This document provides an overview of meat hygiene and quality assurance. It discusses the importance of meat hygiene in ensuring meat safety and suitability for human consumption. Good agricultural practices, good hygiene practices, and hazard analysis and critical control points are described as key components of a meat quality assurance system. Guidelines are provided for maintaining hygiene at various stages of meat production including on farms, during transport, slaughter, processing, and storage.

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UNIVERSITY OF NAIROBI
DEPARTMENT OF PUBLI HEALTH, PHARMACOLOGY AND TOXICOLOGY
FACULTY OF VETERINARY MEDICINE
CERTIFICATE IN MEAT HYGIENE AND QUALITY ASSURANCE
Course Unit
MEAT HYGIENE AND QUALITY ASSURANCE
(Lecture notes - January 2013)

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MEAT HYGIENE AND QUALITY ASSURANCE
Meat hygiene can be defined as the creation of conditions and implementation of
measures necessary to ensure the safety and suitability of meat at all stages of the
meat production chain. Meat safety is the assurance that meat will not cause harm to
the consumer when it is prepared and or eaten according to its intended use. Meat
suitability is the assurance that meat is acceptable (in terms of taste, smell and
nutritional state) for human consumption. Effective hygiene control is vital to avoid the
adverse human health and economic consequences of unsuitable meat and meat
related illnesses. Everyone including farmers, processors and regulators have a
responsibility to assure consumers that meat is safe and suitable for consumption.
The main objectives of Meat hygiene and quality assurance are to:
1. Safeguard the health of the people through production, distribution and
consumption of safe meat;
2. Promote national and international trade of meat;
3. Prevent avoidable meat losses and promote conservation of livestock resources.
Scope of meat hygiene
Meat hygiene programs are aimed at controlling the hygienic standards of the meat at
all levels of meat production chain. This include monitoring the quality of animals being
raised for meat production, their disease status and nutrition level, handling during
transportation to and at the slaughterhouses, methods of slaughter, ensuring thorough
ante-mortem and postmortem inspection, hygienic transportation of meat to butcheries
and/or markets, as well as ensuring the hygienic state of the butcheries and meat
markets, storage conditions before they reach the consumer. In particular, efforts are
directed at prevention of animal diseases at the farm, prudent use of veterinary
medications and other chemicals in food animals and observing the recommended
withdrawal periods, prevention of diseased animals and those under treatment from
getting to the slaughterhouse, ensuring hygienic slaughter of animals and prevention of
contamination of meat from slaughter animals, slaughter personnel, water, vermin
(rodents flies and other insects), equipment and general environment. In addition, there
is need to prevent meat deterioration and spoilage through appropriate measures which
may include controlling temperatures, humidity and or other controls.
Importance of Meat hygiene
1. Prevention of food borne disease outbreaks. Food borne diseases leads to
consumer suffering and loss of life.
2. Prevents meat deterioration that may lead to unnecessary losses
3. Enhances local and international trade in meat due to enhanced consumer
confidence
4. Creates employment as more income is realized from sale of meat.

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Meat Quality Assurance
Meat quality Assurance is the systematic implementation of planned meat hygiene
measures along the meat production chain to provide adequate confidence to the
consumers that meat is safe for their consumption. Meat Quality assurance systems are
a set of regulatory and non-regulatory institutions involved in activities aimed at
ensuring the safety of meat. It includes the official mandatory regulatory activities
enforced by national and local authorities to provide consumer protection and ensure
that meat is safe, wholesome and fit for human consumption.
Meat quality assurance system focuses on hazard prevention, elimination, control or
reduction of hazards to acceptable levels throughout the meat production chain. A
hazard is a biological, chemical or physical agent in food with a potential to cause an
adverse health effect. There are three types of hazards that may be found in meat
1. Biological hazards: which are microorganisms (bacteria, viruses, yeast and
molds) and their toxins that may be found in food
2. Chemical hazards e.g. cleaning chemicals, pesticides, drug residues, heavy
metals, allergens etc.
3. Physical hazards e.g. foreign bodies such as stone, mud, glass, plastics and
metals.
Food hazards are the cause of meat borne diseases that leads to consumer suffering
and loss of life. Food borne diseases are diseases that are transmitted to humans
through consumption of a disease agent (hazard) in the meat. Considerable costs arise
due to associated medical treatment, lost production, recalls and disposal of faulty
products. Outbreaks of meat borne diseases often result in bad publicity, loss of market
for the meat product, and may lead to legal costs related to illness or loss of life.
Meat quality assurance system approach is based on the application of Good
Agriculture Practices (GAP), Good Hygiene Practices (GHP), Good Manufacturing
Practices (GMP), Sanitary Standard Operating Procedures (SSOP), Hazard Analysis
Critical Control Point (HACCP) system, food safety management systems and
traceability/recall systems. The GAP, GHP, GMP and SSOP are considered as
prerequisite systems or programs for implementation of the HACCP system. The
HACCP system for production of meat is a proactive means of controlling production
processes aimed at enhancing the safety of meat.
The GAP, GHP, SSOP are measures that are required to produce safe food. The GAPs
are basic food safety principles associated with minimizing biological, chemical and
physical hazards at primary production stages.

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GAPs related to animal production, health and welfare
Good Agricultural Practices are a collection of principles to apply for on-farm production
and post-production processes, resulting in safe and healthy animal food products and
non-food agricultural products, while taking into account economical, social and
environmental sustainability: Good Agricultural practices therefore include:
 Provide animals with adequate and suitable feed, clean water and appropriate
housing conditions that protect them from adverse weather conditions.
 Prevent outbreak of disease epidemics through regular vaccination programs.
 Alleviate pain in sick animals through provision of appropriate treatment
 Design, construct and use and maintain equipment and handling facilities to
avoid injury and loss.
 Avoid non-therapeutic mutilations, surgical or invasive procedures, such as tail
docking and de-beaking;
 Manage stocking rates to avoid overstocking and the associated negative
impacts on landscape, environment and life:
 Avoiding biological, chemical and physical contamination of land for grazing,
pastures feeds, water and air
 Prevent chemical and veterinary drug residues from entering the food chain
 Minimize non-therapeutic use of antibiotics or hormones
 Avoid feeding animals with animal wastes or animal matter in order to reducing
the risk of alien viral or transgenic genes, or prions such as mad cow disease),
 Minimize transport of live animals (by foot, rail or road) to reducing the risk of
epidemics, e.g. FMD
 Purchase, store and use only approved veterinary products in accordance with
regulations and directions including withholding periods.
 Use methods of pre-slaughter handling and slaughter that are humane and
appropriate for each species.
 Use of appropriate records and traceability methods on the whole production
chain (breeding, feed, medical treatment...) to ensure consumer security and
feedback possibility in case of a food crisis
Good Hygiene practices (GHPs)
Good Hygiene Practices are the conditions and measures necessary to ensure the
safety and suitability of food at all stages of the food chain. They are hygienic rules
translated to work instructions and procedures for employees and machinery
operations.. The GHPs require that all persons working in direct contact with meat, meat
contact surfaces and meat packaging materials conform to sanitation and hygienic
practices and standards to the extent necessary to protect against contamination of
meat from direct or indirect sources. One of the elements of GHPs is that meat
businesses assess their own standards and make decisions regarding the practices and
procedures which ensure good hygiene practices.

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Good hygiene practices at various stages of meat value chain
At farm level:
 Keep animals free of diseases through regular vaccination and treatment.
 Prevent accumulation of chemical and veterinary drug residues in animal tissues
by feed animals with clean feed and prudent use of veterinary drugs.
Lairage: The principal function of the lairage is to provide a reservoir of animals for the
slaughter. For the purposes of food safety, and on animal welfare grounds,
 Facilities must be available for the isolation and removal of any animals showing
signs of illness.
 Pressurized water is preferred to wash animals prior to slaughter
 Animals must be adequately rested before slaughter for 12hrs.
 Food should be withheld12hrs before slaughter.
 Anti-mortem inspection must be diligently carried out to detect sick animals.
Many diseases are often more easy to detect during Am rather than pm
Slaughter and flaying process
• Slaughtering should be done in tables or in hanging rail to reduce contamination
by regurgitation
• The slaughtering knife should be cleaned and sterilized between each carcass at
82°C.
• The head should be removed and after skinning washed separately from the
carcass.
• Tonsils should be removed cleanly as they are heavily contaminated with
pathogenic bacteria.
• During dressing the oesophagus of slaughter animals should be tied to prevent
leakage of ruminal contents.
• Removal of hides or fleece should be carried out so that contact between the
outside of the skin and the carcass is avoided while hands and equipment that
touch the outside of the skin should not come into contact with the underlying
carcass meat.

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• After the initial cut through the skin, sterilize the knife in water at 82°c and then
make all other cuts from the inside out.
• No hair or skin pieces should be left on the skinned carcass.
• No excess blood should appear on the skin of the carcass.
• Prevent contact of dirt from hides and skin to the meat surface.
• Prevent contamination of the carcass with dirty hooks, knives and protective
clothes.
GHP during Evisceration
• It is recommended that a plastic bag is used to seal off the rectum after
loosening; this is sometimes referred to as ‘bagging’.
• Do not puncture the viscera (alimentary tract), uterus, urinary bladder and gall
bladder during separation cuts.
• Prevent contact of viscera with floors, walls or stands.
• Regularly wash hands/aprons and sterilize knives, especially after any possible
contamination has occurred.
• Identify/correlate viscera with the related carcasses
• Careless evisceration must be avoided to prevent contamination of the carcass
with gut contents.
Carcass splitting
• Carcass splitting saws should be sterilized between each carcass.
Carcass washing
• Carcases should be washed after spliting using cold potable water
Post-mortem inspection
• During post-mortem meat inspection, palpation and incision of lymph nodes,
infected tissues or tissues with abnormalities can give rise to cross
contamination.
• Incision should be avoided where possible, and palpation of organs should be
minimal.
Personal Hygiene of slaughterhouse workers
 Appropriate protective clothing should be worn and replaced each day or when
heavily soiled. All meat handlers and persons in a room handling meat should

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wear protective clothing’s (overcoat, cap, plastic apron, and gumboots). They
should be of light color, preferably white and be clean and tidy.
 Hands and arms should be washed and knives and equipments regularly
sterilized.
 Workers must wash their bodies regularly and have the nails trimmed as much
as possible. They should not move from dirty to clean areas, but vice versa.
 Nail polish and dyes on nails and hands should be discouraged.
 Beards should be shaven or covered by a turban.
 The hair should be short, clean and tidy. The hair should either completely
covered with a clean cap or hat or confined by a hairnet to prevent hair falling
into products
 Ornaments such as rings watches and bungles should be removed.
 The following must be avoided: heavy perfumes, use of lipstick, false eyebrows
and eye shadows.
 premises indicate that hygiene is not of high standard.
 Washing facilities should be provided where workers can wash before leaving
and a canteen where they could take refreshment and rest during breaks.
Figure 4.9: Slaughterhouse workers dressed in their protective clothing. The cleanliness
of personnel and the external environment of the slaughterhouse is satisfactory.
Medical fitness of meat handlers

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Meat handlers include slaughterhouse staff involved in all stages of slaughter, loading
and transportation of meat, washing and cleaning of equipment and all slaughterhouse
premises and meat retailers.
(a) A meat handler must be healthy and free from communicable disease e.g.
tuberculosis and salmonellosis.
(b) They must go for medical check-ups after every 6 months
Personnel suffering from boils, nose or throat infections, intestinal disorder (diarrhea)
should not handle meat. Their health status will have an impact on the level of bacterial
contamination of meat and this can spread infection to the whole community. Cuts and
wounds should be dressed up nicely. Dressings on hands must have a waterproof
covering to prevent anything passing through the bandages from the wound. Bacteria
from a wound could easily pass through a bandage.
Slaughterhouse cleaning after slaughter operations
• Proper cleaning and sanitation of the slaughter floor, walls and equipment should
occur daily and frequently during the day to reduce contamination from these
sources and it should be done under skillful and firm supervision.
• Sanitary Standard operation procedures (SSOPs) for cleaning and sanitation of
all parts of the plant should be in-place and well-implemented.
Cold storage of meat
• Move the carcasses into the cooler as soon as possible to speed up the surface
drying and hinder bacterial growth.
• Keep carcasses on rails and without touching floors/walls and other carcasses to
prevent cross-contamination.
• Keep carcasses spaced to allow surface drying limits growth of microorganisms.
• Do not overload the coolers.
• Adjust the cooling régime optimally in terms of air temp., speed and relative
humidity, to achieve rapid refrigeration to a deep muscle temperature of 6-7°C
with no condensation or weight loss.
• Do not open the cooler doors either unnecessary or frequently to avoid
temperature fluctuations.
• It is of extreme necessity that adequate lightening should be maintained inside
the cold rooms

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Personnel Education/Training
Slaughterhouse staff should be made fully aware of and conversant with the need for
occupational hygiene, not only to avoid infection and meat contamination but also from
becoming carriers of infection to their families and communities. Education on good
standards of health and hygiene and its importance is essential. Staff should be
educated on:
(a) Hygienic slaughter e.g. using only properly washed and sterilized equipments
e.g. knives, pangas, axes, saws.
(b) Maintaining their own personal hygiene, cleanliness of the slaughterhouse
and slaughter equipment and the environment.
(c) Thorough washing of hands with soap after visiting toilet, coughing or
sneezing, handling of money,
(d) Proper and hygienic disposal of garbage, soiled or infected material.
(e) Knowledge that negligence may be detrimental to the whole community.
(f) How and why unsanitary practices should be avoided.
Sources of carcass contamination before and during slaughter
1. Contamination a farm level (infection and used of drugs and pesticides in animal
husbandry)
2. Dirty feet, hides and skin due to dirty lairages or trekking routes
3. Butchers tools (knives, pangas, axes etc), hands and clothing which are
contaminated. Butchers should continuously wash and disinfect their tools and

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hands during the slaughter operations. They should wear clean gum booths and
dust coats.
4. Flaying: Hides and skin contain high microbial load. Before slaughter, animals
can pass though a warm shower as they head to the stunning box. In addition
care should be taken to ensure clean flaying operations.
5. Intestinal and stomach content during evisceration. Care should be taken to
avoid puncturing and spilling stomach and intestinal contents during evisceration.
In case a puncture occurs, the spilled contents should be washed off immediately
and knife and hands washed disinfected before proceeding with the operations.
6. Poor quality water used to was carcasses
7. Poor personnel hygiene and habits
8. Contaminated meat carriers
9. Meat loading, transporting and cutting;
10. Dogs, birds and vermin(rodents and insects),
Personnel should be taught to avoid the following unsanitary practices (or bad habits)
 Walking aimlessly while operations are going on
 Unguarded coughing and sneezing which can spread pathogenic respiratory
bacteria:
 Spitting on hands to enable firm gripping of an axe, panga e.g. while splitting
carcass
 Licking of fingers to pick up items e.g. papers, utensils, paper towels or wrapping
papers or to turn over the pages of a book
 Cutting of finger nails using teeth or by using slaughter or meat inspection knife
 Placing the pen, fingers in the mouth
 Any contact with saliva will transfer salivary bacteria to the hands or fingers
 Blowing paper bags to open them
 Shaking of hands in the slaughterhouse
 Eating, Smoking of cigarettes, chewing miraa in the slaughterhouse
 Unnecessary touching of meat and placing of meat on dirty places
 Chewing sweet-gum
 Blowing or wiping of nose using bare hands or protective clothing
 Use of mobile telephones
 Nose picking (removal of dried mucus from nostrils) placing fingers in or around
the nose, mouth etc removal of ear wax using fingers, keys, pen, match stick and
then rubbing it on the hands or protective clothing
 Scratching of head
 Brushing of teeth,
 Urinating in undesignated places within the slaughterhouse environment
 Changing of clothes near meat

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 Confrontations, fighting, playing in the slaughterhouse
 Placing meat, knives, sharpening steel in the gumboots
 Washing the apron and protective clothing on the slaughterhouse floor.
SANITARY STANDARD OPERATING PROCEDURES (SSOPS)
SSOPs describe how GHP is to be achieved i.e. minimizing microbial, chemical and
physical contamination. The objective of SSOPs is to prevent contamination of
carcasses and other food products resulting from employee actions during slaughter
and meat processing.
MODEL SANITARY STANDARD OPERATING PROCEDURES (SSOPS)
Sanitary Standard Operating practices to be followed by all employees
1. No person with illness, or open/infected wounds is allowed to handle foods or
food-contact surfaces.
2. All employees must begin their shift wearing clean garments. Raw product
processing employees must wear hair covers and change or clean/sanitize (or
replace) outer garments when they become soiled. Ready-To-Eat (RTE) product
processing employees must wear hair covers and single-use disposable gloves,
and maintain the cleanliness of all outer garments.
3. Employees must wash hands properly after using the bathroom or handling any
objects that may contaminate products, and before putting on disposable gloves.
4. Employees must not use tobacco, eat, or drink in slaughter or production areas.
5. Employees must not wear jewelry (other than secured wedding bands) or
cosmetic items that could contaminate product.
6. Food, beverages, and medications must be stored in designated employee
locker or storage areas.
7. Hand wash facilities and toilets must be kept functioning correctly and properly
supplied.
Sanitary Standard Operating Practices to be specifically followed during
slaughter
Carcass dressing must be performed under sanitary conditions and in a manner to
prevent contamination of the carcass. The Slaughter Manager is responsible for
ensuring that employee hygiene practices, sanitary conditions and cleaning procedures
are maintained during the slaughter day. He or she must make a visual observation at
least once between each break in work (start, break, lunch, etc.) and record his
observations on the SSOP Inspection form at least once per slaughter day.
The following SSOPs are important during slaughter:
a. Clean hands, arms, gloves, aprons, boots, etc., as often as necessary during the
dressing procedures.

