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Lecture 1. Subject of Microbiology. SHORT HISTORICAL OUTLINE PRINCIPLES OF THE CLASSIFICATION OF MICROORGANISMS. BACTERIAL CELL STRUCTURE. What’s microbiology? Term itself –- mikros – small, bios – life, logos – science. It is the science, which studies a smallest organisms, invisible to naked eye, named microbe. Figure 1.1 Microbiology studies microorganisms from various point of views. There are different subtypes of microbiology according to the subject of investigation: general, technical, agricultural, veterinary, medical, sanitary, and also such sciences as immunology and virology. Medical microbiology: Obtain, isolate and describe the infectious agent. Define and distinguish methods of treatment. Elaborate new methods of diagnostic. Elaborate new methods of prophylactic. The breef history of microbiology. 320 years ago the first microscope was developed by Antony Van Leeuwenhoek, Dutch microscopist. He was the first person, who sow and described microbes. He himself made simple lenses, which magnified 160 – 300 fold. In 1695 he published his work – “ The Secrets of nature discovered by A. V. Leeuwenhoek”, where he discribed all living particles, which he observed in water, soil, feces. Figure 1.2 The microscope of A. V. Leeuwenhoek The discovery of A.V. Leeuwenhuek stimulated the great interest among scientist to invisible world. However, more then 150 years scientists were unable to use these wonderful investigation for learning nature of infectious diseases. But during these times many scientists made the attempts to apply science dates to practical problems being faced in the battle against epidemic diseases. One of them was Russian scientist D. Samoijlovich. He made the conclussion that plaque is transmited by spesial infectious particles. To prove this he carried out a dangerous experiment. He inoculated himself with infectious material taken from a person with bubonic plaque ( 1771). In 1798 the English physician E. Jener published his results of vaccinations against smallpox. He proved that vaccination of humans with cowpox protects them from infections with smallpox. Those discoveries played important role for further development theoretical and practical problems of prophylaxis and struggle against infectious diseases. Figure 1.3. E. Jener Louis Pasteur: the name of great French scientist. Had two Ph.D. deegres – in chemistry and physics, concerning the polarization of liquids; He studied the crystals of wine acid in the microscope Proved microbiological nature of alcoholic, lactic and buturic fermentation Demonstrated a new type of respiration – anaerobic – in some of microbes Then he started to investigate the wine diseases – french wine became sour. He told that when in the wine are presented yeast cells – the wine is good, lactobacteria – bad. He suggested to heat at 560C. It is a method of pasteurization. Figure 1.4 Louis Pasteur Then he presented and proved his famous theory that there is no self-origin of life (microbes). He proved that spontaneous generation of living substases does not exist. First of all he investigated the chicken’ diseases, and namely, anthrax. Also he discovered the nature of rabies and developed the method of producing of antirabies vaccines and began to use this vaccine to treatment (prophylaxis). German school. Robert Koch (1843) made a great progress of medical microbiology. He discovered a solid nutrient media ( gelatine, coagulated serum, meat peptone agar (MPA) and applied them to isolating of pure cultures of microbes. He also introduced aniline dyes and immersion system in practice of microscopy. Proved the bacterial nature (ethiology) of anthrax. Discovered choleric agent and tuberculosis agent. And obtained tuberculin from tuberculoid bacterium. Figure 1.5 Robert Koch English school of microbiology represent Lister, who opened the new way for surgery. He discovered and introduced in surgery practise the methods of antiseptic – cleance with carbolic acid. That is why microbiology opened the new way for surgery. Now – aseptic. Figure.1.6 Lister Also a great role microbiology play in therapy. examples: role of immunology for treatment of rheumatism. Gastric and duodenal ulcer – Helicobacter pilory – decreased number of cancer. Russian Scool. Great role of understanding of inflamation nature was made by I.E. Mechnikov – all basic ideas of immunology: immune status, immune resistance, specific and non-specific factors of defense. The classic works of Mechnikov on the biological theory of immunity opened a new stage in the development of medicine. He discovered and studied the process of intracellular digestion as a mechanism of defence against pathogenic microbes which have penetrated into the body. He discovered that some cells of mesoderma, leycocytes. Those cells was named phagocytes. That is why we now Mechnicoff as a founder of cell- theory of immunity, in 1908 he was awarded Nobel prize. Figure.1.7. I.E. Mechnikov Year 1674 1796 1859 Event Leeuwenhoek discovers microorganisms. Jenner creates a vaccine for smallpox. Pasteur disproves spontaneous generation of microorganisms. 