GENERAL AND FOOD MICROBIOLOGY PART II - L. V. Krasnikova - 2016

TOPIC 1. MICROBIOLOGICAL EXAMINATION OF FRESH MEAT

1.1. Microflora of fresh meat

At meat Processing plants, MICROBIOLOGICAL ANALYSIS OF meat and meat products is carried out for preventive purposes or as a mandatory measure when it is necessary to detect the presence of pathogenic and opportunistic Microorganisms in the meat, as well as when technological process violations are suspected. Raw Materials, finished products, containers, equipment, and the hands of workers directly handling meat products are all subjected to microbiological examination.

Meat serves as an excellent nutrient medium for the vital activity of numerous microorganisms. High values of Water activity (a_w - 0.98-0.99) and pH (from 5.5 to 6.5) are also favorable for the multiplication of microorganisms in fresh meat.

As a rule, microorganisms are not present in the Blood, Muscle tissue, and Internal Organs of healthy animals, whose bodies possess high resistance to their penetration. There are two pathways of animal Organ and tissue contamination by microorganisms: endogenous and exogenous.

Endogenous contamination of meat with microbes is observed in animals suffering from infectious diseases—posing a significant danger to humans—as well as in weakened and exhausted animals. In animals slaughtered in a state of severe exhaustion, microorganisms are found in virtually all organs and Tissues.

Exogenous contamination of meat and organs by microorganisms occurs after slaughter during subsequent operations (skinning, carcass dressing, and Evisceration) and in the course of final meat processing.

The microflora of warm-slaughtered meat from healthy animals is represented by a limited number of microorganisms, yet it is quite diverse in species composition. Bacteria frequently found on fresh meat belong to the genera Pseudomonas, Acineto- bacter/Moraxella, Clostridium, Aeromonas, Psychrobacter, Enterobacteriaceae, Micrococcaceae, Bacillus, Shewanella, Brochotrix, Lactobacillus, Carnobacterium, Leuconostoc, and Weissella. From 10³ to 10⁵ microbial Cells can be counted per 1 cm² of meat surface. The dominant forms are non-spore-forming Gram-negative rods: pseudomonads, flavobacteria, aeromonads, coliform bacteria, and Proteus species. Among Gram-positive bacteria, lactic acid bacteria are most commonly detected, while aerobic and anaerobic spore-forming bacteria, as well as Yeasts of the genera Candida, Cryptococcus, Rhodotorula, Torulopsis, and mold fungal spores, are found in significantly smaller quantities. Many of these microorganisms possess active proteases and lipases that catalyze the Hydrolysis of meat Proteins and Lipids.

Characteristically, out of the large number of contaminant microorganisms in fresh meat, only a few species actively multiply, eventually becoming dominant and forming the "spoilage microorganism association." The species composition of this association depends on the type of biological interactions, internal and external factors, and the technological parameters of meat processing.

Meat can also be contaminated with toxigenic microorganisms, such as Clostridium perfringens, Salmonella typhimurium, Bacillus cereus, and enterococci. If these bacteria multiply significantly, the meat can cause food poisoning.

Bacteriological examination OF meat and meat products is conducted to prevent:

✵ infection of workers involved in livestock slaughter and meat processing;

✵ the consumption of meat and meat products obtained from animals that had infectious diseases;

✵ the release into distribution of products whose consumption could cause foodborne toxicoinfections or toxicosis in humans, as well as the spread of infections.

Microbiological analyses are carried out in the plant's bacteriological laboratory in accordance with current state standards (GOSTs) or regulatory and technical documentation. MICROBIOLOGICAL EXAMINATION OF fresh meat begins with the microscopic analysis of smear-prints (bacterioscopic method), followed by a full bacteriological analysis.

1.2. Bacterioscopic examination of meat

Sampling. To determine the freshness of meat, at least three samples weighing no less than 200 g each are taken as whole pieces from the Muscles of the thigh, shoulder blade, and the region of the 4th-5th cervical vertebrae of each carcass or half-carcass. In addition to muscle tissue, the samples must include tendons and fat.

For bacteriological examination for anthrax, the following are submitted: the Lymph node draining lymph from the site of the affected lesion, edematous tissue, and, in pigs, additionally the submandibular lymph node.

For examination for Listeriosis, the Brain, a lobe of the Liver, and a Kidney are submitted.

When examining a half-carcass or a quarter-carcass, a piece of muscle, Lymph Nodes, and a tubular bone are taken.

Each sample is packaged separately in parchment paper, labeled with the date, sampling Location, animal species, carcass number, and the reason and purpose of the examination, and sent to the laboratory.

