FUNDAMENTALS OF MICROBIOLOGY - E. Yu. Tyumentseva - 2015

TOPIC 5. PHYSIOLOGY OF MICROORGANISMS

Microbial physiology studies the Functions as well as biochemical processes occurring within their Cells and the environment. Specifically, microbial physiology examines their Nutrition, Respiration, reproduction, motility, sporulation, and Transformation of substances.

Nutrition refers to the processes of nutrient uptake into and excretion from The Cell. Nutrition primarily Supports cell reproduction and METABOLISM.

Among the essential nutrients, organogens are distinguished—these are eight chemical elements whose concentration in the bacterial cell exceeds 10-4 mol. These include carbon, oxygen, hydrogen, nitrogen, phosphorus, potassium, magnesium, and calcium.

In addition to organogens, Trace Elements are required. They ensure enzyme activity. These include zinc, manganese, molybdenum, cobalt, copper, nickel, tungsten, sodium, and chlorine.

For the growth of certain microorganisms, special substances are also required—growth stimulants, or growth factors, which are found in Yeast extracts, corn steep liquor, and plant seedlings. These substances contain essential Vitamins, Amino Acids, and related compounds.

Microorganisms obtain oxygen and hydrogen mainly from Water and Organic compounds. Some Bacteria also assimilate free atmospheric oxygen.

Based on their method of carbon utilization, microorganisms are divided into autotrophs (autos - self, trophe - nourishment) and heterotrophs (heteros - other).

Autotrophs, or prototrophs (protos - simple), assimilate carbon from atmospheric carbon dioxide.

Microorganisms assimilate carbon dioxide through Chemosynthesis, i.e., using energy obtained from The oxidation of certain mineral compounds; for example, nitrifying microorganisms obtain energy by oxidizing ammonia to nitric acid, iron bacteria by oxidizing ferrous iron to ferric iron, and sulfur bacteria by oxidizing hydrogen sulfide to sulfur, sulfurous, and sulfuric acids.

Heterotrophs are microorganisms that assimilate carbon only from preformed organic compounds. These include fermentative, putrefactive, and pathogenic (disease-causing) microorganisms. Each microbial species develops in media with a specific hydrogen ion concentration (pH).

Heterotrophic microorganisms, in turn, are divided into:

1) metatrophs (meta - after, and trophe - nourishment), or Saprophytes (sapros - rotten, phyton - plant). They feed on dead organic matter;

2) paratrophs (para - beside, trophe - nourishment), or pathogens. They reproduce only within living organisms. Paratrophs are pathogens of plants, invertebrates, and vertebrates.

Based on their ability to assimilate nitrogen, microorganisms are divided into two groups: aminoautotrophs and aminoheterotrophs.

Aminoautotrophs use atmospheric molecular nitrogen or assimilate it from ammonium salts for cell Protein Synthesis.

Aminoheterotrophs obtain nitrogen from organic compounds, such as Amino Acids and complex Proteins. This group includes all pathogenic microorganisms and most saprophytes.

Based on The Nature of their energy source, microorganisms are divided into phototrophs, which use solar energy for biosynthetic reactions, and chemotrophs. Chemotrophs obtain energy by oxidizing inorganic substances (such as nitrifying bacteria) and organic compounds (most bacteria, including species pathogenic to humans).

Respiration (or Biological Oxidation) is a complex process accompanied by the release of energy required by microorganisms to synthesize various organic compounds.

Based on their type of respiration, microorganisms are divided into aerobes (aer - air) and anaerobes (not requiring atmospheric oxygen).

Aerobes can live and develop only in an oxygen-containing environment. They obtain thermal energy through the oxidation and breakdown of CARBOHYDRATES, whereby the carbohydrate is broken down into water and carbon dioxide, releasing a large amount of energy. The reaction proceeds According to the formula

С₆Н₁₂О₆ + 6О₂ = 6СО + 6Н₂О + 688 ккал.

