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

8. STUDY OF CULTURAL, PHYSIOLOGICAL, AND BIOCHEMICAL CHARACTERISTICS OF BACTERIA

Objective: to examine the cultural, physiological, and Biochemical characteristics of Bacteria grown on various nutrient media.

8.1. Cultural Characteristics of Bacteria

The cultural characteristics of bacteria are identified by the Specific features of their growth on various liquid and solid nutrient media. Each microbial species forms colonies of a specific size and shape on solid media.

Colonies are characterized by size, shape, profile, edge contour, surface, color, Structure, and consistency.

Size of a colony is determined by its diameter, based on which punctiform colonies (diameter less than 1 mm), small (diameter 1-2 mm), medium (from 2 to 9 mm), and large (10 mm or more) are distinguished.

Shape of a colony can be circular, irregular, amoeboid, rosette-like, concentric, etc. (Fig. 8.1).

Class="center">Fig. 8.1. Shapes of microorganism colonies: a - circular; b - circular with a scalloped edge; c - circular with a marginal rim; d, e - rhizoid; f - circular with a rhizoid edge; g - amoeboid; h - mycelial; i - folded; j - irregular; k - concentric; l - complex

Profile of a colony is characterized by its elevation above the nutrient medium surface and the contour of its shape in vertical cross-section. The colony profile is determined by visual inspection from above and from the side. Flat, convex, crateriform, cone-shaped, and drop-like colonies are distinguished (Fig. 8.2).

Fig. 8.2. Profiles of microorganism colonies: a - curved; b - crateriform; c - tuberous; d - growing into the Agar; e - flat; f - convex; g - drop-like; h - cone-shaped

Edge of a colony is determined by examining it through a magnifying Glass or under a Microscope at 8x magnification. The following edge contours of colonies are distinguished: entire, wavy, fimbriate, dentate, scalloped, lobate, Ciliate, villous, branched, rhizoid, etc.

Surface can be smooth or rough, dry or moist, furrowed, folded, wrinkled, velvety, tuberous, with concentric circles, or radially striated.

Optical properties: transparent, translucent, opaque, fluorescent.

Color of a colony is determined by the pigment synthesized by the given microorganism species. Depending on the type of pigment produced, microbial colonies may be colored red, pink, yellow, golden-orange, yellow-green, blue, or black.

Structure of a colony can be uniform, finely or coarsely granular, mealy, membranous, or growing into the agar.

Consistency can be pasty, easily removed with a loop; viscous or mucous, adhering and drawing out into a thread behind the loop; firm; fibrous; dough-like; creamy-like; dry, peeling off the agar surface as an elastic film; brittle, crumbling when touched with a loop. The consistency of a colony is determined by touching its surface with a loop.

When inoculating microorganisms by streaking, one notes the growth intensity (abundant, moderate, weak), streak characteristics (spreading, branched, tuberous), surface optical properties, color, and consistency.

To characterize Bacterial growth in liquid media, cultures are inoculated into meat peptone broth or another medium that ensures good growth. Cultures grown under stationary conditions for 4-7 days are used for description. First, growth intensity is noted (scant, moderate, abundant). Next, attention is paid to the turbidity of the medium (uniform, flaky, with silky waviness), the presence of a pellicle (thick or thin, dry or mucous, smooth or folded, ring-shaped or continuous). If the culture does not produce pigment, the color of the medium remains unchanged, and the sediment usually acquires a grayish-white or yellowish-brown color. Microbial growth in a liquid medium is frequently accompanied by the appearance of an odor and gas evolution. Gas production is detected by the appearance of bubbles or foam.

8.2. PHYSIOLOGICAL AND BIOCHEMICAL Characteristics of Bacteria

When studying the physiological and biochemical characteristics of bacteria, their attitude toward oxygen, enzymatic activity, and ability to ferment various CARBOHYDRATES with the accumulation of specific metabolic products are investigated.

Attitude toward atmospheric oxygen. According to their attitude toward oxygen, microorganisms are divided into obligate aerobes, microaerophiles, facultative anaerobes, and obligate anaerobes. The attitude of bacteria toward oxygen is judged by the growth of the culture when inoculated by stabbing a nutrient agar Column. Aerobes develop in the upper part of the stab, anaerobes in the lower part, and facultative anaerobes evenly throughout the entire stab.

Among the biochemical properties of a culture, determining its enzymatic activity is of primary importance.

