High Power Microscopes and Bacteria Structures
Mesosomes These membrane-associated organelles are more easily demonstrated in gram-positive than in gram-negative bacteria. Mesosomes are usually seen as cytoplasmic sacs that contain whorled, lamellar, tubular, or vesicular structures and are often associated with division septa. Their compositions are obviously observed when utilizing a microscope in studying them. Attachment of mesosomes to both DNA chromatin and membrane has been demonstrated by thin-section electron microscopy. Formation of protoplasts or spheroplasts results in eversion of tubular or vesicular mesosomal components, which remain attached at one end to the outside of the membrane, whereas the enclosing mesosomal sacs disappear and are apparently pulled into the membrane by the stretched protoplast. These are all clearly viewed using the microscope. Mesosomes have been reported to be artifacts of fixation procedures, but it is difficult to explain vesicular mesomal tubules as artifacts.
Cytoplasmic Structures The Nuclear Body Bacterial DNA can be detected as nucleoids or chromatin bodies by light microscopy using Feulgen staining. It is difficult to demonstrate chromatin bodies by direct staining because of the high concentration of ribonucleic acid. Nevertheless, pretreatment with ribonuclease removes all or nearly all of the RNA, and chromatin bodies can then be seen, using the light microscopes, at all stages of the growth cycle. Electron microscopy, with the use of electron microscope or scanning electron microscope, of stained thin sections reveals nuclear material as an irregular, thin, fibrillar, DNA network, which frequently runs parallel to the axis of the cell. A direct attachment to the membrane is sometimes obvious and is seen under an electron microscope. Often the mesosome, itself a membrane-associated structure, appears to serve as a DNA-membrane attachment site. During multiplication, bacterial DNA remains as a diffuse chromatin network and never aggregates to form a well-defined chromosome during cell division, in contrast to the eucaryotic chromosomes. When bacterial cells are very gently lysed, the bacterial chromosome may be visualized by radioautography as a circular molecule. Although bacterial DNA represents only two to three percent of the cell weight, it occupies 10 percent or more of the cell volume. This loose arrangement allows for the ready diffusion of soluble materials to all parts of the nuclear structure.
Ribosomes Negatively stained thin sections allow resolution by electron microscopy, utilizing a scanning electron microscope or an electron microscope, of small cytoplasmic particles that correspond to the ribosomes present in pellets after lysed protoplasts or disrupted bacterial cells are centrifuged at 100,000 g. Ribosomes are composed of approximately 30 percent protein and 70 percent RNA and account for up to 40 percent of the protein and 90 percent of the ribonucleic acid of the cell. Gentle lysis of growing cells yields almost all ribosomes as polyribosome-membrane aggregates that contain all components of the protein-synthesizing mechanism. Polyribosomes are chains of 70S ribosomes or monomers attached to messenger RNA. These chains are lucidly observed under a high-powered electron microscope. Ribosome numbers in the cell vary according to growth conditions. Rapid-growing cells in rich medium contain many more ribosomes than do slow-growing cells in poor medium.
Polyamines and Histone-like Proteins Histone-like proteins have only recently been established in small amounts in association with E. coli DNA, whereas the occurrence of polyamines in bacteria is well known. Polyamines are required for growth of some Haemophilus species, but the lack of them does not retard the growth of other bacteria, which are usually studied under a microscope, that are unable to make them. Polyamines are found associated with ribosomes and membranes. The principal ones are Putrescine and Spermidine. The precise function of these polyamines is not known. Polyamines exert an anti-mutagenic affect, they prevent dissociation of 70S ribosomes to 30S and 50S components, and they increase the resistance of protoplasts to osmotic lysis. The amount of spermidine varies inversely with the amount of Magnesium in ribosomes, and 30S and 50S ribosomal subunits remain associated in the absence of Magnesium if spermidine is present. From this it would appear that charge neutralization of polyanionic polymers, such as nucleic acid, may be at least partly nonspecific.
Cvtoplasmic Granules Granules, identified by appropriate staining procedures, indicate accumulation of food reserves, including polysaccharides, lipids, or polyphosphates. Granules vary with the type of medium and the functional state of the cells. Glycogen is the major storage material of enteric bacteria and may account for as much as 40 percent of the weight of some species. Similarly, some Bacillus and Pseudomonas species accumulate 30 percent or more of their weight as poly-beta-hydroxybutyrate. Finally, polyphosphates are also known as the metachromatically staining Babes-Ernst or volutin granules found in abundance in Corynebacterium diphtheriae, the plague bacillus or Yersinia pestis, mycobacteria such as Mycobacterium tuberculosis, and some other bacteria. Volutin granules stain in various colors, varying from red to blue, with toluidine blue and methylene blue. These bacteria are only observed or studied with the use of the various microscopes such as stereo microscopes and other high-powered microscopes.
Bacterial Endospores Endospore formation is a distinguishing feature of organisms of the family Bacillaceae, which includes members of the aerobic genus, Bacillus, and the anaerobic genus, Clostridium. They are glowingly viewed with the help of the microscopes such as light microscopes. Endospores resist adverse environmental conditions of dryness, heat, and poor nutrient supply. If present in inadequately heat-sterilized canned foods, spores of the anaerobic Clostridium species germinate, grow, and cause spoilage. In the case of C. botulinum, this is especially dangerous because spoilage may not be obvious even though accompanied by the production of deadly exotoxin. Other sporeformers, such as the gas-gangrene bacillus, Clostridium perfringens, or the tetanus bacillus, Clostridium tetani, produce their toxic effects only when a medium of dead or injured tissue affords the spore a nidus for growth in the animal body.
The true endospore is highly refractile body, which is clearly seen in a microscope, formed within the vegetative bacterial cell at a certain stage of growth. The size, shape, and position of the spore are relatively constant characteristics of a given species and are, therefore, of some value in distinguishing, using a microscope, one kind of bacillus from another. The position of the spore in the cell may be central, subterminal, or terminal. It may be the same diameter as the cell, smaller or larger, causing a swelling of the cell. Various protective spore coats are formed within the vegetative cell before death and dissolution of the parent cell. These include a rigid peptidoglycan layer, which differs in composition from that of the parent vegetative cell. Spore surface antigens are usually different from those of the parent vegetative bacilli.