ASSOSA UNIVERSITY

COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES

DEPARTMENT OF APPLIED BIOLOGY

Detection of Escherichia coli & Staphylococcus aurous

Enumeration from Raw Milk in Assosa Town
Submitted to Department of Biology for Partial Fulfillment of Requirement
of Bachelor of Science in Applied Biology

By:

GUTU TESFAYE……………………………………...NCR/157/07

ADDISU AYELE………………………………………NCR/016/07

ETAGEGN ABEBAYEW……………………………..NCR/086/06

SENA MOGOS……………………………………….. NCR/254/07

ELSA WOLDU……………………………………….. NCR/117/07

Advisor: Mohammed T.

June, 2017

Assosa, Ethiopia

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 Acknowledgments

Firstly we are thankful to our God for his mercy & help. Next we are grateful thanks to our
advisor (instructor) Mohammed Tesfaye and biology department head Mr. Asmamaw Abate for
their good assistance and valuable comments starting from proposal developing until the
fruitiness of the final research. We are also grateful to respondents (milk handlers) who
permitted and participated to scarifying their time with us for responding our questions & helped
us in collecting samples and finally to our families for their financial as well as moral support.

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 TABLE OF CONTENT
ACKNOWLEDGMENTS ...............................................................Error! Bookmark not defined.
LIST OF ABBREVIATIONS .................................................................................................... IV
LIST OF TABLES AND FIGURES ........................................................................................... V
ABSTRACT ......................................................................................Error! Bookmark not defined.
1. INTRODUCTION..................................................................................................................... 1
1. 1. Background of the Study .................................................................................................... 1
1.2. Statement of the Problem ..................................................................................................... 2
1.3. Objective of the Study .......................................................................................................... 2
1.3.1. General Objective .......................................................................................................... 2
1.3.2. Specific Objective .......................................................................................................... 2
1.4. Significance of the study ---------------------------------------------------------------------------- 3
1.5. Limitation of the Study ........................................................................................................ 3
2. LITERATURE REVIEW ........................................................................................................ 4
2.1. Raw Milk and Its Contamination with Bacteria ................................................................... 4
2.2. The Two Main Milk Contaminants ...................................................................................... 4
2.2.1 Escherichia coli............................................................................................................... 4
2.2.2 Staphylococcus aurous ................................................................................................... 5
2.3. Safely Handling Raw Milk and Proper Milking Procedures................................................ 5
2.4. Handling Practice of Milk by Milk Handlers ....................................................................... 7
3. METHODOLOGY ................................................................................................................... 8
3.1. The Study Area Description ................................................................................................. 8
3.2. Study Design ........................................................................................................................ 8
3.3. Sample Collection ................................................................................................................ 8
3.4. Bacteriological Analysis ...................................................................................................... 8
3.4.1. Isolation Of E. coli......................................................................................................... 9
3.4.2. Isolation Of S. aurous .................................................................................................... 9
3.5. Assessments of Milk Handling Practices by Milk Handlers ................................................ 9
3.6. Data analyses and Interpretations-------------------------------------------------------------------9
3.7. Ethical Considerations -----------------------------------------------------------------------------10

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4. RESULTS AND DISCUSSION……………………………………………………………..11
5. CONCLUSION AND RECOMMENDATIONS .................................................................. 16
5.1. Conclusions ........................................................................................................................ 16
5.2. Recommendations .............................................................................................................. 16
6. REFERENCES ........................................................................................................................ 18
Appendix ...................................................................................................................................... 21

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 List of Abbreviations

E. coli ----------------------------------------------Escherichia coli

EHEC………………………………… …… Entero hemorrhagic E. coli

EMB…………………………………………Eosin methylene blue agar

HC……………………………………………Hemorrhagic colitis

NAM……………………………………… …Nutrient agar medium

S. aurous …………………………………… Staphylococcus aurous

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 List of Tables and Figures

Table 1. Contamination level and percentage of E. coli from raw milk……………....11

Table 2. Enumeration of S. aurous (Cfu log/ml) ………………………………………12

Table 3. Average Contamination of S. aurous in raw milk …………………;;………13

Table 4. Assessment of milk handling practices among milk handlers and venders….14

