meat 4th year test 2

name safety rules and procedures in lab

1. No food or drinks are permitted in the

laboratory at any time.

2. Only closed-toe shoes are to be worn in

the laboratory. Sandals are not

permitted.

3. Keep hands and other objects (e.g.

phones) away from your face, nose, eyes,

ears, and mouth. The application of

cosmetics in the laboratory is prohibited.

4. Work areas/surfaces must be

disinfected before and after use.

5. Laboratory coats must be worn and

buttoned while in the laboratory.

Laboratory coats should not be worn

outside the laboratory.

what is colony morphology

Bacteria show characteristic types of growth on

solid media under appropriate cultureal

conditions

used as presumptive identification

vary in:

- size

- dimaeter

- outline(circular, wavy, rhizoid)

- elevation(flat, raised, convex)

- translucency(transparent, opaque, translucent)

how do you do a gram stain

1. Place a slide with a bacterial smear on a

staining rack.

2. STAIN the slide with crystal violet for 1-2 min.

3. Pour off the stain.

Note: fingers stain Gram-positive - use

forceps!

4. Flood slide with Gram's iodine for 1-2

min.

5. Pour off the iodine.

6. Decolourize by washing the slide briefly

with acetone/alcohol (20-30 seconds).

7. Wash slide thoroughly with water to

remove the acetone - do not delay with

this step.

8. Flood slide with safranin counterstain

for 2 min.

9. Wash with water.

10. Blot excess water and dry.

what are positive results of gram stain

1 Gram-positive

bacteria, the dark

purple

2 Gram-negative

bacteria, red

3 Bacteria non

staining with Gram

method

taxonomy enterobacterales

>1703 species

families:

yersiniacece(Yersinia, Serratia)

hafniaceae(edwardsiella, hafnia)

morganellaceae(Proteus), morganella

erwiniaceae

pectobacteriaceae

budviciaceae

name enterobacteriaceae

escherichia

cronobacter

salmonella

shigella

klebsiella

enterobacter

plesiomonas

citrobacter

pathogenicity of coliform organisms

-digestive tract infection

- food poisoning

- UTI

- sepsa

digestive: salmonella, shigella, e.coli, yersinia

food poisoning: salmonella, e.coli

UTI: e.coli (fombriae type P or type PAP), proteus, morganella, citrobacter

sepsa: e.coli, k.pmeumonia, salmonella

characteristics enterobacterales

facultatively anaerobic,

gram negative

straight rods

some motile

oxidase-

negative and catalase-positive (except

Shigella dysenteriae type 1).

worldwide

soil, water, plants, animals

many are normal biota of intestinal tract

34 genera

oppertunistic infection - septicemia, pneumonia, UTI

describe salmonella species

s. bongori and s.enterica

all motile except s.typhi produce gas from glucose

produce hydrogen sulphide(except paratyphi A)

describe steps of culture media

1. preenrichment - buffered peptone water

(incubate 37C 18+-2hr)

2. selective enrichment - RVS(malaquite green inhibits growth of accompanying flora) and MKTTn broth(tetrionate inhibit intestinal bacteria, gram neg grows, novobiocine inhibits accompanying flora)

3. plate - XLD agar and salmonella chromogenic agar

4. confirmation - nutrient agar, biochemical test

describe XLD agar

salmonella and shigella

Salmonella also decarboxylate lysine which keeps

the pH neutral or slightly alkaline.

At this pH Salmonella species can produce hydrogen

sulphide from the reduction of thiosulphate.

This is indicated by ferric ammonium citrate

producing black or black-centred colonies.

Some organisms, such as Citrobacter, can also

decarboxylate lysine. However, they ferment

lactose and sucrose which keeps the pH too acid (

for hydrogen sulphide to be produced.

BGA

highly selective medium - isolation of Salmonella other than S. Typhi

from feces and other materials.

dye inhibits gram-positive bacteria

and a majority of gram-negative bacilli.

Phenol red - pH indicator and is

yellow - acid production in the

fermentation of the lactose and/or sucrose in the

medium.

