[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

CHAPTER I
1. INTRODUCTION
1.1 Background
A food may be defined as any substance, which when taken into the body can be utilized
to yield heat and energy, to built up new tissues and to repair worn-out tissues aid to aid
in production of important body compounds. The science of nutrition deals with good
values, good processing, its digestion, adsorption and metabolism, depends to a major
degree, on the use of right kinds of food. Nutrition has been established as one of the
most important environmental factors affecting health. Milk is a diary product, and is
highly nutritive. It is one of the moist essential of all foods as it is one of the most
complete single foods available in the nature for health and promotion of growth.
Milk can be defines in normal terms as the liquid from the mammary gland of healthy
and normally fed cows. Freshly drawn milk varies in chemical composition. Some of the
more important causes of variation are the species of mammals, the breed, the age of the
individual, the stage of her lactation, her feed and the season of the year. The average
chemical composition based on the analysis of a large number of milk sample are shown
in the table 1.
Table 1: Average chemical composition of milk
Constituents
Water
Lactose (Carbohydrate or milk sugar)
Butter fat
Casein (enzyme)
Albumin & Globlulin (Protein)
Mineral

Percentage (%)
87.3
5.0
3.8
2.5
0.7
0.7

Milk also conation vitamin A, B or thiamine and B-2 or riboflavin. It contains small
amount of vitamins C and D with trace quantities of other vitamins. Milk, therefore,
provides essential nutrients for excellent growth of many bacterial species. Sterile skim
milk is routinely employed in the laboratory for the growth and maintenance of bacteria.
Raw milk always source such as bacteria derived from various sources such as mild
dusts of udder and for adulteration, diseased animal (e.g. suffering from mastitis,
tuberculosis, etc). Type of bacteria likely to be found in healthy cow includes following
groups of bacteria;
1. Acid forming Streptococcus faecalis, S. lactis, Staphylococcus, Lactobacillus spp.
1

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

2. Alkali forming Alkaligenes spp., Achromobacter spp.
3. Gas forming Clostridium perfringens, C. butyricum, Proteus vulgaris
Milk is excellent medium for the growth of micro-organisms because of its high
moisture, nearly neutral pH and rich in nutrients. However the milking performed under
hygienic condition with strict attention to sanitary practices will result in a product with
low bacterial content and good keeping quality. Milk born diseases can be described as
following three groups;
1. Infections of animal that can be transmitted to man such as Tuberculosis, Brucellosis,
Streptococcal infections, Salmonellosis, Cowpox, Anthrax, Leptospirosis, Q-fever,
Enterotoxigenis, etc.
2. Infections caused by milk contaminated by excreta of small mammals and other
sources such as thick bone Encephalitis, Streptobacillus moniliformis, etc.
3. Infections primarily of man such that Paratyphoid fever, Cholera shigellosis, E.coli,
Streptococcal infection, Staphylococci, Hepatitis A & E, etc.
Now a days urban people mostly use dairy milk that comprises water and dry milk (in
powder form) and evaporated milk found in cans. This milk is processed to produce the
milk with low bacterial count, good flavour and satisfactory keeping quality following
the process like, clarification, pasteurization and homogenization. Different type of
bacterial disease is shown in table 2.
Table 2: Bacterial diseases due to contaminated milk
Disease
Causative organism
Cholera
Vibrio cholera
Gastroenteritis
Entric E. coli
Typhoid fever
Salmonella typhi
Paratyphoid fever
S. paratyphi
Salmonellosis fevere
S. entheritidis
Shigellosis
Shigella sonnei
Pneumonia
Klebsiella pneumoniae

General public use the packed milk without being aware of its microbiological quality
since it is supposed to be pasteurized. However due to inadequate pasteurization or the
post pasteurization contamination at any stage of processing, handling and packaging
could create havoc in its quality. A very little contamination of such pathogens could
reach to dangerous level as soon as they obtain favourable condition for their growth. In
2