1865 1876 Lister introduces antiseptic techniques. Koch proves that specific microorganisms cause specific diseases. Koch uses agar to obtain a pure culture. Metchnikov discovers phagocytic theory of immunity. lwanowski discovers viruses. Ehriich articulates the principle of selective toxicity. Fleming discovers penicillin. 1881 1883 1892 1894 1929 Subject of microbiology. Microbiology deal with: All what’s invisible (inside the world of living mater). All belong to Protista biologic kingdom: eucariota, procariota (eubacteria and archebacteria), viruses, viriods, plasmids and prions. Figure 1.8. The modern classification: There are 2 Upper kingdom of living mater : procariotes and eucariotes. Group of Procariotes contain: Cianibacterium Arhebacterium Eubacterium (true bacteria) Eucariotes contain: animals; plants; fungi (micota) In this group for microbiology are important: From animals: class Protozoa From plants –- unicellular Water-plants- Algae. From fungi – all representatives. Figure.1.9. That is why Microbiology deal with: Protozoa eucariota have nucleus, chromosomes free living, conditionally pathogenic and human parasites representatives: malaria, trypanosomosys, leishmania etc Fungi eucariotic cells dimorphism (yeast and mold forms) sexual and asexual reproduction growth cycle – reproductive and vegetative phases Bacteria procariota science – bacteriology conditionally pathogenic and pathogenic; normal microflora, antibiotic producents, smallest free-living forms no sexual reproduction, could create spores archebacteria: no peptidoglycan in cell wall; no pathogenic species for human; eubacteria: bacteria, ricketssia, mycoplasma, chlamydia Main classification of bacteria is Bergey’s Manual, which include Two division: 1.cyanobacteria (cyanophyta) Water-plants 2.true bacteria which include 19 parts. Main of them represent: Bacteria ( rod-like, cocci) (aerobic and anaerobic), (endospore forming or no) Spirochetes and spiriles Vibrions Actinomycetes ( – important as producents of antibiotics) Obligate intracellular parasites (Ricketsia and Chlamidia) Viruses (are grouped in an independent kingdom) Viriods, Prions are genetic parasites no cell structures and protein synthesis systems on animals, insects, plants, bacteria (phages) and human contain DNA or RNA are not visible with the light microscope viriods cause plant diseases prions are infectious particles associated with scrapie, a degenerative central nervous system of sheep Principles of classification All bacteria has binary nomenclature: Genus and: Species – pool of microorganisms with common origin, similar genotype (>60% of DNA homology) and maximum adjacent phenotypic signs and properties. Morphological, tinctorial, cultivation, mobility, spores production, physiology, biochemical, phages sensitivity, antigenic, cell wall structure etc. Aditional terms: Clone – a population of microorganisms descended from a single individual by asexual reproduction. Strain – clones that are presumed or known to be genetically different. ( It is specimen of microbic culture the same species, which was isolated from different places, or from one place in different times. Figure 1.10.Main Bacterial Morphology. Cocci: monococci, diplococci (gonococci, meningococci, pneumococci), streptococci, staphilococci, tetracocci, sarcines (8 – 16 cells) Rod-like: mono, strepto, diplo; with or without spores Spirals forms: vibrio, spirillae. Figure 1.11.Structures of bacterial cell. Intracellular structures. What are bacterial cell consist from? – Proyoplast, nucleoid, endospores, plasmides, volutine granuls. Extracellular structures – flagelas as a mechanism of motion (monotrichous, lophotrihous, amphotrihous, peritrichous.) Figure.1.12. Flagelas capsuls – polysaccharide outer mucose layer, which defens cell from outer action.