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

10. METHODS FOR QUANTITATIVE ENUMERATION OF MICROORGANISMS

The number of microbial Cells per unit volume or mass of a product can be determined either by direct microscopic counting, or by counting colonies grown on nutrient media after plating dilutions of the product, or by other Methods.

In addition, microbiological practice often involves determining biomass—the dry weight of cells, expressed in grams, produced in a given volume of nutrient medium. Direct Cell counting is performed using counting chambers, fixed stained smears, and membrane filters. Direct microscopic counting accounts for both live and dead cells, which leads to overestimating the Number of viable cells in the substrate.

10.1. Enumeration of Microbial Cells Using a Goryaev Chamber

Objective: to count cells in a Yeast suspension using a Goryaev chamber.

Goryaev, Thoma-Zeiss, and similar chambers are suitable for counting only large microbial cells, such as Yeasts, unicellular Algae, fungal conidia, and certain large Bacteria.

The Goryaev counting chamber is a thick Glass Microscope slide divided by four grooves into three transverse platforms. The central platform is bisected by a longitudinal groove. A grid is engraved on each half. The area of the grid square and the depth of the chamber are indicated on the slide and are 1/25 (large square) and 1/400 mm² (small square), respectively. The chamber depth is 0.1 or 0.2 mm.

Before counting, the yeast suspension is diluted with Water depending on the estimated cell concentration. A small drop of the test microbial suspension is placed On the surface of the Goryaev grid, covered with a special polished coverslip, and pressed against the side platforms until so-called Newton's rings appear. Cell counting should begin no earlier than 3–5 minutes after filling the chamber to allow the cells to settle and lie in a single plane. Cells are counted using an 8x or 40x objective. To obtain reliable results, counting should be performed in 10 large or 20 small grid squares, moving diagonally across the grid. The number of cells in a large square should not exceed 20, and in a small square—10. Otherwise, the suspension must be diluted with tap water.

The number of cells in 1 cm³ of suspension is calculated using the formula

С = а • 1000 • n/hS,

where C is the number of cells in 1 cm³ of suspension; a is the average number of cells per grid square; 1000 mm² = 1 cm³; n is the dilution factor of the initial suspension; h is the chamber depth in mm; S is the area of the grid square in mm².

10.2. Direct Microscopic Cell Counting (Vinogradski-Breed Method)

Objective: to count Lactobacillus cells in a fermented dairy product.

The advantage of this method over counting cells in a counting chamber is The ability to enumerate small microbial cells, as the count is performed using an immersion objective.

The preparation is made as follows. A thoroughly degreased glass slide is placed over graph paper with a 4 cm² square marked and outlined with a glass marker or india ink. A microbial suspension is prepared by adding 1 cm³ of the fermented dairy product to a test tube containing 9 cm³ of physiological saline (n = 10). Then, a precisely measured volume of the test microbial suspension (usually 0.01 or 0.02 cm³) is applied to the slide using a micropipette. The suspension is evenly distributed with a bacteriological loop over the entire area of the square outlined on the slide. The preparation is air-dried, fixed in a spirit lamp flame, stained with methylene blue for 2 minutes, rinsed with water, and blotted dry with filter paper. A drop of cedar oil is applied to the preparation, and it is examined under an immersion objective. For reliable results, cell counts should be taken in at least 20 fields of view. The total number of counted Cells must be at least 600. Microorganisms are distributed unevenly in the smear, being more concentrated in the center than at the edges. Therefore, to obtain an accurate mean value, counts should be conducted along the diameter of the smear, shifting the field of view from one end of the diameter to the other.

The number of cells in 1 cm³ of suspension is calculated using the formula

С = а • S • n/s • V,

where C is the number of cells in 1 cm³ of suspension; a is the average number of cells per field of view; S is the area of the prepared smear (400 mm²); n is the dilution factor of the initial suspension; s is the area of the field of view (0.02 mm²); V is the volume of the microbial suspension applied to the slide (0.01 or 0.02 cm³).

10.3. Direct Enumeration of Microorganisms by Fluorescence Microscopy

Fluorescence microscopy is based on the ability of certain biological objects to luminesce, i.e., to emit light when illuminated with ultraviolet or blue light, due to the emitted luminescence having a longer wavelength than the absorbed light (Stokes' shift). In this process, objects glow yellow-green or orange. This is intrinsic or primary luminescence. Since most microorganisms lack intrinsic luminescence, several pretreatment methods are used to observe them under a fluorescence microscope. Primarily, this involves staining with special fluorochromes—highly diluted solutions of fluorescent Dyes. Among synthetic fluorochromes, acridine yellow or orange, coryphosphine, primuline, and rhodamine yield the best results. Unlike primary luminescence, this type of luminescence is referred to as induced (secondary).

