FUNDAMENTALS OF MICROBIOLOGY - E. Yu. Tyumentseva - 2015

TOPIC 9. MICROBIOLOGY OF NON-FOOD COMMODITIES

Water used in food Processing facilities must comply with the sanitary regulations (SanPiN 2.1.4.1074-01) established for drinking water.

To assess the sanitary condition of water, the following parameters are determined: total microbial count - not exceeding 50 CFU/cm³; thermotolerant coliform Bacteria - not allowed in 100 cm³; total coliform bacteria must also be absent in 100 cm³; spores of sulfite-reducing clostridia - not allowed in 20 cm³;

coliphages - not allowed in 100 cm³. Drinking water testing is performed once per quarter when connected to a municipal water supply system, and once a month when using private water sources.

Total viable count of water (TVC) - the number of mesophilic aerobic and facultatively anaerobic microorganisms capable of forming colonies on nutrient Agar at 37 °C within 24 hours.

Total coliform bacteria comprise Gram-negative, non-spore-forming rods lacking oxidase activity, which ferment lactose or mannitol with The production of aldehyde, acid, and gas at 37 °C within 24 hours.

Thermotolerant coliform bacteria possess all the characteristics of total coliform bacteria and, in addition, are capable of fermenting lactose to acid and gas at 44 °C within 24 hours.

Sulfite-reducing clostridia (primarily Clostridium perfringens) are spore-forming, anaerobic, rod-shaped bacteria that reduce sodium sulfite on iron sulfite agar within 24 hours at 44 °C.

Coliphages - bacterial Viruses capable of lysing Escherichia coli and forming lysis zones after 18±2 hours at 37 °C on a bacterial lawn grown on nutrient agar. Coliphages serve as indicators of drinking water Treatment efficiency regarding enteroviruses.

Methods for water disinfection include chlorination, ozonation, and ultraviolet irradiation.

Laboratory Procedure

Objective: to master microbiological Methods for Assessing the sanitary condition of water, containers, and packaging Materials.

Materials, Reagents, and equipment: thermostats; microscopes; spirit burners; hot plates; swab test tubes for preparing washings; forceps; test tubes with Kessler medium; Hajna (Koda) medium; sterile Petri dishes; sterile 1 cm³ pipettes; sterile 500 cm³ flasks; culture media: nutrient agar, Endo agar, wort agar or Sabouraud agar, lactose peptone broth, iron sulfite agar; bacteriological loops; Glass slides; Petri dishes; Gram staining kit; filter paper; membrane filters; 96% ethanol.

Experiment No. 1. Assessment of the Sanitary Condition of Water

a) Sampling. The tap or the end of the discharge pipe is sterilized by flaming with a cotton swab soaked in alcohol. The tap is opened and the water is allowed to run for 10-15 minutes, after which a sample is collected into a sterile flask (sample volume not less than 500 cm3). The flask is sealed with a cotton-gauze plug near the flame.

b) Determination of the total viable count (TVC). Performed According to the procedure described in Section 7.6.

For inoculation, 1 cm³ of water is transferred into each of two Petri dishes and mixed with molten nutrient agar (NA) cooled to 45-50 °C. Following incubation, the colonies in each dish are counted, and the results are averaged by summing them and dividing by two.

c) Determination of total and thermotolerant coliform bacteria using the Membrane filtration method. This method is based on filtering a specific volume of water through membrane filters, cultivating the microorganisms on a differential diagnostic medium, and subsequently identifying the colonies grown.

For the analysis, precisely 100 cm³ of water is measured and filtered through a sterile membrane filter. Upon completion of filtration, the filter is carefully lifted with flamed forceps and transferred to a Petri dish containing Endo agar. The surface of the filter with the retained microorganisms must be

facing upwards. The dishes with filters are placed upside down in a thermostat and incubated at 37 °C for 24 hours.

The result is considered negative if no colonies grow on the filters at all, or if colonies with irregular edges and surfaces appear.

In the presence of typical lactose-positive colonies leaving an imprint on the reverse side of the membrane filter and the medium (dark red, red with a metallic sheen), as well as lactose-negative pink colonies without imprints, the number of colonies of each type is counted.