Salmonella (other than S. typhi and S. paratyphi)

- White to red, opaque colonies surrounded by

red zones in the medium

Escherichia coli and Enterobacter/Klebsiella -

Yellow to greenish-yellow colonies surrounded

by intense yellow-green zones in medium

Pseudomonas - Pink to red colonies

S. typhi and S. paratyphi, Shigella, Proteus,

Gram-positive bacteria - No growth to trace

growth

p.aeruginosa - pink(not ferment lactose/glucose)

e.coli - yellow(low pH, produce acid)

bacteria on BGA

Salmonella (other than S. typhi and S. paratyphi)

- White to red, opaque colonies surrounded by

red zones in the medium

Escherichia coli and Enterobacter/Klebsiella -

Yellow to greenish-yellow colonies surrounded

by intense yellow-green zones in medium

Pseudomonas - Pink to red colonies

S. typhi and S. paratyphi, Shigella, Proteus,

Gram-positive bacteria - No growth to trace

growth

what is plating out

Using the RVS culture,

inoculate by means of a

sterile loop one plate of

xylose lysine deoxycholate

agar (XLD) so that well-

isolated colonies are

obtained.

plate.

Repeat for the second

selective agar (BGA) using

a fresh sterile loop.

how to serologically confirm salmonella

agglutination - test An

how to eliminate auto-agglutinable strains

one drop of saline - slide

Disperse part of the colony to be tested/colony from a pure

culture

want to obtain a homogenous and turbid suspension

Rock the slide gently for 30-60 seconds.

Observe the result against a dark background

bacteria have clumped together into

more or less distinct units - the strain is

considered auto-agglutinable, detection of antigens will be impossible.

clumped together in distinct units = auto-agglutinable, detection of antigens

In practice, auto - agglutinating strains of Salmonella are rare

more economical to perform poly O, H and Vi serology first.

examination for O antigen

Using one pure colony, recognised as

non-autoagglutinable, proceed as above,

using one drop of the anti O serum

instead of saline solution.

If agglutination occurs, the reaction is

considered positive for the presence of

that antigen.

exam for H antigens

Inoculate a Semi-Solid Nutrient Agar

(SSNA) slope with a pure

nonautoagglutinable colony from the

XLDA or BGA plate.

Incubate at 37± 1°C for 24± 3 hours.

Use this culture for examination for H

antigens, proceeding as above, but using

one drop of the anti H serum instead of

saline solution.

If agglutination occurs, the reaction is

considered positive for the presence of H

antigen.

how to examine HM antigens

Using one pure colony, recognised as non-

autoagglutinable, proceed as above, using

one drop of the HM serum instead of

saline solution.

If agglutination occurs, the reaction is

considered positive for the presence of

that antigen - Salmonella spp.

describe klebsiella

5 subspecies, 4 subspecies

k.pneumoniae subsp. aerogenes

describe e.coli

6 species

some produce enterotoxins or other virulence factors

some strains are capsulated with K antigen

commonly found in gut

water/food

Symptoms of disease include abdominal cramps and diarrhoea,may be bloody.

Fever and vomiting may also occur.

Most patients recover within 10 days, although in a few cases the disease may become life-threatening.

pathogenicity of e.coli

• urinary tract infections (UTI),

• neonatal meningitis, and

• intestinal diseases (gastroenteritis).

The diseases caused by a particular strain

of E. coli depend on distribution and

expression of an array of virulence

determinants, including adhesins,

invasins, toxins, and abilities to

withstand host defenses.

Pathogenic strains cause severe diseases

such as gastroenteritis, dysentery,

hemolytic uremic syndrome

(HUS), urinary tract infection (UTI),

septicemia, pneumonia, and meningitis.

pathogenic/diarrheagenic e.coli

EAEC - enteroaggregative

DAEC - diffusely adherent

EIEC - enteroinvasive

EPEC - enteropathogenic

EHEC - enterohaemorrhagic

ETEC - enterotoxigenic

describe ETEC

causes bacterial diarrheal illness - travelers diarrhea

underdeveloped nations

food /water

toxins which stimulate the lining of the

intestines causing them to secrete excessive fluid,

thus producing diarrhea.

ST and LT toxins

profuse watery

diarrhea and abdominal cramping.

Fever, nausea with or without vomiting, chills,

loss of appetite, headache, muscle aches and

bloating can also occur but are less common.

Illness develops 1-3 days after exposure and

usually lasts 3-4 days.

Some infections may take a week or longer to

resolve. Symptoms rarely last more than 3

weeks.

Most patients recover with supportive measures

alone and do not require hospitalization or

antibiotics

describe EHEC

include E. coli O157:H7, produce Shiga-like

toxin that can cause diarrhea - mild and nonbloody to bloody

diarrhea

main pathogen responsible

for causing hemorrhagic colitis

Complications of EHEC infection can include

- hemolytic uremic syndrome (HUS) and

- thrombotic thrombocytopenic purpura (TTP).

Outbreaks have occurred in:

- nursing homes,

- child care centers,

- schools, and the community.

