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Файл:Veterinary and sanitary examination of meat and meat products. Study aid
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Methods for staining smears.
Gram staining of smears. A strip of filter paper is placed on the fixed smear
and a solution of carbolic henician violet is poured. Leave for 1–2 minutes, after
which the paper is removed, the paint is drained, the smear is washed with water and
Lugol’s solution is poured onto it for 1–2 minutes. The solution is drained and ethyl
alcohol is poured in for 0.5–1 min, dried, and examined under a microscope.
Staining of capsule-forming microorganisms. In some types of bacteria, a
capsule is formed around the cell, which is a mucous substance containing mucin and
polysaccharides. Depending on the staining method, the capsule acquires a different
color than the protoplasm of the microbial cell, and this makes it possible to
determine its presence. There are methods for coloring capsules according to
Romanovsky-Giemsa, according to Mikhin, with a solution of safranin.
Romanovsky-Giemsa staining method. The fixed preparation is placed smear
down in a glass bacteriological dish on sticks, a solution of Giemsa paint is poured
under the smear, left for 30–40 minutes, lightly washed with water, and dried.
A microscope reveals a pink capsule and a blue body of the microbial cell.
Mikhin staining method. The fixed preparation is stained for 6–7 minutes with
a 1 % solution of methylene blue with slight heating in the flame of an alcohol lamp.
Wash with water, dry with filter paper and microscope. The capsule is pink, the body
of the microbial cell is blue.
Staining of spore-forming bacteria. To color spores, use strong dye solutions or
pre-etch the spore shells with chromic acid or other substances.
Staining of spore-forming bacteria according to Möller. A smear is prepared
from the bacterial culture under study and fixed on the flame of an alcohol lamp. A
5 % aqueous solution of chromic acid is poured onto the smear for 3–4 minutes,
washed with water, and dried with filter paper. Place a strip of clean filter paper on
the smear, pour in a solution of carbol fuchsin, heat the preparation from below with
a burner flame until vapor appears, paint for 8–10 minutes. The paint and paper are
drained without washing with water and decolorized with a 5 % sulfuric acid
solution. Wash with water and stain with methylene blue solution for 4 minutes.
Wash with water, dry with filter paper, and microscope. Spores are painted red,
vegetative cells — blue.
3. Sowing on MPB and MPA nutrient media.
It is carried out to identify pathogens of zooanthroponoses: anthrax bacilli,
listeriosis bacteria, swine erysipelas, etc., pathogens of food toxic infections and
anaerobes.
To identify anthrax bacilli, culture is carried out from a heated broth. Take 1 g of
meat and place it in a test tube with 1 cm3 of sterile MPB, boil for 5 minutes (all non-
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spore-forming bacteria die), cool and pour onto the MPB, spreading evenly over its
surface.
Preparation of nutrient media: MPB — meat water + 1 % peptone (a product of
hydrolysis of meat proteins), to enrich the medium with nitrogenous substances
+ 0.5 % chemically pure sodium chloride. The medium is alkalized (pH = 7.4–7.5),
boiled, filtered, and sterilized.
MPA — 1 dm3 MPB + 2–3 % chopped agar-agar in a Koch apparatus, cool and
adjust the pH to 7.4–7.6. To clarify, add chicken egg white, mix, boil, filter, and
sterilize in an autoclave.
4. Sowing on differential diagnostic nutrient media.
These media are used to distinguish one type of bacteria from another. Endo’s,
Levin’s and Ploskirev’s media are used to differentiate Escherichia coli from
Salmonella. Differentiation is based on the ability of bacteria of the Escherichia coli
group to ferment lactose with the formation of lactic acid, which acidifies the
environment and restores the discolored indicator. Thus, on Endo’s medium, E. coli,
decomposing lactose, restores fuchsin discolored with sodium sulfite and forms red
colonies with a metallic sheen.
Ploskirev's medium includes, in addition to MPA with lactose, bile salts,
brilliant green, and mineral salts. It is not only differential diagnostic, but also
selective, as it inhibits the growth of certain microbes and promotes better growth of
salmonella. On Ploskirev's medium, salmonella form transparent, colorless or pale pink
round colonies. On this medium, E. coli forms pink colonies, on Levin's medium —
blue or black colonies.
Method: a piece of meat is burned twice in alcohol, and a cut is made with a
flambéed tool. With the cut side, impressions are made on the Endo’s medium
4–6 times, distributing them evenly. The cup is placed in a thermostat at a
temperature of 30–37 °C for 16–24 hours.