Figure 1. Percentage of milk contamination with E. coli………… ………….……... 11

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 Abstract
Milk is supposed to constitute a complex ecosystem for various microorganisms including
bacteria since milk is reach in all types of nutrients. Contamination of milk and milk products
with pathogenic bacteria is largely due to processing, handling, and poor hygienic practices
in which they can cause high virulence. Among microbes in raw milk, E. coli and S. aurous
are common especially in unhygienic milk and milk products and they have a great impact
throughout the world on a children and young populations. The main objective of this study
is to isolate and study the presence of E. coli and S.aurous in raw milks, and to assess the
handling practice of raw milk handlers in Assosa town, Benishangul Gumuz regional state. A
cross sectional study was done on a total 45 samples of raw cow milk and each of 15 samples
which taken from cattle udder (teat), milk storage containers and milk venders (merchants)
from March 2017 to June 2017. The isolation and confirmatory test of E. coli and S. aurous
enumeration was conducted following the standard procedures by using EMB agar and
mannitol salt agar, respectively. And finally, all of the data was organized and analyzed
through Excel software in terms of frequency and percentage with tables and chart for simple
reporting of the findings. The study shown that from the total of 45 samples of raw cow milk
collected from three different sites (udder, storing bucket and market place), 28.89% is
contaminated with E. coli and 48.89% of raw milk samples were contaminated with S.
aurous. The present study revealed that there is medium contamination level of sample by S.
aurous <5 log10 to pose pathogenic effect on the body of milk users. But, still the result
suggests that there is a need for hygienic safety and analysis from E. coli suggests that the
milkier and utensils for milking purpose should be kept clean as they are the major sources
of contamination.

Keywords/phrases/: Assosa, Benishangul Gumuz, E. coli, Enteropathogenic, Raw milk,

S. aurous

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 1. INTRODUCTION

1. 1. Background of the Study

Milk and milk products consist of high moisture, nearly neutral in pH and are rich in
carbohydrate, protein, fat and vitamins. Hence, milk easily favors the growth and multiplication
of many bacteria, even pasteurized or refrigerated. These bacteria may significantly influence the
quality of the milk and milk products. Milk contains relatively few bacteria when it is secreted
from the udder of a healthy animal. However, during milking process, raw milk gets
contaminated from the exterior of the udder and the adjacent areas, dairy utensils, milking
machines or the hands of the milking man, from the soil and dust. In these way, bacteria, yeasts
and molds get entry into the milk and thus constitute the normal flora of milk. Milk might also be
contaminated with pathogenic bacteria or bacterial toxins which may serve as vehicle for the
transmission of diseases to humans such as salmonellosis, diarrhea, food poisoning, tuberculosis
etc. (Uddin et al., 2011).

The milk of domesticated cattle is the most popular to human consumption in which milk is
approximately contains 87% water, 4.9% carbohydrate (lactose), 3.4% fat, 3.3% protein and the
remaining 1.4% is vitamins and minerals (Https://www.havemilk.com/delicious-dairy/milk-
varieties). The primary carbohydrate found in the milk is lactose. Lactic acid is produced when
bacteria in milk metabolize lactose (Saurav, 2013). The quality of milk is evaluated by its
composition and hygienic properties. It serves as excellent culture medium for the multiplication
of many different bacteria such as E. coli and S. aureus in laboratory. They could colonize diary
plant premises and consequently contaminate dairy products. According to Singh and Prakash
(2008), milk is supposed to constitute a complex ecosystem for various microorganisms
including bacteria. Milk products like cheese and curd are widely consumed and market for them
has existed in many parts of the world for many generations. Contamination of milk and milk
products with pathogenic bacteria is largely due to processing, handling, and unhygienic
condition in which Escherichia coli frequently contaminating food organism and it is a good
indicator of fecal pollution (Diliello, 1982: Soomro et al., 2002).

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Pathogenic bacteria in milk have been a major factor for public health concern, since, many
diseases are transmissible via milk products. Traditionally, raw or unpasteurized milk has been a
major vehicle for transmission of pathogens (http://www.sciensage.info/jasr). The health of dairy
herd and milking condition basically determines the milk quality. Another source of transmission
of microorganism is unclean teats. The use of unclean milking and transporting equipment also
contributed to the poor hygienic quality (Hafsa et al., 2014).

