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

12. SANITARY BACTERIOLOGICAL ANALYSIS OF WATER

Objective: to study Methods for Assessing the sanitary and bacteriological state of drinking Water and water from natural reservoirs.

Water used in food Processing plants must comply with the requirements for drinking water set forth in current regulatory documents. The epidemiological safety of water is determined by the total microbial count and the number of coliform Bacteria in a given volume of water.

The water quality of centralized drinking water supply systems is determined in accordance with sanitary rules and norms. Drinking water must be safe from epidemiological and radiation Perspectives, chemically safe, and possess favorable organoleptic properties (Table 12.1).

Class="center">Table 12.1. Epidemiological safety of drinking water (microbiological and parasitological indicators) SanPiN 2.1.4.1074-01

* PFU - plaque-forming units.

12.1. Sampling and Sample preparation for Analysis

Water samples for sanitary and bacteriological testing are collected in a volume of 500 cm³ into bottles previously sterilized in paper bags, featuring a cotton-gauze plug covered with a paper cap.

Before sampling, the tap or pipe edge is sterilized by flaming with a cotton swab soaked in alcohol. Open the tap and let the water run for 10-15 min, then collect the sample. The water must be analyzed no later than 2 h after sampling.

Water samples from open bodies of water—wells, pools, rivers, lakes—are collected using bathometers, which are metal frames with a heavy lead sinker at the bottom. A bottle is inserted into the metal frame. The bathometer is submerged to a specified depth and the bottle is opened by pulling a string tied to the stopper. After the bottle fills, the bathometer is retrieved, and the bottle is sealed with a sterile stopper.

Chlorinated water samples are collected in bottles containing a dechlorinating agent, as chlorine kills microbes in the water. Sodium thiosulfate is used as a dechlorinating agent at a rate of 10 mg per 500 cm of the test water.

Collected water samples are accompanied by a document indicating the relevant data. Drinking water samples are transported in cooler containers at temperatures ranging from 4 to 10 °C.

12.2. Determination of the Total Microbial Count in Water

Total viable count (TVC) - is the number of mesophilic aerobic and facultatively anaerobic microorganisms forming colonies on meat-peptone Agar when inoculating 1 cm³ of water, followed by incubation of the cultures at 37±0.5 °C for 48 h. The TVC should not exceed 50 CFU/cm³.

Depending on the expected degree of contamination, at least two different volumes of water are inoculated, calculated so that between 30 and 300 colonies grow on the plates. Tap and artesian water are inoculated undiluted in 1 cm³ portions. Bacteriological examination OF contaminated waters requires inoculation of diluted water. Dilutions are prepared as described in Section 8.3.

From the test sample and its dilution tubes, According to the estimated level of microbial contamination, 1 cm³ aliquots are taken, placed into sterile Petri dishes, and poured with 10-12 cm of meat-peptone agar melted and cooled to 45 °C. Using circular hand motions and rotating the dishes on a flat table surface, the contents are distributed in an even layer across the entire bottom area. After the agar solidifies, the inoculated plates are placed in an incubator at 37 °C for 24 h. Following incubation, the grown colonies are counted.

Determining the microbial count by this method allows for the detection of mesophilic aerobic and facultatively anaerobic microorganisms only.

12.3. Determination of Coliform Bacteria Content in Water

From an epidemiological standpoint, detecting pathogenic microorganisms in water—specifically the causative agents of intestinal infections (typhoid fever, dysentery, cholera, etc.)—is of paramount importance. However, due to the extreme difficulty of detecting pathogens in routine bacteriological analyses, researchers rely on determining so-called sanitary-indicator microorganisms (SIMs). SIMs are microorganisms constantly found in the natural cavities of humans or animals. The presence of SIMs in various environmental objects indicates human-induced contamination. The higher the concentration of SIMs in the environment, the greater the likelihood of specific infectious disease pathogens being present.

Coliform bacteria hold the greatest significance as SIMs. The coliform group includes coliform bacteria of the genera Escherichia, Enterobacter, Citrobacter, Klebsiella, and Serratia.

When determining the quantity of SIMs in water, the following parameters are used:

• coli-titer - the smallest volume of water in which a single coliform bacterium is detected. For treated drinking water, the coli-titer must be at least 300 cm³;

• coli-index - the number of coliform bacteria per 1 dm³ of water. The coli-index for drinking water must not exceed 3.

Coliform bacteria in water are detected using the Membrane filtration method or the Fermentation method.

Fermentation method. The Essence of the fermentation method is to inoculate specific volumes of the test water, incubate

the cultures at 37 °C in enrichment media, followed by subculturing onto Endo agar, differentiating the grown colonies, and determining the most probable number (MPN) of coliform bacteria per 1 dm³ of water.

When testing centralized water supply sources, the sample is inoculated in triplicate across three volumes: 100, 10, and 1 cm³. For river, lake, or pond water, tenfold dilutions of 1:10, 1:100, and 1:1000 are prepared, and additional 10 cm³ and 1 cm³ volumes are inoculated undiluted. Water inoculation is performed into fermentation vessels (flasks, bottles, or tubes with Durham tubes) filled with glucose-peptone Eijkman medium. The cultures are incubated in a thermostat at 37 °C for 24 hours.