Major sources of infection have been:

- ground beef, and salami

- unpasteurized milk and juice,

- sprouts and lettuce.

Waterborne transmission occurs through

swimming in contaminated lakes, pools, or

drinking contaminated water.

Since low numbers of organisms can cause

infection, EHEC is easily transmitted from

person to person and has been difficult to

control in child care centers.

EHEC also produces Shiga toxin that destroys Vero cells

therefore, it is also known as verotoxin-producing E.

coli (VTEC).

describe EPEC

Mostly pathogenic to infants and young children

causing diarrhea mainly in developing and

tropical countries with poor sanitation.

It also affects farm animals, dogs, cats, pigs and

rabbits.

Symptoms include diarrhea, abdominal cramp,

vomiting, headache, fever and chills.

EPEC do not produce toxins like ETEC but some

strains of EPEC invades tissue cells and

produce Shiga toxin.

describe EAEC

Enteroaggregative Escherichia coli (EAEC) is a

subgroup of diarrhoeagenic E. coli (DEC).

EAEC have been isolated from children and

adults worldwide.

As well as sporadic cases, outbreaks of EAEC-

caused diarrhoea have been described.

EAEC produces cytotoxin that damages the

mucosa membrane of the intestine

destabilizing the host cell cytoskeletal

structure.

ability of the

microorganism to adhere to epithelial cells

such as HEp-2 in a very characteristic

'stacked-brick' pattern

EAEC K1 - gram neg causes neonatal meningitis

describe EIEC

invades into epithelial cells

and spreads cell to cell

related to species that cause watery diarrhea and dysenetry

Abdominal cramps, profuse watery diarrhea and

fever are the common symptoms but some

may develop dysentery and bloody mucoid

diarrhea.

describe shigella

dysenteriaea, flexneri, boydii, sonnei

cause shigellosis

describe proteus

normal biota in humans along with e.coli and klebsiella

hospitals - skin and oral mucosa

p.mirabilis - food spoilage and UTIs

describe yersinia

enterocolitica, pseudotuberculosus, pestis

rod shaped

yersiniosis - undercooked pork

reservoir for Y.

enterocolitica strains that cause human

illness is pigs, but other strains are also

found in many other animals including

rodents, rabbits, sheep, cattle, horses,

dogs, and cats.

In pigs, the bacteria are most likely to be

found on the tonsils.

enterocolitica - young children - fever, abdominal pain, diarrhea, bloody

y.pestis - black death

detecting yersinia

horizontal methods detection

CIN agar - y.enterocolitica

deep red center with a

transparent margin, or "bull's-eye" appearance

of Yersinia and Aeromonas colonies is

important for identification, and is due to the

presence of mannitol.

Y. enterocolitica ferments the mannitol in the

medium, producing an acid pH which gives the

colonies their red color and the "bull's eye"

appearance.

Sodium deoxycholate, cefsulodin, irgasan, and

novobiocin are added as selective agents.

describe serratia

s.marcescens - red,water insoluble pigment

s.marcescens - nosocomial outbreaks, urinary tract,

wound infections, pneumonia, and

keratitis, oppertunistic - food poisoning

characteristics of pseduomonadaceae

p.aeruginosa

g neg

aerobic rod

free living - soil, water

oppertunistic

motile - single polar flagellum

produce two

types of soluble pigments, the

fluorescent pigment pyoverdin and

the blue pigment pyocyanin.

pyocanin - blue pus - suuporative ibfection on blood agar

jasmine poets smell

causes UTI, resp infections, dermatitis, soft tissue infectioms, bacteriemia, bone and joint infections, GIT infections

characteristics of Burkholderia

pseudomonodaceae

gram neg aerobic rod

b.mallei - nonmotile, grows on McC - glanders

B.pseudomallei - meliodosis - pain in chest,

bones, or joints, cough, skin infections, lung nodules

and pneumonia

Characteristics of Bacillaceae -

Bacillus

produce

endospores; most are

gram-positive, motile

by lateral or

peritrichous flagella

(having flagella over

the entire surface) or

non-motile, and are

aerobic, facultative, or

anaerobic

B.subtilis - hay/grass bacilus

umbonate,

edge - entire with

undulate, internal

charactaristics -

rough, color - creamy,

white.

bacillus pathogenesis

raw foods - soil/veg

spores survive cooking

B. cereus is responsible for a minority of

foodborne illnesses, causing severe

nausea, vomiting and diarrhea.

Bacterial growth results in production of enterotoxins, ingestion

leads to two types of illness, diarrheal and emetic (vomiting)

syndrome.

