Section 2 Bacteria and Actinomycetes
Bacteria and actinomycetes are both single-celled prokaryotes.
Bacterial cells are tiny and transparent, making them difficult to see clearly even with a microscope. It is usually dyed with an appropriate dye to increase the difference in refractive index between the specimen and the background for easier observation. Living cells that cannot be stained can be observed with a phase contrast microscope. Actinomycetes are a type of prokaryotes that grow in the form of hyphae. They are similar to bacteria in many aspects including cell structure. It can be said that actinomycetes are a type of bacteria with filamentous branched cells.
In the biochemical treatment of pollutants, bacteria play a very important role. For example, Pseudomonas has the ability to metabolize a variety of compounds, and some strains can utilize more than 100 compounds, so it has a very high degradation activity against pollutants; Corynebacterium is a genus that cleaves heterocyclic compounds and The main bacterial species of the hydrocarbon chain; Arthrobacter can oxidize hydrocarbon compounds, steroidal compounds, etc.; Alcaligenes is involved in the decomposition and mineralization of substances in fresh water, seawater and terrestrial environments. Actinomycetes such as Nocardia attack a wide variety of complex organic compounds, including phenols, pyridines, glycerols, steroids, unchlorinated and chlorinated aromatic compounds, paraffins, and even lignin. They decompose organic matter, generate various metabolic products, and then mineralize them under the action of other microorganisms.
1. Shape and arrangement of bacterial cells
(1) Cocci
The cells are spherical or oval. The new cells produced after division often maintain a certain spatial arrangement (Figure 2-1A), which is of great significance in classification and identification.
1. Monococci Cell division occurs along a plane, and new individuals exist separately.
2. Diplococcus Cells divide along a plane and new individuals are arranged in pairs.
3. Streptococcus cells divide along a plane and the new individuals join together in a chain.
4. Tetragenococcus cells divide along two mutually perpendicular planes, and after division four new cells are connected to form a field shape.
5. Sarcina cells divide along three mutually perpendicular planes, with eight new cells characteristically stacked together to form a cube.
6． Staphylococcus aureus Cell division proceeds in an undirected manner, with multiple new cells forming clusters like a bunch of grapes.
(2) Bacilli
The cells are rod-shaped or cylindrical, and their morphology is more complex than that of cocci. The thalli are straight or slightly curved, short or slender. The end is blunt, pointed, enlarged or truncated. Bacillus cells often divide along a plane, and the cells are scattered individually or arranged in short chains, long chains, palisades, splays, filaments, etc. (Figure 2-1B).
(3) Spiral bacteria
The cells are spiral-shaped. If the bacterial body has only one bend and the degree is less than one circle, it is called Vibrio (Figure 2-1C).
Cocci, Bacilli and Spiralis are the three basic forms of bacteria. Cocci are not necessarily perfectly round; diplococci may be triangular, bean-shaped, or kidney-shaped; Sarcina may be nearly square or rectangular; cocci that are about to divide may also be oval.
In addition, there are some bacteria with other forms. For example, stalk bacteria have rod-shaped or spindle-shaped cells with a thin stalk (Figure 2-1), which can be fixed on the substrate. Another example is sheath bacteria. The rod-shaped cells are arranged in a chain and are surrounded by a sheath membrane that is close to the bacterial body (Figure 2-1).
The morphological arrangement of bacteria is sometimes caused by growth stages or culture conditions. For example, in the genus Arthrobacter, young bacterial cells are rod-shaped, while old cells are spherical. When culture conditions are abnormal, cells often exhibit abnormal shapes. Some rod-shaped cells swell and appear pear-shaped, some branch, and some elongate and even become filamentous (Figure 2-2). Transfer them to fresh culture medium and, under appropriate conditions, return to their original normal form.
2. The size of bacteria
The size of bacteria can be measured with a microscope micrometer, or can be measured by making a picture through projection or photography and then calculating it according to the magnification.
Bacterial size is often measured in microns (μm). The diameter of cocci is generally 0.5 μm to 2 μm, the width of bacilli is 0.5 μm to 1 μm, and the length is 1 μm to 8 μm. The width of spiral bacteria is 0.5 μm to 5 μm, and its length (the distance between the two ends of the bacterial body) is 5 μm to 50 μm.
