Download efficacy of distilled water and lactic acid as wash solution in

EFFICACY OF DISTILLED WATER AND LACTIC ACID AS WASH SOLUTION IN
PREVENTING RECONTAMINATION OF SOME SPECIFIC MICROBES ON
REFRIGERATED CHICKEN CARCASSES
BY
LAWAL RAMAN AKINYANJU
MATRIC NO: 2005/0160
DEPARTMENT OF ANIMAL PRODUCTION AND HEALTH
A PROJECT REPORT SUBMITTED TO THE
COLLEGE OF ANIMAL SCIENCE AND LIVESTOCK PRODUCTION
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF
BACHELOLOR OF AGRICULTURE DEGREE (B. Agric.Hons)
UNIVERSITY OF AGRICULTURE, ABEOKUTA, OGUN STATE, NIGERIA
OCTOBER, 2010
1
ABSTRACT
This study was carried out to look at the efficacy of using lactic acid and distilled water as wash
solution to reduce rate of recontamination of some specific microbes on refrigerated chicken
carcass. Frozen chicken wings was obtained from an open commercial market (Kuto), Abeokuta,
in Ogun State, Nigeria. The skin from the lean meat was removed and a certain measured amount
was washed with Distilled Water and (0.4%) Lactic Acid and Agitated with stomatcher blender.
1ml of the rinsate was transferred into sterilized Petri-dishes in triplicate. Specified Agar was
added into the rinsate in the sterilized Petri-dishes and incubated at 370C. Microbial Counts was
carried out at 24hours and 48 hours respectively. The experiment was repeated at day 6
Meanwhile, meat sample washed with lactic acid was able to reduce some microbes to minimal
level and eliminates some during the process. However, when washed with distilled water, the
microbes was very much compare to when wash with lactic acid.
2
ACKNOLEGDEGMENT
All praises and adoration to God Almighty for bringing me this far. He made me to dine among
the best, he brought me from grass to grace, you are infact incredible God.
I can’t but to appreciate my father Fr. I. A Lawal whom through his mystical support, advice,
motivation and for always wanted to me to be ‘possible’ where it seems not, my success today is
attributed to you. I love you.
To my dear, ever caring and loving mother, your spiritual and financial support made me the best
of my kind. I shall always bring peace and joy to you. I love you.
To my sister who actually happens to be my supervisor, your advise irrespective of subject of
discussion, valuable suggestion and indeed constructive critism has helped me a lot. You are a
listening leader. God alone can pay you. Thank you ma.
My siblings, Toibat, Saidat, Sakirat, Mariam, Iretunde, and Aramide as well as the baby of the
house Ramota, thank you all for the understanding and support, we will grow old together with
peace profound
My project was full of fun with intelligent but naughty colleague ever, Akanni, Ak pumping, and
Loveling, you guys are too much.
I personally I appreciate the following people, Hon, Adekola Festus (future Oyo State Governor),
Bamigbade Femi (Baba aladiye), Adekola Dorcas (my little but naughty sister), Kolawole
Victor, my cover brothers Tolu and wife, Bunmi (T n B) as well as Tope, you guys are too much.
My blossom friend, Idowu Olabode you’r the best among the best
Chief and Chief (Mrs) Niniola Abiona, Damilola, Jibola, Bukola, Mayowa, Sister Biola and
Sister Kemi thank you for the love and support. My big brothers, Don p, St. lee, and their
manager, you guys have all it takes. Go for it.
Wao! Should I say my half, myself, my heart, my best, my companion, my precious, or my
partner. Oh! Whatever that be among all, I just know that you Niniola Doyinsola remain and will
always remain my lady. If I say I love you one million times, it is not enough. You’r the secrete
behind my success. Anyway, I love you
3
CERTIFICATION
I certify that this project was carried out by Lawal Raman Akinyanju in the department of
Animal Production and Health, College of Animal Science and Livestock Production, University
of Agricultre, Abeokuta, Ogun State
______________________
____________________
Dr. (Mrs.) K.A SANWO
DATE
4
DEDICATION
This project is dedicated to my father Fr. I. A Lawal
5
TABLE OF CONTENT
CONTENT
Pages
Title
i
Abstract
ii
Acknowledgement
iii
Certification
iv
Dedication
v
Table of Content
vi
List of Tables
ix
CHAPTER ONE
1.0
Introduction
1
1.1
Justification
2
1.2
Broad Objective
2
1.3
Specific Objectives
2
CHAPTER TWO
2.0
Literature Review
3
2.2
Some specific microbes
4
2.3
Source of contamination
5
2.4
Method of reducing contamination
7
CHAPTER THREE
3.0
Materials and method
11
3.1
Collection of Samples
11
3.2
Experiment Sites
11
3.3
Materials used
11
3.4
Preparation of Media
12
6
3.5
Experiment Procedures
12
3.5.1 First phase (washing of meat at different levels)
12
3.5.2
Second Phase (Microbial load counting and identification)
12
The Serial-dilution Process
12
Pour Plate Method.