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b. Clean and then sanitize (with 180ºF water or another approved sanitizer), knives
and other hand tools, saws and other equipment, as often as necessary during the
dressing procedures to prevent contamination of the skinned carcass.
c. Before using the brisket saw, rinse it to remove meat and bone particles. Then
sanitize it using 180° F water or another approved sanitizer.
d. Keep your hands, arms, clothes, aprons, boots and knives clean during the
evisceration process. If contamination occurs, step away from the evisceration area
to clean apron, boots, and knives. Sanitize knives after cleaning. It may also be
necessary to clean hands and arms with soap and water. In cases of contamination
from an abscess or other extensive contamination, you may need to shower and
change clothes before going back to work.
e. If you see any edible product that is contaminated with readily identifiable Specified
Risk Material (SRM), remove it by trimming with a knife. SRM’s are the distal ileum
and tonsils of all cattle and the brain, skull, eyes, trigeminal ganglia, spinal cord,
vertebral column (excluding the vertebrae of the tail, the transverse processes of the
thoracic and lumbar vertebrae and the wings of the sacrum) and the dorsal root
ganglia of cattle 30 months of age and older. The knife you use MUST be cleaned
and sanitized before it is used again on edible product.
f. Clean and sanitize (using a designated approved chemical sanitizer or 180ºF water)
the carcass splitting saw after it is used on a suspect carcass or when contamination
occurs. NOTE: cleaning off tissue debris and sanitizing the splitting saw are
MANDATORY when moving to a beef carcass that is younger than 30 months after
splitting a beef carcass that is 30 months or older.
g. When equipment is visibly contaminated, remove the contaminants and sanitize
the affected equipment before re-starting production. Inspect the carcass (es) to
determine if they were contaminated or adulterated.
h. Try to determine the cause of the contamination and take corrective action. This
may require adjusting equipment, retraining employees, temporarily stopping the
kill process, etc.
Role of government in quality assurance of meat
The Government through the veterinary authorities is obligated to:
a. Control the registration and use of veterinary drugs, pesticides and feedstuffs so
that residues do not occur in meat at unsafe levels that make the meat or its
products unsafe for human consumption;

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b. Control animal and zoonotic diseases in livestock meat for slaughter through
vaccinations, animal treatment.
c. Provide and enforce monitoring and surveillance systems that establish baseline
data of livestock diseases and levels of harmful chemical residue;
d. Take such other measures from time to time that will ensure production of safe
meat and meat products (e.g. control of livestock movements, certification of
slaughter facilities, provision of livestock and meat inspection services, control of
meat transport activities, hygiene of meat transport carriers, control of the
hygiene of meat markets).
Role of Meat Inspectors in Quality assurance of meat
The Veterinary Meat Inspector shall ensure that:-
a. All animals brought to the slaughterhouse are accompanied by a movement
permit indicating the origin, species, number of animals and mode of transport
used
b. a thorough ante-mortem inspection is done to all animals
c. Animals presented for slaughter are sufficiently and thoroughly cleaned before
slaughter in order not to compromise hygienic slaughter and dressing;
d. The animals are held under conditions that minimize cross-contamination with
food-borne pathogens and facilitate efficient slaughter and dressing;
e. The animals are subjected to ante-mortem inspection;
f. Enough facilities for ante-mortem and post-mortem inspection.
g. The slaughter facilities are hygienic and fully prepared for the slaughter.
h. All slaughter and carcass dressing operations as well as any further meat
handling at the slaughterhouse are carried out in a hygienic manner
i. Any information gathered during ante-mortem inspection which is considered
appropriate for the producer for future or immediate action is sent to him in
reasonable time;

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FOOD BORNE DISEASES
Food borne diseases are diseases that are transmitted to humans through
consumption of a disease agent in the food. Food borne diseases are
classified into two categories
1. Food borne infections
2. Food borne intoxications.
Foodborne infections are caused by the entrance of pathogenic
microorganisms contaminating food into the body, and the reaction of the
body tissues to their presence. These can either be fungal, bacterial, viral or
parasitic. Bacterial food borne infections are the most common. Food borne
infections tend to have long incubation periods and are usually characterized
by fever. They include bacterial (salmonellosis, cholera, typhoid fever,
shigellosis, Yersinia enterocolitica infection, E. coli infections,
campylobacteriosis, Listeria monocytogenese, mycotic (Candida spp.,
Sporothrix spp., Wangiella spp. etc), viral (hepatitis A and poliomyelitis),
parasitic infections (hookworms, roundworms and tapeworms) and
protozoan (balantidiasis, amoebiasis etc)..
Salmonellosis
The salmonellae organisms are capable of causing disease in animals and
man when taken into the body in sufficient numbers. The salmonella species
involved in food poisoning include; Salmonella typhimurium, Salmonella
enteritidis, Salmonella dublin, Salmonella softenburg, Salmonella virchow,
Salmonella montevideo, Salmonella infantis, and salmonella newport. These
species are also involved in causing diarrhea in animals. In some cases, they
are nosocomially transmitted between people in hospitals.
Clinical signs in man
Salmonella food poisoning is characterized by symptoms which appear 12
hours after consumption of food. The illness is due to the growth of the
organisms within the body. The ordinary symptoms are abdominal pain
diarrhea, chills, fever, vomiting, headache, prostration, and malaise.
However, when a severe case of the disease is encountered, more symptoms
may be involved like septicaemia with leucopenia, endocarditis, pericarditis

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or other types of illness, such severe cases are encountered in babies, young
children and sick or elderly people. The mortality is up to 13 %.
Reservoir
The primary reservoir of salmonella bacteria is the vertebrate intestine,
which means that almost all animals could excrete the organisms. Animals
and human beings might harbor salmonella bacteria without showing any
signs of the disease, thereby acting as carriers of the disease.
Transmission
Salmonellae reach food in many different ways; either directly at slaughter
from animal or human excreta, from where they are transferred to food
through hands, utensils, equipments, flies etc. Food poisoning is more likely
to occur if the total number of microorganisms present is high. A smaller
number may have no ill effect. If the temperature and other conditions are
suitable, there can be a great multiplication in numbers of organisms i.e. if
food is allowed to cool and gently re-heated, or kept for sometime in a warm
room.
Foods involved
Almost any food may be contaminated with salmonellae and under
conditions of mishandling such food may become involved in the
transmission of salmonella. The foods commonly involved are the various
animal derived foods, like meat and meat products, milk and milk products,
and egg and egg products especially from infected animals. Flies, rats, and
mice may transfer salmonellae from fecal material to food.
Contaminated water can also act as a carrier by contaminating other foods,
but is not directly the vehicle causing the disease since salmonella bacteria
are found in very low numbers in water.
Control
There are many ways that can be employed to reduce the incidence of the
disease especially by improving sanitation and to prevent mishandling of
foods. Salmonella in domestic animals and contamination of foods during
processing must be controlled and salmonella in raw products destroyed by
processing. The following control measures have been shown to considerably
reduce the salmonella carrier rate and the related incidence of food
poisoning cases.

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i).The use in the farm feedstuffs free of salmonellae.
ii). Hygienic standards of animal husbandry including proper control of slurry
disposal, water supply and food protection from insects and rodents.
iii). Proper design of the slaughter lines and the adoption of efficient hygienic
methods of slaughter and carcass dressing which minimizes contamination.
iv). Proper sewage treatment and disposal.
v). Bacteriological monitoring, which should include a presence /or absence
tests for salmonellae.
vi). Efficient refrigeration and hygienic handling of food.
vii). Avoidance of consumption of raw meat, unhygienic handling in the
home, and the use of storage systems which contribute to the proliferation
of bacteria of all types.
viii). Complete thawing of frozen meats and adequate cooking to ensure
destruction of potential pathogens and spoilage organisms.
ix). Heat processing of meat and poultry in a sanitary manner and such as to
avoid cross contamination.
x). Pasteurize egg and milk products and keep them cool.
Typhoid/Paratyphoid fever (Enteric fevers)
Enteric fevers include typhoid and paratyphoid fevers caused by Salmonella
typhi and Salmonella paratyphi A, B and C respectively. The typhoid and
paratyphoid fevers are clinically and pathologically similar regardless of
causal agent and only bacteriological examination can differentiate between
them. The serotypes are similar to other salmonella bacteria, but unlike
them, they are essentially parasites of man..
The disease symptoms
The incubation period is usually about 2 weeks, but might vary between 3
and 28 days for typhoid fever and between 1 and 15 days for the
paratyphoid fevers. The enteric fevers are generalized septicemia infections
with a frequent, if not constant bacteriaemia during the first two weeks of
the disease. The abdominal symptoms are severe, while fever and illness

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may continue for 4-6 weeks. Generally the paratyphoid fevers have a short
duration. The case fatality rate is approximately 10 %.
Reservoir and epidemiology
The typhoid and paratyphoid bacilli are essentially human parasites and are
acquired mostly from human sources, namely, patients and carriers. The
bacteria can be transmitted through contaminated water, milk or food. Only
a few organisms are needed to cause disease.
Control measures
1. Hygienic control of food and water supplies
2. Detection and treatment of chronic carriers and
3. Vaccination using TAB-vaccine. Protection by this vaccine is maintained
for 5-7 yrs.
Campylobacteriosis
Campylobacteriosis is a foodborne disease caused by Campylobacter jejuni
and Campylobacter coli which are associated with acute enterocolitis in man.
The disease can be acquired from a variety of animals including poultry,
pigs, wild animals and birds. The organisms are important causes of diarrhea
illness in all age groups of persons throughout the world, resulting in
diarrhea, abdominal pain, fever nausea, vomiting, and abdominal
complaints. Campylobacter jejuni occur in large numbers in cattle feces, and
poultry as normal flora. Infection in humans is characterised by
gastroenteritis and diarrhea.
Clinical signs
Incubation period ranges between 2-11 days with an average of 3-5 days. It
is preceded by fever, followed by diarrhea, which runs for 3-4 days then it
reduces. Abdominal pain is associated with backache, and a high mortality.
Death only occurs due to other complications. The condition is self-limiting
lasting up to 10 days. The jejunum and ileum are primarily affected with
extension to the colon resulting in acute inflammation and in occasions
abscess formation.
Mode of infection

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Infection occurs by ingestion of organisms in contaminated foodstuffs and
water. Incriminated food includes meat or water, or from contact with
infected animals, unpasteurized milk and possibly cross-contamination from
these sources to foods eaten uncooked or unrefrigerated. Carelessness in
the kitchens e.g. cutting chickens with the same knife used to cut other
foods without prior cleaning. Pork is a major source of C. coli, which is
mostly found as commensals in pig intestines, Contamination of pork occurs
during slaughter. Red meat e.g. lamb and beef are not major causes.
Preventive measures
1. Thorough cooking of all foodstuffs derived from animal sources especially
poultry.
2. Prevention of re-contamination after cooking.
3. Proper refrigeration of foods.
4. Recognition, control and prevention of campylobacter infections in
animals, and
5. Maintenance of high standard of hygiene.
Escherichia coli food borne infection
Escherichia coli are potential food poisoning pathogens which are widely
distributed in food environments in low numbers. E. coli strains involved in
food borne gastroenteritis fall into the following groups :(i).
Enteropathogenic E. coli (EPEC), (ii). Enterotoxigenic E. Coli (ETEC), (iii).
Enteroinvasive E. coli (EIEC) and (iv). Enterohemorrhagic E. coli (EHEC).
Each class is composed of various and often unique O:H serotypes and
posses virulence characteristics of that class. The serotypes are
characterized by using O-somatic and H-flagella antigens.
Enteropathogenic E. coli (EPEC)
Most EPEC strains adhere to the intestinal epithelial tissue and produce a
verocytotoxin. They produce moderate to severe watery, dehydrating
diarrhea, which may be associated with fever, respiratory symptoms and
abdominal distension. Duration of illness on average is about one week, but
some patients may have a longer course. EPEC is one of the few known
causes of chronic diarrhea in infants. Risk factors of death include: young

19. Page | 19
age and virulence of associated EPEC strain. Case fatality can be 70 % in
children less than 2 years.
Enterotoxigenic E. Coli (ETEC)
ETEC strains produce either heat stable enterotoxin (ST) or a heat labile
enterotoxin (LT), or both, as well as colonizing factor antigens represented
by pilli that mediate binding of cells to epithelial cells. These strains colonize
the human small intestines and cause an influx of secretions mediated by
the action of ST and/or LT enterotoxins.
Symptoms. The illness consists of watery non-bloody diarrhea, abdominal
cramps, and little or no fever. The symptoms usually lasts for 4 to 5 days,
and illness is mild in majority of patients, but a dehydrating cholera-like
picture may occur. The disease is however self-limiting, and is common in
children less than 5 years old. ETEC strains are the major etiological agents
of travelers' diarrhea. The illness is common among groups traveling from
low risk areas to high risk areas (e.g. developing countries). A relatively
large inoculum >108 cells/g is necessary to produce disease in adults.
Assurance of safe food and water is the best protection against diarrhea due
to ETEC strains.
Enteroinvasive E. coli
EIEC strains cause illness similar to shigellosis, as they have antigenic
relatedness to shigella. EIEC strains usually produce watery diarrhea in most
patients. In addition, there is fever, nausea, and abdominal cramps. Bloody
diarrhea may occur in fewer than 10 % of patients. The presence of mucus
and polymorphonuclear leucocytes in stool is typical of these strains. Illness
is usually self-limiting, lasting for 2 to 3 days. A relatively high dose (108
cells) is necessary to produce disease in volunteers. The median incubation
period is 18 hours (range 2-48 hrs). Food borne spread appears to be the
usual mode of transmission, although person-to-person spread has also
been reported.
Enterohemorrhagic E. coli
Escherichia coli serotype O157:H7 causes hemorrhagic colitis in humans.
The serotype does not ferment sorbital or may do it slowly, but produce a
verotoxin which is cytotoxic to vero culture cells. Infection involves all age
groups and has been linked to ingestion of contaminated hamburger and
beef in many outbreaks.