• The diarrheal type is associated with a wide-range of foods, has

an 8- to 16.5-hour incubation time and is associated with

diarrhea and gastrointestinal pain. It might be difficult to

differentiate from poisoning caused by Clostridium perfringens.

• The emetic form is commonly caused by rice that is not cooked for

a time and temperature sufficient to kill any spores present, then

improperly refrigerated. It can produce a toxin, cereulide, which is

not inactivated by later reheating. This form leads to nausea and

vomiting 1-5 hours after consumption. It can be difficult to

distinguish from other short-term bacterial foodborne pathogens

such as Staphylococcus aureus.

characteristics of clostridum

Gram-positive rods

(some are Gram-

variable), often

arranged in pairs or

short chains, with

rounded or sometimes

pointed or square

end. They are often

pleomorphic.

growing clostridium

Clostridium novyi type A and Clostridium

haemolyticum are among the strictest of

obligate anaerobes - may require e extended incubation on pre-reduced or freshly prepared plates and total handling in an anaerobic chamber

Clostridium tertium, Clostridium histolyticum and Clostridium carnis are aerotolerant - blood agar atmosphere of air with 5-10% added CO2

describe C.perfringens

G+

anaerobic spore-forming rod

5 types

neurptoxin

toxins(active protein) can be neutralized by antisera

causes food illness

CPE - heat labile(dies@74C)

describe intoxication of clostridium

incubation time 6-24hrs after ingestion

spores withstand cooking

course resolves in 24hrs

symtoms of clostridium

abdominal cramping and diarrhea; vomiting and fever are unusual

rarely - clostridial necrotizing enteritis = Pig-Bel - typce C strainproduces ulcerative beta toxin

c.perfringens food poisoning could be subclinical

describe C.sporogenes

putrefaction - spoilage

swell,burst

putrid odour

partial digestion of food

describe C.botulinum

G+ spore rod

obligate anaerobic

soil/marina

3 groups

neurotoxins

toxin types A-G

botulism pathogenessi

neuromuscular transmission is interrupted by a protein botulin neurotoxin

Paralysis begins with the cranial nerves, then affects the upper extremities, the respiratory muscles, and, finally, the lower extremities in a proximal-to-distal pattern

symtoms of botulinism

onset 18-36hrs after exposure

Initial symptoms can include nausea, vomiting, abdominal cramps or diarrhoea. After the onset of neurologic symptoms, constipation is typical. Dry mouth, blurred vision, and diplopia are usually the earliest neurologic symptoms. They are followed by dysphonia, dysarthria, dysphagia, and peripheral muscle weakness. Symmetric descending paralysis is characteristic of botulism

symmetrical, descending, flaccid paralysis of motor and autonomic nerves usually beginning with cranial nerves (paralysis)

In severe cases, extensive respiratory muscle paralysis leads to ventilatory failure and death unless supportive care is provided

characteristics of botulinum toxin

complex, especially under acidic conditions

dissociates under slightly alkaline - inactive

heat sensitive(spores resistant)

80C for 30 mins/ 100C for 10 mins destroys toxin

Non-acidic foods need to be pasteurized twice, at 24h intervals

destruction of botulinum spores

spores heat resistant @121.1C, expressed at D value

D value is constant for specific bacterial spores when they are subjected to heat at constant temperature

Canners aim to reduce the probability of one spore surviving - thermal process to low level

Experience has shown that a process equivalent in sterilising effect to twelve decimal reductions of the population of Clostridium botulinum is sufficient to protect consumer safety. Such a process is referred to as a "12 D" process and it is equivalent to holding the contents of the container at 121.1C for 2.8 min

describe bacteriological meat exam

- burn surface meat in flame

- cut with sterile knife

- inoculatioe on solid medium

describe bacteria meat exam by dilutions

- 10-20 samples weighing 50-75 g

- grind, weigh 10-20gm into sterile container or Stomachner bag

- add 90 or 180ml water

- shake dilution bottle for 3 mins

- leave fpr 15-30mins in 0-8C

- separate liquid part (used as a x10 dilution for further analysis)

serial dilution continued

Each tube starts out with 9 ml of sterile water. 1 ml of the bacterial culture solution is added to the first tube, and mixed. This dilutes the bacterial solution by a factor of 10. Then, 1 ml of the solution from the first tube is removed, and added to the 9 ml of sterile water in the second tube. This is another 10-fold dilution, making a 100- fold dilution of the original solution

