Summary

This document provides an introduction to microscopes, explaining their types, parts, and uses in various fields of study. It also introduces the concept of cells, their types (prokaryotic and eukaryotic), and their role in living organisms.

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MICROSCOPE A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope. T...

MICROSCOPE A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope. Types of Microscopes: 1. Compound Microscope 2. Stereo Microscope 3. Inverted Microscope 4. Metallurgical Microscope 5. Polarizing Microscope Compound Microscopes A compound microscope may also be referred to as a biological microscope. Compound microscopes are used in laboratories, schools, wastewater treatment plants, veterinary offices, and for histology and pathology. The samples viewed under a compound microscope must be prepared on a microscope slide using a cover slip to flatten the sample. The compound microscope can be used to view a variety of samples, some of which include: blood cells, cheek cells, parasites, bacteria, algae, tissue, and thin sections of organs. Compound microscopes are used to view samples that can not be seen with the naked eye. The magnification of a compound microscope is most commonly 40x, 100x, 400x, and sometimes 1000x. Microscopes that advertise magnification above 1000x should not be purchased as they are offering empty magnification with low resolution. 1 Parts of a Compound Microscope Eyepiece:- The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens. Diopter Adjustment:- Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two eyes. Body tube (Head):- The body tube connects the eyepiece to the objective lenses. Arm:- The arm connects the body tube to the base of the microscope. Coarse adjustment: Brings the specimen into general focus. Fine adjustment:- Fine tunes the focus and increases the detail of the specimen. Nosepiece:- A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses. Objective lenses:- One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. A standard microscope has three, four, or five objective lenses that range in power from 4X to 100X. When focusing the microscope, be careful that the objective lens doesn’t touch the slide, as it could break the slide and destroy the specimen. Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass. The specimen is placed on the glass and a cover slip is placed over the specimen. This allows the slide to be easily inserted or removed from the microscope. It also allows the specimen to be labeled, transported, and stored without damage. 2 Stage:- The flat platform where the slide is placed. Stage clips:- Metal clips that hold the slide in place. Stage height adjustment (Stage Control):- These knobs move the stage left and right or up and down. Aperture:- The hole in the middle of the stage that allows light from the illuminator to reach the specimen. On/off switch: This switch on the base of the microscope turns the illuminator off and on. Illumination: The light source for a microscope. Older microscopes used mirrors to reflect light from an external source up through the bottom of the stage; however, most microscopes now use a low-voltage bulb. Iris diaphragm: Adjusts the amount of light that reaches the specimen. Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed. Base: The base supports the microscope and it’s where illuminator is located. 3 Inclination Joint CELLS All living things are made of cells, and cells are the smallest units that can be alive. Life on Earth is classified into five kingdoms, and they each have their own characteristic kind of cell. However the biggest division is between the cells of the Prokaryote kingdom (the bacteria) and those of the other four kingdoms (Animals, Plants, Fungi and Protoctisa), 4 which are all eukaryotic cells. Prokaryotic cells are smaller and simpler than eukaryotic cells, and do not have a nucleus. Prokaryote – before carrier bag i.e. without a nucleus Eukaryote – good carrier bag i.e. with a nucleus We’ll examine these two kinds of cell in detail, based on structures seen in electron micrographs (photos taken with an electron microscope). These show the individual organelles inside a cell. PROKARYOTES Bacteria are prokaryotes, lacking well-defined nuclei and membrane-bound organelles, and with chromosomes composed of a single closed DNA circle. They come in many shapes and sizes, from minute spheres, cylinders and spiral threads, to flagellated rods, and filamentous chains. They are found practically everywhere on Earth and live in some of the most unusual and seemingly inhospitable places. A prokaryote is a simple, single-celled organism that lacks a nucleus and membrane-bound organelles. The majority of prokaryotic DNA is found in a central region of the cell called the nucleoid, and it typically consists of a single large loop called a circular chromosome. Most bacteria are, however, surrounded by a rigid cell wall made out of peptidoglycan, a polymer composed of linked carbohydrates and small proteins. The cell wall provides an extra layer of protection, helps the cell maintain its shape, and prevents dehydration. Many bacteria also have an outermost layer of carbohydrates called the capsule. The capsule is sticky and helps the cell attach to surfaces in its environment. Typical prokaryotic cells range from 0.1 to 5.0 micrometers (μm) in diameter and are 5 significantly smaller than eukaryotic cells, which usually have diameters ranging from 10 to 100 μm. Animals, plants, algae and fungi are all eukaryotes. EUKARYOTES A eukaryote is an organism with complex cells, or a single cell with a complex structures. In these cells the genetic material is organized into chromosomes in the cell nucleus. Animals, plants, algae and fungi are all eukaryotes. Eukaryotic cells are usually much bigger than prokaryotes. They can be up to 10 times bigger. Eukaryote cells have many different internal membranes and structures, called organelles. They also have a cytoskeleton. The cytoskeleton is made up of microtubules and microfilaments. Those parts are very important in the cell's shape. Eukaryotic DNA is put in bundles called chromosomes, which are separated by a microtubular spindle during cell division. Most eukaryotes have some sort of sexual reproduction through fertilization, which prokaryotes do not use. Eukaryotes have sets of linear chromosomes located in the nucleus and the number of chromosomes is usually typical for each species. 