BASIC MICROBIOLOGY

INTRODUCTION
A microorganism or microbe is a microscopic organism which is a single celled or (unicellular), cell clusters, or having no cell at all (acellular). The study of microorganisms is called microbiology. Microbiology is not a new field of study. In 1676 Anton van Leeuwenhoek had used a simple, self-made microscopes to examine stagnant H2O, where he observed "wee animalcules" a microscopic organisms. There the study of microorganisms had began and gets developed after the discovery of Anton van Leeuwenhoek and now it had become a major branch in science. In a context, the micro-organisms are often a causative agent in a large number of infectious diseases whose presences were easily traced throughout and recorded in the history. Early victims of the microbial infections and epidemics may not have understood the microbiology behind these diseases, but they understood the consequences of the epidemics in their communities. This is a small section which includes the isolation, cultivation, characterization and enumeration of the microbial organisms like viruses, bacteria, fungi, protozoa, and some pathogenic microorganisms. This section will briefly introduces a few core microbiological concepts.

I. VIRUSES
A viruses are small infectious agent that can replicate only inside the living cells of the organisms. They were inert outside of their living host and they are only able to reproduce only within the host cell. They have not been included among any Kingdom or Domain of living organisms. They constitute their own independent group and are classified variously on the basis of their nucleic acid type or characteristic capsid symmetry, presence or absence of envelop, their host, and the diseases caused in animals and plants and hosts.Viruses infect all types of organisms like animals, plants, bacteria and archaea. The study of viruses is known as virology, which is a branch of microbiology. Viruses are usually found in almost every ecosystem on the Earth. Virus particles or the virions mainly consist of two or three parts: the genetic material made-up of either DNA or RNA, long molecules which carries the genetic information, a protein coat that protects these genes, and in some cases there will be an envelope of lipids that surrounds the protein coat when they were outside a cell. The shapes of viruses varies from the simple helical or icosahedral forms to more complex structures. The average virus is of about one one-hundredth the size of an average bacterium. Most viruses are too small to be seen directly with a light microscope. I.1 ISOLATION Viruses are obligate intracellular parasites that require living cells in order to replicate. To isolate a virus take a heterogeneous sample and add it to a graduated density gel, which has to spin in a centrifuge. The contents of the sample settle into separate piles, or bands , at different depths according to their characteristic densities. These bands are called density-purified samples. Because all microbiological entities have characteristic densities, we obtain density-purified samples that contain only certain viruses, and no other material. There is only one way to confirm this a photograph with an electron microscope that contains nothing but identical virus-looking objects. If the micrograph reveals contaminating entities, that

means the sample contained some material that had the same density as the virus-looking objects. In that case, we would have to add additional steps to the isolation process, ones that purify based on other characteristics--like size, or electrical affinity--until they could produce a sample that contained only the virus-looking objects. However, this usually is not necessary. Density purification typically produces true isolates of virus-looking objects. If the density, appearance, and size of these objects match those of a previously characterized virus, we can label the sample a virus isolate. If not, we must subject the sample to a battery of tests to prove that the virus-looking objects are viruses. Looking like a virus is just one feature of a virus. To be a virus, virus-looking objects must behave like a virus, and their constituents must relate to each other in special ways. This can be demonstrated by adding an isolate of virus-looking objects to a culture of suitable cells. If the isolate consists of viruses, they will infect the cells and multiply to numbers much greater than those present in the original isolate. Then we can confirm this by attempting to re-isolate the virus-looking objects from the culture after enough time has passed for substantial viral replication to have taken place. The new isolate should form at the same density as the original, and contain objects that look the same as those in the original sample. But the new isolate should consist of a much thicker band, indicating a larger number of viruses. We also have to examine the constituent molecules of the isolated virus. Among other things we have to confirm that the DNA or RNA codes for all the proteins. Now we can declare that the objects are indeed viruses, and that these viruses are characterized by a certain size, shape, and appearance, and consisting of a particular number of proteins and genetic molecules of certain molecular weights or base pair lengths. Cultured cells, eggs and laboratory animals may be used for virus isolation. Also the embroyonated eggs and laboratory animals are very useful for the isolation of some certain viruses, cell cultures are the sole system for the isolation of viruses in most of the laboratories. The development of methods for cultivating animal cells has been essential to the progress of animal virology. To prepare cell cultures, tissue fragments are first dissociated, usually with the aid of trypsin or collagenase. The cell suspension is then placed in a flat-bottomed glass or plastic container (petri dish, a flask, a bottle, test tube) together with a suitable liquid medium. After a variable lag, the cells will attach and spread on the bottom of the container and then starts get dividing, giving rise to a primary culture. Attachment to a solid support is essential for the growth of normal cells. Many viruses can be isolated as a result of their ability to form discrete visible zones (plaques) in layers of host cells. If a confluent layer of cells is inoculated with virus at a concentration so that only a small proportion of the cells is infected, then plaques may form where areas of cells are killed or altered by the virus infection. Each plaque is formed when infection spreads radially from an infected cell to surrounding cells. Plaques can be formed by many animal viruses in monolayers if the progeny virus in a discrete zone. Plaques can also be formed by phages in lawns of bacterial growth. It is generally assumed that a plaque is the result of the infection of a cell by a single virion. If this is the case then all virus produced from virus in the plaque should be a clone, in other words it should be genetically identical. This colne can be referred to as an isolate, and if it is distinct from all other isokates it can be referred to as a strain. This is analogous to the derivation of a bacterial strain from a colony on an agar plate. There is a possibility that a plaque might be derived from two or more visions so, to increase the probability that a genetically pure strain of virus has been obtained, material from a plaque can be inoculated onto further monolayers and virus can be derived form an individual plaque. The virus is said to have been plaque purified. When a virus is first isolated it may replicate poorly in cells in the laboratory, but after it has gone through a number of replication cycles it may replicate more efficiently. Each time the virus is sub cultured it is said to have been passaged. After a number of passages the virus may be genetically different to the original wild strain in which case it is now a laboratory strain. I.2 CULTIVATION

