CHAPTER FOUR:
ANALYSIS OF MILK AND WATER
SAMPLES FOR QUALITY
Milk is extremely nutrious food
It is an aqueous solution of proteins, fats, and carbohydrates
The PH of milk is about 7.0 and this PH is an excellent medium for
growth of microorganism
87 % of milk is water and 2.5 % is Caseins
In addition Lacto albumin is present
Lactose the commonest carbohydrates in milk and called Milk sugar
Source of Milk for human consumption
Depending on the culture and tradition of societies different
animals could serve as source of Milk
Spoilage of Milk
Lactobacillus and Streptocoous can ferment lactose in milk that
will result in sour curd
Sweat Curdling: This happens when Bacillus, Proteous,
Microcoous attacks caseins enzymatically
It can be also contaminated by gram negative rods, [Link],
[Link]
Ropiness: Formed from Enterobacter, Klebsella and
Alcalegens
Milk Born infection
Mainly occur up on consumption of contaminated milk or poorly
processed milk and milk products
Common microbes that may be transmitted by milk consumption
include:
• M. bovis
• Brucella
• Coxiella burneti
• Campylobacter
• Salmonella
• Listeria spps.
Production of safe milk for human consumption
The quality of milk is highly dependent on proper handling of
animals, proper collection and pasteurization process
Traditional practice of milking and consumption also serve as a
source of infection
Pasteurization
It is the process of eliminating the least microorganism to make
sterile milk (Relative sterility or commercial sterility)
Types of pasteurization
a. LTLT: Low temperature, long Time: This is pasteurization of
milk at 62 o c for 30 minutes .
b. HTST: High temperature Short Time pasteurization: This is
pasteurization of milk at 71 0c for 15 minutes.
c. Ultrapasturization: Pasteurization at 82 degree centigrade for
3 minutes. The shelf life of this type of milk is long. It is
practiced in bulk milk production in diary industry.
MILK STANDSARDS
The quality of milk may vary from place to place in terms of
temperature, bacterial load and other chemical tests
Based on this criteria it could be :
a. Grade A milk: Pasteurized grade A milk
• This type of milk is pasteurized
• The total bacterial limit is 20,000/ml
• Coli forms should not be more than 10/ml
• Phosphatase activity must be less than 1 ug /ml
b. Grade B milk
• Prepasturised Milk
• This type of milk may not be pasteurized
• The total plate count should not exceed
1000000/ml
• The coli form count should not exceed 10/ml
• There should be no inhibitory substances
SOME USEFULL LABORATORY TESTS FOR MILK
QUALITY
• Milk sample has to be collected aseptically and
representatively
• It could be collected from individual houses notably in
developing nations as well as from bulk thank from collection
sites
• It is possible to collect milk sample from retail market or diary
industries
Samples of milk are taken in the milk collecting units
or at producers’ according to the following schedule:
• At least twice a month for fat and protein control
• At least twice a month for somatic cell count
• Drugs and metabolites is determined at least once a
month
• Freezing point of milk is determined according to
circumstances
I. Somatic cell count (SCC)
• SCC is a measure of the white blood cell and other
cells count in milk
• The SCC in milk of an individual indicates her
udder health status, and bulk tank milk
• SCC can indicate the general state of udder health
in a cow/ sheep
• Somatic cells are always present in milk, but the SCC will
rise when an infectious agent enters the udder or when the
udder has been injured
• A major consequence of rising SCC is a decrease in raw
milk quality which has implications for milk processing
• Most studies have found SCC levels for a healthy udder
could range 250,000 to 1,600,000 cells/mL
• It can be done using haemacytometer used for hematology
lab or specially designed for this purpose
• High SCC has negative economic consequences for milk
producers
• A high SCC is a strong signal of an udder infection referred to
as mastitis
• Common clinical signs of mastitis are a swollen udder half,
abnormal color of milk, or clots present in the milk
• High SCC also has a negative effect on milk processing through
decreased yields and off – flavor development in finished
products
• Milk with a SCC>1,000,000 decreases the cheese yield and
increases the development of rancid flavors in the cheese
II. The phosphatase test
• Phosphatase is normally present in milk – heat tolerant enzyme
similar with the phosphatase produced by M. bovis and Coxelella
burntii
• The test is done by mixing milk sample that is incubated with
substrate like Sodium diphenyl phosptae and incubating the
mixture
• Phosphatase is released by the enzymes and the color is changed
and detected with chromogen
• In properly pasteurized milk the test is negative and the color
change is absent and vice versa
III. The standard plate count method
• It is based on the total bacterial colonies that grow on a
culture medium in 1 mL of sample
• A serial dilution of milk sample is done as 1:10, 1:100,
1:1000, 1:100000 and incubated
• The colony that falls between 30 – 300 is selected and