20. Page | 20
EHEC infection is characterized by diarrhea, abdominal pain which may be
severe and vomiting. Few patients develop fever. Illness lasts for 4 to 8
days, although it may extend to 13 days for severe cases. Patients with
complications have bloody diarrhea, acute ulcerative or ischemic colitis and
sub-mucosal edema with severe colonic inflammation. Deaths due to E. coli
O157:H7 infections have been reported only in elderly patients and in
patients who develop the hemolytic uremic syndrome (HUS). HUS can be
recognized by acute renal failure, microangiopathic hemolytic anemia and
thrombocytopenia. Infection also precedes thrombotic thrombocytopenic
purpura (TTP), an illness that shares many clinical features with HUS.
Control: Control measures involve proper cooking of hamburger and other
meats, avoidance of cross-contamination of foods in the kitchen and good
personal hygiene. The organism O157:H7 is heat sensitive, but resistant to
freezing. It grows poorly at 44C-45C, with no growth at 45.5C, suggesting
that its presence may not be detected by fecal coliform assay test.
Listeria monocytogenese infection
Listeria monocytogenese is a gram positive bacterium that is pathogenic to
both animals and human beings. The organism is widespread in nature and
is a transient constituent of the intestinal flora excreted by 1-10% of healthy
humans. It is extremely hardy and can survive for many years in the cold in
naturally infected sources. In animals, it causes abortion and mastitis, while
in man it can cause abortion in pregnant women as well as meningitis in
newborn infants and immunocompromised adults. Pregnant women, infants
and elderly people are at particular risk from listeriosis. The infection is fatal
in susceptible individuals with a mortality of 25-30%.
In the last few years, there has been an increasing number of Listeria
monocytogenese type 1-4 food borne infections caused by consumption of
vegetables, pasteurized milk and soft cheese and meat and products. One
such outbreak occurred in California where a large outbreak was reported in
1988 among pregnant women and or their children that caused by
consumption of Mexican-style cheese in which L. monocytogenese serotype
4B was identified. Deaths were reported in fetuses, neonates and other
individuals with compromised health status. A small portion (3-4%) of milk
used for preparation of cheese was not pasteurized in order to add the flavor
of raw milk to cheese. What flavor was to be added?
In 1981, a large outbreak caused by chilled cabbage occurred in East
Canada. Listeria monocytogenese serotype 4B was isolated from the

21. Page | 21
patients, as well as from cabbage kept in the refrigerator. A total of seven
cases were observed in adults and 34 perinatal cases. The death rate among
babies was 27 %, while 31 % adults suffered from meningitis. The cabbage
originated from the farmer who used cattle manure for the crops. Two cows
had recently died from listeriosis and the cabbage was grown on a field
where manure from the cows had been used as fertilizer. The cabbage was
kept in chilled conditions at the farm until it was sold to a salad producer
immediately before the outbreak.
Vehicle foods
In USA, Canada, Mexico, UK and Switzerland, large-scale outbreaks of
listeriosis have been associated with the consumption of various foods
(including milk, ice cream, cheese, poultry, sauerkraut, salads, sea foods,
meat and meat products contaminated by Listeria. The majority of the cases
of listeriosis in humans are associated with the consumption of Listeria-
contaminated foods. Delicatessens and other ready-to-eat foods may also be
important in causing food Listeria food poisoning. Raw milk or cheese made
of unpasteurized or insufficiently pasteurized milk play an important role in
the epidemiology of the disease.
Preventive measures
Among the measures recommended for reducing the incidence of listeriosis
in humans are serological testing of pregnant women and careful monitoring
of the quality of foods including conditions of processing and storage. It is
almost impossible to eliminated Listeria monocytogenese from the food
supply because of its ubiquitous nature. However, by using a hazard analysis
critical control point approach, health hazards associated with this organism
can be minimized (Harber, 1991).
VIRAL FOODBORNE INFECTIONS
Infectious hepatitis (Viral hepatitis A)
This is cause by hepatitis virus A. The incubation period is long, being an
average of 30 days (range 15-50 days). It is a systemic infection
characterized by gastrointestinal manifestations and liver injury, fever,
malaise anorexia, lassitude, nausea, abdominal discomfort, bile in urine and
jaundice. The duration of the disease could be from a few weeks to several
months.

22. Page | 22
Hepatitis A may be acquired from fecal contaminated food or water, or from
direct contact with infected individuals. Person-to-person transmission is
particularly common between children and between sexual partners. Feces,
urine, blood of infected human cases and persons incubating or convalescing
from the disease are infectios mateials
Foods mostly involved are shellfish, water, milk and orange juices.
Control
1. Foods should be cooked thoroughly, virus survives 50oC for 30 minutes.
2. Shellfish should not be collected or produced in polluted waters
3. Personal hygiene
4. Vaccination of contacts, and
5. Serovaccination with immunoglobulin.
Viral hepatitis E
This was formally called enterically transmitted non-A, non-B hepatitis). It is
a waterborne infection that occurs as epidemics and/or sporadic cases. The
virus has been isolated from hepatitis epidemics in many countries including
Kenya. This disease primarily affects young adults, and is clinically similar to
hepatitis A but does not lead to chronic disease. However, among women in
the second or third trimester of pregnancy who contract the disease, about
15-20 % die of hepatitis.
Control
Proper cooking of food to eliminates the virus
Poliomyelitis
Poliomyelitis is an acute viral illness where the majority of infections remain
inapparent. Manifested illnesses include paralytic and non-paralytic forms.
This disease occur commonly in older people when infected. Symptoms
mostly seen are those of lower extremities, meningitis and general illness.
Reservoirs and epidemiology
Feces and pharyngeal secretions contain the virus. The main route of
transmission is oral-oral and fecal-oral routes. Foods involved include water
and milk which get contamination with feces, and pharyngeal secretions of

23. Page | 23
infected persons. The virus can be cultured from stool, pharyngeal swabs
and spinal fluids of diseased patients. Serology e.g. CFT is frequently used
for diagnosis.
Control
Vaccination against the disease is the most important measure. Live and
killed vaccines are available. Personal and food hygiene must be practiced.
Prevalence in Kenya
Disease occurs in Africa especially where vaccination is taking place. The
threat can be reduced with large scale vaccination campaigns.
RICKETTSIAL FOODBORNE INFECTION
Q-Fever
Q-fever is caused by Coxiella burnetti and has an incubation period of
between 2-4 weeks. Symptoms include a sudden onset of fever, dry cough
and chest pain due to a pneumonitis. Hepatic disorders which include slight
jaundice occur in severe cases. Mortality is low with complete recovery.
Reservoir and epidemiology
O-fever is mostly an occupational disease among people who handle
livestock and raw animal products (e.g. farm and slaughterhouse workers
etc). Consumption of raw milk, contaminated butter and cheese, contact
with infected placentas, contaminated straw beddings and animal carcasses
or slaughterhouse offal are considered important.
Diagnosis
1. Serology including CFT micro-agglutination and FAT.
2. Isolation of agent (in well equipped laboratory due to the high risk of
infection).
Control
1. Pasteurization of milk (heating at 66C for 30 min or 75C for 15 sec).
2. Safe disposal of offal

24. Page | 24
FOOD BORNE INTOXICATIONS
Food borne intoxications are diseases caused by consumption of:
1. Toxicants which are found in tissues of certain plants and animals.
2. Metabolic products (toxins) formed and excreted by microorganisms
(such as bacteria, fungi and algae), while they multiply in food, or in
gastrointestinal tract of man.
3. Food containing poisonous substances, which may be intentionally or
unintentionally added to food as a result of producing, processing,
transporting or storing.
Food borne intoxications have short incubation periods (minutes to hours)
and are characterized by lack of fever. Food borne intoxications can be
classified into:
(1). Bacterial intoxications
(2). Fungal intoxications
(3). Chemical intoxication
(4). Plant toxicants and
(5). Poisonous animals.
BACTRIAL FOOD BORNE INTOXICATIONS
Staphylococcus aureus intoxication
This is a type of food borne intoxication caused by consumption of food
contaminated with staphylococcal enterotoxins produced by certain strains of
Staphylococcus aureus while growing in food. The organism produces
serologically different enterotoxins. Five staphylococcal enterotoxin A(SEA),
Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C (SEC),
Staphylococcal enterotoxin D (SED) and Staphylococcal enterotoxin E (SEE)
have been involved in food poisoning. The recently other staphylococcal
enterotoxins (SEG, SHE, SEI, SEK, SEL, SEM, SEN, and SEO) have been
identified. However, the relationship between these new enterotoxins and

25. Page | 25
human food poisoning is not fully understood. It is known that 95 % of
staphylococcal food poisoning outbreaks are caused by types SEA to SEE.
The remaining 5% of outbreaks may be associated with the new
enterotoxins. Individual strains of S. aureus may produce one or more
enterotoxin types in food or in culture. The percentage of the strains that are
enterotoxigenic differ widely depending on the source of the isolate.
Characteristics and growth conditions
Staphylococcus aureus is a facultative anaerobe, non-spore forming gram
positive cocci. It grows at a temperature between 12-44C (optimum 37C)
and pH range 4.0-9.83 (optimum 7.4-7.6). Growth occurs in an environment
containing up to 18 % sodium chloride and water activity of 0.86 - 0.88
when growing aerobically and 0.9 under anaerobic conditions. Toxin
production occurs at growth temperature 12-44C, pH  4.2 and salt
concentration of  10%. No toxin production occurs at temperatures below
12C, pH < 4.2 and > 10 % salt.
Nature of enterotoxins
All the staphylococcal enterotoxins are heat stable (withstanding heating at
100C for one hour) and ordinary cooking procedures, pasteurization and
drying do not inactivate these enterotoxins. They are insensitive to pH
changes(pH stable) and resistant to most proteolysis enzymes (trypsin,
chymotrypsin renin, papain and pepsin). The enterotoxins are also not
affected by irradiation. All the five enterotoxins have been reported to cause
food poisoning and have the same potency.
Competition with other organisms
Staphylococcus aureus is a poor competitor and therefore grows poorly or
not at all in a mixture of other microorganisms. Majority of S. aureus food
poisoning are due to foods in which the microbial flora is substantially
reduced, where they grow well and produce high yields of enterotoxins.
Foods with substantially reduced flora are such as cured meats (which
contain high salt concentrations that are inhibitory to other microbial flora
selecting for S. aureus), and cooked foods which are contaminated before
consumption.
Reservoirs
Staphylococci are found in varying numbers in air, dust water, food, feces
and sewage. The primary habitat of S. aureus is the mucous membranes of

26. Page | 26
the nasopharynx and skin of man, animals. The organism is found in the
nose, skin, saliva, intestinal contents and in feces. Human carriers of this
organism are numerous and are undoubtedly the source of a number of
outbreaks. Contamination of foods may be traced to food handlers with
minor septic hand infections or severe nasal infections, with subsequent
heavy growth of the organism on the food medium and production of
sufficient enterotoxin to evoke gastrointestinal symptoms in man. The nasal
mucous membrane is another particularly important source of staphylococci
of human origin.
Vehicle foods
Foods involved are those in which the microbial flora is substantially
reduced, such as boiled/cooked or processed milk and meat, cured meats
and meat products which are later contaminated. Milk products involved
include pasteurized milk, yoghurt, butter, chocolate milk, fermented milk.
Other foods include cream filled pastries, poultry, fish, shellfish, non meat
salads, egg and egg products, vegetables and cereal products. Studies have
found that increased handling of foods increases contamination with
enterotoxigenic S. aureus, suggesting that man is a major source. Mincing of
meat for example, increases contamination with enterotoxin producing S.
aureus strains.
Mode of transmission
1. Contamination from food handlers, containers etc
2. Cross contamination from raw foods
3. Improper heat treatment of raw foods
Disease symptoms in man
Symptoms of staphylococcal food poisoning occurs 1-6 hrs after
consumption of food contaminated with at least 1.0 microgram of
enterotoxin. Clinical signs include salvation, nausea, vomiting, abdominal
cramps, sometimes diarrhea with prostration. It has an attack rate of 5-
100%, but fatalities which occurs in children, the old and debilitated victims
are rare. Duration of illness is 24-72 hrs. Dose of 1.0 g or more is needed
to cause disease.
Preventive measures

27. Page | 27
1. Practice good personal hygiene including good personal conduct in food
establishment and when handling food.
2. Fast cooling of cooked food and keeping such foods at low temperatures.
Bacillus cereus food borne intoxication
This is a food borne intoxication caused by consumption of enterotoxins
produced by some strains of Bacillus cereus bacterium. The organism is
facultative anaerobic, spore-forming, Gram-positive and motile by
peritrichous flagella. Germination and growth of spores occur at
temperatures between 4-55oC (optimum 28-38C) and pH range 4.3-9.3.
The minimum water activity for growth is 0.95 and in less than 7 % sodium
chloride. Growth decreases with increasing salt concentration. High salt
concentrations and extreme pH values inhibit growth of the organism.
Spores are highly resistant to heat dehydration and other destructive
factors.
Reservoirs
Bacillus cereus is widely distributed in nature and are normally present in
soil, dust and water. Spores of this organism may contaminate food and
especially of plant origin such as cereals, potatoes, and spices. Spores may
also be found in milk, meat and meat products.
Cause of intoxication
Bacillus cereus food poisoning is caused by ingestion of any of the following
enterotoxins produced by these organisms while growing in food:
1. Diarrhea enterotoxins. The diarrhea enterotoxins are heat labile,
sensitive to enzymes trypsin, and pronase, and pH changes. They are most
stable at pH range of 5.0 to 10.0, but losses activity rapidly outside this
range. They are inactivated at pH below 4.0. Production is favored at pH
6.0-8.5, (optimum 7.0-7.5), and temperature of 18-43oC and low oxygen
and most is produced at the logarithmic phase. The toxin causes diarrhea
syndrome when consumed in food
2. Emetic toxin (cerulide). The toxin is produced in food by some strains
of B. cereus especially H-1 serovas. The toxin is a heat stable (withstand

28. Page | 28
126oC for 90 min) and resistant at pH range 2-11. It is resistant to trypsin
and pepsin and remains active at 4oC for 7 days. The toxin causes emetic
syndrome when comsumed in food. It is cytostatic, i.e. inhibits growth of
HEp-2 cells and is non-hemolytic. Enterotoxigenic strains can grow at 6oC
and 37oC are regarded as potential food poisoning organisms as they can
grow both in the food product and in the ileum. However, no enterotoxin
production occurs at 4oC.
Foods involved
Bacillus cereus is a common soil saprophyte and is easily spread to many
types of foods, especially of plant origin, but is also frequently isolated from
meat, eggs and dairy products. Cereal dishes e.g. rice, spice, mashed
potatoes, herbs, vegetables, minced meat, cream and milk pudding have
been involved in B. cereus poisoning.
Bacillus cereus does not compete well with other microorganisms but spores
can withstand heating at 100oC for 5 min. Spores of some strains can
withstand even higher temperatures (135C for 4 hours) and particularly if
food has a high fat content, which seem to have a protective effect. Spores
grow well after cooking and cooling to 48C during storage. The heat
treatment will cause spore germination, and in the absence of competing
flora, B. cereus grows well and produce enterotoxins. Some additives such
as garlic extract have an inhibitory effect on bacterial growth.
Mode of transmission to foods
1. Contamination due to improper handling.
2. Insufficient heat treatment to destroy spores.
Symptoms of disease in man
1. Emetic syndrome
The syndrome is characterized by nausea, vomiting, abdominal cramps and
sometimes diarrhea that occur 1-6 hrs after consumption of contaminated
food. The syndrome is associated with ingestion of rice and pasta based
foods.
2. Diarrhea syndrome

29. Page | 29
In the diarrhea syndrome, patients experience profuse watery diarrhea,
abdominal cramps and tenesmus, beginning 8 to 16 hours after ingestion of
contaminated food. Fever is absent and symptoms resolve within
approximately 12 hours. Vomiting is not a common symptom in this type of
syndrome.
Prevention
1. Good hygiene should be observed.
2. Low temperature and fast cooling of food.
CLOSTRIDIUM PERFRINGENS FOODBORNE INTOXICATION
This is a food borne intoxication caused by Clostridium perfringens
enterotoxin (CPE) produced in the gastrointestinal tract by enterotoxigenic
strains of C. perfringens. This organism is a spore-forming, anaerobic, gram
positive bacillus. The organism is found in the soil, dust, water, sewage
marine sediments, decaying materials, intestinal tracts of humans and other
animals. Food poisoning strains have a variety of origins including human
and animal faeces, abattoirs, sewage and flies. Spores produced by these
organisms can resist boiling for 4 or more hours. If the spores are present
as contaminants on raw meat they may resist boiling or steaming, and on
slow cooling the spores will germinate into rapidly multiplying bacterial cells
which produce large amounts of toxin.
Cause of intoxication
Clostridium food borne intoxication is caused by the ingestion of food
containing large numbers of vegetative cells of enterotoxigenic C.
perfringens type A and some type C and D strains. These cells multiply in
the intestine and sporulate releasing Clostridium perfringens enterotoxin
(CPE). Sometimes CPE may be pre-formed in food, and once the food is
consumed, symptoms may occur within 1-2 hours. The strains are heat
resistant and survive heating at 100oC for 1 hr).
Characteristics of CPE
Clostridium perfringens enterotoxin (CPE) is a protein of molecular weight
35,000 Daltons, synthesized during sporulation. CPE is heat labile (destroyed
at 60oC for 10 min) and its activity is enhanced by trypsin.