6 EUKARYOTIC CELL. Cells of prokaryotic and eukaryotic organism differ in several significant structural features as shown below; S/N Characteristic Prokaryotic Cell Eukaryotic Cell 1. Nuclear membrane Absent Present 2. Chromosome Usually single, circular Multiple 3. DNA Circular Linear 4. Nucleolus Absent Present 5. Cell Division By mitosis (fission) By mitosis or by meiosis 6. Ribosome 70s is present 80s is present 7. Chloroplast Absent Present 8. Mitochondria Absent Present 9. Cell organelles Absent Present 10. Cell wall Usually present, many Cells, present in plant, no contain peptidoglycan peptidoglycan 11. In flagella No definite arrangement 9+2 filbrillas of fibris arrangement 12. Cytoplasm Amoeboid movement of Amoeboid most of cytoplasm absent cytoplasm present 13. Average size 0.2-2mm in diameter 10-100mm in diameter 14. Plasma membrane No carbohydrates, most Sterols and carbohydrate lack sterols present 15. Number of cell Generally unicellular Generally multicellular 7 THE BACTERIA CELL The most basic thing to discuss about a bacterial cell is its shape. Bacteria come in all sorts of shapes. Round ones are called Cocci. Then there are Bacilli which are rod shaped cells. A cell that is sort of round and not quite a rod (something like an oval) would be called a Coccobacillus. If a rod shaped cell has a bit of a curve to it, like a comma, it's called a Vibrio. Then there are cells with a corkscrew or even a spring shape which are referred to as a Spirillum or Spirochete. Making things even more interesting, these cells often times cluster together into specific patterns that can be seen using a microscope. A pair of cocci are called Diplococci. Sometimes cocci will form chains e.g. Streptococci. Other times cocci may form clusters which is a characteristic of Staphylococci. Bacilli may not form any particular pattern butwhen they do it often looks like a line linked together to form a chain. The different bacterial shapes:a. bacillus (rod), b. coccus (spherical), c. spirillum (spiral), d.spirochaete (corkscrew), e. vibrios (comma), f. chain of cocci, g. cluster of cocci, h. pair ofcocci, i. chain of bacilli EXTERNAL FEATURES 8 Bacteria have a number of cell surface components that differ in look and function. Some of these are for locomotion as bacteria often are found in liquid environments. Others help bacteria attach to surfaces, while still others are protective in nature. These different structures are discussed below. Flagella Some bacteria have long filamentous appendages sticking out of them called Flagella. These structures rotate and function as a propeller screw to move bacteria along in liquid environments. A cell with this ability is said to be motile. Bacteria can have one or many flagella and they can be located in different patterns around the cell. The motility provided by flagella allows bacteria to move around in the environment in search of nutrients. They swim in one direction for a period of time based on the prevailing conditions. If they're swimming in the right direction, they head that way for a longer period of time. If they sense they're going the wrong way they reverse the propeller, tumble around and then head out in a new, random direction. In this way they zigzag around heading ever closer to the most favorable environment. This type of movement is called a random walk. Fimbria These are smaller appendages found all over the surface of some bacteria that allow them to attach to surfaces. The fimbria will stick to just about anything from other bacteria in biofilms to surfaces such as epithelial tissue or glass and plastic. Pili These are also long appendages like flagella but they have a function more similar to fimbria. Some pili allow cells to anchor to surfaces like a grappling hook. The most common function of pili is to connect two bacterial cells together in the process of bacterial conjugation. In this process bacteria are able to share DNA with each other. Glycolax The glycolax layer forms when cells are in harsh environments or when they need to adhere to a surface. It consists of a matrix of carbohydrates and protein, different 9 species have different recipes for this material. If a cell has a general layer of this goop that is not particularly well defined it's called a Slime Layer. A well organized layer of uniform thickness all around is called a Capsule. Pathogenic bacteria rely on a capsule to protect them from the immune system. It's a significant defense mechanism our immune system cannot overcome in many cases of serious illness. While not all pathogens make a capsule, many of the really nasty ones do. Aside from protection, the glycolax allows bacteria to stick to surfaces and form multi species collectives with other bacteria leading to the creation of a biofilm. These are very common in nature where there is a complex interaction going on between all the different species of organisms present. Biofilms can present some significant health hazards when they form on implanted medical devices like catheters or heart valves. Cell Wall Structure Bacteria have a unique cell wall structure that helps protect their cellular membrane from damage in unfavorable conditions. When the conditions (temperature, pH, osmotic pressure) surrounding the cell are right, the bacteria can survive adequately and carry out its metabolic activities adequately. The outer structure of the cell needs to be resilient to withstand/resist the hypotonic environment where they live. Without fortifying the cell membrane, it could burst under unfavourable condition. Bacteria have a cell wall structure outside of the main plasma membrane. Bacteria are surrounded by a layer of Peptidoglycan. This consists of long chains of carbohydrate units called G and M (the actual names are much harder to remember). These carbohydrate chains have protein side chains linking them together. This forms a chain link fence type of structure that surrounds the entire cell. Gram Positive/Negative 10 Bacteria have two different types of cells wall structures. One involves a very thick layer of peptidoglycan, but the other has a very thin layer. In addition, the one with the thin layer of peptidoglycan has a second cell membrane. This second membrane contains lipopolysaccharide (LPS) molecules that play a role in disease. They form endotoxin which is released when the cell is destroyed. Gram positive cells have a thick layer (purple) of peptidoglycan outside the cell membrane. The gram negative cells have a much thinner (pink) layer of peptidoglycan but this is reinforced with a second membrane on top of it. Gram Stain Though the cell wall structure of a cell cannot be seen with a light microscope, it can be shown to be either gram positive or gram negative by a simple differential staining technique. We call it a differential stain because it allows us to see the difference between two options. The stain process takes advantage of the different thickness of the peptidoglycan layers. Gram positive cells with a thick layer will trap dye more effectively than a gram negative cell. Thus it is possible to tell them apart. The gram positive cell retains the crystal violet stain and is purple while the gram negative cell would be clear of stain after rinsing with the alcohol. The pink color comes from adding Saffranin, just to make it show up under magnification. With the gram stain test, the type and shape of a bacterial cell is known which is the first required in bacterial identification. Gram Stain Procedure The Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall). These stain purple. Gram-negative bacteria have walls with thin layers of 11 peptidoglycan (10% of wall), and high lipid content. These stain pink. This staining procedure is not used for Archeae or Eukaryotes as both lack peptidoglycan. The performance of the Gram Stain on any sample requires four basic steps that include: 1. applying a primary stain (crystal violet) to a heat-fixed smear. 