The size of bacterial cells is related to bacterial age and culture conditions. Young bacteria are longer and old bacteria are shorter, but the width of the bacteria is relatively constant. As the osmotic pressure of the culture medium increases, the cells become smaller.
Because bacterial cells are small and transparent, they often need to be stained before they can be observed with a microscope. They must be dried and fixed before staining, resulting in the measured size of the bacterial cells being smaller than that of viable bacteria. If negative staining is used to color the background and set off the bacteria, the specimen will be larger than the viable bacteria.
3. Structure of bacterial cells
Figure 2-3 is a model diagram of bacterial cell structure.
(1) Cell wall
Located on the outer surface of the bacteria, it is tough and elastic.
Its main functions are: fixing the shape of cells; protecting cells from damage by external forces; blocking macromolecular substances from entering cells; making cells have certain antigenicity, pathogenicity and sensitivity to phages; and assisting flagellar movement.
1. Bacterial Cell Wall and Gram Staining Bacteria can be divided into two categories by Gram staining: Gram-positive (G+) and Gram-negative (G-). The former is purple and the latter is red. Gram stain is an important bacterial identification stain.
The chemical composition of the cell wall of G+ bacteria is mainly peptidoglycan, and 75% of the peptidoglycan subunits are criss-crossed to form a dense grid structure. In addition to peptidoglycan, the cell walls of most G+ bacteria also contain teichoic acid (teichoic acid), which creates a negatively charged environment in the cell wall.
The cell wall of G-bacteria is divided into an inner wall layer and an outer wall layer. The inner wall layer is close to the cell membrane and is composed of peptidoglycan. Only 30% of the peptidoglycan subunits are intertwined and connected with each other, and the network structure is relatively loose.
The outer wall layer can be further divided into three layers: inner, middle and outer layers. The inner layer is a lipoprotein layer, the middle is a phospholipid layer, and the outermost layer is a lipopolysaccharide layer. Lipopolysaccharide is the main component of the outer cell wall layer of G-bacteria, which is also the main component of pathogenic bacterial endotoxins. It consists of three parts: polysaccharide core, O-antigen polysaccharide side chains and lipids.
The composition and structure of G+ and G- bacterial cell walls are shown in Table 2-1 and Figure 2-4.
The chemical composition and structure of G+ and G- bacterial cell walls make them react differently to Gram staining. In the Gram stain reaction, an insoluble crystal violet-iodine complex is formed within the cell. This complex can be leached by ethanol from G- bacteria but not from G+ bacteria. This is because the cell wall of G+ bacteria is thicker, the peptidoglycan content is high, and the network structure is tight. Therefore, when eluting with ethanol, the peptidoglycan mesh will shrink significantly due to shrinkage. In addition, it basically does not contain hydrochloric acid. Grease and ethanol treatments cannot dissolve gaps in the wall. Therefore, the complex of crystal violet and iodine is trapped in the cell wall. However, G-bacteria have thin walls, low peptidoglycan content, and loose cross-linking, so their mesh is not easy to shrink when exposed to ethanol. Moreover, G-bacteria have a high lipid content. After being dissolved by ethanol, larger gaps will appear in the cell wall, so that the complex of crystal violet and iodine can be easily leached.
2. Cell wall-deficient bacteria: G+ bacteria can be cultured in a medium containing penicillin, or lysozyme can be added to the culture of G+ bacteria to inhibit cell wall synthesis or destroy the cell wall. The remaining part after removing the cell wall is called protoplast. Bacterial cells of any shape will be spherical after becoming protoplasts. Protoplasts are sensitive to environmental conditions and prone to rupture. Some protoplasts still retain flagella, but they cannot move and cannot be infected by the corresponding phages. Other biological activities remain basically unchanged, and they still grow and reproduce under suitable conditions, forming bacterial colonies.
For G-bacteria, first treat the outer wall with ethylenediaminetetraacetic acid (EDTA), and then treat it with the same method as above to obtain a spheroplast. In the spheroids, the original inner wall layer (thin peptidoglycan layer) is removed, while the outer wall layer remains. Therefore, it is still resistant to adverse external factors and can grow on ordinary culture media.
