isolation of coliforms , esherichia coli and other aerobic bacteria from pasteurized milk samples in

73 Pages·2006·0.45 MB·English

ﻢﻴﺣﺮﻟا ﻦﲪﺮﻟا ﷲا ﻢﺴﺑ

University of Khartoum
Faculty of Public and Environmental Health
Department of Food Hygiene and Safety

ISOLATION OF COLIFORMS , ESHERICHIA COLI AND OTHER AEROBIC BACTERIA FROM
PASTEURIZED MILK SAMPLES IN KHARTOUM AND KHARTIUM NORTH

A Thesis submitted to the Graduate College, University of Khartoum,
as a partial fulfillment of requirement for the degree of Master of
Food Hygiene and Safety

By:

Badria Mohamed Sayed Ahmed Ali, B.Sc. (1998) Faculty of Public and
Environmental Health University of Khartoum

Supervisor:
Dr. Tawfig El Tigani Mohamed

July 2006
Abstract

The present study was to investigate the presence of coliforms and E.
coli and determine the bacterial contamination of pasteurized milk samples
in Khartoum state

Thirty packaged pasteurized milk samples were collected from
Khartoum state shops . The samples were examined microscopically and
cultured to determine if there were coliforms and E. coli bacteria and also
to determine the types of bacteria involved in pasteurized milk samples .

Twenty six samples (86.7%) of milk was found to be positive for
bacteria . Were as only six (13.3%) gave negative bacterial culture. The
isolated bacteria from pasteurized milk were Staphylococcus (82%),
Enterobacteria (15.4%) and Pasteurella(2.6%)

Further more the species of staphylococcus isolated were
haemolyticus(12.8 %) , simulans(12.8%), capitis (9%), aureus(10.3%),
caseolyticus(7.7%), epidermidis (7.7%), hominis (6.4) , intermidius
(6.4%), chromogenes (2.6%), saprophticus(5%) and ludgunensis (1.3%)

The bacteria which was isolated from milk samples other than
Staphylococcus species were serratia marcescens (7.7%), E .coli (2.6%),
proteus mirabilis (3.8%), enterobacter aerogenes (1.3%) pseudomonas
pseudomallei (1.3%) and pasteurella haemolytica (2.6%)
The counts of total coliforms were found to range between 0.9××103.and
6.4 ×103