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

Nepal, epidemics associated with milk and milk products have not been reported. It may
be due to lack of adequate research in this field. However the prevalence of food diseases
like gastroenteritis, dysentery, typhoid fever cant be overlooked.
1.2 Source of contamination in milk
Normally fresh drawn milk is sterile but it gets contaminated from microorganisms
through various sources. The number of microorganisms, as well as the types contributed
from each source may have significant influence upon the quality of milk. It is important
to know the channels through which microorganisms gain access to milk or the products
of milk. The source of contamination may be grouped as interior of the udder, exterior of
cows body, atmosphere, utensils, and various ingredients added to dairy products and
milker or handler. Unless the producing animal is clean, and her udder and teats are
given special sanitary care just before milking, her body can be a source of considerable
consideration. Milk form cow or buffalo with an infected udder is likely to contain a
large number of organisms. Hair, dirt and dust often fall from the body in to the milking
pails or the teat cups of milking machines.
i. Handler or milker: All persons involved in the milking process must be free from
disease and in good health and must give importance to personal cleanliness as well.
They should wash their hands, clean and rinse them with an effective bactericidal
solution and dry them with a clean towel before starting and frequently during the
work. Although workers may not contribute a large number of organisms, these are of
considerable importance since they may well be human pathogens.
The following causal factors can be considered when unsatisfactory plate counts are
observed specifically in milk samples (Department of Health, 1997):

Insufficient cleansing of milking equipment before and/or after milking sessions;

Insufficient cooling of milk and/or maintenance of cold chain thereafter;

Secondary contamination of milk by means of hands/ water used during handling

process, containers and environmental pollution (e.g., dust); Infection of the
udder (mastitis); and

Milk, which has been allowed to age sufficiently (i.e. milk that has become old).

1.3 Microbiological guidelines

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

Routine examination of foods for a range of pathogenic microorganisms is impractical.
In order to assess the microbiological safety from food borne pathogens, widespread use
of groups or species which are easily enumerated and whose presence in foods indicates
exposure to conditions that might introduce hazardous organisms and/or allow their
growth, are used. These groups are referred to as indicator organisms. Indicator
organisms have historically been used to indicate the presence of pathogens of intestinal
origin as a result of direct or indirect faecal contamination. The main aim of using
bacteria as indicators is to reveal conditions of treatment of the product which may imply
a potential hazard that is not necessarily present in the specific sample examined, but
could be present in parallel samples.
Coliform particularly E. coli are frequently used in the microbiological analysis of food
as an indicator of poor hygienic condition. Microbiological examination of milk is
essential to find the degree of contamination with the dictions and enumeration of
indicator organisms (Chatterjee S. N. et al., 2006). The enumeration of E. coli in water
provides a measure of the extent of the pollution. Factors such as, multiplication, dyingoff, or adhesion of the organism to food particles may influence the numbers detected in
foods (Department of Health, 1997). However, substantial numbers of E. coli in foods
suggest a general lack of cleanliness in handling and improper storage. Its presence,
however, does not directly suggest the presence of pathogens, but only implies a certain
risk that it may be present.
The purpose of the original microbiological guidelines for foods sampled at the point of
sale was to standardize the interpretation of the results from the microbiological
(bacteriological) examination of foods by providing peer reviewed guidelines for use by
food microbiologists (Gilber et al., 2000). The purpose of these guidelines is to help food
examiners and environmental health officers to determine the bacteriological quality of
various foods at the point of sale and to indicate the level of contamination that is
considered to represent a significant potential risk to health. The five categories of food
(table 3) are based solely on expected aerobic colony counts, according to the type of
food product and the processing it has received. There are four grades of microbiological
quality (table 4) related to the actual aerobic colony count, number of indicator
organisms, and the presence/number of pathogens determined by the microbiological
examination of the food.