Bacterial L-form is a variant form of bacteria formed under certain environmental conditions. It does not have a complete cell wall, so the cells are polymorphous, and some can pass through bacterial filters, also known as "Filtering bacteria". Because it was first discovered by the British Lister Institute of Medical Research, it is called the bacterial L-type.
(2) Cell membrane
Also known as the cytoplasmic membrane or plasma membrane, it is a soft and elastic semi-permeable film close to the inside of the cell wall. It maintains the relationship between bacteria and It plays an important role in the exchange of external substances. There is a rich enzyme system on the cell membrane, which is an important metabolic activity center of bacteria. The chemical composition of the cell membrane is mainly phospholipids and proteins, and their quantity and type vary with the physiological state of the bacteria.
(3) Nucleoplast
It is the primitive cell nucleus unique to prokaryotes. The bacterial nucleoplast is a large circular double-stranded DNA molecule with a length of 0.25 mm to 3 mm, curled and folded in the nuclear region. Nucleoplasts are the material basis that carries bacterial genetic information.
(4) Cytoplasm and its contents
Both the cytoplasm and the nucleus are differentiated from protoplasts. The latter is based on DNA and is concentrated in the center of the cell. Between it and the cell membrane The middle is the cytoplasm, which is mainly composed of proteins. The cytoplasm is colorless, transparent, and mucus-like. Its main chemical components are water, protein, lipids, nucleic acids, and a small amount of sugar and inorganic salts. The cytoplasm contains ribosomes, bubbles, and other granular inclusions.
1. Ribosomes are granular structures in the form of nucleoproteins composed of approximately 60% RNA and 40% protein. During high-speed centrifugation, the sedimentation coefficient of prokaryotic ribosomes is 70S, which is composed of a 30S subunit and a 50S subunit. The subunits dissociate into RNA and protein molecules under appropriate conditions. Ribosomes are where proteins are synthesized.
2. Bubbles In the cells of many aquatic bacteria that engage in photosynthesis and move without flagella, they often contain a large number of small gas-filled vesicles, called bubbles.
The bubbles are surrounded by a protein membrane only 2nm thick, which has the function of adjusting the specific gravity of cells to float in a suitable water layer.
3. Other inclusions
(1) Metachromatic granule is a polymer of metaphosphate. They are highly basophilic or neutrophilic. After dyeing with blue dye, they turn purple instead of blue, so they are called metachromatic grains. It was first seen in the cells of Spirillum volu-tans, so it is also called roundabout body or bacteriocin. It is a phosphorus source and energy storage, and has the function of reducing osmotic pressure. Corynebacterium diphtheriae has characteristic metachromatic particles at both ends of its body, called polar bodies, which are of certain significance in bacterial species identification.
(2) Poly-β-hydroxybutyric acid (PHB for short) particles are polymers of β-hydroxybutyric acid. They are insoluble in water and are easily colored by fat-soluble dyes. Optical Visible under microscope. The hydroxybutyric acid molecule is acidic. When it polymerizes into poly-β-hydroxybutyric acid, it becomes a neutral fatty acid, thereby maintaining a neutral intracellular environment. It is a source of carbon and a store of energy and can be used directly or indirectly as reducing power. PHB particles are often found in the cytoplasm of many bacteria.
(4) When sulfur granules sulfur bacteria live in an environment containing H2S, they accumulate highly refractive sulfur granules in their cells, which are sulfur storage materials and are also some chemoautotrophic sulfur bacteria. , such as the energy substances stored by Thiobacillus thiooxidans, which can obtain energy by oxidizing elemental sulfur into sulfuric acid.
(5) Polypeptide crystals. Some Bacillus species form a crystallized polypeptide in the cytoplasm during the spore formation stage, called paraspore crystals, which are highly toxic to Lepidopteran larvae and can be used to prevent and control agricultural pests. .
(6) Magnetic particles are unique string-shaped Fe3O4 magnetic particles in the cells of magnetic bacteria. Magnetic bacteria can sense the earth's magnetic field and align the cells in the direction of the magnetic field.
