Deinococcus radiodurans, strange berry that withstands radiation, formerly called Micrococcus radiodurans, is an extremophilic bacterium, and is the most radio resistant organism known. While a dose of 10 Gy is sufficient to kill a human, and a dose of 60 Gy is sufficient to kill all cells in a culture of E. coli, D. radiodurans is capable of withstanding an instantaneous dose of up to 5,000 Gy with no loss of viability, and an instantaneous dose of up to 15,000 Gy with 37 percent viability. It can survive heat, cold, dehydration, vacuum, and acid, and because of its resistance to more than one extreme condition, D. radiodurans is known as a polyextremophile. It has also been listed as the world’s toughest bacterium in The Guiness Book of World Records because of its extraordinary resistance to several tremendous conditions. Studies are being conducted to verify the origin of D. radiodurans as many scientists are speculating that it has originated on Mars. It has been classified as a Gram-positive bacterium.The term Deinobacter has been replaced by Deinococcus based on evaluation of ribosomal RNA sequences. Several other species within the genus have been described, and they are related to heat-resistant bacteria such as Thermus; the group is hence known as Deinococcus-Thermus.D. radiodurans was discovered by A.W. Anderson at the Oregon Agricultural Experiment Station in Corvallis, Oregon. Experiments were being done to determine if canned food could be sterilized using high doses of gamma radiation. A tin of meat was exposed to a dose of radiation that was thought to kill all known forms of life, but the meat subsequently spoiled. D. radiodurans was isolated from the meat.It has been found that these bacteria could survive on another planet. In an Earth lab, Deinococcus radiodurans, in short D. rad can survive extreme levels of radiation, extreme temperatures, dehydration, and exposure to genotoxic chemicals. Amazingly, they even have the ability to repair their own DNA, usually with 48 hours. Known as an extremophile, bacteria such as D. rad are of interest to NASA partly because they might be adaptable to help human astronauts survive on other worlds. A recent map of D. rad’s DNA might allow biologists to augment their survival skills with the ability to produce medicine, clean water, and oxygen. Previously they have been genetically engineered to help clean up spills of toxic mercury. Likely one of the oldest surviving life forms, D. rad was discovered by accident in the 1950s when scientists investigating food preservation techniques could not easily kill it. Pictured above, Deinococcus radiodurans grow quietly in a dish. Deinococcus accomplishes its resistance to radiation by having many copies of its genome and rapid DNA repair mechanisms. It usually repairs breaks in its chromosomes within 12 to 24 hours through a 2-step process. First, D. radiodurans reconnects some chromosome fragments through a process called single-strand annealing. In the second step, a protein mends double-strand breaks through homologous recombination. As a consequence of its hardiness it has been nicknamed Conan the Bacterium after Conan the Barbarian. READ ARTCLE

ENVIRONMENTAL FACTORS

For bacteria to grow and multiply most rapidly, certain requirements must be met. First of this requirement is sufficient food of the proper kind must be present. Then moisture must be available. The temperature must be that most suitable for the species. Likewise, the proper degree of alkalinity or acidity must be present. Another thing to consider is that the oxygen requirements of the species must be met. Light must also be partially or completely excluded. Lastly, the byproducts of bacterial growth must not accumulate in undue amounts. Significant departure from any of these requirements will modify bacterial growth, although bacteria generally possess a greater degree of resistance to unfavorable conditions in the environment than do plants and animals.

Culture media is known as the food materials prepared for the growth of bacteria in the laboratory. It must be noted that some bacteria will grow on practically any properly prepared culture medium. Others grow only on especially nutritious ones, and a few will not grow on any artificial medium. How the bacteria grow in different media can be observed closely with the use of the microscope.

Nutrition

The protoplasm of the bacterial cell is composed of numerous organic compounds, including proteins, fats, and carbohydrates, as well as various inorganic components containing sulfur, phosphorus, calcium, magnesium, potassium, and iron. Proteins comprise about 50% of the dry weight of the cell. It must be mentioned that each species has a type of protein peculiar to itself. Bacterial nitrogen makes up 10%. In some species carbohydrates are plentiful, and important traits of the species depend on these compounds.

Nutrition is the provision of food materials or what we call as chemical substances to bacteria so that they can grow, maintain their constituents, and multiply. For their nourishment bacteria require sources of carbon and nitrogen, growth factors, certain mineral salts, and sources of energy. With the exception of some saprophytic species, all bacteria derive their carbon and nitrogen from organic matter. A number of minerals are required, the most important of the salts being those of calcium, phosphorus, iron, magnesium, potassium, and sodium. Certain minerals are prerequisite to the activation of enzymes.

Many microorganisms can synthesize all the organic compounds of their complex makeup if supplied with the basic nutrients. Many cannot, however, since they require vitamins and certain organic growth factors since growth factor is utilized as the intact substance for their activities. In this respect microorganisms resemble rather closely higher forms of life. In fact, such vitamins as nicotinic acid, pantothenic acid, and para – aminobenzoic acid, biotin, and folic acid requirements for animal nutrition-were first studied and identified as substances necessary for the growth of microorganisms.

