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

15. MICROBIOLOGICAL ANALYSIS OF FOOD PRODUCTS

15.1. Determination of the Total Viable Count (TVC) of Mesophilic Aerobic and Facultatively Anaerobic Microorganisms

MICROBIOLOGICAL ANALYSIS OF food products is performed to detect the TVC by plating onto Petri dishes containing a nutrient medium (meat-peptone Agar) or to quantify specific groups of microorganisms by inoculation onto selective nutrient media. The microbial count is determined per 1 cm³ of a liquid product or per 1 g of a solid product.

Sampling and preparation of the average sample. An average sample is prepared for the analysis of a product batch. Standard Procedures and regulations have been established for sampling various food products. However, in all cases, conditions must be strictly observed to prevent contamination of the test product by extraneous microorganisms.

For liquid and semi-liquid products (milk, sour cream, sauces, etc.), the sample is thoroughly mixed and then transferred into sterile glassware using a sterilized ladle with a capacity of 50–100 cm3. Samples of solid products (meat, sausage, fish, culinary products, etc.) are taken from the bulk of the product at various locations. The surface of the sampling sites is first seared with a red-hot knife, and a deep incision is made through it using a sterile scalpel. The edges of the incision are separated with sterile forceps, and small pieces of the product are excised with sterile scissors. The samples collected from different locations in this manner are homogenized, combined in a sterile container, and used to form the average sample.

When taking average samples of products such as butter, cottage cheese, and cheese, a sterile core borer is used, which is inserted into the product from one edge and pushed diagonally to the opposite side. Then, using a sterile scalpel, small pieces are taken from several spots of the collected sample, minced, and placed into a sterile container.

Preparation of the sample for analysis. When analyzing solid products, a 1 to 10 g test portion is taken from the average sample, transferred to a sterile mortar, and thoroughly ground. If the product is very dense, it is ground in the mortar with pre-ignited quartz sand. The ground product is transferred into a flask containing 90 to 99 cm³ of sterile physiological saline (0.5% sodium chloride solution). The Contents of the flask are gently shaken for 5 min. This yields the first dilution of the product (1:10), from which subsequent dilutions are prepared.

A test portion of butter is melted in a Water bath at a Temperature not exceeding 45 °C, after which 1 cm³ of the product is added with a sterile pipette to 9 cm³ of physiological saline, yielding a 1:10 dilution.

To analyze liquid and semi-liquid products, 1 cm³ of the thoroughly mixed average sample is taken with a sterile pipette and transferred into a test tube containing 9 cm³ of sterile physiological saline.

Preparation of dilutions. Food products may contain a significant number of microorganisms; therefore, to obtain isolated colonies, tenfold dilutions of the product must be prepared.

The resulting primary dilution of the product is thoroughly mixed using a new sterile pipette by drawing the suspension in and expelling it back out. This Procedure is repeated 3–5 times; then, using the same pipette, 1 cm³ of the suspension is withdrawn and transferred to a 2nd test tube containing 9 cm³ of sterile physiological saline. This yields the 2nd dilution (1:100). The pipette must not be immersed deep into the liquid to prevent washing off microorganisms from its outer surface. Using a new sterile pipette, the contents of the 2nd dilution are thoroughly mixed, 1 cm³ is drawn from it and transferred to the next test tube containing 9 cm³ of sterile water, yielding the third dilution (1:1000). Subsequent dilutions are prepared in the same manner until the desired result is achieved.

Attention! A separate sterile pipette must be used for preparing each dilution. Neglecting this rule may lead to erroneous results.

Inoculation of Petri dishes. To determine the TVC, the pour plate method on solid nutrient media is used. The inoculation scheme is shown in Fig. 15.1. The test variant and dilution are marked on the Petri dish lids with a Glass marker. Each dilution is plated in at least 2–3 parallel dishes. Dilutions are selected so that between 30 and 300 colonies grow on a dish. Using a sterile pipette, 1 cm³ of the corresponding suspension dilution is transferred from the test tube into 2–3 empty sterile Petri dishes. After adding the suspension dilution, the dishes are poured with nutrient medium no later than 15 min. To do this, the cotton plug is removed from the test tube or flask containing the nutrient medium (melted and cooled to 45–50 °C) near a spirit lamp flame, and the neck is flamed. Then, the lid of the dish is slightly ajar—just enough to insert the neck of the flask—and 10–15 cm³ of nutrient agar is carefully poured in, maintaining a layer thickness of about 5 mm. The dish is covered with the lid and immediately swirled with gentle rotational movements to mix the nutrient medium with the inoculum, after which it is left in a horizontal position for 10–15 min to allow the medium to solidify. The inoculated dishes are inverted and placed in an incubator at 37±1 °C for 24–48 h.

