Differential Gram’s staining Essay Example for Free

Differential Gram’s staining Essay To Gram stain the given bacterial suspension and to differentiate between gram positive and gram negative organism. Visualization of microorganisms in the living state is very difficult, not just because they are minute, but because they are transparent and almost colorless when suspended in an aqueous medium. To study their properties and divide microorganisms into specific groups for diagnostic purposes, biological stains and staining procedures, in conjunction with light microscopy, have become major tools in microbiology. Chemically, a stain may be defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome. Stains are of 2 types: 1. Acidic stains e.g., picric acid 2. Basic stains e.g., methylene blue. Types of staining techniques: 1. Simple staining. (Use of a single stain)This type of staining is used for visualization of morphological shape (cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads). 2. Differential staining. (Use of 2 contrasting stains)It is divided into two groups: (a) Separation into groups, Gram stain and acid-fast stain. (b) Visualization of structures, Flagella stain, capsule stain, spore stain, nuclear stain. The Gram Stain The Gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between Gram-positive and Gram-negative bacteria. Bacteria that stain purple with the Gram-staining procedure are termed Gram-positive; those that stain pink are said to be Gram-negative. The terms positive and negative have nothing to do with electrical charge, but simply designate 2 distinct morphological groups of bacteria. Grampositive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape, as well as to prevent osmotic lysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan. In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20–80 nm thick and consists of numerous interconnecting layers of peptidoglycan. Chemically, 60% to 90% of the Gram-positive cell wall is peptidoglycan. Interwoven in the cell wall of Grampositive are teichoic acids. Teichoic acids that extend through and beyond the rest of the cell wall are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Some have a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is studded with proteins that differ with the strain and species of the bacterium. The Gram-negative cell wall, on the other hand, contains only 2–3 layers of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10%–20% of the Gram-negative cell wall is peptidoglycan. The phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A: embedded in the membrane, and a polysaccharide portion extending outward from the bacterial surface. The outer membrane also contains a number of proteins that differ with the strain and species of the bacterium. PRINCIPLE The Gram-staining procedure involves 4 basic steps: 1. The bacteria are first stained with the basic dye crystal violet. Both Gram-positive and Gramnegative bacteria become directly stained and appear purple after this step. 2. The bacteria are then treated with Gram’s iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gramnegative bacteria remain purple after this step. 3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex, while Gramnegative are decolorized. 4. Finally, the counterstain safranin (also a basic dye) is applied. Since the Gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, which are now colorless, become directly stained by the safranin. Thus, Gram-positive bacteria appear purple and Gr am-negative bacteria appear pink. With the current theory behind Gram-staining, it is thought that in Gram-positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violetiodine complex within the cell. In the case of Gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the cell is decolorized. It is important to note that Gram-positivity (the ability to retain the purple crystal violet-iodine complex) is not an all-or-nothing phenomenon, but a matter of degree. There are several factors that could result in a Gram-positive organism staining Gram-negatively: 1. The method and techniques used: Overheating during heat fixation, over-decolorization with alcohol, and even too much washing with water between steps may result in Gram-positive bacteria losing the crystal violet-iodine complex. 2. The age of the culture: Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex. 3. The organism itself: Some Gram-positive bacteria are more able to retain the crystal violetiodine complex than others. Therefore, one must use very precise techniques in Gram staining and interpret the results with discretion. Crystal violet (2gm Crystal violet + 20ml 95% ethanol + 0.8gm Ammonium oxalet + 80ml distilled water) Gram’s iodine (1gm iodine + 2gm potassium iodide + 100ml distilled water) Decolorizer (Mix 95% alcohol acetone in equal proportion) Safranin (0.34gm safranin + 10ml absolute alcohol + 90ml distilled water) Clean grease free glass slide Nichrome wire loop Dropper Filter papers Compound microscope Cedar wood oil Miscellaneous PROCEDURE: On a grease free slide prepare smear of bacterial suspension and allow it to air dry. After drying heat fix smear and allow slide to cool. Cover the smear with crystal violet stain and leave for 1 minute Drain stain and wash the slide carefully under running tap water. Flood the smear with Gram’s iodine solution and wait for 1 minute. Wash the slide under running tap water. 