KINDS OF ORGANISMS

Organisms can be classified according to how they obtain their nourishment. Saprophytes are organisms that obtain their nourishment from nonliving organic material. Those that depend on living matter for their sustenance are known as parasites. Facultative saprophytes usually obtain nourishment from living matter but may obtain it from dead organic matter. Facultative parasites on the other hand usually obtain nourishment from dead organic matter but may obtain it from living matter. Some pathogenic bacteria can exist only on living material. For instance the spirochete of syphilis cannot be grown outside a living organism. Most, however, can lead either a parasitic or a saprophytic existence. A few pathogenic bacteria, usually saprophytic, may adapt themselves to a parasitic existence. The best example for this is the bacteria that cause gas gangrene. The organism on which a parasite lives is known as a host.

Organisms that obtain their nutriments by breaking down organic matter into simpler chemical substances are heterotrophic or organotrophic. Those that obtain them by building the organic compounds in protoplasm from the simpler inorganic substances are autotrophic or lithotrophic. All pathogenic bacteria and many nonpathogenic ones are heterotrophic. The different types of organisms can be studied more accurately under the microscope.

Classification

We have heard stories about people who got sick because of bacterial infection. But what is this organism that causes severe ailment when let untreated? Bacteria (sing., bacterium) are minute unicellular microorganisms, the smallest ones having all the necessary protoplasmic equipment for growth and self-multiplication at the expense of available foodstuffs. Bacteria can be observed and studied well using the microscope. They can start with rather simple substances and synthesize them into complicated organic moieties. They use food materials only in solution and excrete waste products in fluids that must diffuse outward. There is no special structure for intake of solids for digestion or for release of solid particles to the environment. In the early days of microbiology, it was believed that bacteria belonged to the animal kingdom, but then for a period of years, it seemed more suitable to classify them with plants.

Unlike plants, bacteria ordinarily do not contain chlorophyll and may be able to move about independently in their environment. Today we classify them as procaryotes. Bacteria are morphologically simpler than the cells of the higher organisms, and as is true for procaryotes, they lack an organized nucleus. Despite their relative simplicity, they have an elaborate and complicate life history.

Distribution

Bacteria are widely distributed in nature They have adapted to every conceivable habitat They are found within and upon our bodies and in the food we eat, the water we drink, and the air we breathe They are plentiful in the upper layers of the soil and no place on earth, except possibly the peaks of snow-capped mountains, is free of them. They are found in frozen Antarctica and in the hot water of the geysers in Yellowstone Park. They can be found in the stratosphere at a height of 20 kilometers (km.) and in ocean sediments at a depth of 11,000 meters (m.); they are able to grow from 5° to -2,° C, the temperature of 90% of the world’s oceans. Under the microscope, bacteria can be examined closely.

When scrutinized under the microscope, our skin has a large bacterial population, and bacteria make up a generous portion of the contents of these alimentary tract. There are several thousand species of bacteria; of this number, about 100 produce disease in humans. The ratio is given as 30,000 nondisease producing bacteria to 1 disease producer. Some of the bacteria that produce disease in humans also produce disease in the lower animals. Others produce disease in the lower animals only, and still others attack only plants. The majority, however, do not attack human beings, lower animals, or living plants and either do not affect animals and plants at all or are actually helpful to them. In fact, if the activities of bacteria were to cease, all plant and animal life would soon become extinct.

After close observation with the aid of the microscope, bacteria can be classified into pathogenic and nonpathogenic. Bacteria that cause disease are spoken of as pathogenic; those that do not cause disease are nonpathogenic.

Morphology

Shape

When viewed under the microscope, bacteria have three well known shapes. Coccus appears as spherical. Bacillus is a rod-shaped bacterium. Spiral shaped are vibrio, spirillum, or spirochete.

Variations exist with these three shapes. Under the microscope cocci are not necessarily perfectly round, but may be somewhat elongated, oval, or flattened on one side. Rod-shaped bacilli may be long and slender or short and plump. Short, thick, oval-shaped bacilli resembling cocci are known as coccobacilli. The ends of bacilli are usually rounded but may be square or concave. The three spiral-shaped bacteria are each distinct. The vibrio is a curved microbe shaped like a comma. The appearance of the other two when viewed under the microscope relates to their movements. In the spirillum, the long axis remains rigid when it is in motion, whereas in the spirochete, the long axis bends when it is in motion.

When bacteria, especially cocci, divide, the manner in which they do so and their tendency to cling together often give them a distinct arrangement. When viewed under the microscope, you will know the distinct appearance of the cocci. Cocci that divide so as to form pairs are known as diplococci. The opposing sides of diplococci may he flattened (examples, gonococci and meningococci). Cocci that divide and cling end to end to form chains are known as streptococci. Those that divide in an irregular manner to form grapelike clusters or broad sheets are known as staphylococci. Other patterns for cocci are in groups of four (tetrads) and cubic packets of eight (sarcinae). No pathogenic cocci are found in the latter group. Bacilli that occur in pairs are known diplobacilli and those that occur in chains as streptohacilli. The diplobacillus and streptobacillus arrangements are unusual. When some bacilli divide, they bend at the point of division to give two organisms arranged in the form of a V. This is known as snapping. In other cases they tend to arrange themselves side by side. This is known as slipping.