2. this is followed by the addition of a mordant (Gram’s Iodine), 3. rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, 4. counterstaining with safranin. Differences between Gram Positive and Gram Negative Bacteria S/N Gram Positive Cell Wall Gram Negative Cell Wall 1. Cell wall is thick Cell wall is thin 2. Few amino acid present Many amino acid present 3. Sulfur containing amino acid absent Sulfur containing amino acid present 4. Teichoic acid present Teichoic acid absent 5. Peptideglyean present Peptideglyean present in less quantity 6. Lipoproteins absent Lipoproteins present 7. Lipopolysaccharide absent Lipopolysaccharide present 8. Polysaccharide present Polysaccharide absent Internal Structures Bacteria have internal structures that are similar to all living cells but are somewhat different from eukaryotes. The genetic material for bacteria consists of a single, circular chromosome rather than the multiple linear chromosomes found in eukaryotes like plant and animal cells. 12 Bacteria also have circular bits of DNA called plasmids that are not found in animal cells. These plasmids contain genes which provides bacteria with genetic advantages such as antibiotic resistance. They also harbor unique enzymes or other proteins with characteristics that may contribute to the pathogenecity of the cell. The genes may code for some toxin for instance. Plasmids can be shared between cells to conferr these abilities on other cells through the mechanism of horizontal gene transfer. Another important internal structure present in microorganisms are the ribososmes. Ribosomesare used to make proteins which are necessary to perform cellular functions. Typically ribosomes are composed of two subunits: a large subunit and a small subunit. The large and small subunits form around a messenger RNA to decode the instructions locked in DNA for building a protein. This occurs when the subunits of ribosomes join together at the point ribosomes attaches to the messenger RNA during the process of protein synthesis. Ribosomes along with a transfer RNA molecule (tRNA), helps to translate the protein-coding genes in mRNA to proteins. It is of note that bacterial ribosomes are not the exact same size and composition as eukaryotic ribosomes.They are different enough that antibiotics can be used to disrupt the function of bacterial ribosomes yet leave your eukaryotic ribosomes alone and unharmed. 1. MesosomesThese are convoluted invaginations within the cytoplasmic membrane. They play an important role during cell division and in the secretion of certain enzymes. 2. Nuclear material: Within the cytoplasmic membrane, the cell itself has the “nucleus” which has no nuclear membrane and lacks a definite shape. It is a single circular strand of deoxyribonucleic acid (DNA) which acts as a “nucleus” (chromosome). 3. Ribosomes: Ribosomes are located throughout the cytoplasm and are the sites of protein synthesis. They are important for conveying the genetic code of the 13 nucleus into instructions in the manufacture of cellular components. They are composed of ribonucleic acid (RNA) and proteins. 4. Cytoplasmic inclusions:These are seen in some bacteria and appear to be sources of reserved food for energy. For example, volution (polymetaphosphate) granules associated with Corynebacteria. 5. Spores These are dense structures produced by the bacteria, e.g. the Bacillus and Clostridiumgroups, that enable them to survive adverse environmental conditions. They develop within and at the expense of the vegetative cell. The spore comprises chromosomal material surrounded by several layers of walls. The location and shape of the spore in the cell may be of diagnostic assistance, e.g. the spores of Clostridium tetani are terminal, and the diameter is greater that of the parent cell, so that they are characteristically of drum stick appearance. The positions of spores are described as terminal, subterminal or central. Spores are resistant to heat, stains, desiccation and disinfectants. Each spore germinates to produce a vegetative cell during favourable growth conditions. TAXONOMY AND IDENTIFICATION (CLASSIFICATION) OF MICROORGANISMS Taxonomy: Taxonomy may be defined as the science or study of the classification of living organisms. It involves separating living organisms (and fossil forms of preexisting organisms) into groups or categories and developing the criteria to be used for determining which organisms fit into which groups. Grouping or categorizing living organisms allows investigators to study and understand them more readily. The categories used in the classification of organisms are intended to show the natural relationships between organisms and to reflect phylogeny, i.e., the evolutionary history of organisms. Recently, new methods for analyzing the biochemical content of organisms have led to the development of new criteria for classification (especially in microbiology), and although this is exciting for taxonomists, it has created inconsistencies in reference sources resulting in considerable confusion for students. It is not unusual to find different 14 authors applying different criteria, and placing the same organisms into different groups. Therefore, if you have studied classification schemas in other classes, it is likely that the one presented in this class will be different. The tendency to categorize (vehicles, foods, clothing, etc.) is common to humans and not restricted to biologists. The information presented here is of a more general nature and includes terminology applicable to taxonomy. Binomial Nomenclature: – Binomial nomenclature refers to the two-part technical name applied to each different type of living organism. It is important to biologists because it provides a system for communicating information about specific organisms named in a language universally recognized and accepted. The development of the binomial system of nomenclature (binomial nomenclature) is credited to Carolus Linnaeus (a botanist/naturalist), in association with his Systema Natural, a manuscript containing a classification of living organisms, first published in 1735. Linnaeus's text contained lengthy descriptions of multiple living organisms, but also included a two-part name for each one, based on key characteristics. Currently the two-part technical name applied to each different type of living organism includes the genus name (which is capitalized) and the specific