The utilization of carbohydrates and alcohols by microbial cultures is determined by inoculating 0.1 — 0.2 cm of the test Cell suspension into tubes containing liquid or semi-liquid media supplemented with a carbohydrate and an indicator. To detect gas production during carbohydrate Fermentation, fermentation tubes (Durham tubes) are placed into the liquid medium. A set of media containing carbohydrates and indicators is known as Hiss's "sugar" series. This name originates from the fact that, depending on the enzymatic activity of the Cells, certain sugars are fermented with the accumulation of acid or alkali—which alters the color of the indicator and the medium—whereas other sugars are not fermented, leaving the color of the medium unchanged. The short Hiss's series includes media with glucose, lactose, sucrose, maltose, and mannitol. The long series additionally incorporates media with arabinose, xylose, rhamnose, galactose, as well as Polysaccharides (inulin, starch, dextrin) and alcohols (glycerol, dulcitol, Inositol).

The inoculated tubes are placed in an incubator at the optimal Temperature. Results are evaluated after 2 – 4 days (or 7 – 10 days for slow-growing microorganisms). Researchers note any color change in the indicator or lack thereof, as well as the presence or absence of gas in the Durham tube.

Based on the data obtained, Conclusions are drawn regarding which sugars are assimilated by the studied bacterial culture.

Proteolytic activity. Microorganisms exhibiting proteolytic activity liquefy gelatin and peptonize milk. To determine this trait, the test culture of bacteria is stab-inoculated into tubes containing gelatin and cultivated for 4–10 days at room temperature, while noting the rate and pattern of liquefaction: layer-by-layer, funnel-shaped, bubbly, etc. (Fig. 8.3).

Fig. 8.3. Patterns of gelatin liquefaction by proteases of the studied bacteria

Catalase activity. The enzyme catalase is produced by many aerobic microorganisms. To perform the test, a drop of a 10% hydrogen peroxide solution is applied to a microbial colony grown for 24 h on a solid medium in a Petri dish. The evolution of oxygen in the form of gas bubbles indicates the presence of catalase in the bacterial cells.

Growth characteristics in milk. Skim milk is diluted with Water in a 4:1 ratio, supplemented with bromocresol purple indicator (2 cm³ of a 1.6% alcohol solution per 1 dm³ of milk) or litmus (10 ^см3 of a 4% solution per 1 dm³ of milk), dispensed into 8-10 cm tubes, and sterilized in an autoclave at 0.05 MPa for 20 min. The milk tubes are inoculated with the test bacterial culture and incubated for 6-14 days at the optimal temperature. Acid production by microorganisms during lactose fermentation is detected by A change in the indicator color. If acid accumulates in significant amounts, a clot forms. Bacteria possessing active proteases break down casein, causing milk peptonization.

Indole production. Some microorganisms have The ability to break down The amino acid Tryptophan with The production of indole, which also serves as a diagnostic feature for identifying bacterial species. The Morel method is used to detect indole. Tubes containing 8-10 cm of sterile meat peptone broth with The addition of 0.01% tryptophan (or without it) are inoculated with the test bacterial culture. A strip of filter paper moistened with oxalic acid is secured beneath the cotton plug. The tubes are incubated at the optimal temperature for 24 h. If indole is produced, the lower part of the strip turns pink.

Ammonia Production. The ammonification of protein substances under METABOLISM/18.html">The Influence of microbial Enzymes is accompanied by the release of ammonia. This bacterial capability is determined by inoculating the test culture into tubes with meat peptone broth, which are incubated for 2-3 days in an incubator at 37 °C. Ammonia production is detected by a color change on a strip of litmus paper secured between the plug and the neck of the tube in such a way that the strip does not Touch the nutrient medium. The release of ammonia is indicated by the litmus paper changing color from red to blue.

HYDROGEN SULFIDE PRODUCTION. During The breakdown of Sulfur-Containing Amino Acids (Cysteine, Methionine) by microorganisms, hydrogen sulfide is produced. To determine hydrogen sulfide production, meat peptone broth tubes are inoculated with the test bacterial culture, and a strip of filter paper impregnated with a lead acetate solution is secured under the plug. The inoculated tubes are placed in an incubator at the optimal temperature for 7-10 days. Hydrogen sulfide evolution is detected by the blackening of the strip due to The formation of lead sulfide.

Based on the morphological, cultural, and physiological-biochemical CHARACTERISTICS OF THE isolated culture, its species identity is determined using Bergey's Manual of Determinative Bacteriology.

The findings from the bacterial identification tests are compiled into a protocol report (Table 8.1).

When necessary, additional characteristics are studied, such as the ability to reduce nitrates, oxidase production, etc.

Table 8.1. Cultural characteristics based on morphological, cultural, and physiological-biochemical features

Review Questions

1. What characteristics are used to identify bacterial species?

2. Based on what features are bacterial colonies characterized on solid nutrient media?

3. How is bacterial growth characterized in a liquid medium?

4. How is the proteolytic activity of bacteria detected?

5. How can one determine the ability of bacteria to ferment sugars?

6. What changes may be observed during the growth of bacteria in milk, and how can they be explained?

7. What features indicate the production of ammonia, hydrogen sulfide, and indole by bacteria?