Plan of Action

The microbiologist may study employ different laboratory equipment like the microscope in the study of microbes. He may study the microbes in various ways. Generally, there are five standard procedures being followed or the identification and evaluation of microbes in the microbiologic laboratory.

First in the procedure is through direct microscopic examination. Microbial cells or their colonies are too tiny to be visible to the unaided eye. The microbiologist must use an instrument of precision. This instrument is the microscope. With the use of the microscope the microbes can be viewed clearly. The microscope magnifies the minute microorganisms, hence they can be studied well. A test specimen is prepared for microscopic study. Some specimen is applied to or spread thinly on a glass slide in a manner that light rays from the light source on the scope can pass through the material and allow or visualization. Sometimes the microscopic preparation is viewed unstained. More often, the slide is stained by one of the several methods o staining before it is viewed under the microscope.

Second is through culture. The microbiologist may make a culture from the test specimen. This is done by allowing microorganisms to multiply sufficiently through media to form visible growth. He then studies the physical pattern of that growth with the use of the microscope. Just like in direct microscopic observation method, the culture may or may not be stained by microscopic preparations.

Third method is done by biochemical test. The colonial growth of the microbes which is obtained from the original specimen is studied under the microscope to determine their biologic properties. The growth is then subjected to a series of biochemical tests from which identifying characteristics of the microbes emerge.

Fourth is through animal inoculation. The microbes recovered from the test specimen by cultural methods are injected into a suitable laboratory animal, just like the rat. The animal is then observed for further reactions.

The fifth method is through immunologic reactions. The microbiologist may opt to use this method which uses antigen-antibody tests for the identification of microorganisms recovered.

Pathogenic microbes must be handled with utmost caution and in accordance with well-known principles of conduct in microbiologic laboratories. These pathogenic microbes are disease-producing agents and can be very dangerous. The accidental laboratory infections can be fatal. In a study conducted which surveyed 1300 infections among laboratory workers, 39 ended fatally. In many instances the infections occurred in research workers and highly trained technologists.

“`Man is a tool-using animal … without tools he is nothing, with tools he is all….” Carlyle.

Tools for the Study

The microbiologist has many instruments of precision and laboratory apparatus at his command. Some are in constant use while others are needed only in special investigations.

The Compound Light (Bright-field) Microscope

The instrument most often used by the microbiologist is his study of various microorganisms is the compound microscope. It is very important that the microscope should be handled with utmost care. Its workmanship should be of highest quality.

There are two kinds of microscopes namely the simple microscope and the compound microscope. A simple microscope is a title more than a magnifying lens. A compound microscope on the other hand incorporates two or more lens systems so that the magnification of one system is increased by the other. Practically, the compound microscope consists of two parts, the supporting stand and the optical system.

The supporting stand includes (1) a base and pillar, (2) an arm to support the optical system and house the fine adjustment, (3) a platform or stage on which the object to be examined rests, and (4) a condenser and mirror fitted beneath the stage. The condenser and mirror focus the light from either an external source such as a special microscope lamp or from an illuminating system fitted into the base of the scope. Where present, the built-in base illuminator which is a self-contained substage illuminator, houses the light source, a collecting lens system, mirror, and a condenser lens. An iris field diaphragm, a variable transformer attached to the system, and properly placed filters permit adjustments of the light.

The optical system consists of a body tube that supports the ocular lenses or the eyepiece at the top end and the objective lenses attached to a revolving nosepiece at the other end. The optical system is connected to the arm of the supporting stand by an intermediate slide, which moves up and down on the arm in response to movement of the fine adjustment. The intermediate slide contains the rack and pinion for the coarse adjustment, which acts directly on the tube of the optical system. The platform of the microscope is usually equipped with a mechanical stage to hold the glass microslide firmly. The object is mounted on the microslide so that it can be moved from place to place by set screws. The advantages of this device are that the specimen can be examined systematically and, unless moved, the specimen remains in a fixed position.

The magnification of an objective is usually designated by its equivalent focal distance in inches or millimeters. By equivalent focal distance is meant the focal distance of a lens having the same magnification as the objective. The higher the number of the objective, the less is its magnification. American microscopes are usually fitted with 16 mm., 4 mm., and 1.8 mm. objectives. The last is an immersion objective because for the best results there must be a liquid either oil or water between the objective and the object being examined. This objective is best known as an oil-immersion objective because only rarely would its lens system be immersed in water. Most modern immersion oil is highly refined mineral oil having the same refractive index, (.52 as glass. The 16 mm. objective magnifies 10 times; most 4 mm.. objectives magnify 43 times; and most 1.8 mm. objectives magnify 97 times.

The oculars of a microscope are given 6x, 10x, and similar designations to indicate that they increase the magnification of the objective 6, 10, or more times, ` respectively. To obtain the magnification of any combination of ocular and objective lenses, multiply the magnification of the objective by that of the ocular. Remember that magnification refers to both the length and the width of an object; that is, a magnification of 100 means that the object is made to appear 100 times as long and 100 times as wide.