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

S. No.

1
2
3
4
5
6
7
8
9
10
11

S. No
1
2
3

Table 3: Guideline levels for determining the microbiological quality

Test
Microbiological result (cfu/g unless otherwise stated)
Good
Acceptable Unsatisfactory Potentially
hazardous
Standard Plate Count/ Aerobic colony count
Category 1
<103
103-<104
>104
N/A
Category 2
<104
104-<105
105
N/A
5
5
6
6
Category 3
<10
10 -<10
>10
N/A
Category 4
<106
106-<107
>107
N/A
Category 5
N/A
N/A
N/A
N/A
Indicators
Enterobacteriaceae
<102
102 to <104
104
N/A
2
2
E. coli
<3
3 to <10
10
N/A
Pathogens
Campylobacter spp. not detected
detected in 25 g
in 25 g
Salmonella spp.
not detected
detected in 25 g
in 25 g
V. cholerae
not detected
detected in 25 g
in 25 g
S. aureus
<20
20-<100
100-<104
>104
Source: Gilber et al., 2000; cited in NSW, 2009
Table 4: Grades of microbiological quality
Microbiological result
Remarks
Satisfactory
Test results indicating good microbiological quality
Acceptable
An index reflecting a borderline limit of microbiological
quality
Unsatisfactory
Test results indicating that further sampling may be
necessary and that environmental health officers may
wish to undertaken a further inspection of the premises
concerned to determine whether hygiene practices for
food production or handling are adequate or not
Unacceptable/potentially Test results indicating that urgent attention is needed to
hazardous
locate the source of the problem; a detailed risk
assessment is recommended.
Source: Gilber et al., 2000

Counts of viable bacteria are commonly based on the number of colonies that develop in
nutrient agar plates which have been inoculated with known amounts of diluted foods
and then incubated under prescribed environmental conditions. Only those bacteria,
which will grow under the chosen environmental conditions, can be counted. The use of
plate counts has a number of advantages. If it is high, or if it varies widely among
samples from different lots or within a lot, microbiological control in processing or
transport was probably inadequate. There are, however, limitations to the value of
5

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

mesophilic counts. High counts have no value in particular foods, e.g., fermented
sausage, cheese, yoghurt, and other dairy products (Department of Health, 1997).
1.4 Justification of study
Food can become microbiologically hazardous to the consumer when the principles of
hygiene and sanitation are not met or when it becomes contaminated by pathogens from
humans or from the environment during production, processing or preparation, or when
it originates from a sick animal, for example, a cow with mastitis or with anthrax. On
subjection to conditions that allow the entry and/or growth of infectious agents, it may
become a vehicle for transmission of diseases such as Salmonellosis or Staphylococcal
food poisoning. Thus examination of quality of food samples allows us to determine the
existence of these hazards.
1.5 Objectives
General objective
The present study was carried out with the objective of studying the quality of different
sources of milk available in the market of Sankhamul, Kathmandu.
Specific objectives
i.

To determine the total bacteria and coliform colony in the milk by standard plate
count (SPC) method.

ii.

To study the quality of milk sample according to microbial guidelines.

1.6 Limitations

i.

The sampling was performed only once hence the result may not be summarised to
accurate value.

ii.

Other factors such as refrigerator temperature, quality of water used for cleaning the
equipments, percentage constitution of water in milk, duration of storage were not
taken into considered.

iii.

Enough biochemical test couldnt be conducted due to laboratory constrains.

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

CHAPTER 2
2. LITERATURE REVIEW
Department of Health (1997) states it is advisable that foods for which standards have
been set, be tested so that further action can be taken. This would include education of
food handlers and food processors as well as the taking of legal action. In terms of raw
foods (meat or poultry) it should be borne in mind that these products are likely to have
high microbiological counts and that certain enteric pathogens are associated with these
products. It is also important to remember that the hygiene of the premises is regarded as
extremely important and this is probably one of the important factors in ensuring a safe
product at the end of the day.
Rimal Poshana (2003) studied the microbial quality of dairy product, curd of Nepal. He
found that the average number of microorganisms observed in the curd sample was
5200 cfu/gm which included gram positive spore former yeast and fungus with blast
spores. The study concludes that the contamination may have happened from inadequate
pasteurization or post pasteurization contamination at any stage of manufacturing,
processing and handling.
Litwiczuk et al. (1999) and Przysucha et al. (2003) documented difficulties to obtain
high quality milk during the summer season. All of them reported that higher air
temperatures favour the increase of bacteria number, especially on the surfaces of no
good enough cleaned up milking equipment which were the potential source of infection.
Joshi DR et al. (2004) accessed microbial quality of some selected national and
international brands of ice cream being sold in Kathmandu valley by studying 72 ice
cream samples of twelve different brands (6 Nepalese and 6 foreign brands). Out of total,
44 samples (61.1%), 49 samples (68.1%) and 16 samples (22.2%) exceeded standard
value of mesophilic aerobic count, total coliform count and Staphylococcal count
respectively. However, only one sample (1.4%) was found to be contaminated with
Salmonella spp. The microbial quality of with additive ice cream samples was poorer
in comparison to plain samples. The overall microbial quality of ice cream samples being
sold in Kathmandu valley was concluded to be poor.
Rajak Sudha (2004) performed qualitative estimation on quality of milk in markets of
Kathmandu. She isolated Escherichia coli in 3 of the milk samples of the total 5 milk
7

