GENERAL BACTERIOLOGY 1. Bacterial cell (morphology, staining reactions, classification of bacteria) The protoplast is bounded peripherally has a very thin, elastic and semi-permeable cytoplasmic membrane (a conventional phospholipid bilayer). Outside, and closely covering this, lies the rigid, supporting cell wall, which is porous and relatively permeable. The structures associated with the cell wall and the cytoplasmic membrane (collectively the cell envelope) combine to produce the cell morphology and characteristic patterns of cell arrangement. Bacterial cells may have two basic shapes: spherical (coccus) or rod-shaped (bacillus); the rod-shaped bacteria show variants that are common-shaped (vibrio), spiral (spirillium and spirochetes) or filamentous. The cytoplasm, or main part of the protoplasm, is a predominantly aqueous environment packed with ribosomes and numerous other protein and nucleotide-protein complexes. Some larger structures such as pores or inclusion granules of storage products occur in some species under specific growth conditions. Outside the cell wall there may be a protective gelatinous covering layer called a capsule. Some bacteria bear, protruding outwards from the cell wall, one or more kinds of filamentous appendages: flagella, which are organs of locomotion, fimbriae, which appear to be organs of adhesion, and pili, which are involved in the transfer of genetic material. Because they are exposed to contact and interaction with the cells and humoral substances of the body of the host, the surface structures of bacteria are the structures most likely to have special roles in the processes of infection. Shape: this can be of 3 main types: round (cocci) - regular (staphylococci) - flattened (meningococci) - lancet shaped (pneumococci) elongated (rods) - straight (majority of them are like this; e.g. E.coli) - short (coccobacilli; e.g acinetobacters) - long (fibres) – these are mainly found in OLD cultures - slender – mycobacterium tuberculosis - robust – lactobacilli, bacillus - with split ends - bifidobacteria - branching - nocardiae, actinomycetes - curved - campylobacters - with flat ends – bacillus anthracis - spindle-shaped - fusobacteria - club-shaped - corynebacteria - pleomorphic – haemophili - spiral – helicobacter, spirilium Spiral bacteria (spirochetes): these are different to the spiral bacteria mentioned above! They are tightly coiled bacteria. - Thick: Spirillium - Uneven: borrelia - Delicate, regular: Treponema - Slender with bent ends: Leptospira STAINING Two basic methods provide foundations for differential staining and detection of bacteria: the Gram stain and acid-fast stains. Bacteria are generally studied when fixed and stained. Smears or films of bacterial cultures and clinical specimens are usually fixed by heat, the slide being first throughly dried in air and then heated gently in a flame. Vegetative bacteria are thereby killed, attached to the surface of the slide and preserve from undergoing autolytic changes. During staining, the coloured, positively charged cation of basic dyes such as methylene blue combines with negatively changed groups in the cell protoplasm, specially with the phosphate groups in the abundant nucleic acids. Acid dyes, having coloured anions, do not stain bacteria strongly except at very acid pH values, and thus can be used for ―negative staining‖. Negative or background staining is of value as a rapid method for the simple morphological study of bateria and yeasts. In the case of bacteria, Gram’s stain has the widest application, distinguisinhg them as ―Gram-positive‖ or ―Gram-negative‖, according to wether or not they resist decoloration with acetone, alcohol or aniline oil after staining with a triphenyl methane dye, such as methyl violet, and subsequent treatment with iodine. The Gram-positive bacteria resist decoloration and remain stained a dark purple colour. The Gram-negative bacteria are decolorized, and are then ounterstained light pink by the subsequent application of safranin, neutral red or dilute carbol fuschin. Gram reactivity appears to reflect a fundamental aspect of cell structure and is correlated with many other biological properties. Gram-positive bacteria are more susceptible than Gram-negative bacteria to the antibacterial actions of penicillin, acids, iodine, basic dyes, detergents and lysozyme, and less susceptible to alkalis, azide, tellurite, proteolytic enzymes, lysis by antibody and complement, and plasmolysis in solutes of high osmotic pressure. The probable mechanism of the Gram stain is attributed to differences in the permeability of the two essential cell wall types. After staining with methyl violet and treatment with iodine, a dye-iodine complex is formed within the cell; this is insoluble in water but