30. Page | 30
Vehicle foods
The food involved are those that are prepared one day and served the next
day. The heating of such foods is inadequate to destroy heat resistant
endospores, and upon cooling and warming the endospores germinate and
grow. Cooking kills the vegetative cells of Cl. perfringens but activates
surviving spores which will germinate and multiply. Foods poisoning occurs
when the level reaches 107-108 cells/g of food, because a certain amount of
cells are needed in order to pass the stomach barrier (low PH) and initiate
growth in the intestinal tract. Foods that have been involved include red
meats, chickens, fish, pork, fruits, vegetables, spices etc. Growth is
enhanced by a low oxidation- reduction potential (Eh) that is achieved after
removal of oxygen by cooking, long slow cooling periods and anaerobiosis
(provided easily in a liquid mass of meat or food). It is important to note
that salted meats/foods are not usually involved in this type of poisoning as
the organisms does not tolerate high salt concentration.
Mode of transmission to foods
1. Directly from slaughter animals
2. Contamination of slaughter meat from containers, handlers dust, water
etc.
Symptoms of disease in man
Symptoms appear 6-24 hours after ingestion of a large number of viable
vegetative cells  5x108/g of food, but not after ingestion of spores. The
symptoms include nausea, intestinal cramps, pronounced diarrhea and
fever. Vomiting is rare and the duration may be one to two days.
Diagnosis
1. Use of clinical signs. Clostridium perfringens is implicated as a cause of
food poisoning outbreak by the appearance of typical symptoms (abdominal
pain and profuse diarrhea), 12-24 hrs following consumption of food.
2. Confirmation of the outbreak requires (a). Enumeration of C. perfringens
in foods and stool using selective media, and the detection of high levels
(counts of  105 cfu/g) of C. perfringens in the food and faeces of patients.

31. Page | 31
This is followed by demonstration of a common C. perfringens serotype.
(b).detection of enterotoxin in food and patient stool using serological
methods e.g. ELISA, RPLA etc.
Prevention
1. Proper cooking of food and eating freshly prepared foods.
2. Thorough washing and sanitation of containers
3. Hygiene handling of cooked food
4. Fast cooling of cooked food and storage in refrigerator in small quantities.
5. Proper re-heating of cold cooked food before consumption
6. Storage of leftovers or unused foods in freezers
CLOSTRIDIUM BOTULINUM FOODBORNE INTOXICATION
Clostridium botulinum food borne intoxication (botulism) is a type of food
poisoning caused by consumption of enterotoxins produced by strains of
Clostridium botulinum. C. botulinum is an obligate, spore-forming anaerobe,
Gram positive bacilli with terminal to sub-terminal spores. The strains are
divided into proteolytic and non-proteolytic according to whether they
hydrolyze proteins or not. The intoxication is caused by botulinal toxins A, B,
E, F and G, produced by C. Botulinum type A, B, E, F and G, while the
organism grows in food. Spores of C. botulinum type A can survive
temperatures of 120oC.
Growth characteristics
Proteolytic strains grow at a temperature range between 10-50oC, while
non-proteolytic strains grow at 3.3-45oC (optimum 35-37oC). Toxin
production occurs at growth temperature range between 25-30oC. Both
strains grow at minimum pH of 4.5. Toxin production occurs in food at an
optimum temperature of 25-37oC, pH  4.5, and water activity (aw)  0.93.
No toxin production occurs at aw < 0.93 and pH < 4.5. Eating habits e.g.
consumption of home canned and fermented products increase the risk of
outbreaks.
Reservoirs
Clostridium botulinum is a saprophyte found in the soil, water, and
decomposing manure. The organism is a natural inhabitant of the surface

32. Page | 32
layers of the soil and thus may easily contaminate fruit vegetables or other
cultivated produce. Spores have been found in animal tissues and
occasionally in the intestines of pig, ox and horse, which feed on
contaminated feed. Where meat foods are infected, soil contamination is the
most likely cause.
Characteristics of botulinal toxins
Botulinal toxins are highly toxic, heat sensitive (inactivated by heating at
80oC for 10 min) neurotoxins,. They are unstable at alkaline pH (but stable
below pH 7.0). They are resistant to pepsin and acidic environment.
Botulinus toxin is one of the most powerful poisons known. The calculated
lethal dose for an adult man is in the region of 10 g. The toxins can resist
the action of the gastric and intestinal juices.
Types of foods implicated
Foods associated with anaerobic conditions and soil contamination such as
home-canned foodstuffs, or hams and bacon stacked without air access, are
particularly liable to be infected. Home made fermented foods have been
incriminated, together with smoked, pickled and canned foods that are
allowed to stand and then eaten without adequate cooking.
Botulism is associated in practically all outbreaks with consumption of
smoked, pickled or canned foods, not because these foods promote toxicity
of the organism, but because they are foods most likely to be eaten straight
out of the can, and without a further heating to destroy the any available
toxin. Uncooked fresh foods are safe because they are eaten before the toxin
has had time to develop, while, if foods are cooked, the toxin is destroyed.
In most, but not all cases of botulism, the preserved foods have been
noticeably spoiled, showing evidence of gas formation, breakdown and
rancidity.
For canned foods contaminated with C. botulinum, the can is often blown,
and the food is soft and disintegrated, with an odor resembling butter or
cheese, although meat foods so contaminated often show evidence of
spoilage. Preserved foods such as beets, olives, spinach or string beans may
contain potent C. botulinum toxin without the presence of obvious change in
the appearance, taste or odor. Infant botulism occurs due to consumption of
C. botulinum spores in honey and syrup. The spores germinate in intestines
where they produce the potent toxin.

33. Page | 33
Role of preservatives in food
Preservatives such as salts especially nitrates/nitrites reduce chances of
growth and inhibit toxin production. The danger of botulism has been the
deciding factor in the formulation of food processing techniques, especially in
meat canning.
Mode of transmission
1. Contamination due to improper handling.
2. Insufficient heating to destroy spores.
3. Spores present in animal tissues e.g. fish.
Symptoms of the disease in man
Symptoms of botulism are produced by the action of a powerful neurotoxin
which possesses an affinity for nerve tissue. The period of incubation in man
is usually under 24 hrs, but may be longer(range 12-72 hrs). Symptoms
include nausea, vomiting, fatigue, dizziness, headache, dryness of skin,
mouth and throat, constipation, lack of fever, nerve paralysis and great
muscular weakness, double vision, respiratory failure and death. Duration of
illness is 1-10 days and mortality is high up to 60-100% of affected persons.
The earlier the appearance of symptoms, the higher the mortality rate.
Diagnosis
1. Use of clinical symptoms
2. Isolation of C. botulinium strain from food.
3. Demonstration of botulinal toxins in suspected food, patient serum, vomit
and stool using the following methods:
i). Biological methods e.g. mouse challenge and protection test
ii). Serological methods e.g. diffusion, electrophoresis, ELISA etc.
Prevention
The following measures can help in the prevention of outbreaks:
1. Ensuring proper manufacturing practices.
2. Increasing salt concentration of food to 10% or more.

34. Page | 34
3. Preserved foods possessing rancid or other odors should be rejected.
4. Ensure proper hygiene of the cans, their transport, handling, storage
5. Proper heating of food before consumption to destroy heat labile
enterotoxins. Food should be heated to  80oC and temperature maintained
for at least 10 min before eating.
6. Ensuring fast cooling of food. This will ensure that spores that may be
remaining do not germinate in food grow and produce toxin.
FUNGAL FOOD BORNE INTOXICATIONS
Fungal intoxications are caused by consumption of metabolites produced
by fungi, when growing in food. These metabolites are called mycotoxins.
Grains, oilseeds, fruits and vegetables are mostly involved if they are stored
at high humidity (> 0.75) or if they are not properly dried before storage.
Poor dry storage practices of grains and other foods leads to mould growth
and production of mycotoxins. Of significance to public health is
aflatoxicosis.
Aflatoxicosis is caused by aflatoxins produced by the fungi Aspergillus
flavus. Four types of aflatoxins have been described i.e. aflatoxin B1, B2, G1
and G2. In addition, M1 and M2 metabolites of B1 may be secreted in milk.
Effects of aflatoxins
1.When consumed in large doses, they are lethal in causing acute
hemorrhagic syndromes
2. Sub-lethal doses cause histotoxic changes
3. Long term consumption of small doses cause liver tumors as these are
potent carcinogens, especially aflatoxin B1.
Prevention of aflatoxicosis
1. Proper drying and storage of grains and other affected foods
2. Quality control of potentially hazardous foods to ensure that they do not
contain above the allowable limits of 20 ppb before consumption by use of
appropriate analytical tests.

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3. use of fungicides as seed dressings to protect stored cereals and other
foods like pulses and potatoes.
CHEMICAL FOODBORNE INTOXICATION
This is a type of food borne intoxication arising from consumption of food
containing poisonous chemicals, which may be intentionally or
unintentionally added to foods as a result of producing, processing,
transporting or storage. A number of substances can enter the food chain
from the environment and through their use as growth promoters or
veterinary therapeutics giving rise to chemical residues. Radionuclide may
contaminate the environment, milk, meat and food crops as it happened in
the Chenobyl nuclear disaster of 1986 in U.S.S.R which contaminated large
areas of Russia and some parts of Europe. Chemical food borne intoxication
involve the following substances:
1. Heavy metals e.g. antimony, mercury, arsenic, flouride, lead, cadmium,
cyanide etc.
2. Pesticides and insecticides e.g. DDT, BHC Organochlorines and
organophosphates.
3. Herbicides
4. Fungicides e.g. organomercurials
5. Preservatives e.g nitrites, nicotinate, etc
6. Antibiotics e.g Pencillin, tetracyclines, chloramphenicol
7. Radionuclides e.g cesium, strontium, radium, molybdenum, barium,
ruthenium, lanthanum, iodine isotopes etc.
These compound usually enter foods through:-
(a) Accidental contamination by (i). Heavy metals, (ii) Pesticides, and (iii)
radionuclides.
(b) Intentional addition e.g preservatives such as nitrite and sodium
nicotinate for color preservation and fungicides used as dressing during
storage.

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(c) Leaching from containers e.g zinc galvanized containers by acid foods,
copper surfaces, lead pipes, asbestos roofs.
(d) Presence of such chemicals in food as a result of use of their use in
animal and crop husbandry
(e). Maliciously added to cause harm (is rare).
Clinical signs and symptoms
Food borne intoxication due to poisonous chemicals exhibit a very short
incubation period, usually a few minutes to a few hours, with an average of
one hour. Symptoms are mainly due to effect on gastrointestinal tract and
central nervous system and include nausea, headache, convulsions,
gastrointestinal irritation, abdominal cramps, vomiting and diarrhea, pallor,
cyanosis, blurred vision, sweating, flushing of skin and collapse. Other signs
may be due to effects on circulatory system.
Symptoms of radionuclide toxicity depend on dose, time and organ affected.
Toxicity causes (i) genetic effects including lethal mutations and reduction of
the lifespan. (ii) somatic effects such as radiation sickness, leukemia,
tumors, malformations and other physical ailments.
Measures to prevent spread by food
1. Discontinue use of utensils or containers that are able to leach chemicals
such as antimony, cadmium, zinc, copper, etc.
2. Use of coloured pesticides and proper storage of the same.
3. Wash leafy vegetables and prevent contamination by protecting food and
utensils when using insecticides.
4. Prevent acid foods or carbonated liquids from contact with exposed
copper.
5. Discontinue use of cyanide silver polishes or exercise care in their use.
6. Discontinue use in food establishments of pesticides containing fluorides.
7. Prevent misuse or avoid use of dangerous additives
8. Education of persons preparing food (e.g. possibility of Zn poisoning).

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9. Ensure that withdrawal periods are observed after use of these chemicals
in animal and crop husbandry.
.

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MEAT BORNE ZOONOSES
Zoonoses are diseases and infections that are naturally transmitted from animals to
humans or from human beings to animals.
Anthrax
This is an acute bacterial infection caused by Bacillus anthracis that occurs frequently in
herbivorous animals. However, all warm blooded animals are susceptible to varying
degrees. Grazing animals are infected when foraging in areas contaminated with spores
of B. anthracis. The disease is typified by sudden death in domestic and wild animals.
Terminally ill animals bleed from the nose, mouth, and bowel thus contaminating soil or
watering place with the bacteria. The disease has a worldwide distribution. Human
infection occurs as a result of contact with animals that have anthrax through:
1. Butchering and skinning
2. Consumption of contaminated meat
3. Contact with contaminated hides ans skins, goats hair, wool or bones.
The following three forms of the disease occur in humans.
i). Cutaneous form
This is characterized by lesions found on exposed areas of skin that results from
introduction of spores into the skin through cuts, abrasions, fly bites. In most (80-90%)
cases, the lesions heal spontaneously. In untreated cases 10 to 20% progress to a
systemic infection characterized by high fever and death
ii). Pulmonary form
It results from inhalation of B. anthracis spores which are deposited directly into lungs.
The spores then spread to regional lymph nodes and cause generalized infection is
followed by and rapid death.
iii). Gastrointestinal form
This form results from consumption of contaminated meat. The symptoms are variable
and include: fever, nausea, vomiting, abdominal pain, bloody diarrhea, sometimes
rapidly development of fluid in the belly, with massive diarrhea in some cases and death
if untreated.
In meat inspection an anthrax case may be seen as:
a) Dead animals on transport vehicle on arrival at slaughterhouse
b) Dead animals in the lairages

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In both cases, there is blood oozing from the natural openings. Great care should be
taken when suspect material is handled.
The diagnosis is established by:
a) Direct microscopy of blood smear or organ samples (always done when suspects
are found)
b) Cultivation – characteristic microscopic appearance
c) Inoculation of material intra-peritoneally into mice – will die within 3 days. Sub-
culturing is then done from dead mice.
d) Serology (Ascolis thermo-precipitation test)
When an anthrax case is diagnosed, the carcass and other materials should be burned
or deep buried with lime on top. If material is to be transported, care should be taken
not to contaminate during transport (head of the animal covered with plastic bag, etc).
The contaminated areas should be thoroughly disinfected, and people who have been
in touch with infected material should be given doses of penicillin and vaccinated.
Staining for presence of capsules
1) Blood smear fixed, either by good heat fixation or with 1% mercury chloride.
2) Staining with polychromethylene blue shall act on the bacteria for 3-5 minutes, then
washed off and dried.
3) Microscopy with oil immersion. Look for blue bacteria with purple capsule.
Control measures
1) In slaughterhouse – all operations must be halted until diagnosis is confirmed. If
positive, all exposed carcasses must be destroyed and premises carefully
disinfected with caustic soda within 8 hours.
2) Deep (6 feet) burial or incineration for anthrax unopened cadavers. The grass
and soil in the vicinity of the cadaver is buried and area covered with quicklime.
3) Affected herds should be quarantined for 2 weeks from last case with no animal
or product allowed out of the farm or slaughterhouse.
4) Environmental and personal hygiene where animals products are handled
5) Prompt treatment of persons in contact with infected carcasses
Tuberculosis
Tuberculosis is a communicable disease of worldwide occurrence that presents itself
with a respiratory problem in all animals including man. The disease is caused by

40. Page | 40
organisms of the genus Mycobacterium. The main species are: M. Tuberculosis (human
TB), M. bovis (Bovine TB) which is zoonotic .), M. microti, M. avium Two major types of
tuberculosis syndromes are recognized: (1). Pulmonary tuberculosis (PTB) and (2)
Extra-pulmonary TB. Epidemiology of human TB shows increasing incidence in both
developing and developed world. This is due to Increasing population density in poor
areas and HIV pandemic. TB in cattle leads to infection of various body organs including
the mammary glands which makes infected milking cows to shed mycobateria
organisms in milk urine, uterine secretions, faeces and sputum. The figure 3.1 below
shows multiple tubercles on the ribs and diaphgram of a bovine infected with
tuberculosis.
Figure 3.1: A. Multiple tubercles attached to the diaphgram of a bovine carcass; B.
Multiple tubercles attached onto the ribs of a bovine carcass.
A B

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Figure: Tuberculous pleuritis in cattle
Transmission of zoonotic tuberculosis. Humans can acquire the disease from animal
sources through:
1. Ingestion of raw and undercooked meat from infected animals
2. Inhalation of contaminated aerosols
3. Direct contact with materials contaminated with nose and mouth secretions of
infected animals
4. Postmortem examination of infected carcasses.
Human infection is characterized by chronic cough, weight loss, fatigue, profuse night
sweating, fever, general discomfort, intermittent fever, paleness, swelling of glands and
eventual death.
Diagnosis
Mycobacteria (M. tuberculosis, M. bovis, M. avium, leprae, M. paratuberculosis and
others) are acid fast. They are able to resist the aniline dyes that are commonly used
for staining of bacteria, because these bacteria contain large (74%) amounts of lipid
material that hinder the absorption of these dyes. In order to stain these bacteria, the
bacteria are exposed to the stain for long periods (hours) or heating could be used.
When they have accepted the stain, they are not easily destained, and even strong
mineral acids will not decolorize the bacteria giving the description “acid-fast bacilli”.