epithet (all lower case). Both names are Latinized and include either Latin or Greek roots providing descriptive information. For example the name Staphylococcus aureus, describes a type of organism forming grape-like clusters of spherical-shaped cells, and golden or yellow-colored colonies. Linnaeus's text was in Latin because it was the language used in universities at the time; however, since Latin is no longer a spoken language, terms and their meanings remain stable and provide the basis for universally accepted scientific communication. Currently, the binomial names of organisms are italicized when in print and underlined when written by hand, a convention allowing for easy recognition. The two-part name applied to each type of organism indicates where that organism fits into a larger taxonomic schema as indicated below. Taxonomic Ranks: – Taxonomic ranks are the categories used in the classification of living organisms. These are nested ranks, with each successively lower level being contained within the one above. A group of organisms occupying a specific rank is called a taxon (pleural = taxa) or taxonomic group. The original taxonomic ranks were as follows: Kingdoms: (singular = Kingdom, the largest or most encompassing) Phyla: (singular = phylum) Sometimes called Divisions 15 Classes: (singular = Class) Orders: (singular = Order) Families: (singular = Family) Genera: (singular = Genus) Species: (the most specific category) One of several mnemonic forms = Kids playing chase on freeways go splat! At the time of Linnaeus’s work, living organisms were grouped into two broad categories, the Plantae (plants) and the Animalia (animals). These broad categories were called Kingdoms. Since then, a number of classification categories have been added between the levels of kingdom and genus. Similar organisms with the same genus name, or genera, are grouped within the same family, similar families are grouped within the same order, similar orders are grouped within the same class, similar classes are grouped within the same phylum (or division), and similar phyla are grouped within the same kingdom. The criteria or rules used for the classification of living organisms into taxonomic ranks are quite specific and are determined by groups of biologists from around the world. These international groups called congresses meet at varying intervals to determine how plants and animals are to be categorized. Although most macroscopic organisms can be readily classified into two kingdoms (Plantae and Animalia), microscopic organisms cannot. Following Van Leeuwenhoek’s discovery of microscopic life forms, many new organisms were identified that did not meet the criteria for either kingdom. One way to solve this problem was to establish a new kingdom. In 1866, Ernst Haeckel proposed a third kingdom be established which he called Protista. This kingdom would include all single-celled organisms and those multicellular forms not developing complex tissues. A diverse group of organisms including protozoa, algae, fungi, sponges and slime molds were to be classified within this kingdom, but their relatedness was minimal. In 1957, Roger Stainer and his associates used electron microscopy to demonstrate significant differences between prokaryotic and eukaryotic cells, suggesting more than three kingdoms were required. In 1969, R.H. Whittaker proposed a five-kingdom system to improve classification. This system included three kingdoms of more complex organisms based on three modes of nutrition. The Animalia ingest food, the Plantae make their own food via photosynthesis, and the Fungi (Myceteae) absorb food in a liquid form. The other two kingdoms, Protista and Monera, include organisms without complex structures that are separated based on their cell types. Monera are prokaryotic and Protista are eukaryotic. Although the Whittaker five-kingdom system is included in many modern 16 textbooks, binomial without problems. Recent studies based on biochemical analyses indicate considerable variation among eukaryotic microorganisms, and the need for multiple additional kingdoms. In 1978, Carl Woese and his associates using biochemical analyses demonstrated significant differences within the kingdom Monera; and prompted the addition of a new taxonomic rank called Domain above kingdoms in the taxonomic hierarchy. This work provided evidence that organisms now recognized as Archaea (formerly Archaeobacteria) have multiple important characteristics unlike either bacteria or eukaryotic organisms. The three domains of life currently accepted by most biologists include the Eukarya (all organisms with eukaryotic cells), the Bacteria and the Archaea. Adding domains to the previously established taxonomic ranks generates a slightly modified hierarchy as shown below: Domain: (pleural = Domains, the largest or most encompassing) Kingdom: (pleural = Kingdoms) Phylum: (pleural = Phyla) Sometimes called Divisions Class: (pleural = Classes) Order: (pleural = Orders) Family: (pleural = Families) Genus: (pleural = Genera) Species: (the most specific category) Mnemonic form = Dumb kids playing chase on freeways go splat! Since understanding the phylogeny (evolutionary history) of life on earth is a major goal of taxonomists, numerous methods have been employed to determine the evolutionary relationships between organisms. Since the 1970s, computer technology and a method called Cladistics have provided considerable information relative to evolutionary trends. In cladistics, specific features of organisms are used to determine relatedness. A feature that is common to several different types of organisms, but shows variation within them is assigned a value or form called a character state. Analysis of the character is then conducted to determine which state is primitive (ancestral) and which is derived (evolved from something else). General Principles of Taxonomy 17 Taxonomy hierarchy is a way of organizing the classification of organisms with different levels of similarity. The species is the fundamental unit of the taxonomic hierarchy. The next level is the genus which will contain one or more closely related species. Closely related genera are organized into families; families into classes; classes into divisions or phyla and phyla into kingdom. Although species are the basic taxonomic units, the genetic variability of microorganisms permits a further division into subspecies or types that describe the specific clone of cells. The subspecies or types may differ physiologically (biovar), morphologically (morphovar) or antigenically (serovar). It is often important to differentiate the subspecies of a given microorganism. For example, one strain of a bacterial species may produce a toxin and be a virulent pathogen and other strain of a same species may be nonpathogenic. A strain is a population of cells that are descendents of a single cell. The ability to distinguish correctly between such strains and subspecies of