The use of the microscope requires simple instructions as follows:

1. Keep both eyes open. A very little practice will enable you to do this.

2. Most modern microscopes have self-contained substage illuminators. If yours does not and you must work with the mirror, avoid direct sunlight. North light is advantageous. Good results are obtained with daylight.

3. When you place a slide on the stage, see that it lies flat against the platform. Adjust the light so that the object is evenly illuminated.

4. Learn to focus with the low-power objectives (such as the 16 mm. objective). Use the coarse adjustment to move the low-power objective until it nearly (but not quite) touches the cover glass or upper surface of the mounted specimen. Then focus up until the object comes plainly into view. Complete focusing with the fine adjustment. Keeping the specimen in focus, use very short back and forth strokes of the fine adjustment to produce an illusion of depth in the specimen. When the immersion objective is used, first place a drop of immersion oil on the object so that it can be clearly brought into view. Parfocal objectives are designed so that when switching from one objective to another the microscopist can keep the specimen essentially in focus; most modern microscopes are equipped with them.

5. Keep the microscope clean and handle all parts with care. Do not touch the glass parts of the microscope. Do not allow chemicals to contact the microscope, since they may injure it. Clean the mechanical parts with an application of olive oil on gauze; wipe the oil off with chamois or lens paper. Keep optical glass parts especially ocular lenses, condenser lens system, and non immersion objective lenses of the microscope clean by frequent use of lens paper. Remove immersion oil from oil-immersion objective lens carefully right after you have finished your microscopic study. At times it may be necessary to remove dried immersion oil with lens paper moistened with xylol. Do this as rapidly as possible to prevent injury to the optical settings of the oil-immersion objective lens system.

6. Clean the microscope thoroughly when you are finished. Leave the, lowest power objective in the working position; thus the least expensive objective would be injured should the optical system be jammed down accidentally. Keep the microscope covered when you are not using it.

Size

Bacteria are so small (no larger than 1/50,000 of an inch*) that the highest magnification of the ordinary compound microscope must be used to study them. Cocci range from 0.4 to 2 µtn. (/j,) in diameter. The smallest bacillus is about 0.5 µm. in length and 0.2 µmL in diameter. The largest pathogenic bacilli are seldom greater than 1 µm. in diameter and 3 µm. is length; the average diameter and length of pathogenic bacilli are 0.5 and 2 µm. respectively. Nonpathogenic bacilli may be larger, reaching a diameter of 4 µm. and a length of 20 µm. The spirilla are usually narrow organisms from 1 to 14 µm. in length. Different species of bacteria show great variation in size, and there is some variation within a species, but as a rule the size of each species is fairly constant. With the use of the microscope the different shapes of bacteria can be better compared.

Structure

Bacteria are minute. They are slightly refractile that unless stained with dyes, they are difficult to see even, with the compound light microscope. When stained, they appear homogeneous or slightly granule. With the electron microscope, however, microbiologists can visualize minute details of bacterial structure

Cell wall

The shape of the bacterial cell is maintained by a cell wall. This cell wall is rigid. The protoplasmic substance of bacteria exerts such a high osmotic pressure, equivalent to that of a 10% to 20% solution of sucrose, that in ordinary environments, were it not for the high tensile strength of the cell wall, the bacterial cell would burst. If the bacterial cell is placed in a suitable hypertonic medium and the cell wall dissolved, the remainder of the bacterium is converted into a spherical protoplast. In an isotonic environment prootoplasts remain viable and grow.

The stability of the cell wall is derived from its chemical makeup, residing in the layer composed of a single, giant, complex molecule of peptidoglycan (murein, mucopeptide). Peptidoglycan is composed of a backbone of alternating amino sugars, N-acetylglucosamine and N-acetylmuramic acid; a set of identical tetrapeptide side chains attached to the N-acetyllmuramic acid, and a set of identical peptide cross bridges. For all bacterial species, that backbone is the same, but the side chains and the cross bridges vary from species to species.

The chemical structure of the cell wall is different In gram-positive bacteria from what it is in gram negative ones, a difference expressed .in their diverse Pam-staining reactions. One variation lies in the peptoglycan layer. In gram-positive bacteria, this layer consists of concentric sheets cross-linked chemically in three dimensions. However, in gram-negative ones the peptidoglycan layer forms two-dimensional monolayers. Still another difference is that most gram-positive cell walls contain teichoic acids in large amounts, even as much as 10% of the dry weight of the total cell; gram-negative cell walls contain none. Teichoic acids probably lie on the outer surface of the peptidoglycan layer and participate in the supply of magnesium to the cell by binding magnesium ions.

