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

13. SANITARY AND MICROBIOLOGICAL SOIL ANALYSIS

Objective: to conduct a microbiological study of soil as a source of food contamination by foreign microorganisms.

Soil is densely populated with microorganisms. It contains all forms of microorganisms existing on Earth: Bacteria, Viruses, actinomycetes, Yeasts, Fungi, and Protozoa. The total bacterial count (TBC) in 1 g of soil can range from 1.0 to 10 billion. The concentration of microorganisms varies across different soil layers. In the uppermost layer (0.5 cm), microorganisms are very scarce. At a depth ranging from 1-5 cm to 30-40 cm, the number of microorganisms reaches its maximum, averaging from 10 to 50 million per 1 g. Below 30-40 cm, the TBC gradually decreases, reaching a minimum in the deeper layers.

Soil microflora is divided into 2 groups:

1) autotrophic - utilizing mineral nutrients;

2) heterotrophic - utilizing organic nutrients.

Both groups participate in soil self-purification and mineralization processes. However, the heterotrophic group may harbor pathogenic microflora. When soil is contaminated with feces from individuals suffering from intestinal infections, plant-based products can become contaminated with pathogens of dysentery, cholera, typhoid fever, salmonellosis, and enteroviruses. A direct correlation has been established between the incidence of intestinal infections in humans and animals and the poor sanitary condition of the soil. Soil acts as a transmission vector for pathogens of such infectious diseases as anthrax, gas gangrene, tetanus, etc.

Sanitary and microbiological soil analysis involves determining the total bacterial count, coli-titer, perfringens titer, titer of nitrifying bacteria, as well as the Abundance of Proteus and thermophilic bacteria. The bacterial count characterizes the degree of organic soil pollution. The presence of coliform bacteria indicates fecal contamination. The detection of Clostridium perfringens also points to fecal contamination of the soil. The presence of bacteria of the genus Proteus suggests potential contamination of the soil with organic matter of animal origin or human feces. The presence of thermophilic microorganisms indicates contamination with manure or composts. Thermophilic microorganisms are typically absent in clean soils.

Soil sampling. When studying surface soil layers, samples are collected at a depth of 15-20 cm after removing the top 2 cm layer (at least 10 samples from various locations within the test area). Samples are collected using a small metal trowel or scoop into sterile wide-mouthed jars wrapped in paper and labeled. Each individual sample should weigh 200-300 g, and the composite sample (average sample) should be at least 1 kg.

Preparation of soil for analysis. Soil samples are freed from large inclusions, crushed, passed through a sterile 3-mm sieve, and then poured onto sterile paper, thoroughly mixed, and a 10 g sample is weighed out. The soil sample is placed into a 250 cm³ flask containing 90 cm³ of sterile tap Water. This yields a 1:10 dilution, corresponding to 0.1 g of the tested soil. The flask is shaken for 10 minutes, allowed to settle for 30 seconds to precipitate coarse soil particles, and then 3 to 6 tenfold dilutions are prepared depending on the level of soil contamination.

13.1. Determination of the bacterial count of the soil

Using two sterile Petri dishes with lids slightly ajar, 1 cm³ of the 10^-4 and 10^-5 soil suspension dilutions is added to each, followed by pouring molten nutrient Agar (cooled to 45 °C, either NA or potato glucose agar). After the agar solidifies, the dishes are placed in an incubator for 24-48 hours at 37±2 °C, and then kept for an equal duration at room Temperature. The number of colonies grown on the plates is counted, and the bacterial count per 1 g of soil is calculated taking into account the dilution plated.

13.2. Determination of the soil coli-titer

The presence of coliform bacteria indicates recent fecal contamination of the soil. The coli-titer is defined as the smallest amount of soil (in grams) in which coliform bacteria are detected. The coli-index of the soil refers to the number of coliform bacteria per 1 g of soil.

To determine the coli-titer, 1 cm³ aliquots of soil suspension dilutions ranging from 10^-1 to 10^-5 are inoculated into tubes containing Kessler medium equipped with Durham tubes. The cultures are incubated at 37 °C for 18-24 hours, after which gas accumulation in the Durham tubes is recorded.

13.3. Determination of the soil perfringens titer

The perfringens titer is the titer of Gram-positive obligate anaerobic spore-forming bacilli that reduce sulfites. The presence of C. perfringens (sporal forms) indicates remote fecal contamination.

Iron sulfite agar (Wilson-Blair medium) is used to determine the perfringens titer. The detection of C. perfringens on this medium is based on the microorganism's ability to reduce Na₂S₂O₃ to _Na2S, which reacts with ferric chloride to form black iron sulfide (FeS).

Tubes containing soil suspension dilutions are heated in a water bath at 80-85 °C for 15 minutes. The tubes are then cooled to room temperature, and 1 cm³ aliquots of soil suspension dilutions from 10^-1 to 10^-4 are inoculated into tubes with Wilson-Blair medium melted and cooled to 45 °C. By rolling the tubes between the palms, the soil suspension is evenly distributed throughout the medium, after which the tubes are cooled in running tap water until the agar solidifies. Cultures are incubated for 18-24 hours at 43 °C. The presence of C. perfringens is assessed by the growth of black colonies in the medium-containing tubes.

13.4. Determination of the number of thermophilic Microorganisms in the soil

To determine the thermophilic bacteria index, 1 cm³ of soil suspension dilutions from 10^-1 to 10^-3 is added to sterile Petri dishes and poured with NA melted and cooled to 45 °C. After agar solidification, the dishes are incubated for 24 hours at 60 °C, after which the number of grown colonies is counted and the number of thermophilic bacteria per 1 g of soil is determined factoring in the dilution.

13.5. Detection of Proteus bacilli in the soil

Bacteria of the genus Proteus enter food products from the soil and multiply in them under favorable conditions. Metabolic products of Proteus bacilli can cause food poisoning.

To detect Proteus bacilli in the soil using Shukevich's method, 0.1 cm³ of the appropriate soil suspension dilution is applied to the Condensation water of freshly sloped NA. The tubes are kept in an incubator at 37 °C for 24-48 hours, after which Proteus growth is recorded by The formation of a thin, veil-like film on the slanted agar surface. If such a film is present, a smear is prepared from its surface, Gram-stained, and tested for motility and H₂S production capability.

The results of sanitary and microbiological soil analysis are evaluated using the data presented in Table 13.1.

Class="center">Table 13.1. Scheme for assessing the sanitary condition of soil based on microbiological indicators

Review Questions

1. In which soil layers is the microorganism population the lowest?

2. What groups of microorganisms are identified in soil?

3. Pathogens of which infectious diseases may be present in soil?

4. How are soil samples collected and prepared for analysis?

5. What are the coli-titer and coli-index of soil?