the total bacterial count is estimated by multiplying the
dilution factor
• Disadvantages
– Does not detect Psychrophilic and Thermophilic
bacteria
– Do not detect anaerobic bacteria, viruses and
molds
– The coli form bacteria is used as an important
indicator of the sanitary quality of milk that is
isolated on a violet red bile agar
IV. Serological tests for Brucellosis
• Rose bengel Plate Agglutination tests
• The test is based on an agglutination reaction of
animal / human serum with Brucella antigen
• If there is an exposure there will be agglutination in the
plate
• An agglutination level of ++ and above considered as
positive if not it is considered as negative
• Sensitivity vary from place to place
V. The dye reduction test
• It tell us the relative amount of bacteria in the sample
• Test principle: A sample of milk usually 1 ml is
mixed with dyes (methylene blue or Resazurin) and
incubated at 37 0c
• Up on presence of bacteria in the milk the blue color
is reduced to colorless product in case of methylene
blue or pink to colorless in case of resazurin
Interpretation of results
• Poor quality milk may have complete reduction of
methylene blue after 2 hours
• While good quality milk has minimal color changes
• The test has to be done on lactose fermenter organism
and most useful if the CFU is more than 100,000/ml
of milk sample
• Slow reducer and non reducing bacteria can be missed
VI. The bread count methods
• In this test, 0.01 ml of milk sample is taken using a
breed pipettes and speeded on microscopic slide
• One square cm area is taken and after staining with
Newman – Lampart stains (Methylene blue), the
average number of bacteria per field is reported
• One of the disadvantage of breed counting method is it
does not differentiate live and dead cells , debris and
the procedure is tiresome.
VII. Antibiotic detection test
• The test is useful for animals that are treated for M. bovis,
Brucellosis other microbial diseases
• The milk may have the antibiotic and or drug metabolites
that could inhibit growth of bacteria
• Indicator organism such as B. subtitles are used and zone
of inhibition is measured
• Penicillin antibiotic can be detected using Penicilinase test
• Milk of animals under treatment should not be consumed
QUALITY OF WATER
• Water is a universal solvent and it is rich naturally.
• However, all water can not be used for human,
animals, industrial purposes and recreational
purposes.
• The microbial , chemical and physical appearance of
any water source has to be investigated before
consumption and any other use
Source of water could be:
a. Ground water
• This kind of water found in deep wells and in
subterranean springs
• It is free from microbe due to filtration action of soils
b. Surface Water
• This form of water found in lakes, Streams, and Shallow
wells
• It has certain number of microbes
C. Contaminated water
• Water that is contaminated with chemicals and microbes
d. Polluted water
• Contaminated water and the condition is known exactly
It is characterized by unpleasant test, smell and
appearance.
• Pollution can be different types based on the pollutants
A. Physical Pollution
• When a particulate matters, like sand or soil make
water as cloudy
• Some living materials may also present
B. Chemical Pollution
This is due to introduction of organic and
inorganic wastes
C. Biological Pollution
When microorganisms contaminate water bodies
Potability
• The term that refers to the drinkability of water (Potable or
unpoatble/non potable)
• In unpolluted water (Mountains, lakes or streams) the number
and types of bacteria is low and mainly Actinomyces,
Bacillus, Sterptomyces, Clostridium species are commonly
found
• In polluted water, on the other hand you may have Coli forms,
Enterobacter, Sterptocoous, Proteous and Pseudomonas spices
which may found in high number
Assessment of water samples for potablility
• In order to have a potable water, it should be free from
contaminant microbes, toxic chemicals and physical
appearance
• Water has to be evaluated before consumption in all
parameters
Laboratory tests for water analysis could be:
1. Screening tests
2. Microbiological tests
1. Screening tests
The Biochemical Oxygen Demand
• It refers to the amount of oxygen that microbes require to
decompose organic matter in the water
• It is a critical measurement in polluted water
• The dissolved oxygen is measured at different intervals
usually immediately after collection and after five days
intervals
• The BOD is reported in parts per million (PPM)
BOD
• Is commonly practiced for routine check up of
water sources
• Practiced in municipal water sources
• Not reliable method
• Does not necessarily indicate microbial
contamination
2. Microbiological tests
There are a number of microbiological tests to asses the
quality of water
Sampling issues for water analysis
• As any clinical samples water samples should be collected
according to the standard methodologies
• The sampling procedures may vary from source to source
• Microbes are usually found in water bodies as diluted one
and larger volume should be collected and analyzed
Figure 1. Example of water sample collection sites Map Adapted
from Yesehak et al, 1999.