In the gram-negative cell wall, outside the peptidoglycan layer, are three polymers: lipoprotein, outer membrane, and lipopolysaccharide. The lipoprotein molecules cross-link the outer membrane with the peptidoglycan layer. A typical phospholipids bilayer, the outer membrane is relatively permeable to small molecules but hinders the penetration of larger ones. This discriminatory capacity in the outer membrane is believed to explain the relative resistance of gram-negative bacteria to certain antibiotics. Tightly bound to the outer membrane and the cell surface, the lipopolysaccharide consists of a complex lipid (lipid A) to which is attached a polysaccharide. This polysaccharide as the 0 antigen represents a major surface antigen of the bacterial cell. It is made up of a constant chemical core and a terminal series of repeat units, a repeat unit being unique to each gram- negative species. Lipopolysaccharide is very toxic to animals, is released only with lysis of the bacterial cells, and is the endotoxin of gram-negative microorganisms.

The viability of bacteria is directly dependent on the integrity of the cell wall. The complex chemical sequence of events, for the biosynthesis of the cell wall provides several possibilities for interference by antimicrobial compounds. Any such compound that inhibits any step can cause the wall to be weakened and consequently the cell to lyse. The sites of action are well known for certain antibiotics that inhibit cell wall synthesis.

Plasma Membrane

The cell wall is so narrow that it cannot be seen with the ordinary compound light microscope. In ultrathin sections it is revealed by the electron microscope as a well-defined structure surrounding a distinct layer, the cell membrane or plasma membrane which separates it from the cytoplasm of the bacterial cell. Composed of phospholipids and proteins, the plasma membrane is the site of important enzyme systems, including the respiratory enzyme system (cytochrome enzymes). In fact, in bacteria it corresponds to the mitochondria of higher organisms. In regulating the passage of food materials and metabolic by-products between the interior of the cell (where metabolic activities are carried on) and the surroundings, it functions osmotically both as a barrier and as a link. It blocks the entry of certain substances and catalyzes the transport of others into the cell.

Capsule

Surrounding many bacteria is a mucilaginous envelope or capsule Indistinct in most bacteria, it is well developed in few Streptococcus pneumoniae, Clostridium perfringerts, and Klebsiella pneumoniae). The capsule is formed by an accumulation of slime excreted by the bacterium. This material is usually a complex polysaccharide. If it is present about the cell in only small amounts, a distinct capsule does not appear.
Capsule formation is most prominent in organisms taken directly from the animal body, for when grown on artificial media, the same organisms often lose their ability to form capsules. A capsule does not stain with the ordinary bacteriologic dyes but may appear as a clear halo around the bacterium, even two or three times broader than the bacterium. It is stained by special methods. The presence of a capsule appears to increase the virulence of an organism by protecting it against phagocytosis, and in some cases the capsule gives the organism its specific immunologic nature. For instance, relative to the nature of their capsules, pneumococci are divided into at least 82 types. The specific antigenic nature of a capsule depends on its carbohydrate content.

Granules

Within some bacteria (such as Corynebacterium diphtheriae) at the ordinary magnification of the light microscope is seen granules that stain more deeply than the remainder of the cell. Known as metachromatic granules and enzymatically active, they are reserves of inorganic phosphate stored as polymerized metaphosphate (volutin). Metachromatic granules may be arranged irregularly within the bacterial cells or located in one or both ends of the cell where they are known as polar bodies. Sulfur-oxidizing- bacteria convert excess hydrogen sulfide from the environment into intracellular granules of elemental sulfur. At times, carbon source materials on reserve, converted by some bacteria to osmotically inert, neutral polymers, are stored in their cytoplasm as insoluble granules, available if needed.

Electron microscopy reveals a dense packing of ribosomes in bacterial cytoplasm and the presence of spherules and various submicroscopic granules. Ribosomes are tiny, uniform, beadlike granules found free in the cytoplasm of a cell and are rich in ribonucleic acid. Ribonucleic acid (RNA) is one of the two nucleic acids of physiologic significance, the other being deoxyribonucleic acid (DNA). RNA is similar to DNA chemically except that its sugar is a different pentose-ribose, one of its nucleotide bases is uracil instead of thvmine, and its chemical pattern is laid out as a single coil, not a double one. The ribosomes play an important role in protein synthesis. The submicroscopic granules are known to be biochemically complex and active; they may be an integral basic unit of the bacterial cell with a definite role in cell metabolism.

No relation exists between these various granules and the ability of the bacteria containing them to produce disease.

Staining

Bacterial cells are so small that, when examined in hanging drop preparations, little of their finer structure can be made out to be studied more closely. They must be colored with some dye. This process is called staining. The dyes most often used are aniline dyes which are derivatives of the coal tar product aniline. It is good that dye-impregnated paper strips used for staining bacteria are now available commercially. The different types of bacteria when stained can be studied well in their different colors under the microscope.

For a stained preparation, place a small amount of the material to be examined on a clean glass slide and spread out into a thin film with a wire loop or swab. Subsequent flame sterilization of the inoculating loop is a must. The film is known as a smear. Let the film dry in the air; then slowly pass the slide, smear side up, through a flame two or three times. Flaming kills the bacteria in the smear and causes them to stick to the slide. The smear is fixed through the process called is fixing. Other methods of fixing, such as immersion in methyl alcohol or Zenker’s solution, are sometimes used, but heat fixing is the most suitable for routine work. Apply the desired stain to the fixed smear. Wash off excess stain with water. Blot the slide dry between sheets of absorbent paper. Observe stained smear with the microscope.