The present study concluded that the pasteurized milk distributed in
Khartoum State was of low quality . Hence it was recommended that
quality assurance programmes should be started to ensure that good quality
milk and milk products are produced and consumed in the country.
ﺔﻓﺮﻌﻣو ىﻻﻮآﺎﻴﺸﻳﺮﻜﺳﻻا و ﺔﻴﻧﻮﻟﻮﻘﻟا ﺎﻳﺮﻴﺘﻜﺒﻟا دﻮﺟو ﺔﻓﺮﻌﻤﻟ ﺔﺳارﺪﻟا ﻩﺬه ﺖﻳﺮﺟا
. مﻮﻃﺮﺨﻟا ﺔﻳﻻﻮﺑ ةﺮﺘﺴﺒﻤﻟا نﺎﺒﻟﻸﻟ ﺔﺛﻮﻠﻤﻟا ﺎﻳﺮﻴﺘﻜﺒﻟا عاﻮﻧأ
ﺖﻋرزو ﺎﻳﺮﻬﺠﻣ ﺖﺼﺤﻓو مﻮﻃﺮﺨﻟا ﺔﻳﻻﻮﺑ تﻻﺎﻘﺑ ﻦﻣ ﺮﺘﺴﺒﻣ ﻦﺒﻟ ﺔﻨﻴﻋ نﻮﺛﻼﺛ ﺖﻌﻤﺟ
ﻪﻨﻴﻋ نوﺮﺸﻋو ﺔﺘﺳ ﻲﻓ ﺎﻳﺮﻴﺘﻜﺒﻟا ﺖﻤﻧ .ﺮﺘﺴﺒﻤﻟا ﻦﺒﻟا ﻲﻓ ةدﻮﺟﻮﻤﻟا ﺎﻳﺮﻴﺘﻜﺒﻟا عاﻮﻧأ ﺔﻓﺮﻌﻤﻟ
.(%13.3) تﺎﻨﻴﻋ ﻪﻌﺑرأ ﻲﻓ ﻮﻤﻨﺗ ﻢﻟو (% 86.6)
ﺔﻳﻮﻌﻤﻟا تﺎﻳﻮﺼﻌﻟا، (%82 )ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا : ﻰﺗﻻا ﺎﻬﻟﺰﻋ ﻢﺗ ﻲﺘﻟا ﺎﻳﺮﻴﺘﻜﺒﻟا عاﻮﻧأ
.(%2.6)ﻼﻳرﻮﻴﺘﺳﺎﺒﻟا و،(%15.4)
،(%12.8) مﺪﻟا ﺔﻟﺎﺣ ﻪﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا :ﻲه ﺎﻬﻟﺰﻋ ﻢﺗ ﻲﺘﻟا ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا عاﻮﻧأ ﺎﻣا
تارﻮﻜﻤﻟا ،(%9) capitis ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%12.8) ﺔﻬﺑﺎﺸﺘﻤﻟا ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا
ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%7.7) ﻪﻨﺒﺠﺘﻤﻟا ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%10.3) ﻪﻴﺒهﺬﻟا ﺔﻳدﻮﻘﻨﻌﻟا
ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%6.4) hominis ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%7.7) ﻪﻳوﺮﺸﺒﻟا
(%5) ﻪﻴﻣﺮﻟا ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%2.6) ﻪﻴﻐﺒﺼﻟا ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟا ،(%6.4)ﻪﻴﻄﺳﻮﻟا
(%1.3)ﻪﻴﺳﺪﻌﻟا ﺔﻳدﻮﻘﻨﻌﻟا تارﻮﻜﻤﻟاو
serratia, E. coli, proteus, : ﺎﻀﻳأ ﻪﻟوﺰﻌﻤﻟا ﺎﻳﺮﻴﺘﻜﺒﻟا عاﻮﻧأ ﻦﻣ ﺖﻧﺎآ
enterobacter, pseudomonas , and pasteurella
6.4 ×103. ﻰﻟإ 0.9×103. ﻦﻣ ﺔﻳﻮﻌﻤﻟا ﺎﻳﺮﻴﺘﻜﺒﻠﻟ ﻰﻠﻜﻟا دﺪﻌﻟا حواﺮﺗ
ﺾﻌﺑ ﺔﻄﺳاﻮﺑ مﻮﻃﺮﺨﻟا ﺔﻳﻻو ﻰﻓ ﺞﺘﻨﻤﻟا ﺮﺘﺴﺒﻤﻟا ﻦﺒﻠﻟا ةدﻮﺟ ﻰﻧﺪﺗ ﻰﻟإ ﺔﺳارﺪﻟا ﺞﺋﺎﺘﻧ ﺖﺼﻠﺧ
ﻰﻓ ﺎﻬﺘﺒﻗاﺮﻣو ةدﻮﺟو ﺔﺤﺼﻟ ﻞﻣﺎﻜﺘﻣ ﺞﻣﺎﻧﺮﺑ ﻊﺿﻮﺑ ﺔﺳارﺪﻟا ﺖﺻوأ اﺬﻟ .ﻚﻠﻬﺘﺴﻤﻟا ﻰﻠﻋ تﺎآﺮﺸﻟا
ةدﻮﺟو ﺔﺤﺻ نﺎﻤﺿ ﻰﻟإ فﺪﻬﻳ ﻞﻣﺎﻜﺘﻣ ﻪﻠﻣﺎﺷ ﻩدﻮﺟ ﺞﻣﺎﻧﺮﺑ مﺎﻴﻘﺑ ﻪﺳا رﺪﻟا ﺖﺻوأ ﺎﻤآ .دﻼﺒﻟا
.كﻼﻬﺘﺳﻻﺎﺑ ءﺎﻬﺘﻧاو جﺎﺘﻧﻹﺎﺑ ﺔﻳاﺪﺑ ﺎﻬﺗﺎﺠﺘﻨﻣو نﺎﺒﻟﻷا
LIST OF CONTENT