Anaerobes can live and develop only in the absence of atmospheric oxygen; for anaerobes, oxygen is toxic. During respiration, these microorganisms obtain energy and the bound oxygen required for cell construction by breaking down organic compounds.

The complex processes of Microbial Nutrition and respiration are carried out with the help of Enzymes.

Enzymes are biological catalysts, high-molecular-weight protein substances produced by living cells. They are highly specific and play a crucial role in microbial metabolism. Their Specificity is determined by active sites formed by a group of amino acids; that is, each enzyme reacts with a specific chemical compound or catalyzes one or several closely related Chemical Reactions. For example, the enzyme lactase breaks down lactose, maltase breaks down maltose, and so on.

The enzymatic profile of microorganisms is constant, and different microbial species are clearly distinguished by their set of enzymes. Therefore, studying the enzymatic composition is of great importance for identifying various microorganisms.

Structure/179.html">Practical Applications OF microbial enzymatic properties include Fermentation processes, The Use of Fungi in brewing and winemaking, hide Processing for softening, and preservation. They are also used to produce bio-additives for laundry detergents to remove protein stains, as they break down proteins into water-soluble compounds.

Enzymes secreted by microorganisms into the environment are called exoenzymes, while those tightly bound to the cell are called endoenzymes. The former prepare nutrients for absorption through The Cell wall, while the latter convert the absorbed substances into cellular components inside the cell.

Enzymes are classified into 6 classes:

1) oxidoreductases;

2) transferases;

3) Hydrolases;

4) lyases;

5) isomerases;

6) ligases (synthetases).

Microorganisms reproduce rapidly. Under favorable conditions, bacterial cells divide every 20-30 minutes. At this rate of division, a single bacterial cell would yield 1024 cells in 5 hours, and within 5 days, the biomass of bacteria could fill all the seas and oceans. In reality, however, this does not happen. Their growth is limited by several unfavorable factors, primarily the lack of an adequate amount of nutrient medium.

Phases of bacterial cell growth in a liquid nutrient medium:

1) initial stationary phase; the initial quantity of bacteria introduced into and present in the nutrient medium;

2) lag phase; lasting 3-4 hours, during which bacteria adapt to the nutrient medium, active cell growth begins, but active division has not yet started; during this time, The amount of Protein and RNA increases;

3) logarithmic growth phase (log phase); active Cell Division occurs within the population, with reproduction outstripping cell death;

4) maximum stationary phase; bacteria reach their maximum concentration, i.e., the maximum Number of viable individuals in the population; the number of dying bacteria equals the number of newly formed ones; no further increase in the cell count occurs;

5) phase of accelerated decline (death phase); death processes outpace reproduction as nutrient substrates in the medium become depleted. Toxic metabolic products accumulate. This phase can be avoided by using continuous cultivation Methods, where metabolic products are constantly removed from the nutrient medium and nutrients are replenished.

Laboratory Procedure

Objective: to study the composition of nutrient media and the conditions for their preparation.

Materials, Reagents, and Equipment: incubators; funnels; alcohol burners; hot plates; test tubes; sterile Petri dishes; filters; pipettors; sterile 1 cm3 pipettes; solutions and dry substances for preparing nutrient media: meat-peptone Agar, Endo agar, wort agar or Sabouraud agar, lactose-peptone medium, iron sulfite agar; 96% ethyl alcohol.

Experiment No. 1. Preparation of Media

Glassware and containers used for preparing media must not contain foreign substances, such as alkalis released by certain types of Glass, or iron oxides that can contaminate the medium if boiled in rusty pots. It is best to use glass, enameled, or aluminum containers. Large volumes of media (tens and hundreds of liters) are prepared in special boiling kettles or bioreactors. Before use, the containers must be thoroughly washed, rinsed, and dried. New glassware is pre-boiled for 30 minutes in a 1-2% Hydrochloric acid solution or soaked in this solution overnight, followed by rinsing in running water for an hour. (Containers intended for media preparation must not be used for other purposes, such as storing chemical reagents or disinfectant solutions—even traces of these substances can inhibit Microbial growth.)