(5) Capsule
It is a mucus-like substance formed outside the cell wall by some bacteria during their metabolism. According to the thickness and shape of the cell wall it covers, there are the following situations: it has a certain shape, is relatively stably attached to the outside of the cell wall (Figure 2-3), is about 200nm thick, is called a capsule or macrocapsule, and is less than 200nm thick. It is called microcapsule; it has no obvious edge and spreads loosely to the surrounding environment, which is called mucus layer. If the mucus layer is localized to one end of the cell, it is called an adhesive, which allows the cells to specifically attach to the surface. The capsule materials on the outside of each bacterial cell fuse with each other and join together to form a unique capsule, in which multiple bacterial cells are embedded to form a bacterial gel mass.
The chemical composition of the capsule varies from strain to strain and is mainly polysaccharides.
The capsule can protect cells from desiccation, serve as an extracellular carbon source and energy storage material, and can also enhance the pathogenic ability of certain pathogenic bacteria. Some capsules are poisonous. The pathogenic effect of some encapsulated pathogens is not that the capsule itself is toxic, but that the capsule protects the pathogenic bacteria from the host's phagocytes, which is conducive to the growth and reproduction of the pathogens in the host.
The colonies formed by capsule-producing bacteria on agar medium have a moist, shiny, mucus-like surface and are called smooth (S-type) colonies. Colonies formed by bacteria that do not produce capsules have dry and rough surfaces and are called rough (R-type) colonies.
The production of capsule is a genetic characteristic of microorganisms and a characteristic of the species. However, capsule-forming bacteria do not remain capsuled throughout their lifespan. The formation of capsule is closely related to environmental conditions. For example, Leuconostoc mesen-teroides only produces a large amount of capsule material in culture media with high sugar content and low nitrogen content; pathogenic bacteria such as Bacillus anthracis only They form capsules in the animals they infect. The capsule can be lost through treatment or due to mutation. Losing the capsule does not affect the normal growth of the bacteria. After pathogenic bacteria lose their capsule, their pathogenicity is greatly reduced.
Capsulating bacteria grow and multiply in sugar juice, making product processing difficult and reducing yield. Encapsulating bacteria can also be used to synthesize glucan and produce dextran, which is the main component of plasma substitutes.
(6) Flagella and pili
Flagella (flegellum) are slender, wavy filaments that grow on the body surface of some bacteria. The length of the flagellum is often several times longer than the bacterial body, but the diameter is very thin, generally 10nm to 20nm, and requires an electron microscope to observe. After special dyeing, the complex of mordant and dye is attached and accumulated on the flagellum. If its diameter is enlarged, it can be observed with an ordinary optical microscope. The number of flagella ranges from one to dozens, and they have the function of movement.
After the bacteria with flagella are punctured and inoculated into the semi-solid medium, the culture spreads and grows around the puncture line. When flagellates are examined under a hanging drop microscope, cells can be seen tumbling or shuttling, from which the flagellum insertion method can be preliminarily determined.
The flagella are attached in various ways, one end is solitary, such as Pseudomonas aeruginosa; both ends are solitary; one end is clustered, such as P. fluorescens; There are clusters at both ends, such as Spirillumserpen, and periphylla, such as Escherichia coli (Figure 2-5).
The way and number of flagella are the characteristics of the species and are therefore important indicators for bacterial classification and identification.
The chemical composition of flagella is mainly protein, accounting for more than 99%, and the sum of carbohydrates, lipids and minerals does not exceed 1%.
Bacteria use flagella to move toward nutrients and suitable living environments or to avoid harmful substances. Motile bacteria change their original movement mode due to stimulation by environmental conditions and exhibit a new movement characteristic. This movement is called avoidance movement. According to different influencing factors, it can be divided into two types: chemical avoidance and phototactic avoidance.
The flagellum attachment situation may be related to the growth stage and culture conditions. For example, nitrosifying bacteria are inoculated into fresh culture medium and exist in the form of non-motile brevibacilli; when nutrients are almost exhausted, the bacteria appear flagella and become motile; finally, when nutrients are exhausted, the bacteria lose their flagella and settle in the bottom.
Another example is that when bacteria such as Rhizobium, Aeromonas, and Alcaligenes faecalis are in liquid culture media, flagella often grow on the end of the solid culture medium. But it is easy to grow peritrichous flagella on it. Another example is Listeria, which can cause meningitis, sepsis and abortion in pregnant women. When the growth temperature is 37°C, 90% of the strains have no flagella, but when cultured at 20°C, 80% of the strains have 1 From the root, 3 flagella grow.