content Page
Dedication I
Acknowledgements II
Abstract. III
Arabic Abstract IV
List of contents V
List of tables X
List of figures XI
CHAPTER ONE: GENERAL INTRODUCTION 1
CHAPTER TOW:REVIEW OF LITERATURE 3
2.I. Milk composition 3
2.2. Milk and it's nutritional value 3
2.3. Source of raw milk contamination 5
2.4. Bacteria in milk 6
2.4.1. Bacteria of importance in milk 7
2.4.2. Spoilage bacteria in milk 8
2.4.2.1 coliform bacteria 10
2.4.2.2. Escherichia coli (E. coli) 12
2.4.3. Pathogenic bacteria in milk 13
2.4.4. Methods of preserving milk 14
2.4.4.1. Pasteurization 14
2.4.4.2. Sterilizations 16
2.4.4.2.1. Ultra High Temperature (UHT) 16
2.4.5. Keeping quality of pasteurized milk 16
2.4.6. Hygienic quality of pasteurized milk 17
2.4.7. Temperature and storage 17
2.4.8. Shelf life of pasteurized milk 18
2.5. Quality control measures 19
2.5.1. Measurement of microbial quality 19
2.5.1.1. Coliform count 19
CHAPTER THREE MATERIALS AND METHODS 20
3.1 Collection of samples 20
3.1.1. Samples for bacteriological examination 20
. Preparation of culture media3.2 20
3.2. Collection of blood 20
3.3. Sterilization and asepses 21
3.3.1 Sterilization of equipments 21
3.3.2.Sterilization of culture media and solutions 21
3.3.3. Laboratory precautions and sterilization by 21
disinfectants
3.4. Culture media for bacteriological examination 21
3.4.1. Solid media 21
3.4.1.1. Nutrient agar 21
3.4.1.2. Blood agar 22
3.4.1.3. MacConkeys agar 22
3.4.1.4. Urea agar G/L 22
3.4.2. Semi solid media 23
2.4.2.1. Hugh and leifsons media (Oxidation Fermentation 23
test ) G/L
2.4.2.2Cragie tube medium (Barrow and Feltham 1993) 23
3.4.3. Liquid media 24
3.4.3.1. Nutrient broth 24
3.4.3.2. Carbohydrate fermentation media 24
3.4.3.3. Peptone water medium (Oxoid) G/L 24
3.4.3.4. Nutrient broth medium (Barrow and Feltham 1993) 25
3.4.3.5. Peptone water sugar (Barrow and Feltham 1993) 25
3.4.3.6. MR-VP medium (Oxoid) G/L 25
3.4.3.7. Koser citrate medium (Oxoid) G/L 25
3.4.4. Solid selective media 26
3.4.4.1. Violet Red Bile agar 26
3.5. Reagents 26
3.5.1. Hydrogen peroxide 26
3.5.2. Kovac's Reagent 26
3.5.3. Voges- Proskaur( VP) 27
3.5.4. Nitrate reagent 27
3.5.5. Oxidase reagent 27
5.6. Methyl red solution 27
3.4.7. Lead acetate papers 28
3.6. indicators 28
3.6.1. Methyl red 28
3.6.2. Human rabbit plasma 28
3.7. Culture methods 28
3.7.1. Media used for primary isolation 28
3.7.2. Subculture of primary isolates 28
3.7.2.1. From solid to solid media 29
3.7.2.2. From solid to liquid media 29
Incubation of culture 29
3.7.4. Examination of culture 29
3.7.5. Purification and preservation 29
3.7.6. Staining technique 30
3.7.6.1. Preparation of smears 30
3.7.6.2. Gram-staining 30
3.7.7. Identification of bacteria 30
3.7.7.1. Catalase test 31
3.7.7.2. Oxidase test 31
3.7.7.3. Sugar fermentation test 31
7.7.4. Tube coagulase test 32
3.7.7.5. Urease test 32
3.7.7.6. Oxidation fermentation test (O/F) 32
3.7.7.7. Citrate utilization 32
3.7.7.8. Nitrate reduction 32
3.7.7.9. H S production 33
2
3.7.7.10. Indol test 33
3.7.7.11. Voges-proskauer VP test 33
3.7.7.12. Methyl red test 34
3.7.7.13. Novobiocin sensitivity test 34
3.7.14.Motility test 34
3.7.7.15. Total coliforms count 35
CHAPTER FOUR : RESULTS 36
4.1 Survey 36
4.2. Bacteriological findings 36
4.2.1. Species of bacteria isolated from packaged pasteurized 36
milk samples
4.2.1.1. Species of Gram-negative bacteria isolated from 37
packaged pasteurized milk samples
4.2.1.2. Staphylococcus spp isolated from milk samples 37
CHAPTER FIVE : DISCUSSION 53
RECOMMENDATIONCHAPTER SIX: CONCLUSION & 56
6.I. CONCLUSION 56
6.2. RECOMMENDATIONS 56
REFERENCES 58
INTRODUCTION
Milk, being the sole meals of sustenance provided by nature for the
young mammals, is perfect food (Martin, 1978). Keeping hygienic
qualities of the milk is the first priority of human beings. Moreover its use
among infants, elders and disable persons makes its public health
importance a major issue (international dairy Federation= IDF, 1994).
Hence efforts are made globally to produce milk from healthy animals and
to preserve its quality all over the way from farm to table (Giovannini,
1998). He also added that milk most therefore, be protected from
contamination and deterioration from the site of the production, farms, to
the table of the consumers.
Milk is magnificent medium for growth of microorganisms and
therefore risk for quick microbial deterioration of quality is present from
time of milking to the time of use in the milk processing plants (IDF,
1994). The heat treatment of milk prior to packaging for liquid
consumption, or manufacture in to milk based products, is an important
critical control point to ensure that pathogenic organism are killed. It also
ensures that spoilage organisms are eliminated, or at least reduced in
number, for optimum keeping quality (IDF, 1994). In order to improve the
quality control of milk and milk products and to increase the shelf life of
milk, eliminating source of post contamination and proper cleaning should
be followed (Gruetzmacher and Bradley, 1999).
In Sudan, Urban milk supply largely comes from village herds
and it’s marketing is by milk venders who distribute raw milk to household
on donkeys. Organized dairy establishment are limited.
The Sudanese dairy industry therefore, in a primitive and early stage
of development and faced with several problems of transportation and
distribution, lack of infrastructure, high temperature, lack of principle of
quality control, bad or improper handling of raw milk, poor husbandry
practices and neglecting sanitary standard by the distributors and the
major impediments. Therefore, it is to be expected that hygienic quality of
the milk would be low.
Hence the objectives of this study are to:
1. Investigate the presence of Escherichia. coli bacteria in pasteurized
milk in Khartoum state
2. Estimate number of total coliform in pasteurized milk.
3. Estimate the level and types of microbial load of pasteurized milk
distributed in Khartoum state.