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

samples (raw, packet, and powder milk) collected. This result draws attention to the poor
microbial quality of dairy product which poses serious threats to the public health.
Chatterjee S. N. et al. (2006) assess the milk quality in Tarakeswar, India by standard
plate count (SPC) method. Out of 10 raw milk samples, the microbial colonies were
found to be high in 6 samples. In pasteurized milk samples, the colonies were low in 7
samples and high in 3 samples. Out of 10 pasteurized samples, 9 samples were of good
quality and 1 was found to be excellent. Bacterial colony was found to be opaque and
metallic sheen in colour prepared and by biochemical characterization it was identified
as Escherichia coli.
Dahal Lekh Raj et al. (2010) studied the quality of raw milk in eastern Terai region of
Nepal. Altogether, 520 milk samples for total bacterial count were analysed at plant and
farm level. They found that the results of mean TBC at farm (9.03 x 105) was nine fold
of international standard (1 x 105), and mean TBC at plant (104.71 x 105) reached 104
folds the international standard. Hence the research concluded that the situation was
critical and needed a real improvement from production point to processing plant.
Mubarack H. Muhamed et al. (2010) investigated the microbiological quality of raw
milk samples from some of the villages and the surrounding areas of Coimbatore district,
South India. Among the 80 raw milk samples used for the study, bacteriological
identification revealed a definite dominance of Lactobacillus spp. Besides it, the other
genera Staphylococcus, Escherichia, Bacillus, Salmonella and Pseudomonas were also
isolated on selective agar and broth.

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

CHAPTER 3
3. METHODOLOGY
3.1 Method
Various methods can be employed for determining of total bacteria as well as coliform in
a food sample. Total plat count method was followed to study the total bacteria in the
milk samples whereas solid medium plating method was used for coliform count. This
was followed by bio-chemical tests such as IMViC, triple sugar iron test (TSIA), gram
staining. Method described by K.R. Aneja (2005) and Manandhar & Sharma (2006) were
adapted for enumeration.
3.1.1 Sampling
Around 15 milk sample were collected from various retailer shops, dairy farms in the
Sankhamul area of Kathmandu in the sterile sampling bottles. The collected samples
belong to various registered as well as unregistered brands of pasteurized and raw milk
as well as branded pasteurized milk. The sampling bottles were sterilized by autoclaving
it for 15 minutes at 1210C (15lb/in2). The samples were collected in an ice box and
carried to laboratory as soon as possible for microbial analysis. The collected samples
were handled aseptically in the laboratory to minimise the contamination. The milk
samples were collected between the periods of 2nd Shrawan 2068 to 10th Shrawan 2068.
Table 5: Sample categorization
Sample Number
Code
Type
Sample 1
RT 1
Unbranded pasteurized milk
Sample 2
RT 2
Unbranded pasteurized milk
Sample 3
RT 3
Unbranded pasteurized milk
Sample 4
RT 4
Unbranded pasteurized milk
Sample 5
RT 5
Unbranded pasteurized milk
Sample 6
PK 1
Branded pasteurized milk
Sample 7
PK 2
Branded pasteurized milk
Sample 8
PK 3
Branded pasteurized milk
Sample 9
PK 4
Branded pasteurized milk
Sample 10
PK 5
Branded pasteurized milk
Sample 11
PK 6
Branded pasteurized milk
Sample 12
RW 1
Raw milk
Sample 13
RW 2
Raw milk
Sample 14
RW 3
Raw milk
Sample 15
RW 4
Raw milk