Three classes of stains are used in bacteriology today, namely the simple stains, the differential stains, and the special stains. In addition, there is the process known as negative staining.

SIMPLE STAINS

A simple stain is usually made up of an aqueous or alcoholic solution of a single dye. It is applied to the fixed smear from 1 to 5 minutes and washed off. The stained preparation is then ready for microscopic examination. Widely used simple stains include Loffler’s alkaline methylene blue, carbolfuchsin, gentian violet, and safranine. The length of time that the stain remains on the smear depends on the avidity with which it acts. Sometimes a chemical to make it stain more intensely is added to the solution. Such a chemical is called a mordant.

With simple stains most bacteria stain easily and quickly, some do not stain so readily, and a few do not stain at all. Capsules and spores are not stained with these simple stains but may give contrast as clear unstained structures. Flagella cannot be demonstrated at all in this way. Stains help in distinguishing one kind of bacteria from another and these can be viewed well with the aid of the microscope.

DIFFERENTIAL STAINS

More complex staining methods divide bacteria intro groups, depending on their reaction to the chemicals used for staining. Of these, the Gram stain and the acid fast stain are most often used.

The method of staining introduced by Hans Christian Gram divides bacteria into two great groups: those that are gram-positive and those that are gram-negative. This method depends on the fact that, when bacteria are stained with either crystal violet or gentian violet (Gram I) and the smear is then treated with a weak solution of iodine as a mordant (Gram II), some bacterial cells combine with the dye and iodine to produce a color that cannot be removed easily by alcohol, acetone, or aniline (Gram III), whereas the color is readily removed from certain other bacteria by these solvents. In the last step of the staining procedure a counterstain (Gram IV), such as safranine, is applied to give a contrasting color to the decolorized bacterial cells. Stains used to give contrast in color are counter stains. The ones most often used in the Gram stain are safranine and dilute carbolfuchsin, both of which give a red color, and Bismarck brown, which, as its name implies, gives a brown color. Many modifications have been devised for the original Gram’s Method.

Bacteria from which the blue color of the stain-iodine-bacterial cell complex cannot be removed are designated gram-positive, and those from which it can be removed and which are stained with the counterstain, as gram-negative. Gram-positive bacteria do not stain with the counterstain because they are already completely stained. A few bacteria that sometimes retain the stain and that at other times do not are referred to as gram-variable. The reactions of the bacteria to the Gram stain are very important in the study of pathogenic bacteria.

It must be noted that the chemical composition of the cell wall in gram-positive bacteria being different from that in gram-negative bacteria explains the alternate reactions of the two groups to gram staining. However the reason that the cell wall of gram-positive bacteria prevents their decolorization in the staining process is not clear. Certain physiologic differences are generally correlated with Gram staining. Gram-positive bacteria tend to be more resistant to the action of oxidizing agents, alkalis, and proteolytic enzymes than are gram-negative ones. They are more susceptible to the acids, detergents, sulfonamides, and antibiotics, such as penicillin, than are gram-negative ones.

Another method of differential staining is the acidfast or Ziehl-Neelsen stain, for which method several standard modifications exist. When most bacteria and related forms are stained with carbolfuchsin, they stain easily, but when the smear is treated with acid-alcohol, they are completely decolorized. It is relatively difficult to stain certain other microbes with carbolfuchsin, but once stained, they retain the dye even when treated with acid-alcohol. Those that retain the stain are spoken of as being acid-fast. That property of being acid-fast derives from the presence of many complex lipids, fatty acids, and waxes within the bacterial cell wall, not possessed by cell walls of non-acid fast bacteria.

Important examples of acid-fast organisms encountered in medicine, some of which are nonpathogenic include e Mycobacterium tuberculosis, Mycobacterium leprae, and other of the Mycobacterium species, and asteroides. These different species can be examined well with under the microscope.

SPECIAL STAINS

Important special stains are those for capsules, spores, flagella, and metachromatic granules. Stains primarily designed to demonstrate metachromatic granules are especially valuable in identifying Corynebacterium diphtheriae and in differentiating it from related bacteria. Important stains of this type are Albert’s stain, using toluidine blue and malachite green, and Neisser’s stain, using methylene blue.

Negative (relief) staining. Microorganisms such as Treponema pallidum, not stained by ordinary dyes, may be made visible by the process known as negative or relief staining, in which the background, but not the microorganisms, is stained. The microorganisms are mixed with India ink or 10% nigrosin both of which are black. The mixture is spread out into a thin smear and allowed to dry. The microbes appear as colorless objects against a gray-black background. Negative staining may be done with the dye Congo red and this has been used to display spiral organisms and Chlamydia (bedsoniae). By a technique of negative staining using an electron-dense material, such as phosphotungstic acid, viruses are prepared for visualization in the electron microscope.