1.2. Statements of the problem

Milk is a host for many microorganisms like Escherichia coli and staphylococcus aurous. This
two major bacterial species have a great impact throughout the world on a children and young
populations who use unhygienic milk and milk products and are considerable causative agents of
disease worldwide. Microbiological assessments have an important role to play in the dairy
industry to protect the public health and can reduce economic losses by the early detection of
inadequate processing, packaging or refrigeration. (Uddin et al., 2010; EATA, 2015).

Presence of E. coli in the milk product indicates the presence of entero-pathogenic

microorganisms, which constitute a public health hazard. Entero pathogenic E. coli can cause
severe diarrhea and vomiting in infants and young children (anon., 1975). Particularly in Assosa
area the distribution and virulence of this pathogenic organisms is believed to be higher, because
of poor hygienic practices of residents. Hence the present study investigates the isolation and
detection of E. coli and S. aurous enumeration from raw milk.

1.3. Objective of the Study

1.3.1. General Objective

 To isolate Escherichia coli and enumeration of staphylococcus aurous from raw milk.
1.3.2. Specific Objectives
 To isolate and determine E. coli from raw milk
 To isolate and enumerate S. aurous from raw milk
 To assess the handling practice of raw milk in Assosa town

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1.4. Significance of the Study
In Ethiopia including Assosa, there is high annual milk production with large number of cattle
resource. But still the handling and consumption of this milk is in a traditional and unhealthy
way. To assess this problem such kind of study is very useful as they help individuals in
understanding of what the milk properly handled as it is not harm to health. In addition, it helps
further study by providing as base line information. Therefore, the present study investigates to
isolate E. coli and Staphylococcus aurous from raw milk sold in Assosa city

1.5. Limitation of the Study

In did this study we faced some problems like shortage of time, scarcity of laboratory
equipment’s and media which makes unable to conduct of biochemical test.

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 2. LITERATURE REVIEW

2.1. Raw Milk and Its Contamination with Bacteria

Milk is among the most perishable foods and it is potentially susceptible to contamination with
microorganisms because it is an excellent medium for the growth of bacteria. Some of these are
harmless but some may be pathogenic to humans. Contaminated or spoiled milk is a one that
causes a remarkable discomfort on individual. This contamination (spoilage) may be either by
contaminated hands of workers or unsanitary utensils, contaminated with flies, and use of
polluted water for cleaning the utensils of storage materials. The sanitary quality of milk can be
checked by bacterial isolation and count in the milk (perihar and perihar, 2007; Dubey and
Maheshwari, 2002). In the recent years there has also been a steady increase in the production
and consumption of processed milk products worldwide because of their high nutritive value and
convenience. However, un processed milk products may at a time constitute a public health
hazard due to the possible presence of food pathogenic bacteria which cause illness, intoxication
and sometimes outbreak of death (Doaa El-Hadedy ; Salwa Abu El-Nour, 2012).

2.2. The Two Main Milk Contaminants

According to priyanka and alka (2012), the most important pathogens found in milk and milk
products are Escherichia coli and staphylococcus species. Most strains of E. coli are harmless but
several strains are extremely pathogenic like E. coli 0157:H7 causing complication and death
associated with hemorrhagic colitis and acute renal failure, especially in children (Robins brown
et al., 2004). The infective dose of E. coli is estimated to be about 10 cells in contrast the
infective dose of S. aurous is high because the actual cause the food poisoning is not the
organism itself, but rather a number of heat stable protein toxins produced by the bacterium and
capable of withstanding 100c for >30 minutes (presscott et al., 2002).

2.2.1. Escherichia Coli

It is considered to be a fecal contamination indicator in foods, because of its presence in the gut.
The presence of E. coli in foods is a matter of concern because some strains may be pathogenic
(Thaker et al., 2012). E. coli 0157;H7 serotypes, identified as enterohaemorrhagic E. coli
(EHEC) and grouped as verotoxin-producing E. coli (VTEC), are recognized as the primary

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cause of hemorrhagic colitis (HC) and the diarrhea associated form of hemolytic uremic
syndrome (HUS) (Radii et al., 2011). Fermented dairy products made with unpasteurized milk
are potential vehicle for the transmission of E. coli to consumers. It has been shown that the
pathogen is present in raw milk, although it will survive proper pasteurization, if it occurs after
post pasteurization the organism has been shown to survive the manufacturing and ripening stages of
fermented dairy products (Coia et al., 2001).