12
3.6
Morphological Identification.
14
3.7
Biochemical test.
15
3.8
Data collection
17
3.9
Statistical Analysis
17
CHAPTER FOUR
4.0
RESULTS AND DISCUSSION
18
4.1
Tables showing the microbial counts in cfu/ml;
19
4.2
Graphical representation of microbial counts in cfu/ml
20-24
CHAPTER FIVE
5.0
CONCLUSION AND RECOMMENDATION
25
5.1
Conclusion
25
5.2
Recommendation
25
REFERENCES
26-31
7
8
CHAPTER ONE
1.0
INTRODUCTION
Meat is an important source of fats, vitamins, and essential mineral. It is therefore animal flesh
suitable for use as food. It’s a high quality protein source that contains almost all the essential
amino acid required in the human body (Lawrie, 1985)
Meat is highly perishable foods that provide for the growth of hazardous microorganism.
However, the growth of these microbes reduces meat quality; shorten shelf life thereby resulting
in economic loss and probably health hazards (kalalou et al., 2004).
In the time past, different method have being in use for meat preservation and microbial control.
These include drying, smoking, salting, refrigeration, freezing, use of chemicals e.t.c.
Furthermore, chemicals such as citric, acetic, lactic, and sodium benzoate acid as well as their
salts ( Ke et al., 2009; Signorini et al., 2006; Jimenez-Villarreal et al., 2003; Jensen et al., 2003;
Bostan et al., 2001), have been used for meat preservation through dipping, spraying, washing.
Among the mentioned chemicals above, lactic acid and sodium benzoate is the most abundant
metabolite produced by lactic acid bacteria while sodium benzoate is the first chemical
preservatives permitted in food by the food and drug administration (FDA) USA (Villareal et al.,
2003). Research has shown that when lactic acid is used as preservatives agent at less than 3%, it
reduces the population of bacteria load on meat and growth rate of other pathogens such as
Escherichia coli, Clostridium botulinum, salmonella spp, campylobacter jejuni, (Signorini et al.,
2006) lactic acids is considered to be a natural constituents of meat and generally recognised as
safe substance (Borpuzari et al.,, 1995).
9
1.1
Justification
Since research has shown that some specific bacteria such as Shigellia spp, Escherichia coli,
Salmonella spp and staphylococcus aurens e,t.c are present in some poultry meat obtained from a
local market in Abeokuta ( Sanwo, et al., 2009 ), and success have being made in reducing some
of the bacteria load using Distilled water and Lactic acid (Sanwo et al., 2009). Therefore, the
need to see the effective of this washing solution in preventing rate of re-contamination on
refrigerated poultry carcasses.
1.2
Broad objective
To carry out the efficacy of distilled water and lactic acid as wash solution in preventing
recontamination of some specific microbes on refrigerated chicken carcasses
1.3
Specific objective
1. To determine the efficacy of washing solution such as distilled water and lactic acid in
preventing re-contamination of some microbes on washed and refrigerated chicken
carcasses
2. To identify the rate of growth of some specific spoilage bacteria such as Shigellia spp,
Escherichia coli, and staphylococcus aurens in washed and refrigerated chicken
carcasses
3. To carry out microbial load counts on washed and refrigerated chicken
10
CHAPTER TWO
LITERATURE REVIEW
2.1
Meat
Meat is an edible portion of domestic mammals such as cattle, calves, sheep, lambs and
swine. It can be applied to edible portions of poultry, wild birds and other animals such as
crustaceans and reptiles that are eaten by humans. It is a nutritious food containing quantities of
essential amino acids in the form of protein and certain minerals especially iron. Meat also
supplies nutrients which contribute significantly to the dietary balance of meals (Alonge, 1981).
Thus, meat give us nutrients, to sustain our health and normal growth.
Certain meats, especially liver, contains vitamins A and D. Although lean meat has a high water
content (about 75%), it is a good source of protein – 20% on a wet basis compared with 8-12% in
cereal protein, 5% of fat, and 1% mineral ash and the rest is water (FAO, 1985). However, it was
discovered that one of the major factors which brings about low protein consumption leading to
malnutrition, is food spoilage and food contamination (poisoning) which is caused mostly by
pathogenic bacteria such as Salmonella spp, staphylococcus aureus, Escherichia coli etc. (Bryan,
1980).