describe bacteriological exam of p.aeruginosa in meat

Inoculate nitrofurantoin broth with (1ml) of serial dilution 1:10, 1:100, 1:1000. Incubate in temp. 37C, 18-24 h. If growth is seen transfer culture on solid medium King B with nitrofurantoin. Identify colonies from King B. P. aeruginosa: Gramm negative; pyocyanin +; glucose +; arginin +; TTC (triphenyl tetrazolium chloride ) +, fluorescein +

how do you detect aerobic spore-forming bacteria

serila dilutions of sample in 80C 10 min

cool immediately

inoculate 0.5ml from each dilution on two solid agar plates

incubate 30C for 72hrs

gram stain - G+, spore forming rods

what is Schaeffer-Fulton method for staining endospores

Malachite green stain (0.5% (wt/vol) aqueous solution) Decolorizing agent Tap water Safranin counterstain 1. Air dry and heat fix the organism on a glass slide. 2. Add few drops of malachite green stain solution and heat to first steam 3 times. Alternatively, the slide may be steamed over a container of boiling water. 3. Wash the slide in tap water. 4. Counterstain with safranin for 30 seconds. Wash with tap water; blot dry. 5. Examine the slide under the oil immersion lens (1,000X) for the presence of endospores. Endospores are bright green and vegetative cells are brownish red to pink

how do you detect anaerobic spore-forming bacteria

Inoculate two Wrzosek's broths with 1g of sample. One of them heat in temp 80C 10min. Both broths incubate in temp. 37C 72 h. From broth with growth prepare bacterioscopy Gramm stained slides. When Gram positive rod shaped bacteria are detected transfer one drop of Wrzosek,s broth on sterile petri dish and add Wilson-Blair medium heated to 45C. Mix carefuly and leave for few minutes

Add add fick layer of water agar heated to 45C to make sure that bacteria will grow in anaerobic conditions. Incubate in 37C, 24-48h. Check results after 24 and 48 hours. Black colonies or black color of medium is confirmation of

detection of botulinum toxin

methods based on immunological methods can be very specific

simple - biological test on mice/guinea pig..

Extract from tested material is injected intra peritoneum or i.v. in two groups and in one group additionally specific antitoxin is injected. Result is positive when all animals from control group died after 96 hours and all animals will survive from group injected with antitoxin

describe Staphylococcus

family Micrococcaceae

G+ spherical

clusters like grapes

aureus(pathogen), epidermidis(most are non-pathogenic, coagulase negative), may be oppertunistic, saprophyticus

catalase +

oxidase -

faculative anaerobes

describe staph.aureus

produce coagulase

grow at 15-45C and at NaCl concentration up to 15%

hemolytic on blood agar

coagulase detection of staph

S. aureus - coagulase (+)

S. epidermidis - coagulase (-)

S. saprophyticus - coagulase (-)

coagulase - heat stable enzyme

Two forms of coagulase exist: one is bound to the cell,

and the other is excreted from the cell as an enzyme.

Bound coagulase, also called "clumping factor", acts

directly on the fibrinogen in plasma and causes the

bacteria to clump.

When the coagulase is released as an enzyme from the

organism, also called "free coagulase", it converts

prothrombin to a product that then acts on

fibrinogen in the plasma to form a fibrin clot.

describe s.epidermidis

most are non-pathogenic, coagulase negative

may be oppertunistic

small, white colonies

non-hemolytic

identification of s.aureus

Chapman - mannitol salt agar • selective medium for the isolation and enumeration

of Staphylococci

• it differentiates mannitol fermenting and mannitol non-

fermenting species

• fermentation of mannitol inducess acidification turning

the medium yellow

aureus - yellow

saprophyticus - yellow

epidermidis - pink

Giolitti-Cantoni Broth

• Liquid medium for the enumeration and

selective enrichment

Baird Parker Agar

• moderately selective and differentia medium for the

isolation and enumeration of Staphylococcus aureus

idenitification of staphylococcus

Liquid medium - Giolitti-Cantoni

enriches s.aureus from food during isolation procedures

incubate 35C for 40-48hrs

Blackening of the medium - a positive reaction. No blackening - a negative reaction.