a particular microbial species is of obvious importance in medical, pharmaceutical and industrial microbiology. Classification of Microorganisms In the past, biologists divided living things into plants and animals, which were later accepted as two broad kingdoms of living creatures. This classification was made before the discovery of microorganisms. During the course of studies, it becomes evident that the old and common divisions of the living world is insufficient because microorganisms show the similarity with plants (some are green and do not eat other organisms) on the one hand and on the other hand with animals (highly motile). Bacteria were included in plants. Slime molds were regarded as middle path between plants and animals because they were considered as plants by botanists and animal by zoologists. In 1866, Ernst Haeckel, a German biologist, created a new kingdom as protista. This kingdom included all those microorganisms which did not possess any extensive development of tissue. On the basis of this character, biologists included all the unicellular microscopic organisms in this kingdom, although a few multicellular organisms have also been included in the same kingdom. Therefore, the kingdom protista includes the bacteria, algae, fungi and protozoa. The kingdom protista was further divided into two large groups on the basis of cellular differentiation, namely lower protista which includes bacteria and cyanobacteria and higher protista, which includes algae, fungi and protozoa. The lower protista are 18 prokaryotic in their cellular organization, ie, the nucleus of the cell is devoid of nuclear membrane while the higher protista are eukaryotic, ie, the nucleus of the cell is surrounded by nuclear membrane. Lower Protista Lower protista are prokaryotic in nature and include bacteria and cyanobacteria (blue green algae). Bacteria are grouped into 19 sections on the basis of structure, genetic data and biochemical, nutritional, staining and ecological characteristics. 19 Sections I The photo trophic bacteria II The gliding bacteria III The sheathed bacteria IV Budding and/or appendaged bacteria V The spirochetes VI Spiral and curved bacteria VII Gram negative aerobic rods and cocci VIII Gram negative facultatively anaerobic rods IX Gram negative anaerobic bacteria X Gram negative cocci and coccobacilli XI Gram negative anaerobic cocci XII Gram negative chemolithotrophic bacteria XIII Methane producing bacteria XIV Gram positive cocci XV Endospore forming rods and cocci XVI Gram positive Asporogenous rod shaped bacteria 19 XVII Actinomycetes and related organisms XVIII The Rickettsias XIX The mycoplasmas Higher Protista The higher or eukaryotic protista include a highly divergent group of organisms and these can be subdivided into three major groups namely the protozoa, the algae and the fungi. Protozoa The protozoa are colourless, non photosynthetic, unicellular, motile organisms with complex life cycles. They are found in a variety of habitats and require a large amount of moisture for growth and activity. Various species are found in freshwater, marine water, sewage or terrestrial environments. Several protozoas are parasitic on other organisms. Protozoa are classified into four major classes: 1. Mastigophora (flagellate protozoa) - They move by one or more flagella. Cells divide by longitudinal fission. 2. Rhizopoda (amoeboid protozoa) - motility is generally by pseudopodia, but some form flagella. Reproduction is by binary fission. 3. Ciliata (Ciliate protozoa) - motility is by numerous cilia. Divide by transverse fission. Cell contains two nuclei. 4. Sporozoa - Generally non motile, some show creeping or gliding motility. Multiply by multiple fission. Parasitic. Algae The algae are a ubiquitous group of aerobic photosynthetic organisms characterized by the presence of photosynthetic pigments and their ability to carry out a plant like photosynthesis. They are abundant in water, soil and other terrestrial environments. It occurs in a variety of forms such as rhizoidal, filamentous, mucilagenus colonies. Different algae species secrete various kinds of mucilages which are polysaccharides in composition. The mucilage may aid in motility or provide anchor to the organism. Based on the structure, pigments, cell wall composition, motility, reproduction and nature of reserve material, algae can be classified into six classes: 20 1. Chlorophyta (Green algae) - Found in fresh water and salt water, soil etc. Chlorophyll a, b, carotene and xanthophyll present. Stores starch as reserve material. It divides vegetatively or asexually to form flagellated zoospores. 2. Euglenophyta (Euglenoids) - Contain both fresh and marine water forms. Unicellular flagellates. Chlorophyll a, b, carotene present. Stores fat and starch like carbohydrates. It divides by longitudinal binary fission and rarely by sexual method. 3. Chrysophyta (diatoms, golden algae) - Consists of a group of several diverse non- flagellar, unicellular algae. Some have calcium containing cell walls and others contain silica in their cell wall (diatoms). Stores oils and lecucosin. Reproduce by asexual or sexual method. 4. Pyrrophyta (Dinoflagellates) - Primarily marine organisms. Two flagella with different structure and arrangement. Most are unicellular and contain carotenes and xanthophylls. Asexual reproduction and cell stores oils and starch. 5. Phacophyta (Brown algae) - Multicellular and contain xanthophyll and carotene pigments. Found in sea water and structurally complex. Reproduce asexually by zoospores and sexually by gametes. 6. Rhodophyta (Red algae) - Contains phycobilins and chlorophyll a. Nonflagellate, unicellular or multicellular with complicated modes of sexual reproduction. Stores starch. Grows in slat water. Polysaccharides agar-agar carrageenan and alginic acid are obtained from red algae. Fungi Fungi include the yeast and molds. They are characterized by the presence of a typical eukaryotic cell, a mycelial (thread like) thallus, and are non-photosynthetic. Most are saprophytes but some are parasitic on plants and animals. Most are mesophilic but some are thermotolerant. Fungi are aerobic, while some yeast are facultatively anaerobic. Reproduction occurs both sexually as well as asexually. Their classification is based on morphology, physiology, the mode of reproduction and the nature of reproduction and the nature of the reproductive structures. They are divided into four major classes: 1. Phycomycetes - Mycelium nonseptate, aquatic or terrestrial, sporangiospores (asexual spores) motile or non motile. Sexual spores are oospores or zygospores. 21 2. Ascomycetes - Mycelium well developed and septate. Sexual spores are borne in asci. Parasitic and saprophytic. 3. Basidomycetes - Characteristics spores borne on basidia (club shaped structure)- basidiospores. 