2.2.2. Staphylococcus aurous

Staphylococcus aurous is the most prevalent and economically significant pathogen causing
inflammatory infections in dairy ruminants. S. aurous can get access to milk either by direct
excretion from udders with clinical or subclinical Staphylococcal mastitis or by contamination
from the environment during handling of raw milk. Staphylococcus aurous is a pathogenic
bacterium that induces several of human illnesses. The ability to cause a wide range of diseases
may be associated with its production of a large spectrum of extracellular toxic compounds and
other virulence factors such as toxic shock syndrome, exfoliative toxins and Enterotoxins
(mueena et al., 2015).

However, despite genetic up gradation and modern methods of rearing livestock production per
capita has remained low. One reason for this could be mastitis especially for the sub clinical
type. Mastitis remains one of the most common economic problems of dairy industry worldwide.
It is mainly caused by bacteria, and the infection results in inflammation and pathophysiological
changes in the udder tissue, resulting in compromised milk quality and decreased amount of milk
production. Depending on the severity of the inflammation the disease can be categorized in to
sub clinical or clinical. Clinical mastitis can be readily detected whereas asymptomatic
conditions make detection of sub clinical mastitis difficult nonetheless (mueena jahan, et al.,
2015).

2.3. Safely Handling of Raw Milk and Proper Milking Procedures

Always try and maintain a healthy animal: Sick cows will not produce healthy milk. Still, it’s
important that your animal have access to fresh water, fresh grass/high-quality hay, clean living
conditions, and mineral supplements/salt licks/etc. (http://theelliot thomestead.com/2014/04/
handling-raw-milk-procedures/).

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Prepare a proper milking area: Wet and dirty ground is not good. So, keep the milking area
clean and as free of feces as possible (http://theelliotthomestead.com/2014/04/handling-raw-
milk-procedures/).

Having proper and Clean equipment: In order to keep the milk clean and free of contaminates,
it’s absolutely essential that the equipment is clean and stainless steel buckets for the milk
storage, filter equipment (for filtering the milk). On the other hands, all of the equipment’s
should be easy to maintain, inexpensive, and easily sterilized to ensure its cleanliness (
http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/)

When it’s time for milking, clean animal properly: Using the warm water to dissolve anything
crusted on the teat we usually only wash for 15-30 seconds. Enough time to get nice and clean
and stimulate milk let down (http://theelliotthomestead.com/2014/04/handling-raw-milk-
procedures/).

Milk cleanly: Make sure the milker’s hands are clean. Dirty hands dirty milk
(http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/).

Cool the milk quickly: This is one of the most important steps in raw milk handling. As soon as
a cow is milk, the bacteria in the milk beginning eating and digesting the sugar in the milk.
Cooling the milk helps to deter the growth of bacteria, allowing for fresher milk for longer. We
carry our bucket up to the house, where it is then filtered into the jugs. The jugs are then placed
in the freezer for an hour. After an hour of speed of cooling, the milk is transferred to the
refrigerator where it remains until it is drunk. The freezing step helps to get a jump start on the
cooling process (http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/).

Handle with care: it’s still important to handle the milk with care and respect for the product
since it is a perishable product. Just like any other agriculture products, has the potential to be
produced well and safely. Handled improperly also has the potential to make consumers sick.
(http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/).

Poor hygiene, practiced by handlers of milk and milk products, may lead to the introduction of
pathogenic micro-organisms into the products. Since they do not undergo further processing
before consumption, these foods may pose risk to the consumers. Therefore, provision of milk

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and milk products of good hygienic quality is desirable from consumer health point of view
(Yohannis and Mesfin , 2015; STTA Report – Vladimir Kokarev, 2006).