6. Decolorize the smear with alcohol-acetone for 20-30 seconds (continue till purple stain just stops coming off the slide). 7. Gently wash the slide under running tap water and drain completely. 8. Counter stain the smear with safranin for 1 minute. 9. Wash the slide with water and air dry (or dry it carefully by using a blotting paper). 10. A drop of cedar wood oil is put over smear and the slide under oil-immersion lens. 1. 2. 3. 4. 5. Determine the growth curve of Escherichia coli by using colorimeter. The increase in the cell size and cell mass during the development of an organism is termed as growth. It is the unique characteristics of all organisms. The organism must require certain basic parameters for their energy generation and cellular biosynthesis. The growth of the organism is affected by both physical and Nutritional factors. The physical factors include the pH, temperature, Osmotic pressure, Hydrostatic pressure, and Moisture content of the medium in which the organism is growing. The nutritional factors include the amount of Carbon, nitrogen, Sulphur, phosphorous, and other trace elements provided in the growth medium. Bacteria are unicellular (single cell) organisms. When the bacteria reach a certain size, they divide by binary fission, in which the one cell divides into two, two into four and continue the process in a geometric fashion. The bacterium is then known to be in an actively growing phase. To study the bacterial growth population, the viable cells of the bacterium should be inoculated on to the sterile broth and incubated under optimal growth conditions. The bacterium starts utilising the components of the media and it will increase in its size and cellular mass. The dynamics of the bacterial growth can be studied by plotting the cell growth (absorbance) versus the incubation time OR log of cell number versus time. The curve thus obtained is a sigmoid curve and is known as a standard growth curve. The growth curve has four distinct phases 1. Lag phase When a microorganism is introduced into the fresh medium, it takes some time to adjust with the new environment. This phase is termed as Lag phase, in which cellular metabolism is accelerated, cells are increasing in size, but the bacteria are not able to replicate and therefore no increase in cell mass. The length of the lag phase depends directly on the previous growth condition of the organism. When the microorganism growing in a rich medium is inoculated into nutritionally poor medium, the organism will take more time to adapt with the new environment. The organism will start synthesizing the necessary proteins, co-enzymes and vitamins needed for their growth and hence there will be a subsequent increase in the lag phase. Similarly when an organism from a nutritionally poor medium is added to a nutritionally rich medium, the organism can easily adapt to the environment, it can start the cell division without any delay, and therefore will have less lag phase it may be absent. Exponential or Logarithmic (log) phase During this phase, the microorganisms are in a rapidly growing and dividing state. Their metabolic activity increases and the organism begin the DNA replication by binary fission at a constant rate. The growth medium is exploited at the maximal rate, the culture reaches the maximum growth rate and the number of bacteria increases logarithmically (exponentially) and finally the single cell divide into two, which replicate into four, eight, sixteen, thirty two and so on (That is 20, 21, 22, 232n, n is the number of generations) This will result in a balanced growth. The time taken by the bacteria to double in number during a specified time period is known as the generation time. The generation time tends to vary with different organisms. E.coli divides in every 20 minutes; hence its generation time is 20 minutes. 3. Stationary phase As the bacterial population continues to grow, all the nutrients in the growth medium are used up by the microorganism for their rapid multiplication. This results in the accumulation of waste materials, toxic metabolites and inhibitory compounds such as antibiotics in the medium. This shifts the conditions of the medium such as pH and temperature, thereby creating an unfavorable environment for the bacterial growth. The reproduction rate will slow down, the cells undergoing division is equal to the number of cell death, and finally bacterium stops its division completely. The cell number is not increased and thus the growth rate is stabilised. If a cell taken from the stationary phase is introduced into a fresh medium, the cell can easily move on the exponential phase and is able to perform its metabolic activities as usual. 4. Decline or Death phase The depletion of nutrients and the subsequent accumulation of metabolic waste products and other toxic materials in the media will facilitates the bacterium to move on to the Death phase. During this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to die due to the unfavorable conditions and the death is rapid and at uniform rate. The number of dead cells exceeds the number of live cells. Some organisms which can resist this condition can survive in the environment by producing endospores.