Compared to conventional light microscopy, fluorescence microscopy allows one to:

✵ combine color imaging with high object contrast;

✵ examine both transparent and opaque live specimens;

✵ study the Morphology of both live and dead microbial cells;

✵ to investigate cellular microstructures that selectively absorb various fluorochromes, and to determine the functional and morphological changes in cells;

✵ to study various life processes in the dynamics of their development.

Microbial cells are stained with acridine orange and concentrated on filters by centrifugation or filtration. Under transmitted light with a wavelength of 450 nm, live bacteria fluoresce green, while dead ones glow orange-red. The enumeration of bacterial cells during microscopy is performed simultaneously by individual morphological traits, making it possible to characterize both the COMPOSITION OF THE community and the total Abundance of microorganisms (cells/cm³) in the test sample.

Epifluorescence microscopy is widely used in medical microbiology for diagnosing the causative agents of tuberculosis, diphtheria, Gonorrhea, relapsing fever, etc.

State Standard GOST R 52415-2005 has been developed: Raw natural cow's milk. Luminescent METHOD FOR DETERMINING the count of mesophilic aerobic and facultatively anaerobic microorganisms.

Currently, the following models of fluorescence microscopes produced domestically are used for research: LYUMAM 3-8, MLD-2, ES BIMAM R-11, and ES BIMAM R-13.

The main disadvantages of fluorescence microscopy include low resolution when counting small bacterial cells (less than 1 µm in size) and, consequently, missing a large portion of them, significant eye strain when counting small cells, and darkened microscopy conditions.

10.4. Flow Cytometry Method

Flow cytometry (FC) is a modern technology for the rapid optical measurement of the parameters of a cell, its Organelles, and the processes occurring within it. THE PRINCIPLE OF the FC method is based on the detection of fluorescence and light scatter from each individual cell. A cell suspension, pre-labeled with fluorescent dyes, is pressurized into a flow cell, where hydrodynamic focusing aligns the cells in a single-file chain within a laminar stream. As a cell crosses the laser beam, highly sensitive detectors record its fluorescence intensity and scattered laser radiation. During the analysis, the fluorescence level of chemical compounds intrinsic to The Cell (autofluorescence) is also taken into account. The resulting signal is fed into a computer, processed, and the data is displayed as various graphs or histograms. An FC device allows the determination of up to 5-10 different cell parameters, such as size, enzyme activity, and the content of Proteins, DNA, Lipids, and antigenic substances.

The FC method finds diverse Applications, ranging from simple cell counting and viability determination to more complex studies in immunology, oncology, Cytology, hematology, and pharmacology.

10.5. Determination of Microorganism Counts by Membrane filtration

The Essence of the method is that a specific volume of the test sample (drinking water, stable soft drinks, pasteurized beer) is filtered through membrane filters with a pore size of 0.15 to 0.25 µm (see section 10.3). Microorganisms retained on the filter are stained and counted under a microscope using an ocular reticle micrometer across several fields of view over a defined area of the preparation. The enumeration of bacterial cells during microscopy is carried out simultaneously for individual morphological groups, which makes it possible to characterize both the composition of the microbial community and (after summing the data) the total abundance of Microorganisms in the test object.

10.6. Determination of Microorganism Counts by Colony Counting (Plate Method)

Objective: to determine the count of mesophilic aerobic and facultatively anaerobic microorganisms in a food product.

The plate method is widely used to determine the population of viable microorganisms in various natural substrates and laboratory cultures. The method is based on Koch's principle, according to which each colony is the progeny of a single cell. However, it must be taken into account that for microorganisms that form chains or other cell clusters, the results are always somewhat underestimated. Therefore, when using the plate method, the result is expressed not as the number of cells per unit mass or volume, but as the number of colony-forming units (CFU). Unlike direct microscopic cell counting, this method makes it possible to determine only the number of viable cells.

Determining the number of microorganisms using the plate method involves the following stages: sampling and preparation for analysis, preparation of dilutions of the microbial suspension, inoculation into Petri dishes containing solid medium, incubation of cultures at an optimum Temperature, counting grown colonies, and Processing the results.