• In developing countries water sources could be:
• Tap water
• River water
• Well water
• Ground water
• Spring water
• The proportion of tap water could be less and the
likelihood of having clean and safe water is less
• Tap water is chlorinated it could inhibit or interfere with
growth of bacteria
• To avoid the effect of chlorine 10 % sodium thiosulphate
is added in tap water
• While non-treated water sources such as river and wells
may not be treated
• The recommended volume of water sample is 200 – 500
ml
• The larger the volume of water sample it is more
preferable
METHODS OF MICROBIAL DETECTION
I. Membrane Filtration Technique
• This method is a popular method, where sample of water
(100 ml) is passed in a filter by negative pressure and the
filter is plated on different medium
• The bacterial colony is counted and reported per 100 ml of
sample
Fig. 2. growth of coliforms in chromagar
II. Standard Plate Count Method
• A sample of water is diluted in a sterile buffer
solution and specified amount will be inoculated in
agar medium and incubated
• The total colony will be enumerated and multiplied
by the dilution factor for final report
• One can utilize selective and differential media for
plate count methods
• Eosin Methylene Blue Agar ( EMB) is commonly
used
• EMB can distinguish lactose fermenter and non
fermenter.
• Lactose fermenters has golden shiny colonies with
blue centers specially for E. coli
• Non lactose fermenter grow as color less colonies
Figure 3. Plate count method that involve serial dilution of water
sample
Detection of Parasites and Viruses from water bodies
• Water bodies and sources could be contaminated with viruses
and parasites.
• Filtration / concentration methods is recommended for isolation
and detection.
• Parasites like Girdia ( cyst), E. histolytica ( cyst),
Cryptosporidium parvum and other Cyclospora spps are
potential parasites that causes water borne illness
• Similarly rota viruses, HAV, and Polio virus can be transmitted
through water system
Table 1: Annual averages of physical and chemical
characteristics of water samples collected from November 1996
to October 1997 from sites stated. Adapted from Yesehak et al,
1999.
Analyte
Sites
Akaki Bulbula Aba Samuel W 17 Bole Gefersa Raw Gefersa Legedadi Raw Legedadi Treat Nefas Silk W 18 Gulele
Treated
pH 7.64 7.54 7.67 7.47 7.12 7.16 7.37 7.44 7.43 7.7
T 6.00 8.25 18.67 18.75 17.25 16.33 17.75 16.83 16.83 17.5
Con 486.33 876.75 377.33 93.33 60.25 108.25 84.50 90.75 97.25 423.2
TDS 386.41 652.83 341.58 76.58 56.00 79.75 81.50 72.83 77.17 140.2
Na +
35.75 50.10 32.08 2.23 2.33 2.29 2.27 2.24 2.26 6.55
K+ 15.35 26.70 8.13 1.68 1.42 1.07 2.30 1.64 1.64 1.63
Mg2+ 9.14 15.5 9.33 2.42 2.06 2.08 3.11 2.46 2.54 5.33
Ca2+ 41.83 53.17 36.08 10.28 5.62 12.35 11.00 9.87 10.83 15.5
Cr2+ 0.10 0.10 0.10 0.10 0.10 0.10 0.18 0.10 0.10 0.10
Cd2+ 0.10 0.10 1.93 0.10 0.10 0.10 0.10 0.10 0.23 0.10
Fe3+ 0.11 0.41 0.38 0.06 1.93 0.11 4.20 0.00 0.10 0.10
Pb2+ 0.10 0.10 0.10 0.10 0.10 0.10 0.18 0.09 0.10 0.10
Zn2+ 0.10 0.10 0.10 0.46 0.10 0.13 0.13 0.10 0.36 0.50
CI- 43.92 97.41 23.83 6.42 0.87 3.25 0.72 5.67 5.83 2.95
F- 0.38 0.61 0.63 0.16 0.08 0.06 0.46 0.16 0.16 0.24
CO2 22.78 24.3 12.6 4.58 5.58 4.17 4.92 4.00 4.55 5.67
HCO3 -
212.08 255.25 179.08 38.33 27.33 20.50 42.42 36.58 36.58 79.25
NO3- 2.45 10.96 22.28 2.93 2.41 1.20 3.66 2.90 2.81 9.38
SiO2 23.25 28.67 34.00 10.08 11.50 6.83 15.08 10.25 9.92 27.58
SO4= 7.07 29.08 8.50 2.81 5.06 24.5 8.12 2.20 3.85 1.09
HBO2 0.33 1.15 0.41 0.42 0.39 0.34 0.50 0.72 0.26 0.36
Cu2+ 0.10 0.08 0.10 0.10 0.10 0.10 0.10 0.05 0.11 0.10
Mn4+ 0.10 0.58 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
Summary
• Milk and water are major part of human nutrition.
• It could be easily contaminated with microorganism and
toxin
• There are criteria for consumption of milk and water.
• A number of laboratory procedures are available for milk
and water analysis.
• The quality of milk and water is assessed not only from the
microbiological point of view.
• It should be checked also physically and chemically.
• SCC, Dye reduction tests, Plate count methods, membrane
filtration are commonly used to assesses milk quality
• BOD is routinely practiced as screening test for water quality
• Membrane filtration , plate count methods, Multiple tube
fermentations, MPN methods are more common for assessment
of water quality to be potable.