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PROCEDURE
1) Fix smear by heat
2) Flood the slide with carbolfuschin using a piece of filter paper to hinder
evaporation, heat over bunsen flame (not to hard). Repeat at intervals and
stain for 10 mins. Do not let the slide dry, add more stain if necessary.
3) Wash in running tap water
4) Pour 20% sulphuric acid on the preparation and leave for 15-30 seconds.
Repeat till the preparation has got a weak reddish colour = 10 min.
5) Wash in running water.
6) Counterstain with malachite green (Solution of malachite green, 0.5% in
water, or of methylene blue 0.5%) for 1 min.
7) Wash in water
8) Blot dry and examine in microscope using oil immersion.
Control
 Proper meat inspection to identify, isolate and condemn TB cases
 Proper cooking of meat before consumption.
Brucellosis
An infection caused by bacteria of the genus Brucella. Four species of Brucella cause
infection in man. These are: Br. Melitensis (affecting mainly sheep and goats), Br.
Abortus (affecting mainly cattle), and Animals acquire the disease by sexual
transmission or ingestion of contaminated feed..
The disease causes abortion in cattle, sheep and camel. Goats only develop lameness
and mastitis. Brucella organisms are continuously shed in milk of lactating animals,
uterine exudates, aborted fetuses and fetal membranes of aborting animals.
Disease in Human
The disease is occupational affecting human beings who work closely with animals such
as butchers, herders, meat inspectors, and veterinarians. Other people may be infected
through:
1. Consumption of raw or improperly pasteurized or raw milk from infected animals.

43. Page | 43
2. Consumption of dairy products such as cheese, and butter prepared from raw
milk from infected animals.
3. Contact with infected animals, aborted fetuses, or infected tissues and products
of abortion e.g. when assisting in difficulty calving.
4. Consumption of contaminated water and vegetables
Disease symptoms
The following three forms of the disease are recognized:
1) acute
2) localized and
3) chronic
Acute form. This is characterized by slow onset after an incubation period of between 7
and 21 days. Symptoms include headaches, fever alternating with chills (cold), severe
night sweats, joint pains, general muscular pain, aches, general body weakness, chest
pains and sometimes a non-productive cough. The signs of brucellosis can be confused
with those diseases that present with flu-like symptoms such as malaria, typhoid fever,
rheumatic fever, influenza and psittacosis. Most patients are anorexic and loose weight
Localized form. This may occur at any anatomic location. The most common signs
are: osteomyelitis, splenic abscess, gastroenteritis, pneumonia and endocarditis.
Endocarditis is the most common cause of mortality in brucellosis patients
Chronic form. The illness persists for more than 1 year following onset of brucellosis
symptoms with varied and mixed manifestations including joint pains, undulating fever
and night sweats among other symptoms..
Control in animals
1. Slaughtering of serologically positive animals and vaccination of young ones
2. Indiscriminate vaccination of all the animals in a region or country
3. Implemention of effective quarantine to restrict animal movement during
outbreaks.
4. Hygienic disposal of aborted fetuses, placentae and uterine materials thereafter
disinfection of contaminated areas.
5. All animals introduced in a farm should be isolated and tested before joining the
rest of the herd.

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Prevention of human infection
1. Employees of the meat industry should wear protective clothing, treat and dress
all wounds on hands and arms.
2. Ensure proper dressing and disinifection of wounds especially those on the
hands that may come into contact with brucella organisms.
3. Proper cooking of meat before consumption.
Rift valley fever
RVF is a viral disease transmitted by mosquitoes. Mosquitoes are numerous during
heavy rains and they transmit the infection among animals by biting. The disease has
been confirmed in at least 24 African countries.
Transmission: Principal mode of transmission among animals is by mosquito bites.
Human beings gets infected when they come into contact with infected animals
during: slaughter, necropsy, food preparation and laboratory activities.
Disease in animals
- Disease has a short incubation period of 20-72hrs.
- There is 95% mortality in newborns
- Abortion In pregnant ewes is common in sheep,
- In cattle there is abortion, fever, anorexia, profuse salivation, abdominal pains
and diarrhea. However, mortality in cattle is low.
Control
- In animals vaccination is the method of choice using inactivated vaccines
- Vaccination of pregnant females can cause abortion
- Should not be used in newborns or areas free of infection -could turn virulent
- Precautions in handling of sick/dead animals - use of protective clothing.
- Precautions in laboratories where work on virus is carried out.
- Care when conducting postmortem especially during outbreaks
Echinococcosis/hydatidosis
Echinococcosis/hydatidosis is a parasitic disease caused by Echnococcus granulosus.
The disease occurs worldwide.

45. Page | 45
Life cycle: E. granulosus adult worm is found in the domestic dog and a wide range of
wild carnivores who are the definitive hosts. The definitive hosts will pass out in their
faeces eggs which are highly resistant to environmental conditions and which remain
infective for a long time in favourable climates. However, desiccation and high
temperatures affect the longevity of the eggs. The intermediate hosts which include
domestic herbivores and a wide range of herbivores acquire infection by ingesting
infective eggs. In the stomach and small intestines enzymes act on the eggs to release
oncospheres from embryophored eggs.
Disease in Human: The clinical signs depend on the site and size of the cyst.
Abdominal swelling due to presence of cysts in the lungs, liver or any abdominal organ,
swelling of one or both eyes when the cysts affects the eyes.
Diagnosis: Done through meat inspection. Hydatid cysts are found in various internal
organs including lungs, liver and peritoneal cavity.
Hydatid cyst in the lung

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Hydatid cyst in liver
Figure: Hhydatid cyst in the lung and liver of cattle. Such cysts are also found in the
lungs and livers of camel, sheep and goats.
Preventive measures
 Creation of public awareness through health education,
 Control of livestock slaughter by centralization of slaughtering,
 Efficient meat inspection and proper disposal of offal
 Dog treatment and control and
 Periodic diagnostic testing of dogs.
 Dog owners to control movement of their dogs.
 Regular deworming of dogs with suitable anthelmintics e.g praziquantel
 Reduction of dog numbers by elimination of stray and surplus dogs
Tapeworm infection (taeniasis)
Taeniasis is a parasitic disease caused by Taenia Saginata. Adult tapeworm is found in
human intestines from where eggs are shed to contaminate the environment such as
pastures. Cattle come into contact with tapeworm eggs while grazing in these pastures
and consume them with the pasture. The eggs develop into larvae, which then lodge in
their muscles where they occur as cysts. When man ingests cattle meat having cysts,
larvae are liberated from the cyst which then develop into adult worms.

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The lifecycle of Taenia. saginata

48. Page | 48
Prevention and control measures
1. Proper meat inspection to identify infected meat
2. Proper disposal of sewage and other wastes to contamination of pastures
3. Construction and use of toilets/latrines
4. Regular de-worming of humans and cattle
5. Adequate cooking/roasting of meat to destroy cysts in the meat
Test for viability of cysticerci in meat
Low temperature treatment is in many countries enforced by law to render moderately
parasite infested meat safe for consumption. Best known is the low temperature
treatment (freezing at –10°C for 10 days) to kill Cysticercus bovis. To make sure that
the required treatment is performed in a satisfactory manner, the viability of parasites
present should be controlled.

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Cysticercus bovis ( C. bovis) in beef muscle
a) Carefully remove cysts from meat to petri dish
b) Add a saline solution of 30% ox bile, or a saline solution of 5% sodium taurocholate
c) Incubate at 37°C for 1-2 hours, cysts are considered dead of the scolex. If no
evagination within 2 hours cysts are considered dead.
Ruptured and fluid filled cysticerci

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Cysticerci in tongue muscle

51. Page | 51
HAZARD ANALYSIS AND CRITICAL CONTROL POINT (HACCP) SYSTEM
The HACCP system is a systematic approach that identifies and evaluates food safety
hazards and put in place measures and/or procedures that will either prevent, eliminate,
control or reduce the food safety hazard to acceptable levels. HACCP is the foremost
means of assuring food safety throughout the food chain, from primary production to
final consumption, particularly when used in combination with the pre-requisite
programs for GHP.
For efficient and effective application of the HACCP system, the establishment operator
must apply the seven HACCP principles namely: (1) Conduct a hazard analysis; (2)
Identify critical control points (CCPs); (3) Establish critical limits for each CCP; (4)
Establish CCP monitoring requirements; (5) Establish corrective actions; (6) Establish
procedures for ensuring the HACCP system is working as intended, and (7) Establish
record keeping procedures.
Principles of HACCP
There are seven principles of HACCP on which a food safety management system is
based. A food safety management system based on the principles of HACCP will
enable hazards to be identified and controlled before they threaten the safety of food
and your customers.
Identify the hazards and determine risks: Involves identification of hazard and
determination of the severity of the hazard associated risks. Hazard usually means the
contamination, growth or survival of microorganisms related to food safety or spoilage, a
dangerous chemical contaminant or foreign objects (glass or metal fragments). Risk is
an estimate of hoe likely it is that the hazard will occur. The risks are associated with
growing, harvesting, processing, distribution, preparing and /or using a raw material or
food product. Look at each step (e.g. purchasing, delivery, storage, preparation,
cooking, chilling etc.) in your operation and identify what can go wrong e.g. Salmonella
in a chicken product (biological hazard), detergent in a chicken product (chemical
hazard) or a piece of glass in a salad (physical hazard).
Determine the critical control points (CCPs) required to control hazard: A critical
control point is a location, practice, procedure or process which can be used to minimize
or prevent unacceptable contamination, survival or growth of food borne pathogens or
spoilage organisms or introduction of unwanted chemicals or foreign objects. Identify
the points in an operation that ensures control of the hazards e.g. Cooking burgers to a
minimum of 70°C for 2 minutes will kill E. coli O157 and other pathogens.

52. Page | 52
Establish critical limit(s): Determine and set critical limits to enable you to identify
when a CCP is out of control e.g. the temperature at the centre of a beef product
following cooking must reach a minimum 70°C for 2 minutes.
Establish a system to monitor control of the CCP: When CCPs and critical limits
have been identified it is important to have a way to monitor and record what is
happening at each CCP. Monitoring procedure enable one to determine that each CCP
is under control. Monitoring systems must be able to effectively determine if a CCP is
under control. Typically monitoring will involve measuring parameters such as
temperature and time. However, how you monitor and how often will depend on the size
and nature of your business. Monitoring should in all cases be simple, clear and easy to
use e.g. recording the final cooking temperature and time for a cooked chicken product.
Establish the corrective action to be taken when monitoring indicates that a
particular CCP is not under control: When monitoring indicates that a CCP is not
under control, corrective action must be taken e.g. the temperature of the food in a
refrigerator rises to 10°C due to a technical fault. Discard the food and repair the
refrigerator using the manufacturer’s instructions to ensure the correct temperature of
5°C is achieved.
Establish procedures for verification to confirm the HACCP system is working
effectively: Review and correct the system periodically and whenever you make
changes to your operation e.g. microbiological analysis of a chicken product to verify
that it is free of Salmonella bacteria before and after cooking. Verification is a procedure
that provides the guarantee to any customer and to the public that the product in
question is of the quality the producer is claiming, since it has bee based on specific
GAP, GMP and HACCP principles that are documented. If verification is performed by
independent agencies, bodies or companies that are accredited by nationally or
internationally approved quality assurance organizations, the procedure becomes a
certification procedure.
Establish documentation of all procedures and records appropriate to these
principles and their application: For the successful implementation of HACCP,
appropriate documentation and records must be kept and be readily available. It is
unrealistic to operate HACCP or to demonstrate compliance with the current legislation
without providing evidence such as written records. As with HACCP itself, the
complexity of the record keeping will very much depend on the nature and complexity of
the business. The aim should be to ensure control is maintained without generating
excessive paperwork e.g. cooking temperatures, delivery or cleaning records.

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Prerequisites for HACCP (Safety Support Measures)
Before implementing HACCP, food businesses must already be operating to standards
of good hygienic practice by having in place appropriate prerequisites These
prerequisites include where appropriate:
1. Appropriate design, layout and construction of slaughter and processing facilities
2. Appropriate equipment and layout
3. Establishment of routine cleaning and sanitation procedures
4. Personnel hygiene and training
5. Established pest control strategies
6. Support services (compressed air, ice, steam, ventilation, water etc)
7. Appropriate meat storage and transport facilities
8. Established waste management systems
9. Zoning (physical separation of activities to prevent potential food contamination)
The Benefits of HACCP to Food Businesses
HACCP provides businesses with a cost effective system for control of food safety from
ingredients through production, storage and distribution to sale and service of the final
consumer. The preventive approach of HACCP not only improves food safety
management but also complements other quality management systems. The main
benefits of HACCP are:
1. Saves business money in the long run
2. Avoids poisoning of meat customers
3. Meat safety standards are increased.

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FOOD SPOILAGE AND PRESERVATION
Food spoilage can be defined as food that has been damaged or injured, hence no longer
good for human use. Food must be considered spoiled if it is contaminated with pathogenic
microorganisms or various poisonous agents, like pesticides, heavy metals etc. In most cases
there does not need to be an evident sign of spoilage, the food might look normal and only after
eating if ,or by careful bacteriological and toxicological investigation, one is able to realize the
defect. Food decay or decomposition is implied when the term spoiled is used. The following are
the causes of food spoilage:
(a). Growth and activity of microorganisms. Bacteria, yeasts and molds are the microorganisms
that cause food spoilage. They produce various enzymes that decompose the various
constituents of food.
(b). Enzyme activity. Action of enzymes found inherently in plant or animal tissues start the
decomposition of various food components after death of plant or animal. These enzymes if
present in the food must be inactivated in order to preserve the food.
(c). Chemical reactions. These are reactions that are not catalysed by enzymes. Such reactions
include oxidation of fat, maillard reaction in milk between proteins and lactose which result in
unpleasant flavour and brown discoloration of food,a nd reactions between the food product and
packaging materials.
(d). Vermin. Vermin includes weevils,cocroaches, ants, rats, mice, birds, larval stages of some
insects. Vermin are important due to: (i). Aesthetic aspect of their presence, (ii) Possible
transmision of pathogenic agents, (iii). Consumption of food.
(e). Physical changes. These include those changes caused by freezing, burning, drying,
pressure etc.
Microbial spoilage of food
Bacteria, yeasts and molds are the major causes of food spoilage. They produce various
enzymes that decompose the various constituents of food. Molds are the major causes of
spoilage of foods with reduced water activity such as dry cereals and cereal product, while
bacteria spoil foods with relatively high water activity such as milk and products.
Factors that affect growth of microorganisms
pH. Most microorganisms grow best at pH near to neutrality (6.0-7.5). Few
microorganisms grow below pH 4.5. Bacteria grow in a narrow pH range between 4.4
and 9.0, while moulds and yeast grow in a wide pH range between 1.5 and 11.0.

55. Page | 55
Moisture content. The effect of moisture is in terms of water activity, which is the
vapor pressure of food substance to that of water at the same temperature. Most foods
have water activity of 0.99. Microorganisms require different water activity values. Most
spoilage bacteria need 0.91, yeast - 0.88, moulds - 0.80, halophiles - 0.75, xerophiles-
0.65 and osmophiles - 0.65 as the minimum water activity.
Type and level of nutrients in the food: Microorganisms require water, energy,
nitrogen, vitamins and minerals for growth. Different food have specific types of
nutrients required by microorganisms hence are susceptible to spoilage. Microorganism
grow well in high protein foods such as meat and milk.
Temperature: Microorganisms grow in a wide range of temperatures but most grow at
between 20-40oC. Microorganisms can be classified according to their temperature
requirements into: (i) Psychrophiles - grow between 20-30oC, (ii) mesophiles -grow
between 30-45oC, and (iii) thermophiles - grow at 55-65oC. Meat should be kept below
4oC to arrest rapid spoilage. Frozen meat have extended shelf-life of up to one year
Relative humidity: Relative humidity is related to water activity. Foods with low water
activity placed in high humid environment take up water and get spoiled easily. Meat
has high relative humidity, hence can easily be spoiled. Drying meat will reduce relative
humidity and this will arrest the rate of spoilage.
Oxidation-reduction potential: Various microorganisms require either increased
oxygen tension (are aerobic) or low oxygen tension (are anaerobic) for growth. Meat
kept under aerobic conditions are spoiled by aerobic and facultative anaerobes, while
canned meat is spoiled by anaerobic microorganisms including Clostridium botulinum
and Clostridim perfringens.
Meat preservation
Meat preservation is where chemical or physical agents prevent biological deterioration
of meat. The various characteristics that affect growth of microorganisms in meat can
be used to prevent growth of these microorganisms in food by inhibiting growth or killing
(destruction) of the organisms.
Inhibition of microorganisms can be through reduction of water activity (by salting and/or
drying), reduction of pH (achieved by fermentation or addition of organic acids), use of
preservatives, low temperatures storage (chilling or freezing) and smoking. Preservation
by smoking occurs through impregnation of chemicals such as phenols on the surface
of microbes in food, and drying effect due to associated heat.
Destruction of microorganism in food can be through heat treatment, irradiation and
uses of ethylene oxide or propylene oxide gases. Two methods of heat treatment are
commonly used in food preservation, i.e. pasteurization and sterilization.