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

3.1.2 Requirements
Table 6: Materials, chemicals and reagents used for microbial study
Materials
Chemicals and reagents
Electric balance
Test tubes
Distilled water
Ethyl alcohol
Petriplates
Pipettes
Plate count ager
Violet red bile agar
Autoclave
Bunsen burner
Lactose broth
TSIA slant
Inoculating loop
Conical flask
Kovacs reagent
Methyl red reagent
Durhams tubes
Incubator
Barritis reagent
Crystal violet
Cotton
Aluminium foil
Grams
iodine Safranin
solution
Glass slides
Compound
microscope
3.2 Standard plate count (SPC) method
The working area was cleaned with ethyl alcohol; Bunsen burner was burned to maintain
aseptic condition. 1 mL of milk sample was poured to test tube containing 9 mL of sterile
saline (0.85% NaCl) and shaken well; this formed 10-1 dilution. Dilutions up to 10-8 were
prepared. 1 mL of sample from each of 10-3, 10-4, 10-5, 10-6 and 10-7 dilution was pipette
out with sterile pipette in two different sterilized, labelled petri plates. Plate count agar
(PCA) and violet red bile agar (VRBA) media were cooled to 480C before use. The
number of CFU per mL was calculated using formula stated below.
No. of colonies dilution factor
No. of CFU/mL or gram = --------------------------------------Weight of sample
About 15 to 20 mL of medium was poured to each plate and it was swirled to mix and
left to solidify. The solidified plates were incubated at 370C for 18 to 24 hours. To
confirm the colonies are coliform, 10 representative colonies were picked from VRBA
medium and each was transferred to a tube of lactose broth, this was incubated at 37 0C.
It was then examined for 24 to 48 hours for gas production.
3.3 IMViC test
Biochemical and physiological reactions of the microorganism reveals the characteristics
of the enzymes present and the metabolic activity of certain microorganism. On the basis
of these characteristics, bacteria can be categorized into different types or groups.
1.4.1 Indol production (I) test: The peptone water broth medium was inoculated with
test organisms. It was then incubated at 370C for 24 hours. Few drops of Kovacs

10

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

reagent was added after incubation. The presence of red ring on culture broth
medium on addition of Kovacs reagent is detected as positive test.
1.4.2 Methyle Red (MR) test: The medium tube containing MR-VP medium was
inoculated with test organisms then it was incubated for 24 hours at 370C. 5 drops
of methyl red indicator were added after incubation. The turning of medium red
is detected as positive test.
1.4.3 Voges-Proskauar (Vi) test: The medium tube containing MR-VP medium was
inoculated with test organisms then it was incubated for 24 hours at 37 0C. A few
drops of barritis reagent were added after incubation. Formation of red colour
ring in the medium indicated positive test.
1.4.4 Citrate utilization (C) test: A loopful of bacterial suspension was streaked on the
citrate slant and it was incubated for 24 hours at 370C. The change in colour of
slant to blue with growth on surface indicated positive test.
3.4 Triple sugar iron ager (TSIA) test
A few cells from the centre of each colony were picked using sterile inoculating needle.
This was then inoculated to a TSIA slant by first streaking the surface of slant and then
stabbing the medium in the butt region. The tubes were incubated at 350C for 24 hours.
The change in colour in the slant and butt region was observed as well as formation of
gas was also noted.
3.5 Gram staining
Firstly thin smear of bacterial culture was prepared on a glass slide with help of sterilized
inoculating loop and was allowed to air dry. 2 drops of Crystal violet was poured over
the smear and it was washed with distilled water after a minute. 2 drops of Grams iodine
was poured to it and then it was washed with 95% ethyl alcohol and water respectively. 2
drops of Safranin was added to it and was washed with distilled water after a minute.
This slide was allowed to air dry and finally it was observed under a compound
microscope.