Second day
1. Accounting for growth on MPA in order to exclude anthrax.
Anthrax bacilli on MPA grow in the form of branched, curl-shaped, rough
colonies of gray-white color with fringed edges, reminiscent of a “jellyfish head.” If
necessary, microscopy is performed and the mobility of bacteria in a hanging drop is
determined (the causative agent of anthrax is Gr+, immobile rods with chopped ends,
located in smears in the form of short chains). The diagnosis of anthrax is made based
on the detection of capsule-forming bacilli in smears, characteristic growth on
nutrient media, lack of motility, and a positive biological test.
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The detection of small transparent dewdrop colonies in crops from meat and
organs on MPA raises suspicion of the presence of pathogens of listeriosis, swine
erysipelas, and pasteurellosis. Differential diagnosis of these pathogens is carried out
by studying their morphological, cultural, and biochemical properties.
The appearance of small transparent or cloudy colonies on the MPA,
sometimes with various pigments, gives reason to suspect the presence of coccal
microflora: streptococci, diplococci or staphylococci.
Bacteria of the genus Proteus are capable of forming a thin veil-like bluishtransparent smoky film on the surface of nutrient media.
2. Accounting for growth in differential diagnostic media (Endo’s
environment).
Growth in Endo’s medium is measured to differentiate Salmonella from
Escherichia coli.
Salmonella do not decompose lactose, on Endo’s agar the color of the medium
and colonies is white or pale pink, the colonies are round, small, translucent, the
edges are even, smooth (S-shaped) or round, rough, dull (R-shapes).
Escherichia coli ferments lactose, and fuchsin is reduced under the influence of
lactic acid, so the colonies and the environment around them turn red-violet. The
detection of round, convex colonies with smooth edges, red, and with or without a
metallic sheen on Endo’s agar gives reason to suspect the presence of Escherichia
coli bacteria.
Examining colonies in passing daylight, determine: shape (round, irregular,
elliptical), size (dew-shaped, small, medium and large), color, relief (flat, convex,
with a raised or depressed center), surface (smooth, wrinkled, rough, tortuous),
transparency (transparent, translucent, opaque), the nature of the edges of the
colonies (smooth, wavy, curl-like, blurry), consistency (dry, slimy, ointment-like,
mushy).
3. Preparation of a “pure culture” suspension (“microbial suspension”).
Find a colony similar to a salmonella colony, remove it with a sterile
bacteriological loop and place it in a test tube with 1 cm3 of saline solution. This
suspension is the starting material for further determination of the serological and
biochemical properties of Salmonella.
4. Morphological typing of isolated microorganisms.
Bacterioscopy of colonies suspicious for salmonella. Using a bacteriological
loop, take a drop of “microbial suspension” and place it on a glass slide, stretch it, dry
it, fix it, and Gram stain it. Microscopy using immersion oil (vol. × 90), determine the
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shape, length, thickness, Gram stain of microorganisms, the presence of spores and
capsules.
Determination of bacterial motility in a hanging drop. A drop of “microbial
suspension” is applied to the middle of the cover glass. Take a slide with a hole,
apply a thin layer of Vaseline along the edge of the hole and, turning the hole down,
apply it to the cover glass so that the drop is in the center of the depression. The glass
is turned over, the drop appears hanging in a hermetically sealed chamber. To study
under a microscope, narrow the diaphragm and find the edge of the drop under low
magnification. The found drop is examined with an immersion lens, and the
movement of bacteria in a straight line with slight oscillatory movements is observed.
5. Serological typing of isolated microorganisms.
To quickly determine whether a microbial culture belongs to the Salmonella
genus, an agglutination test (RA) is performed on a glass slide. A drop of undiluted
polyvalent salmonella serum containing antibodies to the main groups of salmonella
(A, B1, C1, C2, D1, E1) is applied to it with a Pasteur pipette, then a small amount of
“microbial suspension” is added to the drop of serum with a sterile bacteriological
loop; The control is a drop of saline solution, into which a drop of “microbial
suspension” is also added. If the reaction is positive, in a drop of agglutinating serum,
after a few seconds, granularity on a dark background or uniform turbidity visible to
the naked eye appears. In a drop of saline solution, the bacterial suspension remains
unchanged.
microbial suspension
saline salmonella serum
6. Sowing on “variegated row” media.
The general characteristics of the bacteria belonging to the genus Salmonella
are the following: they do not ferment lactose, do not form indole, do not break down
urea, ferment (with a few exceptions) mannitol, sorbitol, arabinose, maltose, break
down glucose with the formation of gas, the majority produce hydrogen sulfide,
reduce nitrates.
Salmonella treat differently arabinose, dulcite, inositol, rhamnose (in Bitter's
medium, xylose, glycerol (in Stern's broth), which allows them to be divided into
different biochemical types.
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Biochemical typification of Salmonella is based on their enzymatic properties:
the ability to decompose sugars and alcohols, as a result of which an indicator is
restored that colors the medium in the corresponding color, or a gas is released.