Meat surface contamination by microorganisms has been a major problem in storing carcasses in
semitropical climates or when meat is subject to high temperature conditions in industrialized
areas. Inexpensive methods of reducing these undesirable populations have been designed, such
as sprays containing chlorine or organic acids. Of these, treatments with lactic acid have proved
to be very efficient. The sensory characteristics of meat are determined by a number of chemical
factors which may be affected by treatments to reduce meat contamination. Any potential
11
treatment for the extension of shelf-life of raw meat should take into consideration a minimum
alteration of its sensory characteristics. From work done at refrigeration temperatures, it is
known that addition of organic acids could be a way to reduce microbial population, increasing
raw meat shelf-life. However, a viable alternative is to promote a controlled lactic fermentation
in the meat surface by applying selected strains of lactic acid bacteria, which reduce undesirable
microbial populations, without considerable alteration of its sensory characteristics.
2.2
Some specific microbes
2.2.1 Penicillium
It is a genus of ascomycetes fungi of major importance in the environment, food and drug
production. It produces penicillin, a molecule that is used as an antibiotic, which kills or stops
the growth of certain kinds of bacteria inside the body. The thallus, (mycelium) typically consists
of a highly branched network of multinucleate, septate, usually colorless hyphae. They are often
green although they exist in different colors ranging from blue to red. They are commonly
known as molds and are among the main causes of food spoilage. Many species produce highly
toxic mycotoxins. Some species have a blue colour, commonly growing on old bread and giving
it a blue fuzzy texture. (De Hoog et al., 2000).
2.2.2 Aspergillus
This organism is commonly isolated from soil, plant debris and indoor air environment. They are
filamentous , cosmopolitan, ubiquitous and are not known with any sexual spore production.
The genus Aspergillus includes over 185 species. Around 20 species have so far been reported as
the causative agents of opportunistic infections in man. Among these, Aspergillus fumigatus is
the most commonly isolated species followed by Aspergillus flavus and Aspergillus niger.
Aspergillus clavatus, Aspergillus glaucus group, Aspergillus nidulans, Aspergillus oryzae,
Aspergillus terreus, Aspergillus ustus and Aspergillus versicolor are among the other species less
commonly isolated as opportunistic pathogens. (Bennett, 2010) .
12
The growth of the Aspergillus sp and the production of aflatoxins are dependent on factors such
as the fungal strain, competing flora, substrates, temperature and relative humidity conditions.
2.2.3 Saccharomyces
Saccharomyces is a yeast commonly isolated from human, mammals, birds, wine,
beer,fruits,trees,plants, olives and soil. It is a unicellular fungi which produces a yeast-like odour.
Colonies are white on malt extract agar and potatoe dextrose agar.
Colonies of Saccharomyces grow rapidly and mature in 3 days. They are flat, smooth, moist,
glistering or dull, and cream to tanish cream in color. They have the ability to
ferment various carbohydrates and an inability to utilize nitrate. (De Hoog et al., 2000).
2.2.4 Shigella species
The genus shigella belongs to the family enterobacteriaceae and only bin are recognized: shigella
dysenteries, Shigella flexneri, shigella boydii and shigella sonnei (Jay, 1992); the shigella species
of concern are typical of most other enteric bacteria in their growth requirement with growth
reported to occur as low as 100C and as high as 480C (Smith, 1998).
2.3
Sources of meat Contamination
2.3.1 Animal Contamination
Live animals are often highly contaminated, or are carriers of pathogenic bacteria (Letellier et
al., 1999) and can serve as sources of subsequent meat contamination. Animal cleanliness is
influenced by climate, geographic location, method of transportation and holding conditions. For
example, animals raised on pastures may carry more bacteria of soil origin, while micro
organism of intestinal origin may be more common on carcasses from animals finished in
feedlots (Sofos et al., 1999). Every feasible effort should be made to prevent accumulation of
excess mud and dung on the animals, because it may introduce bacterial pathogens into the plant
environment. Therefore, there is a need to determine risk factors in order to develop management
13
practices that will help in the control of the prevalence of pathogens in animals and their
products. Factors to be considered include animal fasting, feeding and stressing practices such as
those applied during confinement and transportation, amount of roughage and dietary
components, animal cleanliness.
2.3.2 Carcass contamination
In general, the muscles of live healthy animals are sterile, while lymph nodes, some
organs, and, especially, surfaces exposed to the environment, such as external hide, pelt, or
fleece, the mouth and the gastrointestinal tract carry extensive contamination (Gill, 1998). These
are major sources of plant, carcass and meat contamination during slaughtering and processing.
Beef carcass contamination may vary with season, plant design and operation, geographic area,
location within the plant, and, to some extent, anatomical site (Sofos et al., 1999). Overall, levels
of carcass contamination after 24 hours of carcass chilling were 2.55, 0.27 and 0.12 log colony
forming units (CFU)/cm2 for aerobic plate counts, total coli form counts and Escherichia coli
counts, respectively for seven plants (Sofos et al., 1999). Overall, levels of carcass chilling were
2.55, 0.27 and 0.12 log colony forming units (CFU)/cm2 for aerobic plate counts, total coliform
counts and Escherichia coli counts, respectively for seven plants (Sofos et al.,1999).