4. Fungi imperfecti - Reproduction by asexual process only. Mostly found on decaying wood. Viruses Viruses are the most important microbial enemies of human beings. They are very simple and tiny. Viruses are an intermediate stage between living and non-living. Viruses are called acellular as they do not have cellular organization like other living organisms. The genetic material in viruses is either DNA or RNA but never both. It's an obligate parasite and it can not be cultured on media. But it can be cultured on cell media like bacteria, chick embryo cells, etc. Viruses are made up of only nucleic acid and proteins. But they lack the enzymes necessary for their synthesis. For this, viruses depend on the host enzymes. They can be crystallized. They can be transmitted from one host to another. The capsid of viruses is mostly made up of proteins. Except in some animal viruses, polysaccharides are also present. Classification of viruses is based on a variety of criteria. Based on their hosts, viruses were classified into the following types: 1. Plant viruses - Infect plants 2. Animal viruses - Infect animal 3. Bacteriophage - Infect bacteria 4. Cyanophages - Infect cyanobacteria 5. Myco viruses - Infect fungi 6. Mycoplasma viruses - Infect mycoplasma 7. Phyco viruses - Infect algae. Beside phylogenitic classification, the microorganisms are classified on various criteria, as explained below: 22 CLASSIFICATION OF BACTERIA Bacteria are classified and identified to distinguish one organism from another and to group similar organisms by criteria of interest to microbiologists or other scientists. The classification of bacteria serves a variety of different functions. Because of this variety, bacteria may be grouped using many different typing schemes. The grounds for the classification commonly used may be: Morphologic Characteristics Both wet-mounted and properly stained bacterial cell suspensions can yield a great deal of information. A. Classification on the basis of Gram Stain and Bacterial Cell Wall. Of all the different classification systems, the Gram stain has withstood the test of time. Discovered by H.C. Gram in 1884 it remains an important and useful technique to this day. It allows a large proportion of clinically important bacteria to be classified as either Gram positive or negative based on their morphology and differential staining properties. Slides are sequentially stained with crystal violet, iodine, then de-stained with alcohol and counter- stained with safranin. Gram positive bacteria stain blue-purple and Gram negative bacteria stain red. The difference between the two groups is believed to be due to a much larger peptidoglycan (cell wall) in Gram positives. As a result the iodine and crystal violet precipitate in the thickened cell wall and are not eluted by alcohol in contrast with the Gram negatives where the crystal violet is readily eluted from the bacteria. As a result bacteria can be distinguished based on their morphology and staining properties. Some bacteria such as mycobacteria are not reliably stained due to the large lipid content of the peptidoglycan. Alternative staining techniques (Kinyoun or acid fast stain) are therefore used that take advantage of the resistance to destaining after lengthier initial staining. B. Classification of Bacteria on the Basis of Shape In the year 1872 scientist Cohn classified bacteria to 4 major types depending on their shapes are as follows:i) Cocci:- These types of bacteria are unicellular, spherical or elliptical shape. Either they may remain as a single cell or may aggregate together for various configurations. They are as follows: Monococcus: – they are also called micrococcus and represented by single, discrete round Example:- Micrococcus flavus. Diplococcus: – the cell of the Diplococcus divides ones in a particular plane and after division, the cells remain attached to each other. Example: Diplococcus pneumonia. 23 Streptococcus: – here the cells divide repeatedly in one plane to form chain of cells. Example: – Streptococcus pyogene. Tetracoccus: – this consists of four round cells, which defied in two planes at a right angles to one another. Example: – Gaffkya tetragena. Staphylococcus: – here the cells divided into three planes forming a structured like bunches of grapes giving and irregular configuration. Example: – Staphylococcus aureus. Sarcina: -in this case the cells divide in three planes but they form a cube like configuration consisting of eight or sixteen cells but they have a regular shape. Example:- Sarcina lutea. ii) Bacilli: – These are rod shaped or cylindrical bacteria which either remain singly or in pairs. Example: – Bacillus cereus. iii) Vibro: – The vibro are the curved, comma shaped bacteria and represented by a single genus. Example: – Vibro cholerae. iv) Spirilla: – These type of bacteria are spiral or spring like with multiple curvature and terminal flagella. Example: – Spirillum volutans. Others Actinomycetes: are branching filamentous bacteria, so called because of a fancied resemblance to the radiating rays of the sun when seen in tissue lesions (from actis meaning ray and mykes meaning fungus). Mycoplasmas are bacteria that are cell wall deficient and hence do not possess a stable morphology. They occur as round or oval bodies and as interlacing filaments. C. Classification of Bacteria on the Basis of Mode of Nutrition 1. Phototrophs: Those bacteria which gain energy from light. Phototrops are further divided into two groups on the basis of source of electron. Photolithotrophs: these bacteria gain energy from light and uses reduced inorganic compounds such as H2S as electron source. Eg. Chromatium okenii. Photoorganotrophs: these bacteria gain energy from light and uses organic compounds such as succinate as electron source. 2. Chemotrophs: Those bacteria gain energy from chemical compounds.They cannot carry out photosynthesis. Chemotrops are further divided into two groups on the basis of source of electron. Chemolithotrophs: they gain energy from oxidation of chemical compound and reduces inorganic compounds such as NH3 as electron source. Eg. Nitrosomonas. Chemoorganotrophs: they gain energy from chemical compounds and 24 uses organic compound such as glucose and amino acids as source of electron. eg. Pseudomonas pseudoflava. 3. Autotrophs: Those bacteria which uses carbondioxide as sole source of carbon to prepare its own food. Autotrophs are divided into two types on the basis of energy utilized to assimilate carbondioxide. ie. Photoautotrophs and chemoautotrophs.Photoautotrophs: they utilized light to assimilate CO2. They are further divided into two group on the basis of electron sources. i.e. Photolithotropic autotrophs and Photoorganotropic autotrophs Chemoautotrophs: They utilize chemical energy for assimilation of CO2. 