The basal body of the flagellum is connected to the bacterial cell membrane. When the cell wall is removed, the flagella remain but lose their ability to move.
Pili (fimbria or pilus): is a slender (diameter 7nm ~ 9nm), hollow, short and straight, large number (250 ~ 300) protein appendage that grows on the surface of bacteria (Figure 2-3), mainly found in Gram-negative bacteria, but also in a few Gram-positive bacteria. There are many types of pili with different functions, which are mainly related to adsorption but not to movement. A special kind of pili with properties between flagella and the above-mentioned ordinary pili is called sex pili. Each cell has 1 to 4 pili. Its function is to transfer DNA fragments between strains of different genders. Some sex bacteria Hairs are also adsorption receptors for RNA bacteriophages.
(7) Spore
When certain bacteria grow to a certain period, the cytoplasm condenses and condenses, gradually forming a round, oval or cylindrical stress-resistant dormant body , called spores or endospores. The spore wall is thick and dense, has strong refraction, is not easy to color, has poor permeability, low water content, low enzyme content, low metabolic activity, and is extremely resistant to bactericidal factors such as high temperature, dryness, radiation, acid, alkali and organic solvents. . Usually, Bacillus can be successfully isolated by treating it in boiling water at 100°C for 10 minutes or 80°C for 15 minutes.
Whether spores can be formed, the shape, size and position of the spores within the cell (Figure 2-6) are characteristics of bacterial species and have certain significance in classification and identification.
There are not many types of bacteria that can form spores, the most important ones are Bacillus and Clostridium. They are both Gram-positive bacteria. The spores of the latter are enlarged and significantly wider than the bacteria, but not the spores of the former. In addition, Gram-positive bacteria of the genus Sporosarcina and Gram-negative bacteria of the genus Vibrio can form spores. Bacteria of the genus Sporosarcina and the Gram-negative genus Desulfotomaculum (rod-shaped) and a few genus Vibrio can form spores.
The spores germinate and produce new cells. One bacterial cell can only form one spore, and one spore can only produce one vegetative body. Therefore, spores are not the reproductive form of bacteria.
4. Reproduction of Bacteria
Fission is the most common and main reproduction method of bacteria. The way cocci divide is closely related to the arrangement of their cells (see section 1). Bacilli and Helicobacteria elongate the thallus before dividing, and then divide perpendicular to the long axis. After division, the two daughter cells are basically equal in size, which is called homotypic division. If the two daughter cells are unequal in size after division, it is called atypical division. This situation occasionally occurs in old culture media.
A small number of bacteria undergo budding and reproduction. There are also a few bacteria that can undergo sexual union and transfer genetic material through sexual pili, but the frequency is very low.
5. Culture characteristics of bacteria
(1) On solid medium
Grown on solid medium and originate from one or a few cells The group of microorganisms visible to the naked eye is called a colony. The characteristics of colonies formed by various microorganisms under certain conditions have certain stability and specificity, which is an important basis for measuring the purity of strains, identifying and identifying strains. The characteristics of bacterial colonies are shown in Figure 2-7.
The characteristics of the colony are related to the cell structure and growth behavior of the colony (aerobicity, motility, culture conditions, culture time, etc.). Colony morphology and size are also affected by neighboring colonies. The morphology of the colonies growing on the surface of the plate is also different from that inside the culture medium.
Plant bacteria-containing samples or strains on a flat plate or draw lines on a slope. A large number of microbial cells grow densely on a solid medium, which is called a lawn. As a reference for strain identification, a straight line should be drawn on the sloped bacterial lawn when inoculated. Its culture characteristics are shown in Figure 2-8.
(2) In semi-solid medium
The characteristics of bacterial culture in semi-solid medium in test tubes are shown in Figure 2-9.