11

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

CHAPTER 4
4. RESULTS AND DISCUSSION
4.1 Results
The experimental results of number of colonies obtained in various medium as well as
bio-chemical tests were graphically analyzed. The bacterial load in PCA medium is
shown in figure 1. It was recorded to be high in raw milk samples with highest value
being 27.04105 cfu/mL in RW 1 followed by 24.82105 cfu/mL in RW 3 and
23.21105 cfu/mL in RW 4 respectively. The least value were recorded for both branded
pasteurized milk samples as well as in unbranded pasteurized milk samples with
3.83105 cfu/mL in PK 4, followed by 6.23105 cfu/mL in RT 1 and 7.35105 cfu/mL in
RT 4 respectively.

Figure 1: Bacterial load in PCA medium

The coliform load in VRBA medium is shown in figure 2. It was recorded to be high in
unbranded pasteurized milk samples together with raw milk samples. The highest value
was recorded to be 8.24105 cfu/mL in RT 3 followed by 7.39105 cfu/mL in R 3 and
7.06105 cfu/mL in RW 4 respectively. The least value were recorded for branded
packed milk samples with lowest value being 3.8104 cfu/mL in PK 4, followed by
4.3104 cfu/mL in PK 5 and 5.0104 cfu/mL in PK 6 respectively.

12

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

Figure 2: Coliform load in VRBA medium

The comparision between bacterial and coliform load in PCA and VRBA medium is
shown in figure 3. The correlation coefficient value was found to be 0.623 which
indicated that there is positive medium pearsons correlation coefficint between bacterial
load in the two mediums. The standard deviation for number of bacterial colonies in milk
samples for PCA medium was found to be 7.42105 cfu/mL whereas in VRBA medium
it was found to be 2.82105 cfu/mL.
The biochemical test was carried out to isolate the coliform from the selected milk
sample which are shown in table 7.

Sample
No.

Table 7: Result of biochemical test

Test
Indol

MR VP Citrate

TSAI

Gram
staining

Remark
Colony
character

RT1

Acid, gas, no
H2S production

Gram
negative

Red colour,
rod shaped

RT5

Acid, gas, no
H2S production

Gram
negative

Red colour,
rod shaped

PK1

Acid, gas, no
H2S production

Gram
negative

Red colour,
rod shaped

PK5

Acid, gas, no
H2S production

Gram
negative

Red colour,
rod shaped

RW3

Acid, gas, H2S

production

Gram
negative

Red colour,
rod shaped

13

E. coli,
Shigella
dysnteriae
E. coli,
Shigella
dysnteriae
E. coli,
Shigella
dysnteriae
E. coli,
Shigella
dysnteriae
Salmonella
typhimurium

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

Bacterial colony was found to be opaque and metallic sheen in colour in 4 out of 5 milk
samples. The bacteria were found to be gram negative in nature and were small straight
rods. Biochemical tests showed that the bacteria were indole positive, methyl red
positive, Voges-Proskauer test negative, citrate utilization test negative, and lactose
fermentation positive except for Salmonella typhimurium which showed positive methyl
red and citrate test. The biochemical tests indicated the presence of E.coli, Shigella
dysnteriae and Salmonella typhimurium in the samples. E.coli and Shigella dysnteriae
didnt produce H2S in TSAI test whereas Salmonella typhimurium produced H2S with
acid and gas bubbles.
The result of microbial quality of the milk sample was performed according to guideline
given by Gilber et al., 2000; NSW, 2009 which is shown in table 8. Milk is placed in
category 2 along with other milk products like cheese, milk shake, ice cream, yoghurt
and so on (Gilber et al., 2000).
Table 8: Microbial quality analysis of milk
Sample
Mean CFU/mL
Microbial result
Coliform