Take interest, I implore you, in those sacred dwellings which one designates by the expressive term: laboratories. Demand that they be multiplied, that they be adorned. These are the temples of the future-temples of well-being and of happiness. There it is that humanity grows greater, stronger, better.
- Louis Pasteur

One of the favorite places on earth for many of us is the beach. When you go down the seashore on some quite beach and admire the beauty of nature, what do you see? The blue sky and of course, the sea, sand, pebbles and rocks. Perhaps a few clam shells and little crabs. Not a sign of life? If you look closer at a little slime from the rocks, a seaweed, a little wet sand or perhaps a small jar of water from the sea, you may see nothing extra-ordinary. With your naked eyes you may see nothing alive. Under the microscope you will be astonished with what you will see. There are sensitive minute living organisms swimming or creeping in all of them. A mere droplet of water from the sea contains hundreds or even thousands of the smallest known types of animal life. There you will find other creatures clinging to the seaweed. From the slime in the rocks you will discover some of the simplest plants, and beautiful at that. The seaweed itself is just as fascinating and marvelous. These very minute organisms are called microbes.

Definition

The science which deals with the study of microbes – living minute-sized organisms is referred to as Microbiology. It is the branch of biology dealing with microbes, usually structured as one cell and studied with the use of the microscope. Within the province of microbiology lies the study of certain kinds of microbes classified as bacteria (bacteriology), viruses (virology), fungi (mycology), and protozoa (protozoology). The different shapes and appearances of these microorganisms can be best viewed with the aid of the microscope. Microbiology considers the occurrence in nature of the microscopic forms of life, their reproduction and physiology, their participation in the processes of nature, their helpful or harmful relationships with other living things, and their significance in science and industry.

Although human beings have lived with microorganisms from time immemorial and have used certain of their activities, such as fermentation, to advantage, the science of microbiology is a product of only the last hundred years or so. With the use of the microscope, the studies of Anton van Leeuwenhoek in the seventeenth century had shown the existence of microscopic forms of life, but it was not until the work of Louis Pasteur toward the end of the nineteenth century (some 200 years later) that the science of microbiology really took shape. The new science stated the germ theory of disease, demonstrated patterns of communicable disease, and gave human beings a measure of protection they had not known in their struggle against the injurious forces in the biologic environment. In its time this very young science influenced practically every type of human endeavor. Studies were conducted with the help of the microscope to observe and examine the processes involved in the so called germ theory of disease.

For scientific knowledge to bring results, such as in the organization of public health programs, it must be disseminated. Such is the purpose of health education. To the individual it explains the mechanisms by which he can protect himself against microbial hazards. To the social group it designates the available community resources.

Among many intracellular bacteria, it has been found out through bacteria identification that Listeria monocytogenes presents a rather uncharacteristic way of destroying the host cells. Many deadly microbes have learned that the key to launching an infection is not to kill your host – at least not too quickly.Scientists at the University of California, Berkeley, have discovered how one microbe, Listeria monocytogenes, is able to manage this. In a paper in this week’s issue of Science, Daniel A. Portnoy, professor of molecular and cell biology in the campus’s College of Letters & Science and professor of infectious diseases in the School of Public Health, along with post-doctoral fellow Amy L. Decatur, describe the trick these bacteria use to live comfortably inside a cell until they’re ready to break out and spread the infection to other cells.

The finding could have implications beyond this one bacteria, pointed out through bacteria identification which causes a deadly disease called listeriosis. The world’s top three infectious killers – AIDS, tuberculosis and malaria – all are caused by pathogens that ensconce themselves snugly inside cells and live to wreak havoc. Yet, these intracellular pathogens have been hard to study.There are no effective vaccines for any of these diseases, in part because it is difficult to study intracellular pathogen. Listeria is a great model system for studying the host-pathogen interaction of these intracellular bacteria.

In bacteria identification, Listeria is a common but deadly bacterium that in recent years has made headlines as a contaminant of hot dogs, cheese, cole slaw and other food stuffs, causing more than two thousand infections every year and 500 deaths. Though it hits immune-compromised people the hardest, its overall fatality rate is about 20 percent.Listeria bacteria establish an infection by inducing immune system cells, mostly scavenger cells called phagocytes, to corral and swallow them, so that they end up encased in a bubble within the body of the cell. The bacteria would be benign if they remained isolated in the vacuole, because the cell can kill them there. But they eventually break out and take over the host cell’s machinery to spread the infection. What makes Listeria virulent is a pore-forming toxin that allows the bacteria to break through the wall of the vacuole and enter the cell’s innards.

A big question has always been why the toxin, listeriolysin O, doesn’t also rupture and kill the cell, exposing the bacteria to the immune system?

Several years ago, a post-doctoral fellow in Portnoy’s lab compared listeriolysin O to a similar pore-forming toxin called perfringolysin O, from the extracellular bacteria Clostridium perfringens, which cause gangrene. Sian Jones and Portnoy found that if they substituted perfringolysin O for Listeria’s normal toxin, the altered bacteria were able to punch their way out of a vacuole, but then they killed the host cell. This made Listeria totally avirulent, Portnoy said, because the immune system efficiently mopped up the exposed bacteria.