Processing of analytical results. Upon completion of incubation, the cultures are examined, and the following Conclusions are drawn:

a) if there is no gas production or color change of the medium, a negative result is reported for the presence of coliforms in the tested water volume, allowing the test to be concluded after 24 hours;

b) if acid and gas are produced, material from the fermentation vessels is streaked onto Endo agar. Inoculation is performed using a bacteriological loop with heavy streaking to obtain isolated colonies. The Petri dishes are incubated at 37 °C for 24 hours. After incubation, the plates are examined. The absence of characteristic coliform colonies on Endo agar provides grounds for issuing a negative report and concluding the test;

c) if lactose-positive dark red colonies, with or without a metallic sheen, are found on Endo agar, it is necessary to confirm that the grown microorganisms belong to the Enterobacteriaceae family. For this purpose, a smear from the colonies is examined microscopically, and an oxidase test is performed.

The oxidase test is used to differentiate bacteria of the Enterobacteriaceae family from gram-negative bacteria of the Pseudomonadaceae family and other aquatic Saprophytes, which, unlike coliform bacteria, produce the enzyme oxidase.

To perform the oxidase test, 2–3 colonies of each type are picked from the Endo agar plate using a loop. The microbial mass is streaked onto filter paper moistened with a special reagent (30 g of α-D-naphthol is dissolved in 2.5 cm³ of ethanol, followed by The addition of 7.5 cm³ of distilled water and 40 mg of N,N-dimethyl-p-phenylenediamine. The solution is prepared immediately before testing).

In a negative oxidase test, the paper does not change color upon contact with the colony. If the paper turns blue within 1 minute of contact with the colony, the oxidase test is considered positive.

The presence of gram-negative, non-spore-forming rods in the preparation that lack oxidase activity makes it possible to immediately report the presence of coliform bacteria in the water.

If pink and colorless colonies are found on Endo agar, they are counted, and 2–3 isolated colonies of each type are subcultured into glucose-peptone Eijkman medium. The cultures are incubated at 37 °C for 3–4 hours. The result is considered positive if acid is produced (indicated by a color change of the medium) and gas accumulates in the Durham tube; it is considered negative if no acid or gas is formed.

After completing the analysis, the final results (positive and negative) for each inoculated volume are recorded in the laboratory logbook, and the coli-titer and coli-index are determined.

Membrane filtration method. The essence of the method is concentrating bacteria from a specific volume of water onto membrane filters, followed by cultivating them on Endo agar at 37 °C, differentiating the grown colonies, and counting the number of coliform bacteria in 1 cm³ of water.

Preparation of membrane filters. Membrane filters No. 3 are selected for water filtration, placed in distilled water preheated to 80 °C, and brought to a boil over low heat. Boiling is performed three times for 10 minutes each. After the First and Second boilings, the water is decanted, and after the third boiling, the filters are left in the water until use.

Preparation of the filtration apparatus. The filtration apparatus is sterilized in an autoclave or wiped with a cotton swab soaked in alcohol and flame-sterilized. A membrane filter is placed on the Base of the filtration apparatus using sterile forceps. To prevent damage to the filter, a circle of sterile filter paper is placed underneath it. The upper part of the device—the funnel—is placed onto the filter base and secured (Fig. 12.1).

Fig. 12.1. Determination of microorganism counts using the membrane filtration method

Water filtration and microorganism cultivation. The test volume of water is poured aseptically into the funnel of the filtration apparatus, and a vacuum is created in the receiving vessel using a water-jet pump. When analyzing drinking water entering the distribution network, a volume of at least 333 cm³ must be taken. After filtration, the membrane filter is transferred using flamed forceps onto The surface of Endo nutrient medium in a Petri dish. Filters impregnated with appropriate nutrient media are currently commercially available. The cultures are incubated in a thermostat at 37 °C for 18–24 hours.

Processing of analytical results. Upon completion of incubation, the cultures are examined, and the following conclusions are drawn:

a) the absence of Microbial growth on the filters or the presence of colonies uncharacteristic of coliforms allows the testing to be concluded at this stage with a negative result for the presence of coliform bacteria in the analyzed water volume;

b) if colonies characteristic of coliforms are found on the filter, testing continues. Smears are prepared from several colonies of each type, Gram-stained, and examined microscopically. The absence of small, gram-negative, non-spore-forming rods in the smears provides grounds for terminating the analysis and reporting a negative result for the presence of coliform bacteria in the tested water volume;

c) if gram-negative rods morphologically similar to coliforms are present in the smears, the oxidase test is performed. If uniform lactose-positive colonies (dark red with or without a metallic sheen) that do not produce oxidase are found on the membrane filters, the water analysis is concluded at this stage, and the number of coliform colonies grown on the membrane filter is counted. The result is expressed as the coli-index per 1 dm³ of water;

d) if pink and colorless colonies are found on the membrane filters, their number is counted, and 2–3 isolated colonies of each type are subcultured into glucose-peptone Eijkman medium. After incubation for 3–4 hours at 37 °C, a color change of the medium due to acid production and gas accumulation in the Durham tube is noted. In this case, the result is considered positive. If there are no Changes in the medium, a negative result for the presence of coliform bacteria is reported.

Example of determining the coli-index: three water volumes of 100 cm³ each were filtered. Three colonies grew on the first and second filters, and nine colonies on the third. A total of fifteen colonies grew. Thus, the coli-index of the water sample under study is: (1000 x 15):300 = 50. The coli-index is converted into the coli-titer as follows: 1000:50 = 20.

Control Questions

1. What indicators of the epidemiological safety of drinking water do you know?

2. What are the total microbial count, coli-titer, and coli-index?

3. Which genera of microorganisms are included in the coliform group (CCG)?

4. What methods are used to determine coliform bacteria?

5. What are the main criteria used to establish the presence of coliform bacteria in drinking water?

6. For what purpose is the oxidase test performed?