If blackening occurs, subculture to Baird-Parker

describe pathpgenic staphylococcus identification

beta hemolysis on sheep

blood agar liquefaction of coagulated serum

production of fibrinolysin, hematoxin,

dermotoxin, and lethaltoxin are properties

exhibited only by pathogenic strains, but they

are not exhibited by all pathogenic strains

describe identification of staphylococcus

Standard ISO 6888

Microbiology of food and animal feeding

stuffs - Horizontal method for the

enumeration of coagulase-positive

staphylococci (Staphylococcus aureus

and other species)

describe staphylococcal poisoning

sources:

• the human carrier or individual with clinical

signs

• food of animal origin

• raw milk or • raw milk products from infected animals

Some strains of staphylococci produce

enterotoxin that is comparatively heat-

resistant.

describe streptococcus

family streptococcaceae

G+

alpha hemolysis - oxidation of iron in Hg - green on blood agar

beta hemolysis - rupture of RBC - wide clear area arpund bacteria

gamma hemolysis - misnomer

Beta-hemolytic streptococci are divided on

describe strepto.pyogenes

group A

G+ non-motile

non-spore

chains/pairs

fermetative

catalase negative

aerotolerant

needs enriched medium containing blood

capsole of hyaluronic acid

beta hemolysis

catalase - bubbles

between 5-15% of human

population carries S. pyogenes, usually in the

respiratory tract, without signs of disease

normal oppertunistic flora

pharyngitis and complications such as otitis

media, mastoiditis, peritonsillar abscesses,

meningitis, peritonitis, and pneumonia.

describe enterococcus

family streptoccaceae

intestine commensals in humans

faculatively anaerobic

tolerant to:

- extreme temp 10-45C

- pH 4.5-10

- high NaCl

General coliforms, E. coli, and Enterococcus

bacteria are indicators of microbial

contamination of drinking water and food.

Enterococcus spp. also indicate fecal

low grade pathogens

food intoxication - biogenic amines

reservoir for oppertunistic infections and for virukence traits

describe Nocardia

order actinomycetakes(also Actinomycetaceae, streptomycetaceae)

weak staning G+

catalase +

rod shaped

acid-fast branching filaments

n.asteroides - oppertunistic

s

ubiquitous soil

saprophytes, route of infection can be either by

inhalation or by direct cutaneous inoculation.

what diseases does nocardia cause

slowly progressive pneumonia

cutaneous infections - actinomycetoma, lymphocutaneous disease, cullulitis, subcutaneous abscesse

culturing nocardia

They may be isolated on routine bacterial, fungal, and mycobacterial media. Colonies may appear within 4 days, but may require up to 2-4 weeks of culture.

Nocardia can also be difficult to isolate by culture because of overgrowth by faster-growing nonpathogenic colonizers that may mask its presence.

Nocardia colonies may be smooth and moist, or have a "mold-like" verrucous grey-white waxy or powdery appearance from aerial hyphae. They have a very distinct, strong mildew odor that allows experienced microbiologists to suspect their presence.

describe fungi in meat

molds,yeast

chemoheterotrophs(requiring

organic nutrition) and most are aerobic

saprophytes in soil and water

produce sexual and asexual spores

molds - mycelium - microscopic branching hyphae

cell wall - chitin

recycle organic material

mycotoxins - allergic/immunosuppression/cancer/etc.

describe fungi mycotoxins

Aflatoxins are potent carcinogens and, in

association with hepatitis B virus, are responsible for many thousands of human deaths per annum, mostly in non-

industrialised tropical countries. Ochratoxin A is a probable carcinogen, and may cause urinary tract cancer and kidney damage

in people from northern and eastern Europe. Fumonisins appear to be the cause of oesophageal cancer in southern Africa, parts of China and elsewhere.

Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects in animals and man.

diseases caused by fungi

infections/mycoses classified degree of tissue involvement and mode entry into host:

- superficial(skin, hair, nails)

- subcutaneous (dermis, subcutaneous)

- systemic - deep, internal organs

- opportunistic

humans - candidiasis(thrush), dermatophyte(athletes foot)

immunocompromised - lethal

detection of fungi

Isolation and detection of fungi should be done using cultivation methods and microscopy.

For cultivation are used following media: • Czapek's medium (liquid and solid) • Sabouraud

• Synthetic

Direct inoculation on 16 sq. cm. (Sample prepared as described on previous laboratory)

Incubation in 24-26 C 5 days.

coagulase detection

slide coagulase and tube coagulase

- drop coagulase plasma on dry,clean slide

- drop distilled water near cpagulae(conrrol)

- sterile loop, emulsify isolated colony into each drop

- create smooth suspension

- clumping in coagulase

- clumping in both indicates that organism autoagglutinates

The slide agglutination technique can occasionally generate

describe catalase detection

1. Place a drop of 3% H2O2

on a glass slide. 2. Touch a sterile loop to a culture of the organism to be tested and pick up a visible mass of cells.