Portnoy and Decatur compared the genetic sequences of the two toxins and found that listeriolysin O contains an extra bit of protein that looks just like a tag found in a range of organisms from yeast to humans, and which often tells the cell a protein is trash and should be chopped up and recycled. The tag is referred to as a PEST sequence, signifying the four amino acids characteristic of the tags.

Listeria bacteria apparently stole the tag and placed it on the toxin so that the host cell’s clean-up crew recognizes it and targets it for destruction before it has a chance to make pinholes in the cell membrane. It’s a great example of how bacteria have taken advantage of the host’s biology to enhance their pathogenicity.The two scientists elegantly demonstrated how critical this PEST sequence is to the virulence of Listeria. When they mutated the PEST tag so the cell no longer recognized it, the mutant bacteria quickly killed off the host cells. The mutant Listeria proved 10,000 times less virulent in mice than the wild Listeria bacteria. Apparently, immune system cells eliminated the mutant bacteria once they killed off their host cell.

In order to survive, the pathogen must maintain a protected niche within the host cell. To achieve this, the toxin has co-opted the cell’s own machinery, sprouting a tag that says, Please get rid of me.

Portnoy and his colleagues have discovered the role played by another important protein in the Listeria lifecycle. Once the bacteria break free of their protective vacuoles, they take over the cell machinery and do something amazing. They generate comet-like tails that push them around the cell like a speedboat. Eventually, they slam into the cell membrane and pop from one cell into the next, spreading the infection.

They found that the tail is produced when a protein on the surface of the bacteria, ActA, interacts with a complex of host cell proteins called the Arp2/3 complex. The result is rapid polymerization of a skeletal protein called actin that piles up and physically propels the newly formed bacteria around the cell.Welch, Portnoy and graduate student Justin Skoble dissected ActA even further, and reported details of how different parts of the protein carry out different functions, all of which are critical to the pathogen’s virulence.What’s elegant about this is that over millions of years, the bacteria have evolved individual proteins capable of exploiting complex processes that control host cell biology.READ ARTICLE

Arthur Kornberg in 1990, a Nobel laureate and emeritus professor of biochemistry, and a postdoctoral fellow in his lab, defined the enzyme that produces polyphosphate in E coli bacterium. According to him the enzyme polyphosphate kinase or PPK makes polyphosphate by stringing together many phosphate residues. Members of Kornbergs lab have learned that the PPK enzyme is similar in many bacteria, including several types that cause disease in humans. Organisms such as yeast, plants, humans and other animals have a different kind of enzyme for producing polyphosphate.
Kornberg and his team studied the PPK gene by making mutant strains of bacteria lacking the gene for PPK. Through bacteria identification it was found out that Pseudomonas aeruginosa was used in the study. This bacterium is known to cause dangerous infections in people with a deficient immune system. Without the PPK gene, the bacteria were unable to use their flagella to swim and they were unable to use tiny cell extensions for movement. The team also has found that P. aeruginosa bacteria without the gene are also unable to form microbial communities called biofilms making the bacteria unable to communicate with each other via a process called quorum sensing. Production of the proteins and toxins known as virulence factors, which enable bacteria to cause disease in their host, are also decreased without the presence of the PPK gene. Kornbergs study opens up the possibility of using PPK as a novel antibiotic. Inhibitors of this enzyme could be used as an antibiotic to disrupt cell to xcell communication in bacteria. As of the present, Stanford is already negotiating with pharmaceutical companies to search for potential drugs. READ ARTICLE

This is an article about bacteria found to be following the species area relationship. The article discusses about the nature of bacteria, being the most common organism on earth. They can be found almost everywhere in nature. In bacteria identification, there are an estimated 100 billion kinds of bacteria. Melissa Lage and Jennifer Hughes, along with their Stanford University and the University of Washington colleagues, found that bacteria follow what ecologists call the species area relationship. This species area relationship is the logical notion that the number of species in an area increases as the size of the area increases. Their work was published in the December 9 issue of Nature. It is the first to show that this basic law of nature holds true for bacteria.

According to the article, Lage believes that bacteria respond to their environment. She further explains that if there are changes in the environment, there may be changes in the kinds of bacteria we see. Bacteria have critical roles in the global ecosystem. Bacteria purify water, pump oxygen into the air, and decompose dead plants and animals. The article explains that due to bacteria identification, different bacteria may perform these functions differently, depending on the place. Huges further adds that if the environment changes, the bacteria change too.
Lage and Hughes conducted their experiment in a half-acre of salt marsh on Prudence Island. They used a measured grid and took twenty six one gram samples of soil. Lage used a kit to extract DNA from each sample. A machine then amplified only bacteria from DNA, which was cloned and cultured in E. coli. Lage then randomly tested each of the twenty six plates and counted the different types, using their unique gene sequences.
The findings of the study and bacteria identification revealed that bacterial communities were very similar a couple of centimeters apart, but more different hundreds of meters apart. The study showed that bacteria are not randomly distributed, but they follow the same ordered pattern of distribution as plants and animals.Read article