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Pasteurization: is the process of heat treatment of a food substance at specific
temperatures and times aimed at destroying all pathogenic microorganisms without
affecting the nutritive qualities of such food. Three methods are used in pasteurization:
(a). Low temperature long time (63oC for 30 min),
(b). High temperature short time (72oC for 15 seconds),
(c).Flash method (80oC for 1-2 seconds).
Sterilization. is the use of physical or chemical means to destroy all the
microorganisms that are present in the food. Sterilization can be achieved by (i) heating
at high temperature and pressure, e.g. 100oC-140oC, (ii) irradiation, iii) filtration, using
0.22-0.45µm pore size filter membranes (iv) ethylene oxide or propylene oxide gases.
Irradiation. is a means of food preservation that use ionizing or electromagnetic
radiations to kill microorganisms in the food. The types of radiation which are useful for
this purpose are the gamma rays produced by radioisotopes cesium-137 and cobalt-60
and electron beams generated by electron accelerators. These radioactive radiation kill
bacteria either directly by hitting the bacterial cells or indirectly by creating reactive
compounds in the medium and in the bacterial cell. Application of ionizing irradiation to
foods is to obtain sterilization, extend the shelf-life, and eliminate pathogenic
microorganisms. High amount of irradiation in the food is however dangerous to human
health.
Preservatives. are widely used in the preservation of various food products including
soft drinks, fruit juices and other food products. These preservatives inhibit the
proliferation of microorganisms that might be present in food product, thus preventing
spoilage.
Food processing
Food processing is treating food in such a way as to make it safe for consumption,
increase their shelf-life (preservation) and make various value added products.
The most common method used in food processing is heat treatment, commonly
referred to as cooking by housewives and catering personnel. At industrial level, it is
referred to either pasteurization or sterilization. However In practice, a combination of
various preservation methods are used in the food processing.
Food storage conditions

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Refrigeration (storage of food product at 0C) and freezing (storage at -20C) inhibit
the multiplication of spoilage microorganisms by providing low temperature that are not
favorable for growth and multiplication of these microorganisms. At refrigeration
temperatures, most of food spoilage microorganisms are inhibited, but some of them
(called pyschrotrophs) can grow at that temperature to cause spoilage and sometimes
food poisoning. However, the rate of multiplication is low and it can take up to 4-5 days
before spoilage can take place. Therefore, foodstuffs such as meat and milk should not
be kept in the refrigerator for more than 4 days because such a food will get spoiled by
pyschrotrophs.
Freezing (storage temperatures below 0oC) on the other hand totally suspends any
growth and multiplication of all microorganisms present in the food. So long as food is
kept at freezing temperatures, spoilage by microorganisms will not take place even after
storage for one year. However, some food products may get spoiled as a result of
chemical activity. For example, poultry if stored at freezing temperatures for over 6
months, rancidity starts to develop. It will be of interest to keep any perishable food stuff
in the freezer if it has to stay there for over 4 days.
MEAT TRACEABILITY
Traceability is tracking the movement of identifiable products through the marketing
chain. Traceability can be used to convey information about a product, such as what it
contains, how it was produced and every place it has been. Meat traceability is ability to
follow the movement of a meat product through specified stage(s) of animal production,
slaughter, processing and distribution of meat products. An extensive form of
traceability is the ability to follow meat products forward from their source animal (at
birth) through growth, slaughter, processing and distribution to the point of sale or
consumption, (or backward from the consumer to the source animal). Animal
identification is one component of meat traceability. It is the marking of individual
animals or group of animals so that they can be tracked from place of birth/origin to
slaughter. Producers keep records on identities of each animal. A well designed
traceability system provides accurate data on the origin, sex, age, breed, movements,
and records of treatments that an animal received.
Functions of animal identification and traceability
Commercial production and marketing. Traceability can help to identify and exploit
desirable production characteristics e.g. better yields, fast growth etc. Traceability helps
to coordinate shipments, manage inventories and monitor customer behaviour. Some

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customers prefer milk, meat or eggs from animals raised according to specified organic,
humane treatment or environmental standards. Traceability can help firms separate and
keep records on these unique products to verify production methods. Improved
traceability is viewed as important for maintaining foreign market access as animal
identification systems are being used as technical barriers to trade. Functioning
traceability systems are often a pre-condition for meat producing countries to enter
export markets. Universal bar codes on processed food and meat products are widely
used for tracking.
Animal health. Traceability systems are important tools to prevent the spread of animal
diseases and to enhance biosecurity in general. Animal identification can help track
down more quickly the source of diseases in flock/herds in order to determine origin of
the animals, cause of the disease, control and prevent their spread. When used in
animal health programs, Animal identification and tracing systems are likely to have
both commercial and regulatory functions.
Food safety. In slaughterhouses, meat traceability requires the clear identification of
the live animals through to meat products. A rigorous animal identification and trace
back can prevent potentially serious food safety problems. Traceability system can
facilitate recalls of meat products, enhance process controls, testing and other science
based food safety measures. In addition, it will possibly facilitate identification of the
source of the disease, timely application of control measures to contain its spread, as
well as help authorities prevent future disease incidents. Improved traceability may
enable firms to limit their legal and financial liabilities.
Country of origin labelling. Traceability systems will facilitate country of origin
labeling that is required for animals or products exported to foreign countries.Meat
identification methods include:
 Paper/plastic Tags;
 Ink stamping;
 Bar code labels (Figure 3.1); and
 Microchip (Radio Frequency Identification Device (RFID)).
Figure 3.1: A bar code label

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Costs of the identification and traceability systems vary widely, depending on the
options applied and the level of detail required. An animal identification system will incur
a variety of costs incurred for acquisition of identification devices, data systems and
administration expenses.
MEAT HYGIENE PRACTICALS
STERILIZATION PROCEDURES IN THE LABORATORY
STERILIZATION OF EQUIPMENT
In most microbiological work it is essential that the equipment used is not only clean but
sterile. There are several methods of sterilization but not all can be used for all
purposes.
Heat is generally the most commonly used way of sterilizing equipment for
microbiological work. It has the big advantage that it does not leave any traces of
chemicals as some disinfectants do. We have, however, to take into account that the
heat sometimes can do harm to the equipment or media to be sterilized. We therefore
apply heat in different ways.
Flaming: Is used for sterilizing wire loops and other utensils for example iron, scalpels,
etc. which do not get damaged by this heavy treatment.
Moist heat: can be used by boiling water at 100C which will kill all Non-spore forming
bacteria within 30 min. Addition of 2% Na2Co3 increases the disinfecting power of
boiling water.
Steam or boiling under pressure: This is widely used for sterilization. At a temperature of
121C spores will be killed within 15 min. This is done in a laboratory autoclave or in a less
sophisticated pressure cooker. Some media are often sterilized by intermittent cooking on 3
consecutive days, called “tyndallisation”. By this method the media are steamed for 30 min on
the first day, incubated at room temperature and the treatment is repeated the second and the
third day. This treatment will not kill thermophilic bacterial and anaerobic spore formers unless
the incubation is carried out anaerobically.
Dry heat: Materials and utensils are often sterilized by dry heat. Glassware should be
placed in the oven when cold. The temperature is then raised to 160C for one hour and
allowed to cool slowly before removing the contents. Fans to provide for circulation in
the oven increase the efficiency of dry air sterilization.

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Filters: may also be used for sterilization, especially for fluids that cannot be heat
treated. Membrane filters are most popular in laboratory sterilization. Membranes are
composed of pure and biologically inert cellulose esters, and have porosities ranging from 0.22 -
1.0 microns. They retain all particles of bacteria and cells exceeding the spore size in
dimension. They are easy to handle and are thrown away after use, give little loss of material,
available in great variety of porosities, but are high in cost.
Chemical agents act as sterilizers through protein coagulation in bacteria cell,
disruption of the cell membrane, removal of sulfadryl groups and chemical antagonism.
Among the chemicals are:-
 Alcohols – between 50-70% act as protein coagulants.
 Phenols - (carbolic acid) is a coal tar derivative used at concentrations of 0.5-
2%. Protein coagulation, lysol; = phenol + soap.
 Salts - heavy metal salts (Hg, Cu, Ag) are all protein coagulants at high
concentrations. They are however harmful to human tissues.
 Oxidizing – agents will oxidize free sulfhydryl groups.
o Hydrogen peroxide (H2O2)
o Hypochlorite
o Chlorine
 Detergents – are surface active agents.
STERILIZATION OF ENVIRONMENT
This is absolutely essential in media preparation room. These include:-
a) Fumigation.
b) Cleaning of benches, e.g. using formalin, 70% alcohol
c) Air filtering – using lamina flow cabinets
d) UV lights/radiation.
MICROBIAL CULTIVATION
Aims of microbial cultivation
1. To study microorganisms’ characteristics

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2. To identify different microorganisms
Culture media
They are important in the cultivation of bacteria. A culture medium is a mixture of
nutrients either in liquid or solid form. Different bacteria have different growth
characteristics and therefore have different growth requirements. There is no single
media satisfactory for growth of all bacteria. Bacteria require a source of (i) energy, (ii)
carbon (iii) nitrogen (iv) sulfur (v). Phosphorus (vi) metallic elements e.g. mg, Na, Mn
Zn, Fe etc (vii) vitamins and other growth factors (viii) water. A culture medium should
be able to provide all the required nutrients.
Types of media
Media can be chemically defined or may be complex. Chemically defined media have
known compounds mixed together to produce a medium with a known composition e..
MacConkay medium (broth or Agar), bromothymol blue , nutrient agar etc. Complex
media contain unknown quantities and varieties of substances e.g. peptone water
(contain partially hydrolyzed proteins) cooked meat medium, blood agar, serum agar,
yeast extract (an extract of yeast cells). Complex media are known as broths. Broths
may be turned to solid by adding 1.5% agar. Solid media are referred to as agar. A
medium may be a broth or agar depending on the needs e.g. nutrients broth and
nutrient agar.
Agar is a polysaccharide material obtained from marine algae. Agar melts when heated
to near boiling point of water. When agar suspension is cooled to 43oC, it gels
(hardens to make a porous gel). This makes an important solidifying material in
laboratory preparations. Agar is not a nutrient for microorganisms but a solidifying
material.
Classification of media according to their function
Enrichment media: This has complex material incorporated into the chemically
defined media. The complex materials may be extracts from animals or plants or yeast
and these supply specific nutrients for growth of bacteria. examples of enriched media
are Blood Agar, serum agar, Dorset egg agar etc.
Selective media: These contain chemicals hat prevent some bacteria from frowing
without inhibiting the growth of others. Such chemicals added include

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(a) crystal violet which inhibits gram positive bacteria but allow growth of gram
negative ones
(b) Bile salts which inhibit bacteria that are not normally found in the gastrointestinal
tract.
(c) Selenite – when added to broth will inhibit Shigella.
Selective media are used to isolate a group of organization from a mixture of various
microorganisms.
Differential media: These contain substances that are visibly altered in a characteristic
manner during the growth of certain kinds of bacteria but not other e.g. some bacteria
cause hemolysis of blood agar, while others will not. Media containing blood is therefore
both be altered (color change) depending on the organisms growing in it. The
MacConkay agar contains lactose and bile salts and indicator.
Characterisation Media: These are a wide variety of media routinely used to observe
the type of growth and the chemical changes produced during bacterial growth.
Examples include
(i). Carbohydrate broth- These are used to distinguish fermenting and non-fermenting
bacteria.
(ii). Gelatin agar and Casein agar- are hydrolyzed by some bacteria and not others
(iii). Nitrate agar-reduced by some bacteria and not others
(iv). Simmon’s citrate – Used by some bacteria as source of carbon but not others.
Maintenance media: These are media used to keep bacteria viable during temporary
storage e.g. dorset egg agar, cooked meat medium, nutrient broth
Microbial growth requirements
Temperature: Bacteria require varying conditions of growth.
1. Psychrophiles grow best at temperature range < 20 oC
2. Mesophile grow best between 20-40oC. They incclde most pathogenic bacteria
3. Thermophiles – grow best between 45-650C. they are found in hot springs,
decomposing matter such as composite.
Gaseous requirements

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a. Aerobes: require free oxygen for growth e.g . Bacillus cereus
b. Anaerobes: grow only in the absence of oxygen.
c. Microaerophiles: require some free oxygen, butthey are inhibited by excess
oxygen e.g. Erysipelas insidiosa, lactobcilli
d. Facultative anaerobes: grow either in the presence or absence of oxygen.
Culture media inoculated with microorganisms need to be kept under appropriate
temperature and gaseous conditions of growth to enhance growth.
SPECIFIC MICROBIAL MEDIA
PLATE COUNT AGAR (PCA)
PCA is the medium recommended for the plate count of microorganisms in milk and
other dairy products and will provide a satisfactory total count from other foods e.g.
meat and meat products.
Preparation: Suspend 17.8g in 1 litre of distilled water. Dissolve by bringing to boil with
frequent stirring, mix and distribute into final containers. Sterilize by autoclaving at
121C for 15 minutes. Basically methods applying PCA consists of the preparation of
pour-plates.
BLOOD AGAR (BA)
BA is a non-selective general purpose medium widely employed for the growth of
pathogenic as well as non-pathogenic bacteria. With the blood added the medium is not
only enriched, but becomes suitable for the determination of typical hemolytic reactions
which are important diagnostic criteria for streptococci, staphylococci and other
organisms. 7% sterile blood is added to the sterile medium cooled to 45C-55C.
Preparation: Suspend 40g in 1litre of distilled water. Bring to the boil to dissolve
completely. Sterilize by autoclaving at 121C for 15 minutes.
VIOLET RED BILE AGAR (VRB)

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VRB is a selective medium for the detection and enumeration of coli-form organisms.
The medium has been used for the determination of coli-aerogenes coliform in water,
milk and other dairy products, dairy equipment and food products, etc. Coliform
organisms form dark red colonies which are 1 to 3 mm in diameter, usually surrounded
by a reddish zone caused by the acid production of these organisms.
Preparation: Suspend 38.5 g in 1 litre of distilled water. Bring to the boil to dissolve
completely. No further sterilization is necessary or desirable. Mix well before pouring.
SLANETZ AGAR (SA)
The SA was originally devised to detect and enumerate fecal streptococci by the technique of
membrane filtration, but it has also proved useful for direct plating. The medium contains is very
selective for streptococci due to sodium azide and maroon colonies may be accepted as fecal
streptococci.
Preparation: Suspend 42 grams in 1 litre of distilled water and bring to the boil to
dissolve the agar completely. Excessive heating must be avoided. Dispense into petri
dishes and allow the medium to solidify. The medium should not be re-melted, but
poured into plates enclosed in sealed containers, protected from light. Prepared plates
can be kept at 4C for up to 6 months.
IRON SULPHITE AGAR (IS)
The medium is used to detect thermophilic anaerobic organisms causing sulphide
spoilage in foods. The medium contains tryptone (10g), sodium sulphite (0.5g), iron
citrate (0.5g) and agar (12g).
Preparation: Suspend 23.0 g of powder in 1 litre of distilled water. Dispense 10 ml
amounts into test tubes and sterilize at 121oC for 15 minutes. Maintain tubes in a
molten state at 45oC in a water bath. Inoculate sample and mix to distribute organisms
in the medium a for deep-shape cultures, allow to settle and incubated at 35C for
thermophilic species. Clostridium spp. produces distinct black spherical growth in the
depth of the medium.
POTATO DEXTROSE AGAR (PDA)
PDA is a suitable medium for the isolation and count of yeast and molds occurring on
the surface of fresh meats, cured meats and sausage products.

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Preparation: Suspend 39 g in 1 litre of distilled water. Bring to the boil to dissolve
completely. Sterilize by autoclaving at 121C for 15 minutes. Mix well before pouring.
In order to suppress bacterial growth it is sometimes desirable to acidify the medium to
pH 3.5 by adding 1 ml. of Tartaric acid 10% to each 100 ml. of medium at 50C. Do not
re-heat after acidification. Make pour plates in the usual manner and incubate for 5 days
at 121C, and count the number of yeasts and mold colonies.
DE MAN, ROGOSA, SHARP AGAR (MRS)
The MRS formulation was developed to provide a medium which would support good growth of
lactobacilli in general, even those strains which showed poor growth in existing media. The
nutritional requirements for lactobacilli are complex and optimal growth is obtained in media
such as MRS which contain a fermentable substrate growth factors. The lactobacilli are
microaerophilic and generally require plates for aerobic cultivation on solid media. Submerged
or surface colonies may be compact or feathery, and are small opaque and white.
Preparation: Suspend 62 grams in 1 litre of distilled water. Boil to dissolve the medium
completely, mix and distribute into final containers bottles or flasks. Sterilize by
autoclaving at 121C for 15 minutes
COOKED MEAT MEDIUM (CMM)
CMM is a well established substrate for the cultivation and maintenance of anaerobic
and aerobic organisms. It is particularly suitable for the cultivation and differentiation of
Clostridium species, and stock cultures remain viable for several years. Clostridia may
be divided into two groups of their action upon the medium.
Saccharolytic organisms: these organisms rapidly produce acid and gas, but do not
digestion of the meat. The cultures may have a slightly sour smell, and the meat is often
reddened.
Proteolytic organisms – They ferments, decomposes and blackens the meat with the
formation of foul-smelling sulfur compounds. The protein is broken up into amino-acids,
and the small, white, feathery masses of tyrosine crystals may also be seen.
For the isolation of anaerobes, it is important to use solid media in conjunction with
CMM.