For identification, at least 5 colonies of each type are selected and subcultured onto nutrient agar slants. The cultures are incubated at 37 °C for 16-18 hours. Subsequently, biochemical tests are performed on the pure cultures: the oxidase test and tests for acid and gas production via lactose Fermentation at 37 °C for total coliform bacteria and at 44 °C for thermotolerant coliform bacteria.

d) Determination of total and thermotolerant coliform bacteria by the titration (most probable number) method. The titration method (fermentation tube method) relies on the enrichment of bacteria after inoculating a defined water volume into a liquid enrichment medium, followed by subculturing onto a differential diagnostic medium to identify the grown colonies.

The titration method can be used:

- in the absence of materials and equipment required for analysis by the fermentation tube method;

- when analyzing water with a high content of suspended solids;

- in cases where extraneous microflora predominates in the water, preventing the isolation of total coliform colonies on filters.

The First stage of the study involves inoculating 3 volumes

of water, 100 cm³ each, into concentrated lactose peptone broth. The cultures are incubated at 37 °C for 24–48 hours to determine total coliforms, and at 44 °C for 24±2 hours to determine thermotolerant coliforms. If no signs of growth are detected in the flasks (indicated by the absence of gas in the fermentation tubes), coliform bacteria are absent in 100 cm³.

At the Second Stage of the study, from the test tubes and flasks showing growth and gas production, loop inoculations are streaked onto Endo agar sectors. Cultures on Endo agar are incubated at 37 °C for 18–20 hours. The absence of colonies typical of coliform bacteria yields a negative result regarding the presence of these Microorganisms in the tested water volume.

d) Determination of spore-forming sulfite-reducing clostridia by direct inoculation. From each water sample, 20 cm³ is inoculated into iron sulfite agar. For this purpose, either 10 cm³ is added to each of 2 test tubes with a capacity of 30 cm³, or 5 cm³ to each of 4 test tubes with a capacity of 15 cm³. The cultures are overlaid with hot (75–80 °C) iron sulfite agar in an amount exceeding the water volume by a factor of 2. The medium is poured along the wall of the test tube to avoid The formation of air bubbles. Afterward, the test tubes are rapidly cooled by placing them in a container with cold water to create anaerobic conditions. The cultures are incubated at 44 °C for 24±2 hours. If no blackening of the medium is observed in the test tubes after incubation, it is considered that sulfite-reducing clostridia

are absent in 20 cm³.

Experiment No. 2. Quality control of glassware and equipment

To analyze the sanitary condition of glass bottles and jars, a rinse sample is prepared by washing the inner surface of 10 consecutive items with a total of 20 cm³ of water. The sanitary condition of barrels, milk cans, and cisterns is checked by inoculating the final rinse water. Rinses from small equipment (stirrers, samplers, thermometers, etc.) are prepared by swabbing the entire surface with a sterile swab, whereas for shelves, trays, buckets, and shovels, a template method is used. Total bacterial count and the presence of coliforms are determined in the rinses. Coliform bacteria must be absent in the rinse samples.

Experiment No. 3. Quality control of packaging materials

Parchment, foil, film, and combined food packaging materials are unwrapped, and a rinse sample is taken from the inner surface using a sterile cotton swab (from 100 cm³ of surface area). The presence of microscopic Fungi and coliform bacteria is determined. Coliforms must be absent in the rinses, and the mold count must not exceed 5 per 1 cm³ of rinse.

Presentation and analysis of research results

Perform microbiological analyses and draw Conclusions regarding the microbiological safety of water, packaging materials, glassware, and equipment.

Upon completing this topic, students will be able to assess the microbiological safety of water, packaging materials, glassware, and equipment.

Review Questions

1. What microbiological parameters are determined in drinking water according to state standards (GOST) to assess its sanitary condition?

2. How often is drinking water tested to evaluate its sanitary condition?

3. How are rinse samples prepared from equipment to assess its sanitary condition?

4. What microbiological indicators are determined in rinses from glassware?

5. How is MICROBIOLOGICAL CONTROL OF packaging materials carried out?

6. What is the total viable count (TVC)?

7. Define coliphages.

8. What water disinfection methods do you know?

9. How are the coli-titer and coli-index determined in drinking water?

10. What is the titration METHOD FOR DETERMINING the content of total and coliform bacteria based on?