4. Heterotrophs: Those bacteria which uses organic compound as carbon source.They lack the ability to fix CO2. Most of the human pathogenic bacteria are heterotropic in nature.Some heterotrops are simple, because they have simple nutritional requirement. However there are some bacteria that require special nutrients for their growth; known as fastidious heterotrophs. D. Classification of Bacteria on the Basis of Temperature Requirement Bacteria can be classified into the following major types on the basis of their temperatures response as indicated below: Psychrophiles: Bacteria that can grow at 0°C or below but the optimum temperature of growth is 15 °C or below and maximum temperature is 20°C are called psychrophiles, Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid nature to the cell membrane even at lower temperature. Examples: Vibrio psychroerythrus, vibrio marinus, Polaromonas vaculata, Psychroflexus. 2. Psychrotrops (facultative psychrophiles): Those bacteria that can grow even at 0°C but optimum temperature for growth is (20-30)°C3. Mesophiles : Those bacteria that can grow best between (25-40) C but optimum temperature for growth is 37C Most of the human pathogens are mesophilic in nature. Examples: E. coli , Salmonella , Klebsiella, Staphylococci. 4. Thermophiles: Those bacteria that can best grow above 45C. Thermophiles capable of growing in mesophilic range are called facultative thermophiles. True thermophiles are called as Stenothermophiles, they are obligate thermophiles, Thermophils contains saturated fattyacids in their cell membrane so their cell 25 membrane does not become too fluid even at higher temperature. Examples: Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus. 5. Hypethermophiles: Those bacteria that have optimum temperature of growth above 80C. Mostly Archeobacteria are hyperthermophiles. Monolayer cell membrane of Archeobacteria is more resistant to heat and they adopt to grow in higher temperature. Examples: Thermodesulfobacterium, Aquifex, Pyrolobus fumari , Thermotoga. E. Classification of Bacteria on the Basis of Oxygen Requirement 1. Obligate Aerobes: Require oxygen to live. Example: Pseudomonas, common nosocomial pathogen. 2. Facultative Anaerobes: Can use oxygen, but can grow in its absence. They have complex set of enzymes. Examples: E. coli, Staphylococcus, yeasts, and many intestinal bacteria. 3. Obligate Anaerobes: Cannot use oxygen and are harmed by the presence of toxic forms of oxygen. Examples: Clostridium bacteria that cause tetanus and botulism. 4. Aerotolerant Anaerobes: Cannot use oxygen, but tolerate its presence. Can break down toxic forms of oxygen. Example: Lactobacillus carries out fermentation regardless of oxygen presence. 5. Microaerophiles: Require oxygen, but at low concentrations. Sensitive to toxic forms of oxygen. Example: Campylobacter. F Classification of Bacteria on the Basis of Number of Flagella On the basis of flagella the bacteria can be classified as: 1. Atrichos: – These bacteria has no flagella. Example: Corynebacterium diptherae. 2. Monotrichous: – One flagellum is attached to one end of the bacteria cell. Example: – Vibro cholerae. 3. Lophotrichous: – Bunch of flagella is attached to one end of the bacteria cell. Example: Pseudomonas. 4. Amphitrichous: – Bunch of flagella arising from both end of the bacteria cell. Example: Rhodospirillum rubrum. 5. Peritrichous: 26 – The flagella are evenly distributed surrounding the entire bacterial cell. Example: Bacillus. G. Classification of Bacteria on the basis of Spore Formation 1. Spore forming bacteria: Those bacteria that produce spore during unfavorable condition.These are further divided into two groups:i) Endospore forming bacteria: Spore is produced within the bacterial cell. Examples. Bacillus, Clostridium, Sporosarcina etcii) Exospore forming bacteria: Spore is produced outside the cell. Example. Methylosinus 2. Non sporin bacteria : Those bacteria which do not produce spores. Eg. E. coli, Salmonella. H. Classification on of Bacteria on the Basis of pH of Growth 1. Acidophiles: These bacteria grow best at an acidic pH. The cytoplasm of these bacteria are acidic in nature. Some acidopiles are thermophilic in nature, such bacteria are called Thermoacidophiles. Examples, Thiobacillus, ferroxidans, Thermoplasma, Sulfolobus 2. Alkaliphiles: These bacteria grow best at an alkaline pH. Example: Vibrio cholerae optimum ph of growth is 8.2. 3. Neutrophiles: These bacteria grow best at neutral pH (6.5-7.5). Most of the bacteria grow at neutral pH. Example: E. coli I. Classification of Bacteria on the Osmotic Pressure Requirement 1 Halophiles: Require moderate large salt concentrations. Cell membrane of halophilic bacteria is made up of glycoprotein with high content of negatively charged glutamic acid and aspartic acids. So high concentration of Na+ ion concentration is required to shield the –ve charge. Ocean water contains 3.5% salt. Most such bacteria are present in the oceans. Archeobacteria, Halobacterium, Halococcus. Extreme or Obligate Halophiles: Require a very high salt concentrations (20 to 30%). Bacteria in Dead Sea, brine vats. Facultative Halophiles: Do not require high salt concentrations for growth, but tolerate upto 2% salt or more. 27 GENERAL MECHANISM OF DRUG RESISTANCE Bacteria are said to be resistant to an antimicrobial drug if the maximal level of the agent that can be achieved in vivo or tolerated by the host does not halt their growth. Some organisms are inherently resistant to antibiotics, for instance, because they lack the target of the antimicrobial agent. However, microbes that are normally responsive to a particular drug may develop resistance through spontaneous mutation or by acquisition of new genes followed by selection. Some strains may even become resistant to more than one antibiotic by acquisition of genetic elements that encode multiple resistance genes. A. GENETIC ALTERATIONS LEADING TO DRUG RESISTANCE Acquired antibiotic resistance involves mutation of existing genes or the acquisition of new genes. 1. Spontaneous Mutations in DNA Chromosomal alteration may occur by insertion, deletion, or substitution of one or more nucleotides within the genome. The resulting mutation may persist, be corrected by the organism, or be lethal to the cell. If the cell survives, it can replicate and transmit its mutated properties to progeny cells. Mutations that produce antibiotic-resistant strains can result in organisms that proliferate under selective pressures such as in the presence of antimicrobial agents. Example is in the emergence of rifampicin-resistant Mycobacterium tuberculosis when rifampicin is used as a single antibiotic. 2. DNA Transfer of Drug Resistance Of particular clinical concern is the resistance acquired due to DNA transfer from one bacterium to another. Resistance properties are often encoded on extrachromosomal plasmids, known as R, or resistance factors. DNA can be transferred from donor to recipient cell by processes including transduction (phage mediated), transformation, or bacterial conjugation. 28 B. ALTERED EXPRESSION OF PROTEINS IN DRUG RESISTANT ORGANISMS Drug resistance may be mediated by several different mechanisms including an alteration in the antimicrobial drug target site, decreased uptake of the drug due to changes in membrane permeability, increased efflux of the drug, or the presence of antibiotic-inactivating enzymes. 