The raw materials for preparing most media are products of animal or plant origin: meat and meat substitutes, milk, eggs, potatoes, soy, corn, yeast, etc.

Basic nutrient broths are prepared using meat infusion or various digests obtained through acid or Enzymatic Hydrolysis of raw materials. Digest-based broths are 5 to 10 times more economical than those made from meat infusion. Digest-based media are richer in amino acids and, consequently, more nutritious; they also exhibit higher buffering capacity, meaning they maintain a more stable pH. In addition, preparations can be made from meat substitutes (Blood clots, Placenta, casein, etc.).

Currently, the supply of meat infusion and digests to laboratories is centralized. Hottinger's pancreatic digest, casein hydrolysates, or fodder yeast hydrolysates are most commonly used. The required media are prepared from these intermediates according to specific formulations.

Stages of media preparation: 1) boiling; 2) adjusting to the optimal pH; 3) clarification; 4) filtration; 5) dispensing; 6) sterilization; 7) quality control.

Media are boiled over an open flame, in a water bath, in an autoclave, or in steam-heated kettles.

Approximate pH adjustment of the media is performed using pH indicator paper. A potentiometer is used for precise pH determination.

Media are dispensed into test tubes (3–5 ml or 10 ml), vials, flasks, Roux flasks, and bottles to no more than 2/3 of their capacity, as the plugs may become wet during sterilization, causing the media to lose sterility.

Media sterilized at temperatures above 100 °C are dispensed into clean, dry glassware. Media sterilized at lower temperatures must be dispensed into sterile glassware.

Media are dispensed using a funnel with a rubber tube and a Mohr clamp attached to its end. For volumetric dispensing, graduated cylinders, burettes, dispensers, syringe pipettes, etc., are used.

Glassware containing the medium is usually closed with cotton-gauze plugs, which are then covered with paper caps. It is important that the medium does not wet the rims of the glassware during dispensing; otherwise, the plugs may stick to them. A label indicating the name of the medium and its preparation date must be attached to each vessel.

Sterilization. The sterilization regime depends on the COMPOSITION OF THE medium and is specified in its formulation.

Quality control of prepared media:

1) to test for sterility, the media are placed in an incubator for 2 days, after which they are inspected. If no signs of growth appear, they are considered sterile, and several samples from each batch are sent for chemical control;

2) chemical control: the final pH, total and amino nitrogen, peptone, and chloride content are determined (their amounts must correspond to those specified in the formulation). Chemical control of the media is carried out in a chemical laboratory;

3) for biological control, several media samples are inoculated with specifically selected cultures of microorganisms, and their growth is used to evaluate the nutritional (growth-promoting) Properties of the medium. A label and a certificate indicating the name and composition of the medium, control results, etc., are attached to the finished medium.

Media are stored at room Temperature in cabinets, preferably designated specifically for this purpose. Some media, such as those containing blood and vitamins, are stored in a refrigerator.

Task: prepare the culture media specified by the instructor (Appendix 3).

Recording and analysis of research results

Record the formulations for preparing 5 culture media. Prepare the culture medium specified by the instructor and keep it for further research.

In this section, students can familiarize themselves with the conditions for preparing the culture media required for microbiological control.

Review Questions

1. What is metabolism?

2. What is The chemical composition of microbes?

3. What elements and substances are required for microbial nutrition?

4. What is the Selection/36.html">Biological essence of the processes of nutrition and respiration?

5. How do nutrients enter the cell?

6. What changes occur to the substances that enter the cell from the culture medium?

7. What is The Essence of aerobic nutrition?

8. What is the essence of anaerobic nutrition?

9. How are microorganisms classified by their energy source?

10. List the growth phases of bacterial cells in a liquid nutrient medium.

11. What are enzymes, and what are their key characteristics?

12. What is The Role of enzymes in the life of microorganisms?

13. How are enzymes classified?

14. Define exoenzymes and endoenzymes.

15. What glassware can be used to prepare culture media?