(important espesially for pathogenic microbes inside host organism) Pili (fimbrias) – filamentous structures which take part in processes of conjugation (transmission of DNA )and adhesion. Figure 1.13 Capsuls Figure 1.14. Pili cell envelope So called: Gram+ and Gram– depend from structure of cell-wall Figure.1.15. Structure of cell envelope. Comparision of a Procaryotic cell and a Eucaryotic cell Structure Appendages Glycocalyx Outer membrane and periplasm Cell wall Procaryote Pili, flagella, axial filaments in spirochetes Usually; can be a thin slime layer or thicker capsule Gram-negatives only Eucaryote Flagella; structurally very different from procaryotic flagella In some algae, fungi, protozoa, and human cells that lack walls Never All eubacteria except mycoplasmas; basic component is peptidoglycan Algae, fungi, protozoa, plants, but never with peptidoglycan; no cell walls in animals, including human cells All; a phospholipid bilayer Cytoskeleton and cytoplasmic streaming in all; pseudopods in amoebae Always membrane-bound; include nucleus, endoplasmic reticulum, Golgi apparatus, 80S ribosomes (except 70S in organelles), mitochondria and/or chloroplasts (algae and some protozoa have both) Paired chromosomes; associated with histones Mitosis for growth and meiosis for reproduction Never Plasma membrane All; a phospholipid bilayer Cytoplasm Undifferentiated Organelles Not membrane-bound; they include nucleoid, 70S ribosomes, various inclusions, and the periptasm in Gramnegatives DNA Usually one circular chromosome Cell division Binary fission or budding for both growth and reproduction Principally in Gram-positive bacteria, but in some Gram-negative . Endospores Size Typically one or a few micrometers Typically 10 micrometers or more Modern diagnostic methods in microbiology 1.Microscopic light microscopes: fluorescenes, dark-field, etc. EM – 1939 – thin structure, DNA etc. scanning EM – fimbriae and pili native or stained biological material define cell form, their arrangement ability to be stained by different dyes 2.Bacteriological or physiological methods isolation of pure culture and its identification grow on special nutrient media – for heterotrophes, not for intracellular parasites – cultural properties; 3.Biochemical methods Investigation of fermentative activity of microbes. 4.Serological methods Identification of antigenic composition of bacteria. determine specific antibodies or antigenes in patients blood Investigation of patient serum with serological reactions: agglutination, precipitation, ELISA, immunofluorescence etc. Allergic probes sensitivity of macroorganism to various infectious agents or their metabolits 5.Biological methods contamination (infecting) of lab animal Investigation of Diagnostical triade: Isolation of causative agent from sick human Microscopy of obtained material Isolation of pure culture and its investigation Model of the disease on lab animal 6. Genetics methods Investigation DNA (RNA) sequence. PCR. 7. Sensitivity to bacteriophages and antibiotics Laboratory Equipment and Procedures The microscopy Laboratory equipment and procedures Light microscopes Stained bacteria Ultraviolet microscope Staining reactions Fluorescence microscope Staining techniques Dark-field microscope Preparation of a pure culture Phase-contrast microscope Pure culture techniques Electron microscope Culture media Transmission electron microscope Control of pH and temperature Scanning electron microscope Oxygen requirements Techniques for microscopic study of Sterilization methods bacteria How bacteria are identified There are certain basic techniques that the student of microbiology must learn to use in the laboratory. These techniques are used in growing bacteria, isolating them in pure culture (containing only one kind of bacterium), observing them, and finally in identifying the organisms. THE MICROSCOPE Light Microscopes Accounts in seventeenth-century scientific literature convey the excitement with which the Royal Society of London awaited letters and descriptions from Leeuwenhoek. We have advanced a long way in the last 350 years with respect to the equipment available for the microscopic study of microorganisms. The microscope you will use in the laboratory is no longer the "simple" (single lens) microscope of the type made and used by Leeuwenhoek but rather what we call a compound microscope because it has two sets of lenses (see Figure 2.1). One set is next to the object to be studied and, therefore, is called the objective. The other set, the ocular, is (as the name implies) the one next to your eye. Figure 1.16 Modern binocular (two eyepieces) microscope. Note the mechanical stage, which facilitates the movement of slides. Both objectives and oculars are designed for different magnifications. The objectives usually are mounted in a rotating wheel known as a turret or revolving nosepiece; any one objective may be rotated into place depending on the magnification desired. Oculars are made in different magnifications — that is, X5, X 10, and X 15 (X meaning power or times actual size) — but most frequently