2.4. Handling Practice of Milk by Milk Handlers

Whether milking by hand or machine, good hygiene is essential. This requires that: The milkers
hands and clothes are clean and he or she is in good health. The milking machine and milk
storage equipment such as milk churns are kept clean and are in good condition (i.e. without
cracks or dents which are difficult to clean and can easily harbor bacteria. Immediately after
milking, the milk must be cooled preferably to 4° C. This requires mechanical refrigeration or
milk cooling tanks. These are expensive and can usually be afforded by large scale commercial
farms. For small scale dairy farmers, setting up a milk cooling center centrally may be the ideal
solution. Where farmers bring their milk to a cooling centre through a co-operative, they should
do so as soon as milking is completed. A Milk cooling centre with a capacity of 1000 - 3000
liters will serve up to 300 small holder farmers ensuring that the quality of their milk when
produced under hygienic conditions is well preserved and accepted at the processing plant.
(http://www.fao.org/ag/againfo/resources/documents/mpguide/mpguide1.htm)

To maintain the quality of milk during transportation, adhere to the following guidelines
(Lusato, 2006):

 Ensure that milk containers and the transport vehicle are kept clean.
 Do not use milk containers for storage of other goods.
 Do not transport milk with other goods. The code of hygiene requires that vessels and
carriers used for milk transport should be used only for that purpose and be labeled
“MILK ONLY”.
 Keep the milk as cool as possible and avoid exposing it to high temperatures.
 Keep the milk covered at all times to protect it from light and dust.
 Transport the milk to the sales point or processing factory in the shortest time possible.
 Do not smoke, handle tobacco or other materials with strong odor (e.g. kerosene fuel)
when handling milk.
 Avoid excessive agitation of the milk cans.

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 3. METHODOLOGY
3.1. The Study Area Description
The study was conducted in Assosa town of Benishangul gumuz regional state. The town is
about 661km far from Addis Ababa which is the capital of Ethiopia. The study region located in
north western part of the country between 0917’-1206’ north latitude and 3410’-374’ east
longitude (moreda, 2013) and having a total area of about 50,382 kilometer square (flatei, et al.,
2009). According to 2007 census report, the population size of the region was 670,847 (CSA,
2008). And the region is known by the availability of livestock resource and high milk
production as most of the other regions do.

3.2. Study Design

A cross-sectional study was conducted in the town from April 2017 to May 2017 to collect and
determine E. coli and S. aurous from raw milk, and assess raw milk handling practice of milk
venders and owners in different Keble’s of the town. Samples of raw milk was collected at
morning and evening from dairy cooperative milk producer centers and farmers from
purposively selected three urban Kebeles (03, 02 and Amba 5) which have large number of
customers.

3.3. Sample Collection

Milk sample were collected from points or milk seller who considered to be associated with
contamination. The sampling point was at the teat during milking, milking buckets and at the
market place. Over all 45 samples of raw milk was taken in sterile bottles using ice box, in
which, 15 samples from the teat, 15 from milking bucket or storage, and the remaining 15 have
been collected after arrival to the market in the market place. And all the samples were
transported to Assosa university Biology laboratory for immediate bacteriological analyses.

3.4. Bacteriological Analysis

The milk samples was subjected to serial dilution, where one ml of each sample was added to 9
ml of peptone bacteriological (an enrichment media) and shaken gently. All the samples were
serially diluted up to 10-3 dilution factor and the test tubes were incubated for 24 h at 37° C
(Hafsa, et al., 2013) for enrichment purpose.

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3.4.1. Isolation of E. coli

For the isolation of E. coli, 1ml of the enriched sample from peptone bacteriological water was
cultured on selective and differential MacConkey agar medium and incubated at 37˚C for 24
hours. Typical colonies having pink color from MacConkey plate agar streaked on a nutrient
slant for refreshment and incubated for additional 24 hours. Then, transferred to eosin methylene
blue (EMB) and E. coli is confirmed by its green metallic sheen color on the EMB media.
Morphologically typical colonies having metallic green sheen was taken and confirmed as E. coli
and into for further identification on nutrient agar slants (http://www.sciensage.info/jasr.media
for E. coli isolation).