The bacterioscopic method is a set of techniques for detecting and studying the morphological and tinctorial properties of bacteria (microbes) under laboratory conditions. In laboratory practice, the following types of microscopic preparations are used: smear-print, hanging drop, crushed drop, thin smear, and fixed stained preparation.

Bacterioscopic examination of meat is performed in cases of discrepancies between biochemical and organoleptic evaluation Methods. The Need for bacterioscopic examination of meat freshness is eliminated if the organoleptic analysis yields a negative result.

At least two smear-prints must be prepared from each meat sample.

To prepare an impression smear from the surface layer (at a depth of 2–3 cm), a small piece of meat weighing 2–3 g is cut out using sterile scissors or a scalpel, and its freshly cut inner side is pressed against a previously flamed Glass slide.

When preparing impression smears from deep layers, The surface of the meat sample must first be sterilized (moistened with alcohol and flamed, or seared with a heated metal spatula). Then, using a sterile instrument, small pieces of meat measuring 2 x 1.5 x 2.5 cm are excised from the interior to prepare the impression smears.

The impression smears prepared on glass slides must be air-dried, fixed in the flame of a burner or spirit lamp, and stained using the Gram method. Each impression smear is examined under a Microscope with an immersion objective in at least 25 different fields of view.

During microscopic examination, the number of bacterial cells (cocci and rods) and yeasts is counted separately in each viewed field of view; the result is the average Total Cell Count across the twenty-five fields of view. The presence or absence of muscle tissue decay traces in the microscopic field of view is also noted. The Microscopy results are evaluated in accordance with the data presented in Table 1.1.

Class="center">Table 1.1. Evaluation of the Results of Bacterioscopic Meat Analysis

Note. If gram-positive rods with truncated ends are detected in the impression smears, they are stained with a 2% safranin solution. The presence of rods or chains with capsules in safranin-stained smears indicates the presence of the anthrax pathogen.

1.3. Bacteriological Examination of Meat

If no bacteria resembling anthrax bacilli are found in the impression smears, inoculations are performed from meat and offal samples onto nutrient media to detect foodborne toxicoinfection agents (bacteria of the genera Escherichia, Proteus, Salmonella), zoonotic pathogens (anthrax bacilli, listeriosis bacteria, swine erysipelas, etc.), and anaerobes (pathogenic and toxigenic clostridia).

During bacteriological examination, each sample is stripped of adipose and Connective Tissue, immersed in alcohol, and then sterile scissors are used to excise pieces measuring 2.0 x 1.5 x 2.5 cm from the interior at various locations. All excised pieces are then minced with sterile scissors. For inoculation, 15 g samples are prepared. One sample consists of pieces of muscle and lymph nodes, while the other consists of pieces of parenchymatous organs. A suspension containing 0.5 g of the product per 1 cm³ is prepared from each sample in a sterile mortar.

1.3.1. Determination of Total Viable Count (TVC / Total Mesophilic Aerobic and Facultative Anaerobic Microorganisms)

The total microbial count refers to the quantity of mesophilic aerobic and facultative anaerobic microorganisms (TVC) per 1 g of the product. The determination method is based on the ability of mesophilic aerobic and facultative anaerobic microorganisms to multiply on solid nutrient Agar at a Temperature of 37±1 °C for 72 hours. The amount of the product inoculated is determined taking into account the most probable microbial contamination. When examining fresh meat, dilutions ranging from 10^-1 to 10^-3 are inoculated into the nutrient medium.

To inoculate 0.1 g of the product (10^-1 dilution), the first tenfold dilution of the suspension is prepared: 1 cm³ of the suspension is drawn up with a sterile pipette and transferred into a test tube containing 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.1 g of the product).

To inoculate 0.01 g of the product (10^-2 dilution), the second tenfold dilution is prepared: the Contents of the test tube with the first dilution are mixed using a sterile pipette, 1 cm³ is drawn up and transferred into a test tube with 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.01 g of the product).

To inoculate 0.001 g of the product (10^-3 dilution), the third tenfold dilution is prepared: the contents of the test tube with the second dilution are mixed using a sterile pipette, 1 cm³ is drawn up and transferred into a test tube with 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.001 g of the product).

A 1 cm³ volume of each dilution is inoculated into Petri dishes with pre-labeled lids and poured over with 10–15 cm³ of meat-peptone agar (MPA) melted and cooled to a temperature of 40–45 °C. Immediately after pouring the agar, the contents of the Petri dishes are thoroughly mixed by gentle swirling to evenly distribute the inoculum. After the agar solidifies, the Petri dishes are inverted with their lids facing down and placed in an incubator at 37 °C for 72 hours.