the X 10 is used. Now, how do these two lenses work together to give a higher magnification of the specimen than is possible with one lens alone? Suppose that the objective is X10, which means that it makes an image 10 times as big as the object under study. Here you have, in effect, a simple magnifying glass. Now, suppose that you view this magnified image through a second lens (the ocular) that will magnify it five more times. How much larger than the object is the final image that you see? The answer is 50. The total power of any microscope, then, can be determined simply by multiplying the objective power by the ocular power. The usual laboratory microscope has three objective lenses: low-power, high-power, and oil-immersion. The latter is the highest-powered of them all and is used almost exclusively to study bacteria. A drop of clear oil is placed directly on the cover glass over the specimen, and the oil-immersion objective is lowered into the oil. The light rays traveling from the specimen through glass and oil do not bend as much as they would if they were to pass through glass and air; therefore, the image is clearer. The so-called high-power objective is actually intermediate in the power range; because it does not require oil, it is usually referred to as high-dry. The oil-immersion objective usually magnifies 97 times; with a X10 ocular, the total magnification of the objective would be X 970. It is possible to obtain somewhat higher power with different lens combinations, but there is a definite limit to magnification by this means. The high-dry usually has a X 44 objective; thus, with a X10 ocular the overall magnification is 440. The low-power is usually a 10 X 10, or a hundredfold, magnification. A physical law states that the smallest detail we can see (called the resolving power) is limited by the wavelength of light being used to illuminate the specimen. The limit of resolution in ordinary visible light can be calculated to be approximately 0.2 mkm, and all the lenses in the world will not let you see something smaller than 0.2 mkm unless a different type of light source is used. Ultraviolet Microscope A variation on the ordinary light microscope is the ultraviolet microscope. Because ultraviolet light has a shorter wavelength than visible light, the use of ultraviolet light for illumination can increase the resolving power to twice that of the light microscope. The limit of resolution then becomes 0.1 mkm. Since ultraviolet light is invisible to the human eye, the image must be recorded on a photographic plate. These microscopes use quartz lenses, and they are too intricate and expensive for common use. Fluorescence Microscope Bacteria stained with a fluorescent dye will be a different color under the fluorescence microscope than they are under an ordinary light microscope. The quality known as fluorescence can be observed in the presence of ultraviolet light. Mycobacterium tuberculosis stained with auramine (a yellow dye) will appear bright against a dark background. Because most bacteria do not stain with this dye, this procedure is useful in identifying the tubercle bacillus. Fluorescence microscopy may also be used to detect foreign substances or antigens (such as bacteria, rickettsiae, or viruses) in tissues. In this technique the specific antibody protein is first separated from the serum in which it occurs. It is then combined, or conjugated, with a fluorescent dye. Because antibody-antigen reactions are specific, fluorescence will occur only if the antigen in question is present and binds to the fluores-cently labeled antibody. Antibody-antigen reactions are discussed further. Dark-Field Microscope The dark-field microscope is used for viewing living bacteria, particularly those so thin that they approach the limit of resolution of the compound microscope. Many pathogenic spirochetes fall into this category, particularly the syphilis spirochete, Treponema pallidum. The dark-field microscope differs from the ordinary compound light microscope only in having a special condenser which produces a hollow cone of visible light. The rays of light from this hollow cone do not go directly up into the objective lens. Instead, they are reflected away at a slight angle from the top of the slide (see Figure 2.3). However, any light rays touching the specimen will be reflected directly into the objective, with the result that the specimen looks completely white against a black background. With no specimen in the field, the field would look dark, since there would be nothing to cause the light to be reflected up into the objective. Phase-Contrast Microscope The ideal way to observe living matter is in its natural