2.3.3
Edible offal contamination
Variety meats (edible offal) may carry a higher level of microbiological contamination
than other meat animal tissues, either by nature and origin, or due to poor hygienic and chilling
conditions (Gill, 1998). It was found that bacterial counts in most of 17 types of beef variety
meats examined from six plants increased between packaging and chilling, indicating the
inefficiency of the chilling process (Delmore, 1998). Average total coli form counts for various
14
offal products before and after chilling were 1.3-3.4 and 2.0-3.9 log CFU/g, respectively.
Pathogen incidence in the 830 samples, examined only after chilling, was 0% for E.coli
O157:H7, 0.8% for Salmonella and 4.5% for L.monocytogenes.
2.4
Methods of reducing contamination of meat
2.4.1
Animal cleaning
One, seemingly obvious, approach that may contribute to the reduction of external
animal contamination, and subsequently, carcass contamination is to clean or wash the hide of
the animals before slaughter and dressing. Pre-slaughter washing of sheep has been practiced in
New Zealand (Biss and Hathaway, 1996), while, partial or complete, washing of cattle before
slaughter has been used by some plants in the United States. Individual operations have
evaluated, or applied interventions, such as removal (by cutting or shearing) of hair and faecal
tags from the exterior of the animals or washing of animals before slaughter, but in many
instances the results are generally less than promising (Gill, 1998). In general, animal washing
before slaughter has variable influence on carcass contamination. Furthermore, application of the
procedure may be limited by climate, type of animal, and availability of facilities (Sofos and
Smith, 1998). United state regulatory guidelines require cattle to be dry, or at least not dropping,
when they are slaughtered (reed., 1996).which can be constraint when an animal washing is
considered before slaughtered. However, when animals are wet or excessively soiled, slaughter
speeds should be reduced to minimize accidental transfer of contamination from the exterior of
the animals onto the carcass or the plant environment.
In addition, modification in the steps involved in hide removal, or in equipment used for hide
removal, may help in minimizing transfer of contamination onto the carcass surface (Hadley et
15
al., 1997).One approach that may help in the reduction of carcass contamination with pathogen
may be to process highly contaminated or infected animals separately from cleaner or pathogenfree herds (Gills,1998).This approach, however may be practical in some system of animal
production, marketing, distribution, and slaughtering, or for control of more than one type of
pathogenic microorganisms on the same animals. Nevertheless, highly soiled animals are an
important potential source of plant contamination, and presentation of clean animals for
slaughter is desirable because it reduces the likelihood of pathogen presence and transfer onto
carcass (Bolton et al., 1998).However poor sanitation, hygiene and manufacturing practices
during slaughtering, fabrication and processing can lead to excessively contaminated meat, even
when less heavily soiled animals are processed.
2.4.2
Carcass decontamination
Carcass contamination varies with season of the year, type of animal slaughtered,
anatomical carcass site, and step in the dressing process. However, extent of carcass
contamination is often influenced the most by variation among plants, including plant design,
speed of slaughter and skill of operators (Gill, 1998). Application of decontamination processes
on carcasses, during and following dressing, is generally regarded as an effective intervention to
reduce contamination (Sofos and Smith, 1998). Carcass decontamination processes are based on
immersion, flooding, cascading, deluging, rinsing, or spray-washing with water or chemical
solutions. They are applied to remove visible soil, such as residual hair, faeces and bone dust in
the majority of slaughter plants in the United States and other countries, such as Australia and
Canada. The decontaminating efficacy of these treatments is influenced by water pressured,
temperature, chemicals present and their concentration, time of exposure (which depends on
16
speed of slaughter and length of the application chamber), method of application, and time or
stage of application during carcass dressing (Sofos and Smith, 1998).
Application of spraying/rinsing treatments to carcasses may cause penetration of bacteria
into the meat or spreading and redistribution on the carcass, depending on the spraying pressure.
Other concerns are associated with the influence of time before decontamination on bacteria
attachment, bio film formation and potential from exposure to the decontamination treatment and
injuries to bacterial cells or development of resistance in bacteria during exposure to
decontamination treatments such as acids and hot water or steam. Removal, rather than
redistribution of bacteria on the carcass by spray-washing treatments can be effected through
proper use of spraying nozzles (e.g. type, number, distribution, position, spraying angle, water
output, and operation), spraying pressure and time, size of carcass, and overall design of the
chamber and spraying system. In general, it is believed that carcass decontamination
interventions contribute to the production of carcasses with lower levels of contamination and
that reduced incidence of enteric pathogens helps in meeting regulatory requirements during
slaughter.