Class="center">Fig. 15.1. Scheme of product inoculation for TVC determination

Counting grown colonies. After incubation, dishes containing between 30 and 300 colonies are selected and counted visually or using a specialized colony counter. During counting, the dishes are examined against transmitted light, and the counted colonies are marked with ink or waterproof marker. If more than 300 colonies have grown on the dish with the highest dilution, they can be counted using a magnifying glass and a Plexiglas grid with a 1 cm square side under side illumination. Colony counting is performed in at least 20 squares, the average number per 1 cm² is determined, and it is multiplied by the surface area of the medium in the dish.

The number of microorganisms in 1 cm³ (or 1 g) of the product (TVC) is calculated as the product of the number of grown colonies (N) and the dilution factor (n):

TVC = N •10ⁿ CFU/cm³.

Note. Anaerobic microorganisms are not quantified by the pour plate method, as specific anaerobic conditions must be established for their growth. Cultivation of anaerobes is carried out in anaerobic jars (see Fig. 4.1) or by using nutrient media supplemented with reducing agents.

15.2. Determination of Coliform Bacteria (Coliforms) in Food Products

The determination of coliforms in food products is carried out in accordance with GOST R 50474-93: Food products. Methods for the detection and enumeration of coliform bacteria.

The method for coliform determination is based on detecting gas and acid production when a specific amount of a product (or surface wash) is inoculated into liquid nutrient media containing lactose (Kessler medium), followed by confirming the belonging of the grown microorganisms to the coliform group based on morphological and cultural characteristics.

Testing for the presence of coliforms is carried out in several stages:

Stage I - preparation of product dilutions. If the product is solid (sausage, cheese, butter, cottage cheese, etc.), the result is expressed as the absence of coliforms in a specified mass (g). If the product is liquid (milk, fermented milk drinks, juice, etc.), coliforms must be absent in a specified volume (cm³ or dm³).

Stage II — inoculation of a specified amount of the product or its dilutions into Kessler medium. Kessler medium is dispensed into test tubes or flasks containing a Durham tube (a small glass tube

with one sealed end). After sterilization, the Durham tube must be completely filled with the medium.

A 1 cm³ volume of each product dilution (10^-1; 10^-2; 10^-3) is added to the tubes containing Kessler medium and incubated in a thermostat at (37±1) °C. After 24 hours of incubation, the cultures are examined for gas production in the Durham tube and a color change of the medium from purple to yellow-green. The dilutions showing gas production are recorded.

Stage III — confirmation that the microorganisms grown in Kessler medium belong to coliforms (coli-aerogenes bacteria). Subcultures from the fermented tubes of Kessler medium are streaked onto the surface of Endo agar using an inoculation loop to obtain isolated colonies. For this purpose, the bottom of the Petri dish is divided into 4–8 sectors using a glass-marking pencil. A separate sector is inoculated from each tube. A minimum amount of the test material is taken from the tube with a bacteriological loop and streaked in a zig-zag pattern within the sector, without lifting the loop from the agar surface or damaging it. The Petri dishes are labeled and placed in a thermostat (bottom side down) at 37±1 °C for 18–24 hours.

Following incubation, the plates are examined. Coliform bacteria form red or pink shiny colonies on Endo agar, with or without a metallic sheen. This characteristic growth on Endo agar is due to lactose Fermentation and acid production, which reduces the fuchsin indicator and colors the colonies red.

Smears are prepared from typical colonies on Endo agar, Gram-stained, and examined microscopically. If small, Gram-negative, non-spore-forming rods are observed, an oxidase test is performed concurrently. The Conclusion that the microorganisms found in the product belong to coliforms is made based on the presence of Gram-negative, non-spore-forming rods that ferment lactose with acid and gas production at 37±1 °C and yield a negative oxidase test. The mass (in g) or volume (in cm³) of the tested sample is indicated accordingly. If no colonies typical of coliforms appear on Endo agar, the product is considered free from coliform contamination.

15.3. Determination of Yeast and Mold Counts

To determine the count of Yeasts and Molds in food products, appropriate dilutions are inoculated into Petri dishes and mixed with melted and cooled wort agar (WA) or Sabouraud agar maintained at 45–50 °C. The contents of the dishes are mixed with gentle swirling motions and allowed to solidify for 10–15 minutes, after which they are placed in a thermostat at 30±2 °C for 2–3 days. Following incubation, the mold and yeast colonies that have grown are counted separately. The results are expressed as CFU/cm³ (or g).

Review Questions

1. What is QMAFAnM (total viable count)?

2. Why are dilutions of a food product prepared when determining microbial counts?

3. What culture media are used to determine QMAFAnM, yeast, and mold counts?

4. What are the stages involved in detecting coliforms in food products?

5. What media are used for the detection of coliforms in food products?

6. What indicators are used to confirm coliform growth in Kessler medium?

7. What do coliform colonies look like on Endo agar?

8. Under what conditions is a positive result reported for the presence of coliforms in a food product?