state: unstained and alive. As a rule, however, a microscopic fragment of living matter (such as animal tissue or bacteria) is practically transparent, and individual details do not stand out. This difficulty can be overcome with the use of the phase-contrast microscope. The principle of this device is complicated. For example, when an ordinary microscope is used, the nucleus of an unstained living cell is invisible. Since the nucleus is nevertheless present in the cell, its presence will alter very slightly the relationship of the light that passes through the nucleus with the light passing through the material around the nucleus. This relationship, imperceptible to the human eye, is called a phase difference. However, an arrangement of filters and diaphragms on the phase-contrast microscope will translate this phase difference into a difference in brightness — that is, into areas of light and shade that can be discerned by the eye — rendering the hitherto invisible nucleus (or any other structure) visible. Electron Microscope Transmission Electron Microscope The transmission electron microscope has been a boon to microbiologists in the past 35 years (see Figure). This microscope uses a beam of electrons rather than visible or ultraviolet light. Because the wavelength of the electron beam is much shorter (approximately 0.005 ran, or nanometers) than that of even ultraviolet light, the resolving power of the microscope is increased tremendously. With a resolving power greater than one nm (0.001 /im), magnifications of as much as 1 million diameters are possible. The image produced by the electron microscope is visible when projected onto a fluorescent screen. Figure 1.17. The operation of an electron microscope requires a great deal of experience and skill to obtain clear results. However, the magnification obtainable is many times that possible with the light microscope. There are several major problems involved in using a transmission electron microscope: (1) it requires a skilled technician for operation; (2) it is a very expensive piece of equipment; (3) it requires the use of very thin specimens (which may be easily distorted); (4) the specimen being examined must be contained in a very high vacuum in order that the electrons may move effectively; (5) live specimens, therefore, cannot be examined; and (6) objects show no color. This means that preparations must be dried and fixed with chemicals prior to study, and the drying and fixing process may also result in distortion of some of the cellular components. An important development in electron microscopy is the use of phosphotungstic acid as a negative stain. Phosphotungstic acid is electron- dense, allowing structural details of the cell to be observed while the background and empty areas remain opaque. Shadow casting is a technique for revealing surface details. An electron-dense metal such as platinum or chromium is vaporized under hifh vacuum and deposited at an angle on the preparation. The uncoated area on the opposite side acquires a shadow, and the resulting electron micrograph shows a three-dimensional effect. Scanning Electron Microscope The scanning electron microscope employs a beam of electrons, but — instead of being simultaneously transmitted through the entire field — the electrons are focused as a very fine probe or spot which is moved back and forth over the specimen. As the probe electrons strike the surface of the specimen, secondary electrons are emitted and then collected by a cathode ray tube. The strength of the signal will be seen as dark or light areas on the collector, providing an image of the specimen's surface. Photographs taken of the cathode ray tube appear as threedimensional micrographs, as is shown in Figure 1.19. Figure 1.18 Electron micrograph of the spirochete Spirochaeta stenostrepta shadowed with platinum (X 12,100). Figure 1.19 The three-dimensional qualities of scanning electron microscopy clearly reveal the corkscrew shape of cells of the syphilis-causing spirochete T. pallidum, attached here to rabbit testicular cells grown in culture (X8000). TECHNIQUES FOR MICROSCOPIC STUDY OF BACTERIA Living Bacteria Living bacteria are difficult to see with the average light microscope because they appear almost colorless when viewed individually, even though the culture as a whole may be highly colored. However, it is often necessary and desirable to look at living bacteria under the microscope, particularly to determine whether they are motile. Laboratory Equipment and Procedures One satisfactory means of observing living bacteria is in a hanging drop preparation. A drop of liquid culture (or organisms suspended in water) is placed in the