1. Modification of Target Sites Alteration of an antimicrobial agent’s target site through mutation can confer resistance to one or more related antibiotics. For example, Streptococcus pneumonia resistance to Beta-Lactam drugs includes alteration of one or more of the major bacterial penicillin- binding proteins, resulting in decreased binding of the antimicrobial to its target. 2. Decreased Accumulation Decreased uptake or increased efflux of an antimicrobial agent can confer resistance because the drug is unable to attain access to the site of the action in sufficient concentrations to inhibit or kill the organism. For example, Gram-negative organisms can limit the penetration of certain agents, including beta-lactam antibiotics, tetracyclines, and chloramphenicol, as a result of an alteration in the number and structure of porins (channels) in the outer membrane. Also, expression of an efflux pump can limit levels of a drug that accumulate in an organism. For example, transmembrane proteins located in the cytoplasmic membrane actively pump intracellular antibiotic molecule out of the microorganism. These drug efflux pumps for xenobiotic compounds have a broad substrate specificity and are responsible for decreased drug accumulation in multidrug- resistant cells. The efflux pumps may be encoded on chromosomes and plasmids, thus contributing to both intrinsic (natural) and acquired resistance, respectively. As an intrinsic mechanism of resistance, efflux pump genes allow bacteria expressing them to survive a hostile environment (e.g, in the presence of antibiotics), which allows for the selection of mutants that overexpress these genes. Being located on transmissible genetic elements as plasmids or transposons is also advantageous for the microorganism insofar as it allows the easy spread of efflux genes between distinct species, 29 3. Enzymatic Inactivation The ability to destroy or inactivate the antimicrobial agent can also confer resistance to microorganisms, example of antibiotic-inactivating enzymes include: beta-lactamases that hydrolytically inactivate the beta-lactam ring of penicillins, cephalosporins, and related drugs. Acetyltransferases that transfer an acetyl group to the antibiotic inactivating chloramphenicol, and Esterases that hydrolyze the lactone ring of macrolides.. CULTURE MEDIA To study and identify microorganisms, they have to be cultivated and isolated in pure culture. Most bacteria can be cultured in artificial media if the media meet the nutritional and physical growth requirements of the bacteria. The media used for growing bacteria are either liquid or solid media. They must contain water and sources of nitrogen, carbon, mineral salts and essential vitamins. Some bacteria may require additional specific substances which may be added to the medium. Media are either chemically defined or complex media. Chemically Defined or Simple Media:- are media in which the concentration of each ingredient is known. The ingredients are usually highly purified inorganic salts and simple organic compounds such as glucose or purified amino acids. Here is little or no variation in their composition from batch to batch, hence are valuable. They are used to determine specific growth requirements of bacteria. Complex Media:- are media prepared using natural products such as meat extract or vegetable infusions. The natural products contain essential bacterial nutrients but the exact concentration of nutrient is not known. Complex culture media are used routinely 30 because they are easy to prepare, relatively cheap and able to support the growth of many bacteria. COMPOSITION OF CULTURE MEDIA 1. Water:- Water is the main solvent for most of the ingredients used in preparing culture media. Deionised or distilled water should preferably be used. 2. Peptone:- Peptone is the water soluble product of protein hydrolysis. Proteins in meat, milk and soya beans are hydrolysed by acids or by the enzymatic actions of enzymes such as pepsin, trypsin, and papain. All forms of peptone are heat stable. Peptone from animal protein is the source of nitrogen, the soya bean is the source of carbohydrates. Qualities of a good brand of peptone: i. Must have a neutral pH. ii. Must be light in colour. iii. Must contain large amount of tryptophan for indole production. 3. Meat Extracts:- Lab Lemco, a commercially available meat extract, is incorporated into culture media as a source of amino acids and other essential vitamins. 4. Yeast Extract:- It acts as a stimulant for bacterial growth in the culture media. This is amply done in thiosulphate citrate bile salt sucrose (TCBS) medium. 5. Mineral Salts:- Trace elements required for the enzymatic activities of bacterial growth are derived from some salts such as sulphates, phosphates, sodium chloride and some elements like magnesium, potassium, iron, calcium etc. 31 6. Carbohydrates:- These serve as sources of carbon and energy in the media. They may also be added to perform some special purposes. TYPES OF MEDIA 1. Liquid Media or Broth:- Bacterial can freely move about in liquid media. Growth is shown by turbidity, though some organisms show surface growth.They are used mainly for biochemical testing, blood culture, testing for motility and as enrichment media. The major disadvantage is that purity of growth cannot be guaranteed. 2. Solid Media:- Organisms grow and multiply at the site of inoculation forming visible colonies when grown on solid media. Colonial appearance and change in surrounding identifies growth. Liquid medium is made solid medium by incorporating a solidifying agent which does not change the nutritional content of the medium. The most widely used of such agent is agar. Agar is an inert carbohydrate extract obtained from a type of seaweed found in Japan, New Zealand, and California (USA). It is available in powder form. A good brand of agar must: i. gel at a concentration of 1% ii. melt at 98°C iii. set on cooling at 42°C iv. be easily soluble and maintain clarity in solution. A protein, derived from collagen of skin and bone called gelatin, is also used as solidifying agent. Gelatin gels at a concentration of 12-15% in nutrient broth; melts at 22°C and denatured by prolonged exposure to 100°C. Some bacteria are able to liquefy gelatin at 37°C, and this property is used to identify such bacteria. 32 Agar advantages over gelatin: i. It is solid at 37°C ii. Once solidified, it can be re-melted. iii. It is bacteriologically inert and so it is not attacked by most bacteria. 1. Basal Media:- Media that support the growth of most microorganisms that do not need special nutritional requirements.They contained basic nutrients. They are generally referred to as nutrient broths and may be in the following forms: 33

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