2.4.3 Chemical Decontamination
Warm (50-550C) solutions of organic acids (1-3%), such as acetic and lactic, have reduced
bacterial numbers on carcass tissue by 1-3 logs (Castillo et al., 1998) and are used extensively in
commercial beef slaughter in the United States, while they are not permitted in Europe. In the
form of rinses, before chilling, they are found useful, especially in combination with proceeding
treatments of hot water spraying, and potentially as having a residual antimicrobial effect during
storage. Potential concerns associated with the used of organic acids include selection of acid-
17
resistant organisms that may increase product spoilage, undesirable effects on product
appearance, and equipment corrosion concerns (Gill, 1998). In addition to organic acids, several
other chemical solutions have also been proposed and tested for the decontamination of meat.
They include common chlorine and chlorine oxide, trisodium bisulphide, sodium chloride,
acidified sodium chlorite, potassium solutions have been approved for treatment of beef and
poultry carcasses in the United States (Morris et al., 1997). Hydrogen peroxide and ozonated
water were also found to reduce bacterial counts in experimental trails but their use may be of
concern due to their oxidizing effects on fat and muscle pigments.
2.4.3.1 Use of Lactic acid
When used as a sanitizing agent at less than 3%, it can reduce the number of spoilage bacteria. It
has been reported to control growth rate of Escherichia coli, Campylobacter jejuni, Clostridium
botulinum, Listeria monocytogenesis and Salmonella spp. (Signorini et al., 2006). Lactic acid is
suitable as washing solution because it is a natural constituent of meat and generally recognized
as safe (GRAS) substance (Borpuzari and Borpuzari, 1995).
18
CHAPTER THREE
3.0
MATERIALS AND METHODS
3.1
Collection of samples.
Frozen chicken wings was purchased from an open commercial market (kuto), Abeokuta, Ogun
State. The skin of the chicken wing was removed, weighed and agitated with washing solution.
3.2
Experiment Sites
The first phase of the experiment which is washing by agitation of the chicken sample
was carried out at the Meat Laboratory of the Department of Animal Production and Health
(APH), while microbial load counting and identification was carried out at the Department of
Microbiology, University of agriculture, Abeokuta ogun state.
3.3
Materials used
The following materials were used during the conduct of the experiment;
Petri dishes (disposable), measuring cylinder, cotton wool, gloves, spirit lamp, conical flask
(250ml), paper tape, sample bottles, methylated spirit, autoclave, test tube, incubator, blender,
foil paper, sensitive digital weighing scale, cooker, lactic, Potato dextrose agar (PDA),
Salmonella- shigella agar (SSA), Plate count agar (PCA), Manitol salt agar (MSA), and EMBA.
3.4
Preparation of Media
Media used were Potato dextrose agar (PDA), Salmonella- shigella agar (SSA), Plate
count agar (PCA), Manitol salt agar (MSA), and Eumosine methylene blue agar (EMBA). The
Agars were prepared according to manufacturer procedure., they were then autoclaved at 1200C
19
for 15 minutes except for PDA, which was boiled on the cooker because its low temperature
requirement
3.5
Experiment Procedures
3.5.1 First phase (washing of meat at different levels)
The purchased frozen chicken wings was prepared by removing the skin from the lean
meat, 30g of the skin collected was weighed and washed by agitation in a stomacher laboratory
blender at the same constant speed of 10seconds with distilled water (treatment 1) and 0.4%
lactic acid (treatment 2) by diluting the percent solution in 100ml distilled water. Each treatment
samples were replicated and bacteria identification was done on days 0, and 6.
3.5.2
Second Phase (Microbial load counting and identification)
(i)
The Serial-dilution Process
Each sample was separately analyzed by ensuring homogeneity of the samples using a
sterile pipette. 1ml of each sample was suspended into 9ml sterile water ascetically in a test tube
which was then shaken together. Further dilution was carried out. This was carried out by using a
sterile pipette to take 1ml from the initial dilution into the first test tube of 9ml; the first test tube
was labeled 10-1.After this, another 1ml of the diluents was taken from the 10-1 dilution test tube
to be transferred aseptically into another tube containing 9mls of distilled water to obtain 10-2 ,
dilution of sample. These exercises were continued serially until approximate 10-5 dilution was
obtained.
20
(ii)
Isolation and enumeration of microorganisms
Samples 1ml each were homogenized in 9mls of distilled water to obtain a ratio of 1:9.
Further dilution and the second diluents of each samples was plated using pour plate technique.
Enumeration of organisms were carried out using Potato dextrose agar (PDA), Salmonellashigella agar (SSA), Plate count agar (PCA), Manitol salt agar (MSA), and EMBA (Euosine
methylene blue agar).
(iii)
Pour Plate Method.