(3) In liquid culture medium
Bacteria grow in liquid culture medium, making the culture medium turbid. The turbidity varies depending on the bacteria’s different requirements for O2:
Facultative anaerobic bacteria - the culture liquid is uniformly turbid, aerobic bacteria - the culture liquid is only turbid in the upper part, and anaerobic bacteria - the culture liquid is only turbid in the lower part. Some bacteria form bacterial rings or bacterial films on the surface of the culture solution, or produce flocculent precipitates at the bottom, and some produce bubbles and pigments (see Figure 2-10).
6. The morphology and structure of actinomycetes
The cells of actinomycetes are filamentally branched, and the mycelium (mycelium) is composed of hyphae. The width of hyphae is similar to that of common bacilli (<1μm). During the vegetative growth stage, there are no septa within the hyphae and they are single cells. There are numerous nucleoplasts in the cell. The hyphae of actinomycetes are divided into three categories: basal hyphae, aerial hyphae and spore hyphae according to their morphology and function.
The intrabasal hyphae, also called primary hyphae, grow in the culture medium, and their physiological function is to absorb and excrete metabolic waste. The hyphae in the base are colorless or produce various pigments, which are water-soluble or fat-soluble. The water-soluble pigments make the culture medium and the hyphae appear the same color.
Aerial hyphae, or secondary hyphae, are formed by basal hyphae elongating into space. They are thicker than basal hyphae, have straight or curved branches, and some produce pigments.
Spore filaments are also called reproductive hyphae, which are differentiated from mature aerial hyphae. The various spore filament shapes are shown in Figure 2-11. Spore filaments divide by transection to produce single, double or clustered conidia. The spores are spherical, oval, rod-shaped, melon seed-shaped, etc., with a smooth surface or spiny and hairy surfaces, and come in various colors, depending on the species.
7. Reproduction of Actinomycetes
Actinomycetes mainly reproduce by forming conidia through transverse division of spore filaments.
Actinomycetes can also use fragments of broken hyphae to form new bacterial cells. This method of reproduction is common in liquid culture media.
8. Culture characteristics of actinomycetes
The colonies of actinomycetes on solid culture media are generally round, smooth or have many wrinkles. Because it is tight and solid, it is difficult to pick out with a needle. After sporulation, the surface of the colony appears powdery. The bottom (basal hyphae) and surface (aerial hyphae, spore threads) of actinomycete colonies often have different colors.
When actinomycetes are cultured statically in a liquid culture medium, plaque-like or film-like cultures will form on the liquid surface on the inner wall of the container, or they will settle to the bottom without making the culture medium turbid. If cultured with shaking, spherical particles composed of short mycelium are often formed.
9. Representative genera of actinomycetes
(1) Streptomyces
There are many types of actinomycetes in this genus, including various Streptomyces species. There are different forms of spore filaments, which are important indicators for classification and identification. Of the antibiotics produced by actinomycetes, 90% are produced by the genus Streptomyces. Such as commonly used streptomycin, oxytetracycline, anti-tuberculosis kanamycin, anti-fungal nystatin, anti-tumor bleomycin, mitomycin, Jinggangmycin to prevent rice sheath blight, etc. , are all secondary metabolites of Streptomyces. Some streptomycetes can produce more than one antibiotic. Streptomyces griseus is a strain that produces vitamin B12. The genus Streptomyces has great economic value and medical significance.
(2) Nocardia
Also known as Proactinomyces, it forms typical mycelium on the culture medium. Its characteristic is that within 15h to 4d of culture, the hyphae produce diaphragms, and all branched hyphae suddenly break into rod-shaped, spherical or branched rod-shaped bodies.
Many species of this genus can produce antibiotics, such as rifumycin, which is effective against Mycobacterium tuberculosis and M. leprae, and is effective against viruses and protozoa. Effective metamycin, etc. Actinomycetes of this genus can assimilate various carbohydrates, and some can utilize hydrocarbons, cellulose, etc., and can be used for petroleum dewaxing, hydrocarbon fermentation and sewage treatment. Therefore, Nocardia has important implications for both medicine and environmental protection.
(3) Actinomyces
This genus only has intrabasal hyphae, which have transverse septa and can be broken into a "V" shape or "Y'' Body. There is no aerial hyphae and no spores are formed. Actinomyces are mostly pathogenic bacteria, such as Actinomyces bovis causing bovine jaw disease and Actinomyces israelii. Posterior jawbone tumors and lung infections in humans.