For a more complete biochemical typification of a crop, it is sowed in an
expanded color row. Variegated media consist of Hiss’s media with various sugars:
glucose, lactose, sucrose, mannitol, arabinose, dulcit, xylose; broth with glycerin
(according to Stern), medium with rhamnose (according to Bitter), litmus milk and
meat-peptone broth with indicator paper for hydrogen sulfide. Each of these media
contains two components: the main ingredient (sugar or alcohol) and an indicator —
a substance whose color change indicates the decomposition of the main ingredient.
Method of inoculation on media of the variegated series: at the same time we
hold 2 test tubes in our hand (one with a “microbial suspension,” the second with one
of the media of the variegated series) and, having opened the plugs, use the second
hand to inoculate from the first one using a bacteriological loop test tubes into the
second one, immediately close the caps, sterilize the tube loop over an alcohol flame
after each inoculation. The tubes are incubated for 18–24 hours at 37 °C.
Day three
1. Biochemical typing of Salmonella.
The growth of Salmonella on the “variegated series” media is recorded
according to the scheme shown in Fig. 6.
Veterinary and sanitary assessment of meat and meat products
upon detection of foodborne pathogens
According to the “Rules for Veterinary Inspection of Slaughter Animals and
Veterinary and Sanitary Examination of Meat and Meat Products,” when salmonella
is isolated from the muscle tissue of slaughter animal carcasses, lymph nodes or
internal organs, the latter are subject to technical disposal, and the carcasses are
subject to disinfection by boiling or processing for meat loaves and preserves,
regardless of the type of salmonella. If there are dystrophic or other pathological
changes in the muscles of animals suffering from salmonellosis, the carcass with
internal organs is sent for disposal. Finished food products in which salmonella are
found (brawn, jellies, ham, etc.) are disposed of or destroyed.
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Fig. 6. Scheme for identifying Salmonella by their growth on “variegated series” media
(V.A. Makarov, M.F. Borovkov et al., 1987)
When E. coli and Proteus are isolated from the muscle tissue of slaughtered
animal carcasses, lymph nodes or internal organs, the internal organs are subject to
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technical disposal, and the carcasses are subject to disinfection by boiling or
processing into boiled or boiled-smoked sausage products. Sausages are cooked at a
temperature of 88–90 °C until the temperature inside the loaf reaches at least 75 °C.
If such cooking is not possible, the meat is sent for the production of meat loaves and
canned food. When E. coli is isolated only from internal organs, the latter are boiled
and the carcasses are released without restrictions. Finished food products containing
E. coli and Proteus are destroyed.
If bacteria from the genus Protea are detected in the deep layers of muscle
tissue or in the lymph nodes of the carcass, but with satisfactory organoleptic
characteristics, the meat is sent for boiling or making meat loaves. If organoleptic
indicators indicate putrefactive decomposition of meat, or if there is an unusual odor
that does not disappear when tested by cooking, carcasses and internal organs are
disposed of or destroyed.
Cl. perfringens. Criteria for sanitary assessment of products contaminated with
Cl. perfringens have not yet been developed. The presence of these bacteria is not
allowed in certain meat products (pasteurized canned food). When establishing
contamination with Cl. perfringens of meat and meat products must be boiled. It is
recommended to consider food products that are not subject to long-term storage as
benign if 1 g of the product is contaminated with up to 10,000 vegetative microbial
cells and up to 1,000 spores.
B. cereus. Criteria for the sanitary assessment of products contaminated with
B. cereus have not yet been developed. 1 g of raw material should contain no more
than 100 microbial cells. The presence of B. cereus in pasteurized products is not
allowed.
Control questions
1. What organs and tissues are taken from an animal carcass for bacteriological
research?
2. For what purpose is culture performed on Endo’s differential diagnostic
medium?
3. How to carry out serological typing of Salmonella?
4. What do you do with meat and internal organs if salmonella is detected?
5. How do foodborne toxic infections differ from toxicoses?
6. What is the growth pattern of coliform bacteria on Endo’s medium?
7. How do anthrax bacilli grow on MPA?
8. What is the sanitary assessment of meat if coli bacteria are detected only in
internal organs?
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CHAPTER 7. STUDY OF MEAT FOR TRICHINELLOSIS
Trichinosis is a dangerous anthrosoohelminthosis caused by 2 types of
Trichinella (TrichineIla spiralis and TrichineIla pseudospiralis), occurring acutely and
chronically. The entire development cycle of both species takes place in the body of
one host—the sexually mature stage is localized in the intestines, and the larval stage
is localized in muscle tissue.
Trichinosis affects pigs, wild boars, bears, other omnivores and carnivores
(dogs, wolves, foxes), marine mammals (whales, seals, walruses), insectivores,
rodents, and horses.