moderately soluble and dissociable in the acetone or alcohol use as the decolorizer. Under the action of the decolorizer, the dye and iodine diffuse freely out of the Gram-negative cell, but not from the Gram-positive cell, presumably because the cell wall of the latter is less permeable. Gram-positive bacteria become Gram-negative when their cell wall is ruptured or removed. G+ G- 1. Fixation by flame 3 times 2. Gram stain 20 s 3. Lugol solution 20 s 4. Alcohol máx. 20 s 5. Aqua fontis rinse 6. Safranin 1 min 7. Aqua fontis rinse 8 Drying 2. Anatomy of the bacterial cell I – contents of cytoplasm, cytoplasmatic membrane The cytoplasm, or main part of the protoplasm, is a predominantly aqueous environment packed with ribosomes and numerous other protein and nucleotide - protein complexes. Bacterial nucleoid The genetic information of bacterial cell is mostly contain in a single, long molecule of double-stranded deoxyribonucleic acid (DNA). The cell solves the problem of packaging this enormous macromolecule by condensing and looping it into a supercoloid state. The bacterial nucleoid lies within the cytoplasm. This means that as DNA-dependent RNA polymerase makes RNA, ribosomes may attach and initiate protein synthesis on the still attached (nascent) messenger RNA. Synthesis of mRNA and protein are therefore seen to be directly coupled in bacteria. Cytoplasmic membrane The bacterial protoplasm is limited externally by a thin, elastic cytoplasmic membrane which is 5-10nm thick and consists mainly of phospholipids and proteins. Integral, transmembrane and peripheral or anchored proteins occur in abundance and perform similar functions to those described in eukaryotes (e.g. transport and signal transduction). Prokaryotic cell membranes are relatively protein-rich, allowing relatively little space for phospholipids. 3. Anatomy of the bacterial cell II – cell wall, capsule, flagella, fimbriae, pili Cell wall The cell wall encases the protoplast and lies immediately external to the cytoplasmic membrane. It is 10- 25nm thick, strong and relatively rigid, tough with some elasticity, and openly porous, being freely permeable to small molecules. It supports the weak cytoplasmic membrane against the high internal osmotic pressure of the protoplasm and maintains the characteristic shape of the bacterium in its coccal, bacillar, filamentous or spiral form. Capsule Many bacteria have been demonstrated to possess a more or less continuous but relatively amorphous layer external to the Gram-negative and Gram-positive envelopes. This layer is called a capsule. The capsular gel consists largely of water and has only a small content of solids. In most species, the solid material is a complex polysaccharide, though in some species its main constituent is polypeptide. The capsule have some role in interactions with the external environment. In some cases capsules have been shown to protect against phagocytosis, the lytic action of complement and bacteriophage invasion. Capsules also appear to have a role in protecting cells against desiccation. The production of extracellular polysaccharides in general provides a matrix within which biofilm formation can take place. Flagella Motile bacteria possess filamentous appendages known as flagella, which act as organs of locomotion. The flagellum is a long, thin filament, twisted spirally in an open regular wave form. It originates in the bacterial protoplasm and the structure projects throught the cell envelope. According to the species, there may be one, or up to 20 flagella per cell. The external portion of a flagellum is essentially a polymer of a single protein, flagellin, while the basal region inserted into the cytoplasmic membrane comprises multiple subunits which anchor and power the organ. Motility is clearly important to many bacteria and probably serves mainly to place the cell in environments favourable to growth and free from noxious influences. In some cases possession of flagella is thought to contribute to the pathogenesis of disease. Fimbriae and pili Many bacteria possess filamentous appendages termed fimbriae or pili. These terms are often uses interchangeably, although the latter was originally reserved for structures involved in genetic exchange between bacteria (sex pili). Fimbriae are far more numerous than flagella and more much shorter and only about half as thick. In contrast, sex pili are structurally similar to other fimbriae but are longer and confer the ability to attach specifically to other bacteria that lack these appendages. Sex pili initiate the process of conjugation; they also act as receptor sites for certain bacteriophages. 4. Anatomy of the bacterial cell III – bacterial spores Some bacteria, develop a highly resistant resting phase or endospore, whereby