ISOLATION AND IDENTIFICATION OF BACTERIAL ORGANISMS IN RAW MILK
(CASE STUDY OF AKWUKE CATTLE RANCH IN ENUGU)
ABSTRACT
The isolation and identification of bacterial organisms in raw milk was carried out.  The pour plate technique was employed for isolation using MacConkey agar, blood agar and calf serum agar. Gram-staining and biochemical tests were carried out to identify the bacterial organisms isolated. Out of the fifty samples of raw milk from Akwuke cattle ranch in Enugu analyzed, the total bacterial mean count was 157cfu. The highest bacteria isolation with it’s percentage was Brucella abortus with 35cfu (22.2%). This was followed by Staphylococcus aureus with 33cfu (20.8%), the next was Escherichia coli with 33cfu (20.7%), followed by Pseudomonas aeruginosa with 29cfu (18.3%). while the least bacterial organism isolated was Streptococcus agalactiae with bacterial mean count of 28cfu (17.9%). The results showed that the total bacterial count of the samples and the individual mean counts, plus pathogenic ones identified, were unsatisfactory and enough to cause both food infection and intoxication. This calls for proper pasteurization as well as hygienic practices in milking processes.
TABLE OF CONTENTS
CHAPTER ONE
1.0    Introduction    -    -    -    -    -    -    -    
1.1    Meaning of milk -    -    -    -    -    -    -    
1.2    Composition of raw milk     -    -    -    -    -    
1.3    Spoilage of raw milk -    -    -    -    -    -    
1.4    Sources of contamination of raw milk -    -    -    
1.4.1    Microbial contamination from within the udder -    
1.4.2    Microbial contamination from outside the udder –
1.4.3    Microbial contamination of raw milk from equipments  
1.4.4    Contaminational of raw milk from milk storage temperature and time -    -    -    -    -    
1.5    Aims of the study -    -    -    -    -    -    -    
1.6    Statement of the problem -    -    -    -    -    
1.7    Significance of the study -    -    -    -    -    
1.8    Limitations of the study -    -    -    -    -    -    
CHAPTER TWO
2.0    Literature Review.    -    -    -    -    -    -    
2.1    Bacterial organisms associated with spoilage of raw milk    -    -    -    -    -    -    
2.1.1    Brucella species -    -    -    -    -    -    
2.1.1.1    Signs and symptoms of brucella infections (Brucellosis)    -    -    -    -    -    -    -    
2.1.1.2    Treatment of Brucellosis     -                
2.1.2    Staphylococcus species                     
2.1.2.1    Sign and symptoms of staphylococcus infection    
2.2.2.2 Treatment of staphylococcus disease            
2.1.3    Streptococcal infection                     
2.1.3.1    Sign and symptoms of streptococcus infection    
2.2.2.2 Treatment of streptococcus disease            
2.1.4    Pseudomonas species                    
2.1.4.1    General infections by Pseudomonas spp        
2.1.4.2    Pseudomonas aeruginosa infection             
2.1.4.3    Signs and symptoms of pseudomonas infections  
2.1.4.4    Treatment of pseudomonal infections    -    -    
2.1.5 Escherichia coli                             
2.1.5.1 Signs and symptoms of Escherichia coli         
2.1.5.2 Treatment Escherichia coli diseases             
2.2    Preventive measures against raw milk contamination    
2.3    Laboratory analysis of raw milk o isolate and identify bacterial organisms    -    -    -    -    
2.3.1    Coliform count                             
2.3.2    Standard plate count     -    -    -    -    
2.3.3    Plating method -    -    -    -    -    -    
2.3.3.1    Pour plate technique                        
2.3.3.2    Spread plate technique                    
2.3.4    Gram-staining techniques                 
CHAPTER THREE
3.0    Materials and Method    -    -    -    -    -    
3.1    Materials (See appendix)-    -    -    -    -    
3.2    Methods     -    -    -    -    -    -    -    -    
3.2.1    Sterilization of Materials -    -    -    -    
3.2.2    Collection of sample -    -    -    -    -    
3.2.3    Preparation of milk samples                 
3.2.4    Preparation of culture media                
3.2.5    Culture techniques (serial dilution)            
3.2.6    Bacterial counting                         
3.2.7    Gram-staining -    -    -    -    -    -    
3.3    Biochemical tests for identification of isolated bacterial –
3.3.1    Indole test -    -    -    -    -    -    -    