The following are subject to mandatory testing for trichinosis: carcasses, halfcarcases, quarters of pigs (except for piglets up to 3 weeks of age), wild boars,
badgers, bears, omnivorous and carnivorous animals, as well as nutria.
For post-mortem diagnosis of trichinosis, 2 research methods are used:
microscopic (compressor) and biochemical (digestion method), intravital diagnosis is
carried out by enzyme-linked immunosorbent assay (ELISA).
Meat and animal by-products (having muscle tissue) are examined by
microscopic or biochemical methods.
Lard (with the presence of muscle layers) is examined only by the microscopic
method.
The study of smoked meats, imported pork in blocks (with random control) and
other types of products is carried out only by the biochemical method.
The diagnosis of trichinosis is made based on the results of laboratory tests.
Rules for sampling muscle tissue for research on natrichinosis
For the study, samples are taken from the cruses of the diaphragm (at the
border of the transition of muscle tissue into the tendon), in their absence — parts of
the intercostal, cervical, chewing, lumbar, calf muscles, flexors and extensors of the
metacarpus, as well as the muscles of the tongue, esophagus and larynx; from marine
mammal carcasses — muscles of the tip of the tongue and eye, from horse meat
carcasses — muscles of the tongue or masseter, cruses of the diaphragm (among the
most affected).
The sample weight from each muscle group must be at least 5 g, and the total
sample weight from one animal must be at least 25 g.
Samples of salted and smoked bacon (if there is a cut or layers of muscle
tissue) are taken from each piece; the sample weight must be at least 25 g.
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Samples of smoked products are taken from 3 % of packaging units, making
10–15 recesses from each packaging unit, of which a combined sample is made.
Pork by-products (tongues, heads, legs, tails), in the absence of veterinary
confirmation of their origin from carcasses subjected to trichinoscopy, are examined
as follows: from 3 % of packaging units, take 10–15 recesses from each and make a
combined sample weighing at least 25 g.
In a batch of imported pork (in carcasses, half-carcasses), at least 10 % of the
product units are examined, samples are taken from the remains of the cruses of the
diaphragm or intercostal muscles. The mass of the muscle sample from a carcass or
half-carcass must be at least 1 g, the total mass of the sample for research must be at
least 25 g.
In a batch of imported pork in blocks, at least 1 % of the meat blocks are
examined; samples are taken in 25 pockets (1 g each) from a block with a total
weight of at least 25 g.
Samples are packaged in moisture-proof containers and delivered to the
laboratory on the day of collection.
Methods for testing meat for trichinosis
Microscopic examination (compressor trichinoscopy)
When studying meat and meat products, depending on the epizootic and
epidemiological situations in a particular area, from 24 to 96 sections of muscle tissue
are prepared: from pieces of selected muscles, with curved eye scissors along the
muscle fibers, sections of an oat grain size are made and placed in the middle
compressorium cells. The sections are covered with a second glass, the screws are
tightened, crushing the sections so that they become transparent and convenient for
their high-quality viewing.
When studying bacon with layers of muscle tissue, 24 sections of muscle are
made from each piece and placed in a Petri dish with 0.5 cm3 of a 1 % solution of
fuchsin in a 5 % solution of sodium hydroxide for 5–8 minutes. The sections are then
placed in a compressorium and viewed.
Sections are examined under low magnification (8 × 10) using a microscope or
a projection trichinelloscope.
When viewing the sections, capsules with trichynella larvae are found.
Normally encapsulated trichinella (one or more) are spirally coiled and
enclosed in a cavity surrounded by a capsule. Inside such a cavity contains a clear
liquid. The shape of the capsule can be different depending on the type of infested
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animal: round, oval, pear-shaped, lemon-shaped, spindle-shaped (Fig. 7). In fibers
adjacent to the trichinella cavity, the transverse striation disappears.
The larvae of acapsular trichinella have a specific configuration of location in
muscle fibers, and they are easier to detect at the edges of muscle sections and in the
tissue fluid surrounding the sections.
When trichinella remain in muscle fibers for a long time, they undergo
degenerative changes: calcification and germination by connective tissue.
Calcification begins from the poles of the capsule (Fig. 8); with severe calcification,
solid stones are formed.
а b c
Fig. 7. Trichinella larvae in muscles:
a — pigs; b — foxes; c — bear
Fig. 8. Calcification of Trichinella capsules
To clarify calcified capsules, muscle sections are placed in a Petri dish with a
5–10 % solution of hydrochloric acid. The cup is placed in a thermostat at a
temperature of 37 ± 1 °C for 20–30 minutes. The sections are then transferred to the
compressorium and viewed.
In salted, frozen and smoked meat and bacon, calcification of capsules and
degenerative changes in trichinella usually occur, which significantly complicates
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