Upon completion of cultivation, the number of colonies grown on the MPA plates is counted. A magnifying glass with 4–10x magnification or a specialized colony counter is used for this purpose. If there is A large number of colonies and their distribution in the agar is even, four or more identical sectors are drawn on the bottom of the Petri dish, the number of colonies in two or three sectors is counted (but on at least 1/3 of the dish surface), the arithmetic mean of the colony count is calculated and multiplied by the total number of sectors of the entire dish.

The number of mesophilic aerobic and facultative anaerobic microorganisms in 1 cm³ of fresh meat (X) is calculated using the formula

X = n x 10^m,

where n is the arithmetic mean number of colonies counted on Petri dishes with different dilutions of the product; m is the number of tenfold dilutions.

1.3.2. Determination of Coliform Bacteria (Coliform Group)

Currently, the coliform group includes the following genera from the family Enterobacteriaceae: Escherichia, Enterobacter, Citrobacter, Klebsiella, Serratia.

Coliforms are small, non-spore-forming, Gram-negative, motile rods with peritrichous flagella (with the exception of the genus Klebsiella, which lacks flagella), forming neither spores nor capsules (except for bacteria of the genus Klebsiella, whose cells are surrounded by a capsule), and are facultative anaerobes. Representatives of this family possess catalase and Cytochromes, and can obtain energy through both Respiration and Fermentation processes. They carry out mixed-acid fermentation with the accumulation of CO₂, H₂O, and acids (acetic, lactic, succinic).

The METHOD FOR DETERMINING coliforms is based on detecting The formation of gas or acid in an elective nutrient medium containing lactose (Kessler, Heifetz, or Koda media). The method for enumerating coliforms is known as the fermentation method, The Essence of which consists of inoculating a specific amount of the product or its dilutions into a liquid nutrient medium, followed by incubation at 37 °C and detecting gas in fermentation tubes accompanied by a color change of the medium.

Upon gas detection in the test tubes during the Second Stage of the study, the material from the fermented tubes is subcultured onto Endo agar. The subculture is performed using a bacteriological loop in a dense streak to obtain isolated colonies. Petri dishes with the inoculations are incubated at 37 °C for 24 hours, after which the cultures are examined. On Endo agar, coliforms grow to form bright red, glossy colonies with or without a metallic sheen.

If Endo agar shows no characteristic bright red colonies of coliform bacteria (both with and without a metallic sheen), the result for the presence of coliforms is considered negative, and the testing is discontinued. If colonies typical of coliform bacteria are found on Endo agar, the isolated microorganisms are identified as members of the Enterobacteriaceae family. To confirm this, an oxidase test is performed (which must be negative for coliforms). Next, a fixed smear is prepared from the colonies suspected of being coliforms, Gram-stained, and examined under a microscope. Microscopic examination should reveal small, Gram-negative rods with rounded ends. In this case, the test for the presence of coliforms in the product is deemed positive.

1.3.3. Detection of Bacteria of the Genus Proteus

The presence of large numbers of *Proteus* bacteria in fresh meat indicates putrefactive decomposition of meat proteins.

Bacteria of the genus *Proteus* also belong to the family Enterobacteriaceae and are polymorphic Gram-negative rods measuring 0.4–0.8 × 1–3 µm. They are motile due to peritrichous flagella. Most strains exhibit "swarming" motility—spreading as a uniform film across the moist surface of an agar growth medium. *Proteus* bacterial cultures have a specific putrid odor.

To detect *Proteus* in meat and meat products, Shukevich's inoculation method is used: scrapings from the surface of a freshly cut test sample are inoculated into the Condensation water of a freshly slanted nutrient agar (NA) tube, without touching the agar surface. The tubes are placed vertically in an incubator at 37 °C for 18–24 hours. The appearance of a veil-like growth on the medium, which microscopy reveals to consist of motile, Gram-negative rods, indicates the presence of *Proteus* in the test samples.

The biochemical properties of bacterial species belonging to the genus *Proteus* are presented in Table 1.2.

Table 1.2. Biochemical properties of bacteria of the genus Proteus

* кг — carbohydrate fermentation produces acid and gas; к — carbohydrate fermentation produces acid.

It should be noted that *Proteus rettgeri*, a species formerly included in this group, has now been reassigned to the genus *Providencia*.

During microbiological monitoring, the total count of all *Proteus* species is determined collectively.

General Characteristics of Bacteria of the genus *Proteus* include positive methyl red and Voges-Proskauer reactions, production of indole and hydrogen sulfide, The ability to hydrolyze urea, and the inability to ferment lactose and mannitol.

1.3.4. Detection of Bacteria of the Genus Salmonella

Bacteria of the genus *Salmonella* rank first among microorganisms responsible for foodborne poisoning.