3.3.2    Catalase test -    -    -    -    -    -    -    
3.3.3    Methyl red tes                             
3.3.4    Coagulase test (slide)    -    -        -    
3.3.4.1     Coagulase test (tube)                    
3.3.5    Voges-proskaver test -    -    -    -    -    
3.3.6    Urease test -    -    -    -    -    -    -    
3.3.7    Milk ring test (MRI)     -    -    -    -    --    
3.3.8    Nitrate reduction test -    -    -    -    -    
3.3.9    Citrate utilization test -    -    -    -    -    
3.3.10    Sugar fermentation test -    -    -    -    
3.3.11    Motility test -    -    -    -    -    -    -    
CHAPTER FOUR
4.0    Result    -    -    -    -    -    -    -    -    
CHAPTER FIVE
5.0    Discussion, Conclusion and Recommendation    -    
5.1    Discussion    -    -    -    -    -    -    -    
5.2    Conclusion    -    -    -    -    -    -    -    
5.3     Recommendation    -    -    -    -    -    -    
REFERENCES    -    -    -    -    -    -    -    
APPENDIX    -    -    -    -    -    -    -    
LIST OF TABLES
Table 1:     Total plate count of colonial forms
Table II:    Standard plate count of differential colonies
Table III:    Morphological characteristics, gram staining reactions of bacteria isolated from raw milk
Table IV:    Table showing the results of biochemical test
Table V:    Percentage distribution of organisms identified
CHAPTER ONE
1.0    INTRODUCTION
1.1    MEANING OF MILK
Giffel (2003), defines milk as a whitish liquid substance that is secreted from the udder of a healthy and sterile animal such as cow, goat, pig, dog etc used as food for both human being and animals. He continued to point out that microorganisms have long and interesting association with milk due to the fact that, it is nutritious and serves as an excellent source of food for microbiological growth. While Thompson et al (2004), defines milk as substance that is synthesized by cells within the mammary gland and is virtually sterile when secreted into the alveoli of the udder.
1.2    COMPOSITION OF MILK
Landmark et al, (2003), explains that, the average composition of cow milk is 3.7% lactose, 3.2%, carbohydrate 4.8%, 0.9% minerals, fats 6.14%. Besides casein and lactal albumin. It also have free amino acids that provides a good nitrogen source. He goes further to state that, the occasional over consumption of raw milk has brought a lot of problem to mankind. Since there is no place in the body where excess protein can be stored in the body unless it is delaminated. So knowing the composition of cow milk will help us to know the right quantity to consume at every point in time. Almost all cattle has the same composition of milk, unless the cattle is sick or unhealthy.
1.3    SPOILAGE OF MILK
Capasso (2002), explains that the spoilage of milk involves changes such as alteration in the appearance, colours, pocket of gasses which is seen as swelling, change of texture such as soft and messy odour and flavour or slime formation due to microbacteriological contamination of the milk which makes it unacceptable for consumption. He went further to explain that, the main causes of the spoilage of milk are oxidative rancidity of the fat in milk, the growth and activities of microbacterias such as Brucella species Streptococci, Micrococci, Staphylococcus, Salmonella alcaligens, Flavobacterium and many more which can now react with raw milk produce acetic acid, lactic acids formic acids, carbondioxide, hydrogen sulphide leading to rancidity, curdling or sourcing of the raw milk. The spoilage of raw milk usually affect the quality of the milk and the health of the individuals that consume it.
1.4    SOURCES OF CONTAMINATION OF RAW MILK
Fook et al (2004), explains that milk when still in the udder or mammary gland of healthy animals, used to be virtually sterile. But cow health, environment, milking procedures and equipment sanitation can influence the level of microbial contamination of raw milk. Equally important is the holding temperature and length of time milk is stored before testing and processing that allowed bacterial contaminant to multiply. Further more, he explains that beyond the stages of milk production, bacterial contamination can generally occur from three main sources which includes within the udder, outside the udder and from the surface of equipment used for milk handling and storage.