center of a coverslip. A special slide with a hollow depression in the center is used. The depression is ringed with a thin film of petrolatum and then turned upside down over the cover glass so that the drop of culture is in the center of the depression. The entire slide with the coverslip then is quickly turned over so that the drop of culture actually hangs from the coverslip down into the depression. The slide is placed on the stage of a microscope and the organisms are observed using the highdry or the oil-immersion objective (see One can also use a normal flat slide to determine motility; however, a thin ridge of petrolatum must be applied under the edge of the coverslip to prevent convection currents caused by evaporation from the wet mount. Keep in mind that a bacterium is considered motile only if it seems to be going in a definite direction. Even nonmotile bacteria will bounce back and forth rapidly (brownian movement) due to bombardment from molecules of water. Figure 1.20. This side view of a hanging-drop preparation shows the drop of culture hanging from the center of the cover glass above the depression slide. Stained Bacteria Bacteria are far more frequently observed in stained smears than in the living state. By stained bacteria we mean organisms that have been colored with a chemical stain so as to make them easier to see and study. In general, stained smears of bacteria reveal size, shape, and arrangement and the presence of certain internal structures such as granules and spores. Special stains are used for observing capsules or flagella as well as certain internal cellular details. Stains that reveal chemical differences in bacterial structure may also be employed. These are called differential stains. To prepare bacteria for staining, a small amount of culture is spread in a drop of water on a glass slide. This is called a smear. The smear is dried at room temperature, and the bacteria may be firmly bound (fixed) by passing the slide quickly (smear side up) two or three times through the flame of a Bunsen burner. When cool, the smear is ready to be stained. Alternatively, many laboratories use methanol to fix the bacteria to the slide because heat may cause distortions in the appearance of the stained bacteria. This is done by placing a few drops of methanol on the air-dried smear and again allowing the smear to air-dry. The following sections outline a few of the more common procedures used to stain bacteria. Staining Reactions Stains are salts composed of a positive and a negative ion, one of which is colored. In basic dyes, the color is in the positive ion (i.e., dye+ Cl~), whereas in acid dyes it is in the negative ion (i.e., Na+ dye"). The marked affinity of bacteria for basic dyes is due primarily to the large amount of nucleic acid in the cell's protoplasm. Thus, when the bacterium is stained, the negative charges in the nucleic acid of the bacterium react with the positive ion of a basic dye. Crystal violet, safranin, and methylene blue are a few of the basic dyes commonly used. In contrast, acid dyes are repelled by the overall negative charge of a bacterium. Thus, staining a bacterial smear with an acid dye has the effect of coloring only the background area. Since the bacterial cell is colorless against a colored background, this technique is very valuable for observing the overall shape of extremely small cells. This process is referred to as negative staining. Staining Techniques Simple stain This, as the name implies, is the simplest type of staining. One merely covers the fixed smear with any one of the following dyes: gentian violet, crystal violet, safranin, methylene blue, basic fuchsin, and other basic aniline dyes. After 30 to 60 sec the slide is washed off under the water tap and the smear gently blotted dry. It is now ready to be looked at under the microscope. After locating the stained specimen with the high-dry objective, one can observe it under oil immersion by placing a drop of oil directly on the stained smear and lowering the oilimmersion objective into the oil. Gram stain In 1884 the Danish physician Christian Gram devised a special stain that is probably the most important one used in bacteriology. It is a differential stain, so called because it divides all the true bacteria into two physiologic groups, thereby greatly facilitating the identification of a species. The staining procedure has four steps: (1) the smear is flooded with gentian or crystal violet; (2) after 60 sec, the violet dye is washed off and the smear is flooded with a solution of iodine; (3) 60 sec later, the iodine is washed off and the slide is washed with 95 percent ethyl alcohol for 15 to 30 sec; and (4) the slide is counterstained for 30 sec with either safranin (a red dye) or Bismarck brown. (The Bismarck brown usually is used by people who are color-blind to red.) The length of time that each dye is left on the smear is not critical, and many laboratories have modified this procedure to allow each dye to remain in contact with the fixed organisms for only a few seconds. Also, many people prefer to use a 50:50 mixture of acetone and ethyl alcohol for the decolorization step because this solution acts faster than the 95 percent alcohol alone. Either decolorizing agent should give the same result, but in the discussion to follow we shall refer to 95 percent ethyl alcohol rather than the acetone-alcohol mixture. The violet dye and the iodine form a complex compound. Some genera of bacteria readily give up the stain when washed; in other bacteria the stain resists even a wash of 95 percent ethyl alcohol. Organisms that do not reta in the dye complex after the 95 percent alcohol wash are called gram-negative organisms; those that retain the complex are called gram-positive organisms. Because gram-negative bacteria are colorless after the alcohol wash, one always counterstains with a different color dye before looking at the smear under the microscope. The usual counterstain is the red dye safranin; hence gram-negative bacteria are red. Because alcohol does not wash out the blue dye complex from gram-positive cells, safranin counterstain has no effect, and gram-positive cells appear blue or bluish purple. Table 2.2 summarizes the appearance of both types of cells after each step of the Gram staining procedure. Table 2.3 lists the Gram reactions of some selected common bacteria. Many theories have been proposed to explain why some organisms are gram-positive and others gram-negative. This staining difference results from the fact that the cell walls of gram-positive bacteria are different from those of the gram-negative bacteria; the dye-iodine complex appears to become trapped between the cell wall Table Gram Reaction of Selected Common Bacteria Gram-positive Gram-negative Bacillus Bacteroides Neisseria Clostridium Bordetella Pasteurella Corynebacterium Brucella Proteus Lactobacillus Enterobactc Pseudomonas Listeria Escherichia Salmonella r Micrococcus Franciscella Shigella Staphylococcus Haemophilu Vibrio Streptococcus Kkbsiella Yersinia s and the cytoplasmic membrane of the gram-positive organisms, whereas the removal of lipids from the gram-negative cell wall by the alcohol wash permits the dye-iodine complex to be washed out of the cell. It is essential to know the Gram reaction (positive or negative) as well as the overall appearance of the bacterium in order to identify it. In addition, other general characteristics of an organism are associated with the Gram reaction. For example, most gram-positive bacteria are easily killed by low concentrations of penicillin, gramicidin, or gentian violet, whereas gram-negative bacteria are much more resistant to these compounds but are considerably more sensitive to streptomycin. We discuss the reasons for these differences in more detail in the next Chapters . Acid-fast stain The acid-fast stain (also called the Ziehl-Neelsen stain) is used specifically to help identify organisms in the genus Mycobacterium. This genus contains many non-disease-producing organisms as well as some virulent pathogens, of which tuberculosis and leprosy are the most important. The mycobacteria are called acid-fast because once they are stained with carbolfuchsin (a red dye), their unique chemical properties cause the stain to remain even though the stained smear has been washed with acid alcohol (95 percent ethanol containing 3 percent hydrochloric acid). This treatment removes the dye from all other organisms in a smear. One exception is found in the pathogenic (disease-producing) moldlike bacteria classified in the genus Nocardia. These bacteria are not as strongly acid-fast as the pathogenic mycobacteria are, and prolonged washing decolorizes most nocardia. This property sets the acid-fast group apart from other bacteria and makes it possible to stain mixtures of large numbers of bacteria (such as those present in sputum) and still recognize the acid-fast bacteria. This is a real help in the diagnosis of tuberculosis. Other stains A number of additional specialized staining procedures are used to stain parts of the bacterial cell. They include techniques for staining capsules, cell walls, nucleic acid, flagella, endospores, and other structures. All these staining procedures involve the use of two or more special dyes, but none of these stains is used routinely in the identification of a bacterium.