Salmonella

Raw milk
24.47105
Unsatisfactory
Present
Present

Unbranded pasturized milk

11.87105
Unsatisfactory
Present
Present

Branded pasturized milk

10.21105
Unsatisfactory
Present
Present

4.2 Discussion
Analysis of raw, unbranded pasteurized milk and branded pasteurized milk samples in
the market of Sankhamul showed that the samples were heavily contaminated with both
coliform and bacterial load. Branded pasteurized milk was found to be contaminated by
relatively lower number of bacteria than the number found in other samples. This may be
due to processing of milk such as pasteurization, clarification and homogenization singly
or in combination. Unbranded milk sample although were stored in refrigerator but
appropriate temperature (below 00C) was hardly maintained in the retail shop whereas
the raw milk samples were directly taken to consumers without any pre-treatment. Thus
the bacterial load was found to be more in these samples.
The result of mean TBC in raw milk sample (24.47105 cfu/mL) was 24 fold of
international standard (EU standard) (1x105 cfu/mL) while mean unbrande pasturized
milk (11.87105 cfu/mL) reached 12 folds and branded pasturized milk (10.21105
cfu/mL) reached 10 folds of international standards (Hutchison, 2012). The value of
14

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

mean TCC in raw milk sample (6.35105 cfu/mL) while in unbranded pasturized milk
(5.23105 cfu/mL) and branded pasturized milk (1.6105 cfu/mL) reached many folds of
international standard of 50 cfu/mL (Department of Health, 1997; Hutchison, 2012). This
may be due to combination of factors like improper cleaning of milking equipment after
each milking or neglecting to sanitize equipment before the next milking; mastitis
infections in cows/buffalos due to Streptococcus agalactiae; using the wrong amount or
type of detergent, and problems with debris buildup in receiver jars, plate coolers and
chillers (Hutchison, 2012). Hence the result is consistent with the previous findings of
Rajak Sudha (2004) and Dahal Lekh Raj et al. (2010).
Almost all samples under this study were found to be low in quality. This is revealed by
the result of microbial quality analysis of the milk samples which gave unsatisfactory
result. It further states that the results are outside the expected microbiological levels,
present no food safety concern, but might indicate poor food handling practices
(NSW, 2009). Presence of E. coli can be an indication of inadequate processing and/or
post process recontamination by raw materials, dirty equipment or poor hygienic
handling and presence of Salmonella spp. may be a result of undercooking, poor
handling practices and cross contamination practices (NSW, 2009; Department of
Health, 1997).
The raw milk contained higher number of micro flora probably due to contamination
from the animal. Bacteria found in manure, soil and water may enter milk due to dairy
utensils and milk contact surfaces. If the milk contact surfaces are inadequately cleaned,
bacteria may develop in large numbers. The high numbers of the isolated
microorganisms not only contaminate the milk but also multiply and grow in it. This
might be due to the fact that milk is a good nutritive medium for the growth of
microorganisms, especially with poor sanitary procedures and lack of the cooling
facilities (Mubarack H. Muhamed et al., 2010; Regmi, S. et al., 2012).

15

[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

CHAPTER 5
5. CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
The results obtained during the study on the hygienic quality of raw milk in Sankhamul
indicate that the current situation is critical and needs real improvement. In effect, a great
majority of milk samples had very high level of bacteria and coliform such as E.coli,
Shigella and Salmonella. The presence of those bacteria in milk suggested contamination
from various sources, such as animal, human, environment, utensils and others. Milking
and other handling equipment that are the potential source for increasing the bacterial
load in developing country likes Nepal where the traditional manual method of milking
is still in practice. Contaminated water, transportation time, cleanliness of equipments
and temperature of milk are the important factors for bacterial contamination.
Unsatisfactory hygiene and sanitary conditions of milk samples suggests the existence of
great risk to the health of consumers, especially when the product is taken without being
boiled.
5.2 Recommendations
The bacteriological study has drawn attention to poor microbiological quality of milk
available in the market. In order to safeguard the public health various suggestions are
recommended.

Nepal Dairy Development Board (NDDB) should bring all dairy stakeholders
together to set milk pricing policy in order to stimulate the awareness among farmers
and processors for quality milk production.

There should be a mechanism to educate the food handlers, milkers and factories for
following basic sanitation and health practices especially washing hands, use of
proper method in storing, preparing and serving food products.

There should be implementation and monitoring of Hazard Analysis Critical Point

(HACCP) system to identify the point at which a product mat be subjected to the
bacterial contamination and point at which bacterial growth may occur.

Government should include legal enforcement on TBC of marketed milk considering

Nepalese situation.

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[MICROBIAL STUDY OF MILK] By, Pradip Shrestha

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