3.4.2. Isolation of S. aurous

From each samples of prepared serial dilution, one ml was transferred into triplicate sterile Petri
dishes and Mannitol salt agar (Oxoid, England) was poured and swirled gently and finally
incubated at 37ºC for a maximum of 48 hours (Robers and Greenwood, 2003). Yellow and
orange colonies surrounded by yellow zones due to mannitol fermentation enumerated as the
presence of S. aurous.

3.5. Assessments of Milk Handling Practices by Milk Handlers

The observation checklist and semi structured questioner was used for assessment of the sanitary
conditions and milk hygienic practices of the handlers. A check lists covering topics on the
personal hygiene of the milk seller and milker’s hygiene practices (modes of cleaning and
sanitizing utensils) and hygiene of the selling area to asses weather the area exposed to flies,
insects and animals, presence of solid and liquid waste in vending area. Assessment of the
hygienic practices of farmers, venders and vending environment was a total of 18. Respondents
were selected purposely who engage with milk handling or milk venders and also interview and
a semi structured questionnaires was used.

3.6. Data Analysis and Interpretation

After sample collection from milk handlers, the result was analyzed using tables and charts to
show and make comparison of E. coli contamination or presence/absence among milk samples
taken from different sites. In addition, descriptive statistics such as percentage and frequency

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distribution was done using Microsoft excel software to describe the enumerated S. aurous and
E. coli contamination rate. Finally, short tabular description was used about milk handling
practice among venders and handlers in order to make conclusion

3.7. Ethical Considerations

In conducting this research we put all necessary issues to be considered in to account when doing
every action: all farmers and venders including those who participate in responding
questionnaires had got awareness of benefits of the research. In addition, we have taken every
action with the recommended license from our department and institution. Generally the present
study was done by creating positive attitude with every participant and respondents.

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 4. RESULTS AND DISCUSSION

The present study shows that (Table 1 & figure 1) among the total of 45 samples of raw cow
milk collected in three different sites (udder, storing bucket and market place) 15 from each, 7
(46.66%) is contaminated with Escherichia coli (E. coli) as higher contamination recorded at
storage bucket followed by udder (26.66%) and low contamination at market place (13.33%).
Generally, as shown (Table 1 & figure 1), the total contamination rate of raw milk samples taken
from all sources with E. coli was 13 (28.89%).

Table. 1. E. coli contamination level and percentage of raw milk collected from three sources

No. Source of sample Number of Number of E. coli Percentage (%)

samples positive samples
1 Udder/teat 15 4 26.66

2 Bucket/storage 15 7 46.66

3 Vender/market 15 2 13.33

Total 45 13 28.89

E. coli
13.33% 26.66% udder

46.66% bucket
market

Figure 1. E. coli contamination percentage

Presence of E. coli in milk suggests that the sample is contaminated through transportation,
unhygienic processing or through fecal contamination. Similarly, in the present study, the

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methods of production, transportation, handling and sale of milk were entirely unhygienic and
the study showed that 13 out of 45 milk samples were contaminated with E. coli. On the other
hands, high incidence of E. coli was found in raw milk samples conducted by many researchers,
Martin and others (1986), for instance, reported that 43% of the milk samples were contaminated
with E. coli; and similarly Sharma and Joshi (1992) reported that 50% of the milk samples were
contaminated with E.coli.

According to the results (kumar, 2011), the 16 (29.09%) samples out of 55 showed that the
contamination of E. coli. E. coli is one of the bacteria that exist in the normal micro flora of the
intestinal tract of humans and warm blooded animals. Its contamination with milk and milk
products, is largely due to processing, handling, and unhygienic conditions. In addition, E. coli
is, furthermore, a known causative agent of diarrhea and other food-borne related illnesses
through the ingestion of contaminated foodstuffs. Pathogenic members of the E. coli form group
as well as the enterobacteriacae family are represented by genera such as salmonella and shigella
and are found in the intestines of humans and animals (collins et al., 1995).