(i)    MICROBIAL CONTAMINATION FROM WITHIN THE UDDER
Fook (2004), continues to explain that raw milk as it leaves the udder of healthy cows, normally contains very low numbers of micro-organisms and generally will contain less than 1000 colony-forming unit of total bacterial per milliliter (cfu/ml). He continues to state that in healthy cow, bacterial colonization within the teat cistern, teat canal, and on healthy teat skin, does not significantly contribute the total numbers of bacterial neither in bulk milk, nor to the potential increase in bacterial number during refrigerated storage. This natural flora of the cow generally will not influence the standard plate count or coliform counts. While the healthy udder should contribute very little to the total bacterial count of the bulk milk, a cow with mastisis (inflammation of the breast usually caused in bacterial infection which damages the nipple, it may develop during the period of breast feeding about a month after child birth and sometimes, involves the discharge of pus) has the potential to shed large number of organisms such as streptococcus and rarely staphylococci into her milk thereby contaminating it. Although Staphylcoccus aureus can occur in milk as a result of other factors such as dirty cow, poor equipment cleaning and poor cooling.    
(ii)    MICROBIAL CONTAMINATION OR RAW MILK FROM OUTSIDE THE UDDER
Gonzale (2001), further point out that the exterior of cow udders and teat can contribute micro-organism that are normally associated with the skin of animals as well as micro-organism that are derived from the environment in which the cow is housed and milked. In general, the direct influence of natural inhabitants as contaminants in the total bulk milk count is considered to be small and most of this organisms do not grow competitive in milk, of more important is the contribution of micro-organisms from vat soiled with manure, mild feeds or bedding. The teat also get contaminate while they are lying in stalls or when allowed in dirty lots. The influence of dirty cows on total bacterial counts depends on the extent of soiling of the that surface and the udder preparation procedures employed organisms associated with bedding materials that contaminates the surface of the teats and udder includes streptococci, staphylococci, spore-formers, coliform bacteria, and other gram-negative bacteria.
(iii)    MICROBIAL CONTAMINATION OF RAW MILK FROM EQUIPMENT
Braun et al. (2002), explains that the degree of cleanliness of the milking system probably influences the total bulk milk bacteria count as much, if not more, than any other factor, milk residue left in equipments contacts surfaces supports, the growth of varieties of microorganism. Organism considered to be natural inhabitant teat canal and skin are not thought to grow significantly on soiled milk contact surfaces or during refrigeration milk storage. He added that the organisms found here are usually gram negative rods and some streptococci.
(iv)    CONTAMINATION FROM RAW MILK STORAGE TEMPERATURE AND TIME
Causoud et al. (2007), states that raw milk while preventing the growth of non-psychotrophic bacteria, will select for psychotrophic micro-organisms that enter the milk from soiled cows, dirty equipments and the environment. Minimizing the level of contamination from these sources will help to prevent psychotrophs from growing of significant levels in bulk tank during the on-farm storage period or at processing plant. In general, organisms are not thermoduric and will not survive pasteurization. The longer the milk is held before processing (legally up to 5 days), the greater the chances that pyschotrophs will increase in number. Holding milk near the pmol (legal unit of 45oF allows much quicker growth than milk held below 40oF).
1.5    AIMS OF THE STUDY
The aims of this study is as follows
(i)    To isolate and identify bacterial organisms in raw milk samples.
(ii)    To characterize the bacterial organisms isolated from raw milk samples.
(iii)    To determine the bacterial load of raw milk from Akwuke Cattle Ranch.
1.6    STATEMENT OF THE PROBLEM
Giffel (2003), explains that cow milk secreted from glands of a healthy udder is sterile, but when secreted becomes frequently contaminated by Brucella species, Staphylococci, Micrococcus, Staphylococci and many more normally present in the milk duct and cistern of the udder, the environment, instrument and hand of handlers. Wilchris (2003) added that, the bacterial organisms causes various infections diseases to human. These statement prompted the need for a project work on isolation and identification of bacterial organisms in raw milk.
1.7    SIGNIFICANCE OF THE STUDY
The result of the project work would reveal the bacteriological safety of raw milk. The result would equally reveal if the bacteriological load of raw milk was high or low and if the bacterial types were pathogenic. And if high, then consumption of raw milk by individuals should be discouraged. The work further showed that hygienic practices were not properly ensured by the cattle rearers. Therefore, there is need for proper hygienic practices to be introduced by the milk handlers. Moreover, consumers should pasteurize the raw milk brought from the local cattle rearers before consumption.
1.8    LIMITATION OF THE STUDY
This project work is limited to isolation and identification of bacterial organisms in raw milk. Moreover, I had a handicarp in carrying out this project due to poor financial status on my part as a student. Furthermore, the cost of the media and reagent for project analysis was high, thus, limiting the number of samples runs for the project work.