1ml of the diluents in the last test tube (10^3) was aseptically pipetted into sterilized Petri
dishes. Media such as salmonella shigella Agar (SSA), Mannitol salt Agar (MSA), Potato
Dextrose Agar (PDA), and EMBA (Euosine methylene blue agar) was poured into the sterilized
Petri dishes in order to identify the salmonella and Shigella spp, Staphylococcus aureus and
Escherichia coli and fungi by pouring the media into the diluents in the Petri dishes. Also Potato
Dextrose Agar Media was poured into sterilized Petri dishes in order to carry out total plate
counts on the bacteria for each sample. The media was sterilized in an autoclave at 126ºC for
15mins. They were allowed to cool down but warm to touch before pouring them into the
diluents in sterilized Petri dishes in replicate. The samples were then incubated at 37o C
Colonial growths was counted 24hrs and 48hrs after incubation. The numbers of colonial growth
were recorded in colony forming unit per mil (cfu/ml).
21
3.6
Morphological Identification.
3.6.1 Gram reaction.
One loopful of sterile distilled water was placed on a clean grease free slide using a
sterile wire loop. The wire loop was heated to red hot on spirit lamp and allowed to cool, a small
portion of the pure bacteria culture was picked and emulsified on the slide, the slide was air dried
and heat-fixed by passing the slide over the flame for 3-4 times.
Crystal violet was poured on the slide for 30-60 seconds and rinsed with water, the slide
was flooded with Lugol’s iodine for 30-60 seconds and rinsed with water. It was then
decolorized with 75% alcohol for 30 seconds and washed with water.
The slide was then covered with safrain for 30 seconds and washed with distilled water.
The slide was blotted dried and covered with cover slip, a drop of oil immersion was then
observed under oil immersion microscope. The shape and cell arrangement was seen and
recorded.
Gram positive bacteria were characterized by a purple color, while gram negative were
red in color. (Olutiola et al; 2000 ). This was incubated for 24 hrs at 37oC. 0.5ml of Kovac
reagent was added and shaken gently. Appearance of red color indicate the presence of Indole.
3.6.2 Motility test.
This was determined by hanging drop technique. Using a sterile loop, colony of the
organisms were grown in peptone water for 18hrs and then placed in the grease free slide, and
covered with a vaseline bound cover slip and observer under 100x objective lens. Amotile
organism is seen moving in the drops of liquid.
22
3.7
Biochemical test.
3.7.1 Citrate utilization test
22.3g Simmon citrate Agar powder was weighed into 1000ml of distilled water and was
shaken thoroughly. The medium was divided into clean Mac Cartney bottles, covered and
sterilized in an autoclaved at 121oc for 15minutes. They were placed in a slanting position to
form a slope while solidifying. The prepared Simmons citrate Agar slant was streak inoculated
with the test isolates. The slant was incubated at 370c for 4days. The utilization of citrate resulted
in an alkali reaction which was indicated by color change from green to blue along the streak
organism. Negative test retained the green color with no growth. (Fawole and Oso 1995).
3.7.2
SUGAR FERMENTATION
1.5g of peptone powder and 0.009g of phenol red were dissolved in 100ml of distilled
water. 1g of each sugar (lactose and glucose) were dissolved in 100ml distilled water separately,
9ml of the phenol red peptone solution was pipette into test tube and 1ml of 1% sugar solution
was added and mixed gently. Durham tubes were inverted in the test tubes. The tubes were
inverted in the autoclave at 121oc for 15minutes. After cooling one tube was inaugurated with the
bacteria while others served as control. The tubes were incubated at 37oc for 48 hours. Color
changed from red to yellow indicated thee production of acids. When carbohydrates were
utilized in partial or total absence of oxygen, this process is known as fermentation (Fawole and
Oso, 1995).
23
3.7.3 Oxidation.
1.8g of peptone powder and 0.009g of phenol red were dissolved in 100ml of distilled
water. 1g of each sugar (lactose and glucose) were dissolved in 100ml distilled water separately,
9ml of the phenol red peptone solution was pipette into test tube and 1ml of 1% sugar solution
was added and mixed gently. Durham tubes were inverted in the test tubes. The tubes were
inverted in the autoclave at 121oC for 15minutes. After cooling one tube was inaugurated with
the bacteria while others served as control. The tubes were incubated at 37oc for 48 hours. Color
changed from red to yellow indicated thee production of acids. When carbohydrates were
utilized in partial or total absence of oxygen, this process is known as fermentation (Fawole and
Oso, 1995).
3.7.4 Lacto-Phenolcotton Blue Staining.
One loop full of sterile distilled water was placed on a clean grease for slide using a sterile
inoculating pin. The inoculating pin was heated to red hot on a spirit lamp and allowed to cool. A
small portion of the pure fungal mycelia was picked and emulsified on the slide, the slide was air
dried and heat fixed by passing the slide over flame for 3 – 4 times. 2ml of lacto phenol cotton
blue stain was added to the smear and tilted further. A cover slip was slightly and gently used
over the slide and covered to avoid air bubbles. A blotting paper was used to absorb excess stain
and fungi structure was observed under the microscope.
