % 8.5 — Lactose (%) 3.5 5.5 Protein (%) 2.8 5.0 Ash (%) 0.6 0.9 Dirt (mg/100 ml) — 6

Determination of Ash in Milk Ash is determined by heating the dried residue of milk at a temperature not exceeding 500°C so that the chlorides are not volatilized. Chloride in Ash: } Pipette and weigh 10 ml milk into a 250 ml flask. Then add by pipette 10 ml 0.05 M silver nitrate (Section 5.7.). } Add 10 ml conc. nitric acid and a few anti-bump granules and boil gently for a few min. The liquid should then be pale yellow. } Cool, add 60 ml water and 1 ml ammonium ferric sulphate (Fe (SO ) .(NH ) .SO .24H O) solution as indicator 2 4 3 4 2 4 2 } And titrate the excess silver nitrate with the potassium thiocyanate solution (Section 5.5.). } Perform a blank determination by repeating the procedure with 10 ml water instead of milk. 1 ml of 0.050 M potassium thiocyanate = 0.001773 g chloride.

The rapid determination of protein in the milk can be made by formal titration method. } The protein in Milk can also be determined on 10 g milk by the macro Kjeldahl method using the factor N*6.38. } Formal titration method depends on the fact that when formaldehyde is added to neutralised milk, free acid(which can be titrated by alkali) is produced in proportion to the amount of protein present. } The protein content is then obtained by multiplying the titration by an empirical factor, which depends on the ratio of casein to albumin and also the particular technique employed in which interference due to calcium is prevented by addition of oxalate give reasonably accurate results.

To 10 ml of milk add 0.5 ml of 0.5 % phenolphthalein indicator and 0.4 ml of neutral saturated potassium oxalate ((COOK) .H O or C K O .H O). 2 2 2 2 4 2 } Mix and allow to stand for a few min. and neutralise with 0.1 M NaOH to the standard pink color. } Add exactly 2 ml. of formalin. } Mix and allow to stand for a few minute and titrate the new acidity produced with 0.1 M NaOH (Section 5.3.) to the same pink color (a). } Titrate separately 2 ml. of formalin and 10 ml. of water with the same alkali (b) as blank. } Then the protein content of the milk is % Pr otein = 1.7 *(a -b) } If the oxalate is omitted, the first titration gives the acidity and a higher formal factor is usually appropriate; % Pr otein = 1.95 *(a -b)

The specific gravity of milk varies according to the proportions of fat (sp. gr. 0.93), non-fatty solids, NFS, (sp. gr. 1.614) and water (sp. gr. 1.0). The density of milk can be conveniently measured by means of the lactometer which is a special hydrometer calibrated over the range 1.025-1.035 (25 ° – 35 ° as lactometer degrees). For determination, the milk should be reasonably fresh and be thoroughly but gently mixed, avoiding incorporation of air. The temperature of the sample being measured must always be taken. } If the percentage of fat is determined by Gerber method, the total solid (T) figure can be calculated from the modified Richmond’s formula T = 0.25 *D + 1.22 *F + 0.72 where D is the density hydrometer reading (lactometer degrees) at 20 °C and F is the fat in percentage. NFS = T -F

The acidity value can be expressed in different manners. These are: } Degree of Soxhlet Henkel ( SH° ) : Amount of N/4 NaOH required to neutralise 100 ml of milk. Or use 25 ml of milk then multiply the amount of alkali consumed by 4. Generally, SH° of milk is about 6-7. } Degree of Dornic (D°): Amount of 0.1 N NaOH required to neutralise 10 ml milk multiplied by 9. Generally the acidity of milk is 15-16 D° (1 SH° º 2.25D°). } % Lactic Acid. } } Procedure: } Take 20 g milk into a flask and dilute with twice its volume of CO2-free water. } By using ( 1% in alcohol) phenolphthalein (Section 5.1.1.) as indicator. } Titrate the sample with 0.1 N NaOH (Section 5.3.) to the first persistent pink color. } Calculate the percent acidity as lactic acid. 1 ml of 0.1 N NaOH = 0.0090 g lactic acid

When milk is heated at 80°C or above, all the albumin becomes denatured and if solutions of inorganic salts or acids are added the albumin separates with the casein. Procedure: } Weigh out 4.0 g of ammonium sulphate into a 50 ml conical flask. } Add 20.0 ml of milk sample and shake the mixture for 1 min. to dissolve the ammonium sulphate. } Allow the solution to stand 5 min., then filter it. } When at least 5 ml of clear filtrate have been collected, place the tube in boiling water for 5 min. } Then cool it in cold water and examine the solution for turbidity. } A sterilised milk that has been satisfactorily heat treated gives no turbidity. UHT milk gives a faint turbidity and raw pasteurised milk give a white precipitate.

Procedure: Put some milk into dish. Replace into microvawe oven. Run the program.


Milk Composition and Structure

Milk is defined as the secretion of the mammary glands of mammals, its primary natural function being nutrition of the young. Milk of some animals, especially cows, buffaloes, goats and sheep, is also used for human consumption, either as such or in the form of a range of dairy products. In this book, the word milk will be used for the ‘normal’ milk of healthy cows, unless stated otherwise. Occasionally, a comparison will be made with human milk.

Milk for Liquid

Liquid milk can be delivered to the consumer after various heat treatments: none (raw milk), pasteurized or sterilized, and either packaged or not (although sterilized milk is, of course, always packaged). The properties of liquid milk that require the most attention are safety to the consumer, shelf life, and flavor. Safety is, of course, essential and consumption of raw milk cannot be considered safe. Consequently, the delivery of raw milk is prohibited or severely curtailed in many countries. Likewise, delivering milk that is not packaged may involve health hazards. The relative importance of other quality marks depends on usage.

Milk can be consumed as a beverage, in which case flavor is of paramount importance. Most consumers tend to dislike a cooked flavor and, therefore, low-intensity pasteurization is generally preferred. Others use milk primarily in coffee or tea, in cooking, in baking, etc., where the absence of a cooked flavor is mostly not essential (if not too intense) and shelf life may be the most important quality mark. Consequently, sterilized milk is often favored.

Milk Processing

Milk fresh from the cow is virtually a sterile product. All post-milking handling must maintain the milk’s nutritional value and prevent deterioration caused by numerous physical and biological factors. In addition, equipment on the farm must be maintained to government and industry standards. Most cows are milked twice a day, although some farms milk three or four times per day. The milk is immediately cooled from body temperature to below 40°F (5°C), then stored at the farm under refrigeration until picked up by insulated tanker trucks at least every other day.

The milk tanker driver records the amount of milk and notes the temperature and the presence of any off-odors. If the milk is too warm or has an off-odor, it will not be picked up, and the farmer will have to feed it to his animals or dump it. When the milk is pumped into the tanker, a sample is collected for later lab analysis.

Süt Kimyası – Giriş

Chemistry of Milk

Other Substances in Trace Amounts

1. Pigments

2. Enzymes

3. Vitamins

4. Phospholipids

5. Gases