3. Mix the organism in the drop of hydrogen peroxide. 4. Observe for immediate and vigorous bubbling. 5. Dispose of slide in the contaminated slide

container.

Interpretation: Bubbling

describe titration method of detecting and quantitatively counting enterococci

Prepare serial dilutions of a sample.

Transfer 1 ml from each dilution to

separate probe with selective broth (with

sodium azide and crystal violet)

Incubate 24h in 37C

If enterococci are present broth will change

color into yellow.

If growth can be seen in 1:100 dilution or

higher it means that hygienic status of

meat is unacceptable.

pathogens in carcass organisms

sterile until slaughter

mixed flora - nonspecific organisms of environmental origin

could be food-poisoning organisms

specific pathogens may be

present in organs or tissues such as the

spleen, muscular tissue or lymph nodes

and their presence can only be attributed

to a generalised septic or bacteraemic

infection in the animal at the time of

slaughter

systemic invasion - ill animals

indications for exam of carcass

3 stages:

ante-mortem

gross post-mortem

lab tests

carcass condemned when organs exhibit marked pathological changes of non-infectious nature

A bacteriological examination can never

substitute for a careful organoleptic

examination(detect septiccemia or bacteremia)

when is a bacteriological exam considered obligatory

1. Animal was slaughtered in emergency;

2. Animal was slaughtered on account of a

disease associated with systemic

disturbances;

3. Animal shows pathological changes on

post-mortem inspection questioning the

suitability of the meat for

human consumption, even though the

animal was found healthy on ante-

mortem inspection;

4. Animal was excreting food-poisoning

organisms prior to slaughter, or which

emanate from a herd in which the

presence of food-poisoning organisms

has been officially reported;

5. Animal was not eviscerated within 1

hour after slaughter or the parts of

the slaughtered animal necessary for

postmortem examination are absent or

have been handled in such way that

makes satisfactory judgement

impossible;

6. Animal was slaughtered without the

prescribed ante-mortem inspection.

what materials may be talen for submission to lab for bacteriological exam

1. Two complete muscles, with their

fascia, one from a forequarter, and

one from a hindquarter, or cubes of

muscle, each side measuring not less

than 8cm.

2. The prescaptilar or axillary lymph

node from the other forequarter of the

carcase and the internal iliac node from

the other hindquarter, including the

surrounding fat and connective tissue of

the nodes.

lymph node muscle

muscle lymph node

Material submitted for

examination

3. The spleen, which should not be

incised except in cases where the organ

is considerably enlarged, in which case a

piece as large as the hand should be

taken.

4. A kidney.

5. In the case of small animals, the

whole liver with the gall bladder; in other

animals, a portion of liver twice the size

of a fist and including the portal vein, or

the caudate lobe and including the portal

vein, and also the portal lymph nodes

and gall bladder.

6. Parts showing pathological change

and which, in view of their position, are

suspected of containing pathogenic

bacteria, together with the associated

lymph nodes (e.g. in the case of

pneumonia a portion of lung and

associated lymph nodes).

7. A portion of the small intestine along

with a number of mesentcric lymph

nodes in those cases where animals nave

suffered from enteritis and have been

reported to be excretors of Salmonella

organisms, or animals known to emanate

from a herd infected with such

pathogens.

liver - intestinal bacteria

scheme of examination

solid media:

nutritive agar

baird-parker agar

BGA

blood agar

liquid media:

Rappaport-Vasiliadis modified broth(MRSV)

Wrzoseks broth(2 probes, one signed with "T" after inoculation heat in water bath 80C for 10mins)

muscles, ln.nodes, spleen, liver, kidney

describe sample maintenance in lab

unpack without delay

place on sterile plate

note visible changes in samples

burn surface of sample in flame

cut with sterile knife

inoculate on solid medium

stamp cut surface of sample on 16sq cm of solid medium

inoculate liquid medium with 1g of sample cut from internal part

Interpretation of microbiological

diagnostic of meat - regulations

COMMISSION REGULATION (EC)

No 2073/2005 of 15 November 2005

on microbiological criteria for foodstuffs

article 2:

- "process hygiene criterion"indicates acceptable functioing of production process

Microorganisms in this criterion (regarding meat):

- Aerobic bacteria

- Enterobacteriacae family

- Salmonella

- E. coli

- "food safety criterion" acceptability of product/batch

Microorganisms in this criterion (regarding meat):

- Listeria monocytogenes

- Salmonella

COMMISSION REGULATION (EC)