3.7.5 Coagulase Test
This test was carried out to determine the presence of enzyme, coagulase. This test is used to
distinguish coagulase positive Staphylococcus aureus from coagulase negative staphylococcus
epidermis.
24
A colony of bacteria was emulsified with a sterile saline solution on clean grease free slide. A
drop of plasma was added with the emulsion and mixed. Positive coagulase organisms shows
clumping, while negative coagulase organism showed no clumping (Fawole and Oso 2001).
Coagulase causes plasma to clot by converting fibrinogen to fibrin.
Morphological and biological characteristics of fungi isolate of meat
COLOUR
MYCELIA
CONIDIA
Whit
changing
blue
Septate
White
changing
black
Septate
with foot
cell
White
changing
black
Nonseprate
stolon join
two
hyphea
Branching
Conidiospore
brush-like
appearance
with spore
Conidospore
arising from
foot cell
forming
Vesicle at
apex
-
3.8
SPORANGIA
-
SEXUAL
REPRODUCTION
-
VEGETATIVE
REPRODUCTION
+
SUGAR
FERMENTATION
AG
SUSPECTED
ORGANISM
Penicillum
spp
-
_
+
AG
Aspergillus
spp
+
+
+
AG
Rhizopus
spp
Data collection
Population of bacteria was expressed in cfu/ml on peptone-saline wash solution for each
treatment
3.9
Statistical Analysis
Microbial population count were expressed on tables and graph
25
CHAPTER FOUR
4.0
RESULTS AND DISCUSSION
Table one shows Means of bacteria and Fungi (Aspergillus spp) counts after washing. There was
reduction in the counts of microbes under study ( E. Coli, Salmonella spp, Staphylococcus spp )
as well as specific fungi (Aspergillus spp) on chicken carcass when washed with distilled water
after six days of refrigeration. On the other hand lactic acid either eliminate or reduce the
presence of these specific Microbes and Fungi (Aspergillus spp) on chicken carcass before and
after refrigeration. These observation is however consistent with the report that lactic acid either
eliminates or decreases some specific microbes on broiler carcass (Izat et al., 1989)
Table two shows the cultural, morphological and biochemical characteristics of the bacterial
isolates obtained from the washed refrigerated chicken carcass. Salmonella spp produces black
colour after 48hrs of culture. E.Coli is an acid gas fermenters and the colour observed was
shinning green during culture after 48hrs. Staphylococcus on the other hand has a white colour
cultural characteristics and also an acid fermenters
Table three shows the characteristics of identified fungi, their shape, surface, elevation type,
spore colour, type of mycelium and reproduction. Aspergillus for example is circular in shape
with powdery surface and semi raised elevation. It is black in colour and reproduce sexually.
26
Table 1: Means of bacteria and Fungi counts after washing
E. coli
Salmonella
Distilled
Water
Lactic
Acid
Staphylococcus
Total plate count
Fungi
(proteus)
Day 0
Day 6
Day 0
Day 6
Day 0
Day 6
Day 0
Day 6
Day 0
Day 6
3000
770
3001
1880
3330
460
6000
960
3330
3100
3210
910
3200
2960
3323
480
6200
920
3210
3134
3450
840
3175
2940
3032
470
6100
670
3230
3116
0
0
0
0
430
0
3500
175
2260
0
0
0
0
0
430
0
3645
172
2220
0
0
0
0
0
410
0
3746
132
2240
0
27
Table 2: Shows the cultural, morphological and biochemical characteristics of the bacterial
isolates obtained from the washed chicken carcass.
Suspected
organism
Salmonella
E.coli
Enterobacter
Proteus
Staphylococcus
aureus
Sugar
fermentation
Biochemical
chaeacteristics
-
A
AG
A
AG
AG
AG
AG
+
-
AG
+
+
+
+
+
+
Morphological
characteristics
+
+
+
28
+
+
+
+
-`
+
-
Cultural
characteristics
Black
Green
White
Table 3: Shows the characteristics of Fungi identified
Shape
Surface
elevations
Spore
colour
Type of
mycelium
Type of
septate
reproduction
Suspected
organism
Circular
powdery
Semi
raised
Black
Conidiospore
sexual
septate
Aspergillus spp
Filamentous
Cotton
Raised
Black
Sporangiospore
asexual
Rhizopus spp
Circular
Fluffy
Raised
Greenish
blue
Conidiospore
Sexual
Non
septate
septate
Elevation
Raised
Colour
Creamy
Mycelium
Conidiospore
Shape
Oval
29
Penicillum spp
Saccharromyces
spp
4.2
Graphical representation of microbial counts in cfu/ml
4.2.1 Figure 1: Shows the rate at which the Salmonella spp bacteria grew at day 0 and day 6
on different chemical treatment. At day 0, when the sample was treated with distilled water and
not refrigerated, bacteria counts reduced from 3450cfu/ml to 840cfu/ml at day 6 when washed
with distilled water and refrigerated.