Meat-peptone Agar is used as a nutrient medium for counting bacteria, on which only saprophytic aerobic and facultatively anaerobic bacteria can grow, while obligate anaerobes are unable to grow. Yeasts and Molds are usually cultivated on wort agar.

10.7. Enumeration of Living Microorganisms by the Most Probable Number Method

The serial dilution method is used to determine the population of microorganisms belonging to specific physiological groups (lactic acid bacteria, acetic acid bacteria, coliforms, etc.). The essence of the method is that a series of tenfold dilutions of the test material is prepared. The number of dilutions is prepared based on the expected Bacterial Content in the test object so that the final dilutions contain no such bacteria. A defined number of tubes with nutrient medium are inoculated with material taken from the prepared dilutions. After incubation at an optimum temperature, it is visually determined in what smallest volume of the test material Representatives of the given microbial group are still present.

For example, when determining the count of lactic acid bacteria in a fermented dairy product, a series of its consecutive dilutions from 10^-1 to 10^-10 is prepared. From each dilution, 1 cm³ of the Suspensions is inoculated in parallel into three tubes containing sterilized skimmed milk. The tubes are kept in an incubator at an optimum temperature for 18-24 h, after which it is noted in which tubes a clot has formed due to the accumulation of lactic acid by lactic acid bacteria. Parallel inoculations may yield differing results due to the uneven distribution of cells in the test sample. Subsequently, the titer is determined using special calculation tables.

If a liquid nutrient broth medium is used to determine the number of viable microorganisms, then after incubation, the tubes in which microorganisms have multiplied will appear turbid, while tubes inoculated with dilutions that no longer contained viable microorganisms will remain transparent. The proportion of tubes that turn out to be turbid upon inoculation with a culture of a given dilution depends on the number of living cells in the undiluted culture. Based on the number of turbid tubes in the groups inoculated with suspensions of three consecutive dilutions, the most probable number (MPN) of microbial cells is determined using appropriate tables.

10.8. Nephelometric Method for Biomass Determination

The nephelometric (turbidimetric) method for biomass determination has found widespread application in microbiological laboratory research because it allows a rapid and fairly accurate determination of cell concentration in a microbial suspension.

The nephelometric method is based on the scattering of a light beam by particles suspended in the liquid phase. This method is suitable only for those microorganisms whose growth causes a uniform turbidity of the medium without The formation of mycelium, pellicles, and other clumps. The nutrient medium in which microorganisms are cultivated must be optically clear.

Light scattering caused by microorganisms grown in nutrient broth is most conveniently measured using a photoelectric colorimeter (PEC) or spectrophotometer (SF) at a wavelength of 540 to 650 nm, at which Light absorption by the given suspension is minimal.

The relationship between the intensity of incident light (l₀) and transmitted light I (i.e., light not scattered by the culture) at low bacterial concentrations obeys the Beer-Lambert law:

l= lо 10^-ε/с,

where ε is the extinction coefficient, l is the suspension layer thickness, and с is the bacterial concentration. From this relation, it follows that:

lg (lо/l) = εlс.

The plot of lg (lо/l) versus с (bacterial concentration) is a straight line whose slope is determined by the product εl. Thus, The change in optical density caused by a unit change in bacterial concentration depends on the suspension layer thickness l and the suspension properties characterized by the coefficient ε.

At high bacterial concentrations, the Beer-Lambert law breaks down.

To determine the bacterial count using the nephelometric method, calibration curves are constructed to show the relationship between light scattering and cell count (or dry biomass per unit volume of the medium). To construct a calibration curve, the light scattering of suspensions with varying cell concentrations is measured using a photoelectric colorimeter or spectrophotometer, and the cell count and biomass are determined in each. The resulting relationship is presented graphically by plotting the colorimeter readings on the ordinate axis and the cell count per 1 cm³ of suspension or the biomass per 1 dm³ of culture medium on the abscissa axis. A separate calibration curve must be constructed for each microorganism.

Review Questions

1. What Methods for determining the number of microorganisms in various samples do you know?

2. Why is a series of dilutions of the test sample prepared to determine the total microbial count using the plate-count method? WHAT IS A CFU?

3. Are all bacteria present in the test product accounted for by the plate-count method?

4. What nutrient media are used to determine the count of bacteria and Fungi (yeasts and molds) in a product?

5. What are the Advantages and disadvantages of the fluorescence method for microorganism enumeration?

6. What is the core principle of flow cytometry?

7. What is The basis of the nephelometric method for determining bacterial count or biomass in a suspension?