No 1441/2007 of 5 December 2007

amending Regulation (EC) No 2073/2005

on microbiological criteria for foodstuffs

general criteria fro salmonella in meat - absent in 25g

general criteria for aerobic colony count - <5cfu/sq cm

if above are nit met meat is amrked unfit for human consumtion

decission made by vet on basis:

ante-mortem, post-mortem and results of lab tests

brucellosis etiology

contagious, 3 species;

melitensis(sheep,goat and most pathogenic for humans), abortus(cattle), suis

(others are monopathpgenic)

brucellosis pathogenesis

enter and migrate ti ln.nodes next to infection gate

seeding of brucella bacilli into blood stream

10-21 days bacillemia - fever aswell

bacilli in organs

cause inflammation

disappear frm organs

remain for life-time in reticuloendothelial system

in ln.nodes, spleen, udder, joints for many years

may cause latent infections(no clinical symtoms, may be periodic bacteremia)

may

permenently or periodically excrete

Brucella bacilli in milk, urine, faeces, and

in case of aborting females - in leakage

from vagina, as well as in foetal

membranes and in amniotic fluid.

where do Brucella bacilli exhibit particular relation to

pregnant uterus

cause abortive births in 6-8 months for cow, 4-6 weeks before birth for sheep and goats, sows 4-12 weeks of pregnancy

then disappear from uterus

sources of brucellosis

aborting females - excretions - skin, conjunctivitis, alimentary tract, air passages and genitalia

what is brucella bacilli sensitive to and resistant to

external factors

;

62C for 1 min

disinfecting agemts in few mins

survuve long time in animal tissues

resistance to external factors.

They survive meat acidification, even pH = 4.0,

in corned and smoked meat products they were

detected even after 63 days,

in frozen meat they survive for up to 460 days,

in 25% brine at temperature of 0°C - for 201

days.

preslaughter symptoms of brucellosis

lack typical symptoms

abortion

rarely - inflammation of genital canak and pathogenic changes in vaginal leakage 1-2 weeks after abortion

In males reddening of foreskin and penis swelling, and

sometimes epididymo-orchitis (inflammation of

testicles and epididymis) occurres.

Relatively rarely joint inflammations may be seen in

brucellosis, especially knee-joint and carpal joint

inflamation.

All of those symptoms are not specific and may be

caused by another pathogen.

In latent brucellosis no symptoms of brucellosis

occurrs.

after slaughter lesions in brucellosis

lack of significant

anatomopathologic changes.

In severe form of brucellosis inflammations may

be observed, sometimes spread inflammatory-

necrosis foci in the splen, lymph nodes, less

commonly in the liver and lungs

In pregnant females or after

abortions brucellosis may be

suggested by infections of uterus,

fetal membranes and fetus.

In males epididymo-orchitis, with

presence of necrosis foci and

abscesses may occurrs

sanitary-veteronary proceedings in case of brucellosis

proliferayion, economic effects - special prevention and combatting in many countries

Poland - free

carcass and

internal organs of animals with brucellosis are

considered unfit.

Brucellosis of cattle, goats, sheep and pigs is a state-

combatted disease.

In many countries there is prohibition of slaughtering

during outbreaks of sheep and goat brucellosis.

how do humans become affected by brucellosis

products of animal orogin - direct contact/alimentary - repetitive consumption of raw infected meat(may lead to severe latent)

slaughterhouse staff

alimentary - consumption milk and meat from sick animals

a) relatively low quantitative level of infections

of meat with Brucella bacilli, which in

pathogenesis of brucellosis has a significant

importance,

b) consumption of meat usually after thermal

processing (high temperature destroys the

pathogen).

pasteurellosis etiology

infectious, rod bacteria

p.multocida

pasteurellosis pathogenesis

pathogenic - primary disease in weak individuals

morbid agent

p.multocida in cattle

In cattle:

• hemorrhagic septicemia of cattle, bovine

hemorrhagic septicemia, described earlier as

cattle and wild game plague or Bollinger

disease

• secondcary pasteurellosis of cattle, present in

calves and young cattle

it usually joins primary viral infections

predisposing factors (environmental

stressors) play an important role in

development of thise disease

pasteurellosis in pig and sheep

In pigs:

• swine pasteurellosis, called previously swine

plague

spontaneous occurence of disease is

generally rare, much common are

secondary infections and predisposing

factors play significant role

In sheep:

• sheep pasteurellosis, typically of a chronic

course

resistance of pasteurella

not resistant to

external factors:

• they can be easily destroyed at the

temperature of >60°C

• disinfecting agents destroy them

successfully