However no count was recorded both on day 0 and day 6 when washed with lactic acid even with
or without refrigeration. This observation is consistent with a report that lactic acid either
decreases or eliminated Salmonella spp from broiler carcass (Izat et al., 1989)
3450
3500
3000
3210
3000
2500
2000
Salmonella Day 0
1500
Salmonella Day 6
1000
500
0
0
0
0
Dist
watr
Lact
acd
Figure 1: A graph showing the growth rate of recontamination of salmonella infection using
distilled water and lactic acid as treatment on refrigerated chicken carcass
30
4.2.2 Figure 2: Shows the rate of growth of E. coli both at day 0 and day 6 when washed with
different treatment solution. At day 0, the bacteria counts decline from 3200cfu/ml without
refrigeration and to 2960cfu/ml at day 6 when treated with distilled water and refrigerated.
Result obtained was different when treated with lactic acid both at day 0 without refrigeration
and day 6 during refrigeration. This was because the lactic acid totally inhibited the microbial
growth and 0cfu/mil count was recorded.
3500
3001
3200
3175
3000
2500
2000
E. Coli Day 0
1500
E. Coli Day 6
1000
500
0
0
0
0
Dist
watr
Lact acd
Figure 2: A graph showing the growth rate of recontamination of E. coli infection using
distilled water and lactic acid as treatment on refrigerated chicken carcass
31
4.2.3 Figure 3: The population of Staphylococcus aureus recovered from washed chicken
wing were reduced to 480cfu/ml at day 6 from 3330cfu/ml at day 0 after washed with distilled
water.
When treated with lactic acid at day 0 without refrigeration, the population of microbe counted
was 430cfu/ml but at day 6 when samples were refrigerated, all the staphylococcus aureus
present were eliminated. These could likely due to effect of both the chemicals and storage
temperature.
3500
3330
3323
3032
3000
2500
2000
Staphylococcus Day 0
1500
Staphylococcus Day 6
1000
430
500
430
410
0
Dist
watr
Lact
acd
Figure 3: A graph showing the growth rate of recontamination of Staphylococcus aureus
infection using distilled water and lactic acid as treatment on refrigerated chicken carcass
32
4.2.4 Figure 4: Total plate count of microbes at day 0 was 6200cfu/ml when washed with
distilled water without refrigeration but decline to 960cfu/ml at day 6 under refrigeration. At day
0 when treated with lactic acid, the microbial population was 3745cfu/ml and decline to
132cfu/ml at day 6 under refrigeration.
7000
6000
6200 6100
6000
5000
3500 3645
4000
3746
TotalPlateCount Day 0
3000
TotalPlateCount Day 6
2000
1000
0
Dist
watr
Lact
acd
Figure 4: A graph showing the growth rate of recontamination of Total plate count infection
using distilled water and lactic acid as treatment on refrigerated chicken carcass
33
4.2.5 Figure 5: This shows the rate of growth of Fungi (Aspergillus spp) on refrigerated
chicken carcass. At day 0 when washed with distilled water, the counts reduces from 3330cfu/ml
to 3100cfu/ml at day 6 . When washed with lactic acid, the initial count was as high as
2260cfu/ml and decline to 0cfu/ml at day 6
3500
3330
3210
3230
3000
2260
2500
2220
2240
2000
Fungi Day 0
1500
Fungi Day 6
1000
500
0
Dist
watr
Lact acd
Graph 5: A graph showing the growth rate of recontamination of Fungi (Aspergillus spp)
infection using distilled water and lactic acid as treatment on refrigerated chicken carcass
34
CHAPTER FIVE
5.0
CONCLUSION AND RECOMMENDATION
5.1
Conclusion
The use of 0.4% concentration of lactic acid as a wash solution on chicken carcass, effectively
eliminated the growth rate of Salmonella and E. coli at day 0 and day 6 with or without
refrigeration than when washed with distilled water.
However, lactic acid at 0.4% was not very effective on Staphylococcus, Fungi (Aspergillus spp)
and Total plate count when the carcass was not refrigerated but eliminated the microbes after the
sixth day when it was under refrigeration. This shows that both the refrigeration temperature as
well as washing solution ( lactic acid) was necessary to totally eliminate these microbes on
chicken carcass
5.2
Recommendation
It could be recommended that commercially purchased chicken carcass should washed with
(0.4%) lactic acid and then stored under refrigeration for at least six days to totally inhibit the
presence of microbes and prevent rate of recontamination before consumption.
More so, Federal Government should ensure availability of power supply all through the year
because lack of power supply is the major factors that can encourage the growth of microbes
especially under refrigeration. Unstable power supply perhaps increases rate of recontamination.
35
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40