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Preparation:Add 55 g to 1 litre of distilled water and soak for 15 minutes. Distribute into
narrow containers (tubes) keeping the meat in suspension so that uniform layer is
obtained. Sterilize by autoclaving at 121C for 15 minutes.

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DEXTROSE BROTH (DB)
DB is a highly nutritious broth especially suitable for the initial isolation of microbes
which are fastidious, attenuated, or only present in small numbers. It is recommended
for use as a medium for sterility testing. It is particularly suitable for the initiation of
growth but it is important to subculture after growth take place since many bacteria will
not survive for long in the acid conditions produced by dextrose fermentation.
Add 23 g to 1 litre of distilled water. Mix well, distribute into final containers and sterilize
at 121C for 15 minutes
Testing sterility: Two plates/broth tubes of each batch are incubated at 37C for
24 hours. If there is no growth, the media are sterile and the batch can be used for
isolation of microorganisms.
SELENITE BROTH (SB)
Selenite Botha selective enrichment medium was developed for the isolation of
salmonella from feces, urine or other pathological materials. The development of E. coli
and Proteus species is not indefinitely retarded in selenite media. Where the initial
proportion of these organisms is high, it is often advantageous to subculture on the solid
media after 6 hours as well as after 18 hours. It contains bacteriological peptone,
lactose and sodium phosphate. (10g)
Preparation: Add 19 grams of powder to 1 litre of distilled water to which 4 grams of
sodium Biselenite (Oxoid L121) has been added. Warm to dissolve, mix well and fill out
into containers to a depth of 5.cm. Sterilize in a boiling water bath, or in free flowing
steam for 10 minutes.
TETRATHIONATE BROTH BASE (TB)
TB is a selective enrichment medium for isolation of salmonellae from food, feces,
sewage, etc. Organisms which reduce tetrathionate, such as salmonellae, flourish in the
medium while many fecal microorganisms are inhibited.
Members of the Proteus group reduce tetrathionate and may consequently impair the
value of this medium for the isolation of salmonella, this disadvantage of the medium is
largely overcome by the addition of 40 micrograms of Novobiocin to each millilitre of the
incomplete medium before the addition of iodine.

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Preparation: Add 77g of powder to 1 litre of distilled water and bring to the boil. Cool to
below 45C and add 20 ml of iodine solution. The sterilized base will keep for many
weeks but should be used within a few hours of the addition of iodine solution iodine
(6g, potassium iodine 5g in 20ml distilled water).
MACCONKEY AGAR
This is a highly selective modification of MacConkey medium is suitable for a detection
and enumeration of coliforms and for detection and isolation of salmonella and Shigella
species occurring in pathological and food specimen. The medium gives a good
differentiation between coliforms and non-lactose fermenting organisms whilst Gram-
positive cocci are completely inhibited. Lactose-positive organisms give red colonies
and lactose-negative organisms give colorless and transparent colonies.
BROMOTHYMOL BLUE AGAR (BB)
BB is useful for the isolation of salmonella. On this medium, Salmonella will form blue
colonies with a darker centre, while the lactose fermenting enterobacteria will form
yellow colonies. Prepare the media according to the manufacturers’ instructions and
dispense to plates.
SALMONELLA-SHIGELLA AGAR (S.S. AGAR ()
SS agar is a differential, selective medium for the isolation of Shigella and Salmonella
species from pathological specimens, suspected foods, etc. Gram positive
microorganisms and coliforms are inhibited by the action of a specially prepared bile salt
mixture.
Preparation: Suspend 63 g in 1litre of distilled water. Bring to the boil with frequent
agitation, and allow the media to dissolve the agar (Do not autoclave). Cool to about
50C, mix and pour to petri dishes.
Inoculate the medium heavily with the specimen, spreading a portion of the original
innoculum in order to obtain well separated colonies on some pact of the plate. Incubate
for 18 to 24 hours at 37C: non-lactose fermenters form colorless colonies, whilst
occasional resistant coliforms or other lactose fermenters produce pink red colonies.
TRIPLE SUGAR IRON (TSI)
TSI is a composite medium for the differentiation of Enterobacteriaceae according to
their ability to ferment lactose, sucrose and dextrose and to produce – hydrogen sulfide.

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Because some Proteus and other species may give similar reactions to salmonellae and
shigella, it is necessary to distinguish them by their ability to hydrolyze urea. For this
reason TSI should be used in parallel with Urea Agar.
The three carbohydrates in the medium, lactose, sucrose and dextrose are added as
1% and 0.1% respectively. If only dextrose is fermented this will give yellow color (acid
reaction) down but of the agar and red color (alkaline reaction) on top of the slant. The
yellow color is caused by acid production by fermentation of the dextrose. The alkaline
reaction on top of the slant caused by the rapid fermentation of peptone gives alkaline
end-products. If lactose and/or sucrose is fermented the medium will turn yellow caused
by acid production.
Gas production is detected by its appearance under the agar butt at the bottom of the
tube. (The medium is inoculated by stabbing through the butt). If H2S is produced, it will
react with the iron compound in the medium to form FeS which has a black color. For
Salmonella, the black color will appear as a black band between the red and the yellow
parts of the media.
Preparation: Suspend 65g in 1litre of distilled water. Bring to the boil to dissolve
completely. Mix well and distribute. Sterilize by autoclaving at 121C for 15 minutes.
Allow the medium to set in slope to form a slant of about 1 in long.
UREA AGAR BASE (UA)
UA may be used for the detection of urea splitting organisms such as Proteus vulgaris.
The reaction is usually complete after 3 to 5 hours at 37C. Urea production organisms
hydrolyze the urea to form ammonia, and the medium changes to purple-red.
Preparation: Suspend 2.4g Urea agar base in 95 ml of distilled water. Bring to the boil
to dissolve completely. Sterilize by autoclaving at 115C for 20 minutes. Cool to 50C
and aseptically introduce 5 ml of sterile 40% Urea solution. Mix well, distribute 10 ml of
amounts into sterile bijou bottles and allow the medium to set in the slope position.
SAMPLING PROCEDURES
For bacteriological examination, it is essential that no contamination take place during
or after sampling. No laboratory method can compensate for deficiencies in sampling.
Products of heterogeneous nature create difficult problems in order to make a sample

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which is representative for the product concerned. When sampling food products, the
following factors must be considered:-
a) Number and size of samples required.
b) Type of product:- Liquid, Homogeneous, semi solid, Homogeneous solid
or heterogeneous solid
c) Method of preservation: Fresh, dried, frozen, canned, etc.
d) Expected degree of contamination.
If possible, samples of food products should be submitted to the laboratory in original,
unopened containers. This will prevent contamination and reveal the condition of the
product as offered to the public.
If products are in bulk or in too big containers a representative sample has to be
transferred to sterile containers under strict aseptic conditions. If equipment has to be
used for sampling (spoons, knives, bottles, plastic bags, etc…) they should be
effectively sterilized before use. It might be impracticable to sterilize the sampling
devises in the field, and this should be done in the laboratory and the equipment
transported in the sterile containers. Plastic bags of heavy material are useful for
sampling purposes as they are sterile as a result of the production techniques.
SAMPLE NUMBER AND SAMPLE SIZE
The question as to the adequate, representative number comprising a sample from a
given lot of food products has extensive mathematical involvement. Obviously one
sample cannot fully represent a total lot. The sample itself may also not continuously
uniform or homogenous. Even in fluids, like water, the distribution of bacteria may be
irregular. Increase in homogeneity will be found in foods with increasing viscosity, lack
of previous mixing, presence of different phases, variability in material used, etc. It has
been suggested that the number of samples to be drawn should be the square root of
the number of items in the lot. This however, will be excessive for routine testing.
Experience indicates that from a large lot, at least 10 samples are needed for
representation, particularly if high variability is likely in the food products.
The size of each sample taken is also important for the reliability of laboratory results. If
the product is composed of large particles or if heterogeneity is expected, a rather large
sample should be obtained. This can then be mixed and homogenized in the laboratory
before the final sample is taken out.

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TRANSPORTATION OF THE SAMPLES
Should be performed under the same conditions (temp., etc…) as the products a red
held. The transport time should be as short as possible. Canned or dry products need
no particular precautions during transportation.
Samples of refrigerated products have to be kept under refrigeration and brought to the
laboratory as soon as possible. This can be obtained in an insulated box with ice. Under
long transportation, freezing of the product may be necessary, but this should be
recorded for used- when the lab results are interpreted because certain microorganisms
may be destroyed during freezing.
Frozen products should be kept solidly frozen at all times. Thawed samples of frozen
products should be discarded as results of bacteriological examination of such samples
are biased.
PREPARING SAMPLES FOR EXAMINATION
Great care has to be taken to avoid all contamination when opening sample containers.
Cans, jars and other sealed containers should be washed, dried and the area of
opening sterilized by some suitable agent such as alcohol which is removed by burning
if the package is a can.
Paper cartons, plastic foils, etc, should be wiped with a cloth dipped in a sterilizing
agent to remove surface contamination and special attention given to the area of
opening. From the material in question a truly representative sample has to be
produced. Liquid or free materials are agitated until the components are homogenous. If
the container is too large to handle you may sample by means of sampling tubes or
tiers, making sure that all parts of the package are represented in the sample. If material
is in pieces, or a part must be cut or broken away, obtain material from several locations
in order to represent the true condition of the package.
Large materials should be placed in a large sterile container and broken into smaller pieces by
suitable means and under aseptic conditions before a sample is transferred to a sterile
container.
Frozen material has to be thawed before homogenization. Thawing of bigger samples is
often performed overnight in a refrigerator with temperatures not above 10C to avoid
bacterial growth during this.

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INOCULATION TECHNIQUES
The use of microbial media in public health work has 3 main purposes:-
1. to isolate and identify bacteria from a sample
2. to trace specific bacteria
3. to check the hygienic standard (number of bacteria in a product or of a surface).
It is very important that no contamination takes place during inoculation. Inoculation
must be done under aseptic conditions. The wire loops should be flamed frequently and
only sterile pipettes should be used and discarded after use. You should ensure that all
equipment is sterile when used in contact with inoculation.
When we want to isolate bacteria we usually inoculate on the surface of an agar
medium. After flaming and air cooling of the wire loop it is dipped to touch the material
and the loop is smoothly moved back and forth on the agar surface. To avoid too heavy
inoculation, only half of the plate is covered. The loop is flamed and the inoculated part
and continued on the clean part of the plate. To get even better distribution of the
bacteria the loop is flamed once more and the bacteria are diluted on the last part of the
plate as shown on the figure:-
By this method it should be possible to spread the bacterial of the surface of the agar in
such a way that colonies can be picked for further studies.
If the material is supposed to have a very low number of bacteria, enrichment media are
used. These are nutrient into which some of the material for testing is inoculated. The
enrichment media are then inoculated to let the bacterial multiply before plating.
When we want to trace specific pathogens, we usually use selective media. These are
enrichment media which contain substances that will prevent or retard other bacteria
from growing while the species in question will multiply. The inoculation into such media
is as for the enrichment media to transfer some of the material into the medium.
Remember: All equipment (scapels, scissors, pipettes, etc) used for transferring of the
material must be sterilized before and after use.
When checking the hygienic standard of a product or a surface we want to determine
the amount of bacteria per gram of material or per square cm (or inch) of a surface.
To do this we have to let the bacteria grow on an agar medium assuming that one
bacterium can grow out to one colony after incubation. The colonies can then be
counted on the agar plate. Most of the time the number of bacteria is too high to use the
original material for incubation because the plate will over-grow and the counting will be
impossible. We therefore dilute the sample in sterile saline. If we for example mix 10g of

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the sample with 90g of water, we will in 1g of this suspension find the bacteria which
were present in 1/10g of the sample. As 1g of water is 1 ml we can as well measure the
amount in volume as it now will be a fluid.
If the concentration of bacterial is still too high to do a count of bacteria on an agar plate
after inoculation and incubation, we will dilute even more. We can for example pipette 1
ml of the well mixed suspension into 9 ml of distilled water. 1 ml of this suspension will
after mixing represent 1/100g of the sample. In a sample material with heavy bacterial
contamination, we can continue to dilute the sample each time by factor 10.
Usually we will inoculate from different dilutions to obtain at least one plate which can
give reliable results for counting. Too many colonies on the plate will be impossible to
count. On the other hand a plate with only a few colonies will give unreliable results.
Generally, it is said that you should pick a plate in the range of 30 to 300 colonies for
your counting.
As this is a quantitative method it is very important that we keep track of the different
diluting factors. The pipette used for transferring the material is contaminated with
material containing 10 times the amount of bacteria as compared to the next dilution,
and it is therefore important that we each time discard the pipette and take a sterile one
for the next step.
The inoculation is made by taking 1ml of the appropriate dilution on sterile petri dish.
Molten agar is poured on and the two components are mixed by describing the figure 8
with the plate on table before the agar solidifies. This mixing should be done in slow
motions to prevent the fluid to come over 45C in before hand to prevent killing the
bacteria by heat. You should make clear from which factor you are going to multiply the
number of colonies you count in the plate to establish the number of bacteria per g of
sample.
Sometimes we want to combine the counting of bacteria with a possibility of further
studies of the bacteria in question. Then we can pipette 0.1 of an appropriate dilution on
to the surface of an agar plate and distribute this evenly with a bend glass rod.
TESTING FOR MICROBIAL CONTAMINATION OF SURFACES
One of the tasks for a food hygiene veterinarian is hygiene inspection in
slaughterhouses and meat processing plants. Inspection of premises is a very important

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means to establish the general hygienic situation in the plant. Use the eyes to see if the
floors, walls and equipment are clean or if traces of organic substances are
unsatisfactorily cleaned off. In a food industry or slaughterhouse plant, you will also
smell if the cleaning operations are effectively done.
In addition to this subjective way of supervising the hygiene in a plant, there is need for
more objective methods that can be demonstrated to mangers, foremen and workers.
We therefore want to determine the number of bacteria after cleaning and disinfection of
surface like meat cutting table, conveyor belt, knives, etc. As we cannot establish this
just by using a microscope on the surface, we have to transfer the bacteria existing on
the surface to a medium and then incubate this to permit formation of colonies for
counting.
There are different ways of doing this:-
Agar sausage method
For this purpose we use an agar medium that is filled in an artificial sausage casing.
The medium needs to be more firm than those on agar plates. It therefore contains a
higher percentage of agar). The outside of the agar sausage is disinfected and it is cut
through with a sterile knife. The agar is squeezed 1 cm outside the casing, and the end
surface of the agar is moved to touch the surface to be examined. A slice of the agar is
cut off with a sterile knife and placed in a sterile petri dish. More slices are placed in the
same dish, incubated and colonies counted the next day.
It is obvious that not all the bacteria on the surface will stick to the agar. In fact a small
percentage of the total number will do so. The number of colonies counted on the agar
slice is therefore not the total amount of bacteria on the particular surface. But if 10% of
the bacteria stick on the agar the method will give us an indication of total amount.
Contact plate method
This method, special petri dishes are filled with agar and the surface of the agar is
moved in contact with the surface in question. In principle, it is the same as the agar
sausage
method. It is more expensive in use than the agar sausage method, but each plate
covers a bigger area and it gives better results.