Table 2. Enumeration of S. aurous (Cfu log/ml) in raw milk from Udder, Bucket and Market

Sample site Sample code Cfu/ml Cfu log/ml

Udder 2 1203 3.08
“ 4 1297 3.11
“ 8 1420 3.15
“ 9 1273 3.10
“ 11 540 2.73
“ 13 5527 3.74
Bucket 17 340 2.53
“ 19 30 1.48
“ 20 413 2.62
“ 21 23 1.36
“ 22 3387 3.53
“ 28 617 2.80
“ 29 3483 3.54
“ 30 7 0.85

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 Market 33 280 2.45
“ 34 3420 3.53
“ 35 1950 3.30
“ 37 3197 3.50
“ 38 1200 3.08
“ 42 157 2.20
“ 43 2433 3.39
“ 45 4090 3.61

S. aurous is often found in unprocessed milk and dairy products due to contamination caused by
poor hygiene conditions, or the origin of the milk, which can come from mastitis cows or
through hands or skins of handlers (human being). Staphylococcus is one of the most important
food borne microorganisms responsible for staphylococcal food poisoning (SFP) consumption of
100µg of staphylococcus Enterotoxins (SE ) produced by enterotoxigenic strains causes SFP
(Enquebaher, 2015). When compared to our finding with the study conducted by Aftab (2010)
reported that 11 (50%) raw milk samples out of 22 tested samples were contaminated with
staphylococci, it is somewhat almost similar with the present findings. On the other hands,
contamination of the raw milk with S. aurous is suspected to result from the origin of the milk or
poor hygiene conditions which can be from mastitis cow or unclean utensils. An increase in time
or temperature before consumption could lead to further proliferation of the pathogen and the
production of toxins by enterotoxigenic if any (Enquebaher, 2015).

Table 3. Average S. aurous contamination level of raw milk in each sources

Source of No of No of positive Average log cfu/ml Percentage (%)

sample samples samples
Udder 15 6 3.15 40.0
Bucket 15 8 2.34 53.33
Market 15 8 3.13 53.33
Total 45 22 Average 2.874 48.89

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On the other hands, The present finding showed that, from the total 45 samples at three different
sites; 22 (48.89%) are shown to be S. aurous positive in which 6 (40%), 8 (53.33%), 8 (53.33%)
at udder, bucket and market, respectively were contaminated with S. aurous. The average
number of S. aurous colonies per milliliter of samples as a form of (log cfu/ml) were 2.34, 3.13
and 3.15 at bucket, market, and udder, respectively (Table 3).

Table 4: Assessment of the hygienic practice of milk handlers, venders and the environment

No. Questions Total number of Percentage (%)

respondents (n)= 18
1 Do you check whether your cow is
healthier or not before milking?
a. yes 11 61.11
b. no 7 39.89
2 Have you clean your milking bucket,
teats of milked animal and wash your
hands before milking?
1. Wash hands before milking
a. yes 12 66.67
b. no 6 33.33
2.washing udder(teats) before
milking
A. yes 8 44.44
B. no 10 55.56
3.Clean bucket before milking
a. yes 9 50
b. no 9 50
Stage of selling
1.raw 2 11.11
3 2.after boiling 16 88.89
4 Area of milking
a. large and clean area 1 5.56
b. small corner 3 16.67
c. where ever free area 14 77.78
5 Storage condition at home:
a. at refrigerator 6 33.33
b. at room temperature 12 66.67

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6 Frequency of milking
a. one’s per day 1 5.56
b. two times per day 17 94.44
c. more than two 0 0
7 Habit of washing the teat
with______
a. detergent 1 5.56
b. hot water 1 5.56
c. tap water 6 33.33
8 Presence of separate worker
a. yes 1 5.56
b. no 17 94.44
9 Presence of separate cloth for
milking
a. yes 1 5.56
b. no 17 94.44

10 Type of bucket
a. plastic 11 61.11
b. metal 1 5.56
c. others 6 33.33
11 Practice of cleaning of milking area
a. yes 3
b. no 15 16.67
83.33
12 Cleaning of vending environment
a. yes 4 22.22
b. no 14 77.78

Assessments of milk handlers: A total of 18 milk handlers including venders were interested
and participated in responding the questions rose to access the milk handling tradition and their
knowledge against infectious diseases transmitted through milk and milk products. From 18
participants, 15 were female and the other 3 were males. The age of the respondents ranges
between 24 and 52 years.