The genus *Salmonella* is part of the family Enterobacteriaceae. Salmonellae appear as small Gram-negative rods measuring 0.7–1.5 × 2–5 µm, typically motile via peritrichous flagella. Their METABOLISM is both respiratory and fermentative; they lack oxidase but possess catalase. Characteristic Features of salmonellae include a positive methyl red reaction, a negative Voges-Proskauer reaction, the ability to utilize citrates, and the inability to produce indole.

To detect salmonellae, a 25 g analytical portion of the pooled sample is placed into a bottle containing 100 cm³ of enrichment broth (Kauffmann, Müller, Kilián, magnesium chloride "M", or selenite F broth) and incubated at 37 °C. For selenite F broth, the optimal temperature for salmonella enrichment is 43±1 °C. After 16–24 hours, subcultures from the enrichment broth are streaked onto Endo, Levine, Ploskirev, or bismuth sulfite agar (BSA) (see Appendix, pp. 1.5–1.8), spreading the inoculum evenly across the medium surface using a microbiological spreader. The inoculated plates are incubated at 37±1 °C for 20–24 hours. Salmonellae do not ferment lactose and exhibit typical growth on selective-differential media:

✵ on Endo agar, salmonellae grow as round, colorless or slightly pink, transparent or translucent colonies;

✵ on Levine agar, salmonellae form transparent, pale, soft pink, or pinkish-violet colonies;

✵ on Ploskirev agar, salmonella colonies are colorless, and the medium turns yellow in areas of abundant growth;

✵ on bismuth sulfite agar, salmonellae form black or brown colonies with a characteristic metallic sheen. Additionally, the agar beneath the colony develops a black discoloration.

When suspicious colonies are found on the plates, a portion of a colony is used to prepare a fixed smear, which is then Gram-stained and examined microscopically.

1.3.5. Detection of Anaerobic Bacteria in Meat

Testing for anaerobic infections involves examining smears prepared directly from meat or pathological material inoculated onto nutrient media. When examining bacterioscopic imprint smears stained with methylene blue or by the Gram method, attention is paid to the presence of rod-shaped Gram-positive bacteria, the presence of endospores, and their intracellular position. The detection of thick, Gram-positive rods with rounded ends, or spore-forming rods resembling a candle flame or a tennis racket in the imprint preparations, indicates the potential presence of *C. botulinum*.

For bacteriological analysis, a 10 g meat sample is weighed, ground in a mortar with twice the volume of sterile saline to prepare a suspension. Four tubes of Kitt-Tarozzi medium (see Appendix, p. 1.1) are required per sample; these are pre-heated (regenerated) in a boiling water bath for 20–30 minutes and rapidly cooled to 50 °C. Each tube is inoculated with 3–5 cm³ of the prepared suspension. Afterward, two of the inoculated tubes are heated at 80 °C for 30 minutes to destroy the vegetative microflora and detect spore-forming anaerobes, while the other two tubes are left unheated. The cultures are then placed in an incubator at 37±1 °C for 5–10 days.

When testing for the presence of type E botulinum toxin producers (*Clostridium botulinum*), inoculation is similarly performed in four tubes of Kitt-Tarozzi medium; however, prior to incubation, one tube is heated at 80 °C for 30 minutes, and another at 60 °C for 15 minutes (which preserves the spores of *C. botulinum* type E). The remaining two inoculated tubes are left unheated. The heat-treated tubes are incubated at 28±1 °C, whereas the other two are incubated at 37±1 °C. The cultures are inspected daily. Growth of *C. botulinum* is characterized by gas production and turbidity of the medium. When growth is detected, Gram-stained smears are prepared for microscopic examination.

Microbiological parameters of meat and meat semi-finished products are presented in Table 1.3.

Table 1.3. Microbiological parameters of meat and meat semi-finished products (TR CU 034/2013)

Assignment on the topic:

1. Perform bacterioscopic analysis of fresh meat. Draw a Conclusion regarding the freshness of the tested meat samples.

2. Perform bacteriological analysis of fresh meat. Inoculate nutrient media to determine the total viable count (TVC), coliform bacteria, and bacteria of the genus Proteus using Shukevich's method.

3. Review the methods for detecting bacteria of the genera Salmonella and Clostridium in meat.

Review Questions

1. What microbiological methods are used to determine the wholesomeness of meat?

2. Under what circumstances is bacterioscopic analysis of meat and meat products performed?

3. What does bacteriological analysis of meat entail?

4. Which groups of microorganisms are determined in fresh meat?

5. What are the growth characteristics of salmonellae on Endo agar, Levin's agar, Ploskirev agar, and bismuth sulfite agar?

6. In which cases is it necessary to test meat for the presence of anaerobic bacteria?