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Swab method
This method uses sterile cotton swabs to swab off a certain area, for example 10 square
cm. The swab is transferred to a known amount, for example 100 ml. Of sterile saline
solution and stirred well. From this we get 1ml for inoculation by pour plate followed by
incubation and counting.
CONTROL OF PREPARED MEDIA
The incubate media made the previous day are checked for any microbial colonies. It
colonies are found within the agar medium it indicates that the medium was not sterile.
If colonies are present on the surface of the medium it indicates contamination of the
plates after preparation.
Disposal of cultures: Done by sterilization of cultures followed by disposal
STAINING PROTOCOLS
Gram staining procedure
Gram staining involves three processes: staining with a water-soluble dye called crystal
violet, decolorization, and counterstaining, usually with safanin. Due to differences in the
thickness of a peptidoglycan layer in the cell membrane between Gram positive and
Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer)
retain crystal violet stain during the decolorization process, while Gram negative
bacteria lose the crystal violet stain and are instead stained by the safranin in the final
staining process. The process involves three steps:
1. Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine
and potassium iodide) is added to form a complex between the crystal violet and
iodine. This complex is a larger molecule than the original crystal violet stain and
iodine and is insoluble in water.
2. A decolorizer such as ethyl alcohol or acetone is added to the sample, which
dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal
violet-iodine complex is not able to penetrate this tightened peptidoglycan layer,
and is thus trapped in the cell in Gram positive bacteria. Conversely, the the
outer membrane of Gram negative bacteria is degraded and the thinner
peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-
iodine complex and the color is lost.
3. A counterstain, such as the weakly water soluble safranin, is added to the
sample, staining it red. Since the safranin is lighter than crystal violet, it does not

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disrupt the purple coloration in Gram positive cells. However, the decolorized
Gram negative cells are stained red.
Reagents
 Crystal violet (primary stain)
 Iodine solution/Gram's Iodine (mordant that fixes crystal violet to cell wall)
 Decolorizer (e.g. ethanol)
 Safranin (secondary stain)
 Water (preferably in a running tap)
Procedure
1. Make a slide of cell sample to be stained. Heat fix the sample to the slide by
carefully passing the slide with a drop or small piece of sample on it through a
Bunsen burner three times.
2. Add the primary stain (crystal violet) to the sample/slide and incubate for 1
minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to
remove unbound crystal violet.
3. Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the
crystal violet to the bacterial cell wall.
4. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a
gentle stream of water. The alcohol will decolorize the sample if it is Gram
negative, removing the crystal violet. However, if the alcohol remains on the
sample for too long, it may also decolorize Gram positive cells.
5. Add the secondary stain, safranin, to the slide and incubate for 1 minute. Wash
with a gentle stream of water for a maximum of 5 seconds. If the bacteria is
Gram positive, it will retain the primary stain (crystal violet) and not take the
secondary stain (safranin), causing it to look violet/purple under a microscope. If
the bacteria is Gram negative, it will lose the primary stain and take the
secondary stain, causing it to appear red when viewed under a microscope.

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Gram positive Staphylococcus aureus.
Simple (Direct) Staining with Methylene Blue
1. Prepare a heat fixed smear of the culture you wish to examine
2. Cover the smear with methylene blue
3. Allow the dye to remain on the smear for approximately 1 minute. (Note staining
time is not critical. Somewhere between 30 seconds and 2 minutes should give
you an acceptable stain. The longer you leave the dye on, in general, the darker
the stain.)
4. Wash the excess stain off the slide Pick up the slide by one end and hold it at an
angle over the staining tray. Using the distilled water wash bottle, gently wash off
the excess methylene blue from the slide by directing a gentle stream of water
over the surface of the slide. Wash off any stain that got on the bottom of the
slide as well.
5. Blot off excess stain using a blotting paper. DO NOT rub the slide, rather place
the slide between two sheets of blotting paper and press down gently. Paper will
absorb excess dye.
6. Examine the slide under the bright field microscope.
7. Record the shape, arrangement, and approximate size of the organisms. You
should have data on two cultures which you have stained.

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BACTERIOLOGICAL EXAMINATION (BE) OF CARCASSES
Sometimes the affections seen at post mortem inspection are not enough to get the
whole picture of the condition and the B.E. is a good tool to make decisions about the
fitness of a carcass as human food. The meat of a healthy carcass is supposed to be
practically sterile and there should be no growth of bacteria from a muscle sample.
Under pyemic or septicemic conditions all meat and organs will contain bacteria and
give growth when it is applied.
Meat inspection is responsible for preventing meat hazardous to human health from
reaching the market. On the other hand great economic losses will be the result if
carcasses are condemned without being a threat to human health. In many cases BE
will be of value in making the judgment. BE is intended to be of help to the meat
inspector make a decision on the suitability of the meat for human consumption..
Bacteriological examination of a carcass has to be carried out:-
1) In carcasses derived from an emergency slaughter.
2) In carcasses derived from animals judged “suspect” during ante-mortem
inspection.
3) In carcasses derived from animals with bone fractures with perforation of the hide
or skin.
4) In carcasses derived from animals with external injuries affecting the general
health of the animal.
5) In carcasses derived from animals with a foreign body which has perforated
oesophagus.
6) In carcasses derived from animals with prolapse of uterus, bladder or rectum, if
slaughter has not taken place immediately after the accident. If fever is recorded,
condemnation is done straight away.
7) In cases where there has been a delay in evisceration after killing of more than
45 minutes in cattle or 30 minutes in calves, sheep and goats.
8) In carcasses where there is a diffuse chronic pleuritis or peritonitis (in acute
cases the carcass and organs shall be condemned without doing BE).
9) In cases where animals are found healthy prior to slaughtering but on post
mortem inspection show symptoms of illness which may be dangerous to
humans.
10) In carcasses which are suspected for being symptomless carriers of organisms
which may cause meat borne disease.

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Samples taken for BE
The decision to take samples for bacteriological examination is determined by the meat
inspector when necessary. The objective of BE is to establish whether an infection is
generalized. In such cases the most logic thing is to inoculate from the meat itself. To
ensure that no contamination from outside has taken place, there is need to take 2
whole muscles with their facia intact, and it is important that they are not too big. We
also want to take one from the fore-quarter and one from the hindquarters. It might be
wise to take these diagonally from the carcass.
When you suspect specific infections like salmonella you might choose to inoculate from
the liver or other organs.
If you suspect that the animal may have been treated with antibiotics, you should
sample one kidney for the test for antibacterial activity.
MEDIA USED FOR BE OF CARCASSES
1. Dextrose broth (DB)
Dextrose broth is a highly nutritious and is especially suitable for the initial isolation of
microbes which are fastidious, attenuated, or only present in small numbers. It contains
tryptose, dextrose and sodium chloride. The Dextrose broth is recommended for use as
a medium for sterility testing. It is particularly suitable for the initiation of growth but it is
important that sub-culturing after growth take place since many bacteria will not survive
for long in the acid conditions produced by dextrose fermentations
Preparation: Add 23g of DB to 1 litre of distilled water. Mix well, distribute into final
containers and sterilize at 121C for minutes.
2. Cooked Meat Medium (CMM)
Cooked meat medium is a well established substrate for the cultivation and
maintenance of anaerobic and aerobic organisms. The medium contains peptone, heart
tissue and sodium chloride. It is particularly suitable for the cultivation and differentiation
of Clostridia species, and stock cultures remain viable for several years. Clostridia may
be divided into main groups of their action upon the medium Saccharolytic organisms –
rapid production of acid and gas, but no digestion of the meat. The cultures may have a
slightly sour smell, and the meat is often reddened.

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Proteolytic organisms – Proteolytic ferments decomposes and blacken the meat with
the formation of foul-smelling sulphur compounds. The protein is broken up into
amino-acids, and the small white feathery masses of tryosine crystals may also be
seen. For the isolation of anaerobes, it is important to use solid media in conjunction
with CMM.
Preparation. Add 55g of CMM granules to 1 litre of distilled water and soak for 15 min.
Distribute into narrow containers (tubes) keeping the meat in suspension so that a
uniform layer is obtained. Sterilize by autoclaving at 121C for 15 minutes.
Procedure for BE of a carcass
There are no regulations as to the methods to be used in BE of carcasses. In
establishing whether there is an unspecific generalized infection, which is not often the
question, we incubate in glucose broth to see if there are aerobic bacteria and in cooked
meat medium to test for anaerobic organisms. It is very important that the surface of the
sample is sterilized and that innoculum is taken aseptic condition. The BE can be done
as follows:
3 pieces to glucose broth from
A. One or two muscles each muscle (5 if one muscle)
1 piece to cooked meat medium
(cover with melted parafin)
Loop into blood agar.
Loop onto blood agar
B. Liver Salmonella test
(Enrichment/selective media)
C. Kidney Test for antibacterial activity

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Incubation of inoculated samples is done at 37C for 24 hours.
Interpretation of results of BE of carcasses
a) When the samples prove to be sterile, the carcass and those organs which are
unchanged may be released provided that the inspector is satisfied as to their
fitness as food after further visual examinations. Local condemnation is done of
the affected for parts (Abscesses, fracture areas etc.)
b) When non-specific bacteria are found in more than one of the tubes inoculated
from muscles, it is likely that there is a generalized infection. Total condemnation
should therefore be considered.
c) When specific pathogenic bacteria are found in one or two more of the samples,
the carcass should be condemned, because this indicates that a generalized
septicaemia or bacterial infection has occurred, even though the bacteria
demonstrated may not themselves be transmissible to man.
d) The liver frequently contains intestinal bacteria which have gained entry to the
organ by the way of portal vein. The demonstration of organisms in this organ is
of no significance unless these are of specific pathogenic type.
e) Similarly demonstration of non-specific bacteria in the kidney is also no evidence
of generalized infection as the bacterial invasion of the kidney substance readily
occurs by the way of kidney pelvis and renal tubules.
As stated the results obtained from bacteriological examination are meant to be of help
in judging of the case. The final decision is to be made by taking all findings from ante
mortem, post mortem and B.E. into account.
Bacillus anthracis
This bacterium is the cause of anthrax in many animals including cattle, sheep and
goats. The bacteria also cause acute disease in man.
The disease is very dangerous in meat inspection. Bacillus anthracis is a spore former,
aerobic, gram positive rods that occur in chains. When exposed to air they form spores,

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but do not form spores in vivo (blood smear). If inhaled, spore can cause pulmonary
anthrax leading to death. The bacteria form capsules in the organism or if grown on
50% CO2 atmosphere.
In meat inspection, an anthrax case may be seen as dead animals on transport, on
arrival at the slaughterhouse or in the lairages.
The diagnosis of anthrax is established by direct microscopy of blood smear or organ
samples. A blood smear is always done when anthrax is suspected and great care
should be taken when suspect material is handled
When an anthrax case is diagnosed, the carcass and other materials should be burned
or buried 6ft deep followed by application of lime on top. If the carcass is to be
transported, care should be taken not to contaminate the environment during transport.
The head of the animal must be covered with plastic bag to
avoid leakage of blood that will contaminate the environment. The contaminated areas
should thereafter be thoroughly disinfected. Any human beings who have been in touch
with infected material should be given doses of penicillin and vaccinated.
Staining for presence of capsules
1. Blood smear fixed, either by good heat fixation or with 1% mercury chloride.
2. Staining with polychromethylene blue shall act on the bacteria for 3-5 minutes,
then washed off and dry.
3. Microscopy done with oil immersion. Look for blue bacteria with purple capsule

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Bacillus anthracis showing large rod in chains
Detection of salmonella in meat
The family enterobacteriacea includes a number of genera of Gram negative bacteria.
Some members of this genera are part of the normal intestinal flora, while others are
parasite that are pathogenic to man and animals. Enterobactericea are mostly motile,
aerobic or facultative anaerobes. They ferment glucose, producing acid or acid + gas.
Enterobactericea grow fairly well on ordinary bacterial media. In blood agar, colonies
are mostly circular, convex, smooth, 1-3 mm in diameter and hemolytic in some types.
The most known genera are: Salmonella group, Shigella, Escherichia, Arizona, Proteus
Identification of Salmonella in meat
Samples: Liver tissue, muscle tissue (whole or minced)
a. General Approach
o Direct microscopy: Gram stain and microscopy,
o Homogenization and dilutions (10-2, 10-4, 10-6)
o Plating on PCA or nutrient agar and blood agar.
o Incubation inoculated plates at 37C aerobically
b. Specific approach
o Enrichment in Tetrathionate or Selenite broth, incubate at 37C for
overnight.
o Plating from Tetrathionate or Selenite broth onto Bromothymol blue Agar,
incubation at 37C aerobically overnight.
o Biochemical tests: Identify a single pure colony from Bromothymol blue
agar and inoculate a portion of it onto TSI (triple sugar iron), and another
portion onto Urea agar and Tryptone broth.
Salmonella are gram negative, non-lactose fermenting with close antigenic relationship.
All ferment glucose and mannitol mostly with gas formation. Most are pathogenic for
man and animals.
Results of biochemical tests
Salmonella is a non-lactose fermenter. On bromotymol blue agar, typical colonies are
about 1 mm, raised with entire margins and have dark centres in a blue coloured
medium. One typical colony is tested biochemically with the following results:- TSI (H2S
+ gas); Urea (-); Indole (-); Clark & Lub – Methyl red (+); Voges Prosteuer (-); Citrate

84. Page | 84
(+). Even if these relations fit, we still have to verify our diagnosis through serological
typing using O and H antisera to identify the various serotypes of salmonella. The
photographs below are the microscopic appearance of a gram stained smear at various
magnifications.
Gram stain of gram negative Salmonella. X1000
Salmonella at even higher magnification of X15000
Biochemical characteristics of enterobacteriaceae
BACTERIA TSI INDOL MR VP UREA
Salmonella Red slant, Black centre,
yellow butt, gas
production
- + - -
Proteus Whole tube yellow -/+ ++
Shigella Weakly red slant, yellow
centre; No gas
-/+ =/+

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Detection of Clostridium
This is done inoculating a meat sample onto Iron Sulphite medium and incubating at
37oC for 18-24 hrs. In this medium, the clostridium organism form black round colonies.
Clostridium organism are Gram positive long rods.
Gramstain of Clostridumperfringens
E.coli Yellow + +/-

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Gram positive Clostridium botulinum

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Clostridium botulinum: Types AB E And F cause food poisoining.

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Gram positive Staphylococcus aureus
Mycobacterium
This Is an acid-fast bacterium that is able to resist the aniline dyes commonly used for
staining of bacteria, because these bacteria contain large (74%) amounts of lipid
material that hinder the absorption of these dyes. Mycobacteria spp. include M.
tuberculosis, M. bovis, M. avium, leprae, M. paratuberculosis and others. In order to
stain these bacteria, they have to be exposed to the stain for long periods (hours) or
heating could be used. When they then have accepted the stain, they are not easily
destained, and even strong mineral acids will not decolorize the bacteria giving the
description “acid-fast bacilli”.
Lesion smear can be prepared and stained using the following procedure
Staining Procedure
a) Fix smear by heat
b) Flood the slide with carbolfuschin using a piece of filter paper to hinder
evaporation, heat over bunsen flame (not to hard). Repeat at intervals and stain
for 10 mins. Do not let the slide dry, add more stain if necessary.

89. Page | 89
c) Wash in running tap water
d) Pour 20% sulfuric acid on the preparation and leave for 15-30 seconds. Repeat
till the preparation has got a weak reddish color after 10 minutes
e) Wash in running water.
f) Counterstain with malachite green for 1 min.
g) Wash in water
h) Blot dry and examine in microscope using oil immersion.
DETERMINATION OF ANTIMICROBIAL RESIDUES IN MEAT
In meat inspection we might sometimes suspect that an animal has been treated with
antibiotics or sulphanamide prior to slaughter. In such cases we do test to determine
whether some antimicrobial substances are present in the carcass or organs.
The principle of the test is that if some of these substances are present in the tissue of
for example kidney, it will prevent growth of bacteria when placed onto an agar in which
a test organism is inoculated. Several media may be used for this purpose. They
include: Plate count agar, Blood agar, Mueller-Hinton agar
Mueller Hinton is especially used when sulfonamides are suspected due to low content
of paraminobenzoic acid and folic acid as these substances act antagonistically towards
sulfonamides.
The most used test organism is Micrococcus luteus, Bacillus subtilis, and Bacills
sterothermophilus. From a fresh culture of these test organisms, 2-3 typical pure
colonies are transferred to a tube about 10 ml. Sterile physiological saline and mixed by
shaking. To 100 ml. of melted Mueller Hinton agar, cooled to 45C is added 0.5 ml of
the test organism suspension and mixed well. The inoculated medium is then poured
into sterile petri plates in 3mm. Layer. (The plates may be stored in refrigerator for 1-2
weeks when tightly wrapped in plastic bags). From kidneys to be tested a piece (1.5 x
1.5 cm) is to cut both from cortex and medulla. The pieces are placed tight on the agar
surface with a space of at least 3 cm between each piece and 1.5 cm from the edge of
the plate. The plates are stored in refrigerator for 2 hours to allow diffusion of growth
inhibiting substance into the medium before incubation at 37C for 18-25 hours. If
inhibiting substances is present in the kidney piece, there will be a zone from of growth
of at least 1.5 mm around the piece of the kidney.

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Figure : Testing of meat for presence of antibiotic residues. The first sample has a zone
of inhibition around it which shows that it contains antibiotic residues.

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