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 5. CONCLUSION AND RECOMMENDATION

5.1. Conclusions

The result of the present study revealed that there is medium contamination level of milk
samples by S. aurous, i.e., <5log10 cfu/ml (Enquibahar et al., 2015) to pose pathogenic effect.
But, still the result shows that there is a need for hygiene to keep safety of milk and following up
the health of the animal in order to reduce contamination of milk and its products by this
microbe. In the same manner analysis from E. coli suggests that the milker and utensils for
milking purpose should be kept clean as they are the major sources of contamination. As easily
understood from (table 1 and figure 1) high contamination rate with E. coli is recorded at storage
utensils which indicates low practice of cleaning the materials.

As the data collected from milk handlers and venders, most of the respondents do not use those
necessary measures such as: - a) detergents and hot water to clean the teat of their animal, b) lack
of cleaning their hands before milking, c) absence of separate worker and cloth for safety and d)
shortage of large and clean area which is free from insect exposure might be the main reasons of
much contamination of the milk samples with E. coli and S. aurous.

5.2. Recommendations
Based on the present study we will be recommended the following points:

 The milk handlers and the venders who do not properly protect their hygienic quality and
those who supply low quality milk to the market should be undertake some quality
measures and awareness creation among them about safety handling practice of milk and
milk products
 Poor hygiene, practiced by handlers of milk and milk products, may lead to the
introduction of pathogenic micro-organisms into the products. Since they do not undergo
further processing before consumption, these foods may pose risk to the consumers.
Therefore, provision of milk and milk products of good hygienic quality is desirable from
consumer health point of view.

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  In addition the habit of washing the animal udder by soap or warm water should be
developed among the community as there is high probability of contamination of udder
with those disease posing pathogens from the environment.
 In the present study, due to lack of reagents and culture media we cannot conduct some
biochemical study regarding to E. coli. So, further researchers in the future should
conduct biochemical tests and isolate other pathogens from milk samples.

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 6. REFERENCES

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food by conventional and molecular methods. Journal of genetic engineering and
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6 Dilielo LR (1982). Methods in food and dairy microbiology. Avi publishing co. Inc Westport
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Company LTD New Delhi.
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staphylococcus species in milk and milk products from tigray region, Northern Ethiopia
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group assosa zone Benishangul gumuz regional state, Midwest Ethiopia. J. ethnobiol.
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13 Uddin Md, Hasan Md, Motazzim-ul-Haque, 1 Rashed Noor 1 (2011). Isolation and
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Abdul KT and Sultan A, (2015). Isolation and characterization of Staphylococcus aureus
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jodhour.
18 Presscott LM, Doughari JP, Klein DA (2002). Textbook of microbiology 5th edition brown
publishers.
19 Preethirani PL, Sloor S, Sundareshan S, Nutanala K, Shivi V, Deepthikiran K, Sinha Ay,
Akhauri Y(2015). Isolation, biochemical and molecular identification and invitro
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24 STTA Report – Vladimir Kokarev, August 2006.

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25 Thaker HC, Brahmbhatt MN, Nayak JB. (2012). Study on occurrence and antibiogram
pattern of E. coli from raw milk samples in Anand, Gujarat, India. Vet World. 5 (9):556–559.
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 APPENDIX
1. Do you check whether your cow is healthier or not before milking? A) yes b) No
2. Have you ever clean your milking bucket, teats of milked animal and wash your hands before
milking? A) Yes b) No
3. At which stage you sell the milk
a) At raw stage b) boil and cool back in refrigerator before feeding
4. Which areas you choose to milk the animal?
a) Large b) medium c) small d) clean e) others
5. At what condition the milk is stored after milking (for owners) and after arrival (for sellers)?
a) At room temperature b) in refrigerator C) Others…. if any
6. Frequency of milking per day
a) Ones per day b) two times per day c) more than two times
7. The habit of washing the teat is:-
a) Detergent b) hot water c) tap water D) If any, others
8. Presence of separate worker for milking
a) Yes b) No
9. Presence of separate cloth for milking purpose
a) Yes b) No
10. Type of milking utensil
a) Plastic b) metallic c) others
11. Practice of cleaning milking environment.
a) Yes b) no
12. Cleaning vending environment.
a) Yes b) no

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