Virologists need to be able to produce the objects of their study, so a wide reange of procedures has been developed for cultivating viruses. Virus cultivation is also referred to as propagation or growth, all terms borrowed from horticulture. A few techniques have been developed for the cultivation of viruses in cell free systems, but in the vast mahority of cases it is necessary to supply the virus with appropriate cells in which it can replicate. Phages are supplied with bacterial cultures, plant viruses may be supplied with specially cultivated plants or with cultures of protoplasts, while animal viruses may be supplied with whole organisms, such as mice, eggs containing chick embryos or insect larvae. For the most part however animal viruses are grown in cultured animal cells. I.2.1 Animal cell culture Animal cell culture techniques are well developed and most of the cells used are from continuous cell lines derived from humans and other animal species. Continuous cell lines consist of cells that have been immortalized either in the laboratory or in the body, they can be subcultured indefinitely. The HeLa cell (an immortal cell line used in scientific research) line is a widely used continuous cell line that ws initiated in the miccle of the 20th century from cells taken from a cervical carcinoma. Sometimes it is difficult to find a cell line in which a virus can replicate. For many years no suitable cell culture system could be found for hepatitis C virus, but eventually a human hepatoma cell line was found to support replication of an isolate of the virus. Cells are cultured in media that provide nutrients. Most media are supplemented with animal serum which contains substances that promote the growth of many cell lines. Other important roles for the medium are the maintenance of optimum osmotic pressure and pH for the cells. Viruses can be cultivated in cells growing on the surface of a variety of plastic vessels with the cells bathed in the growth medium. Most cells grow on a plastic or glass surgace as a single layer of cells known as a monolayer. Alternatively the cells can be suspended in the medium, which is stirred to keep them in suspension. Contamination with bacteria and fungi can cause major problems in cell culture work, inorder to minimize these problems work is normally done in a sterile cabinet and most media contain antibiotics. Many cell types require a relatively high concentration of carbon dioxide which can be supplied in a special incubator. I.3 CHARACTERIZATION Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time consuming and unexpected or unknown viruses may escape detection. Therefore a random PCR (Polymerase Chain Reaction) approach was developed that allows sequence independent amplification of viral nucleic acids from virus isolation positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3' end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-

PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses. I.4 ENUMERATION There are essentially two different ways to enumerate the viruses: the indirect viable count and the direct total count. Viable count techniques are based on the lysis of a cultivable host while the direct counts involve counting the viruses in the environmental sample without the need for a cultivable host. The methods available for performing viable counts are the plaque-forming unit (PFU) and most probable number (MPN) techniques. Direct counts may be performed using TEM, epifluorescent microscopy (EFM), or flow cytometry (FCM). PFU and MNP techniques are both used to quantifu the number of particles released from a lysed host. This means that the host must be cultivable which is not the case for most of the microbes in the environment. The PFU method is used to determine the number of viruses that cause lysis of bacteria, cyanobacteria, or an algae that can grow on a solid medium. Initially the host has to be grown in a liquid medium and then it is mixed with a sample that contains the virus. The mixture of virus and host is then combined with molten agar or soft agar and poured on to a plate in which the agar content is high and contains a medium that the host can utilize. After incubation the plaques which are the areas in wich cells have been killed or transformed can be counted and number of infective virus particles in the original suspension estimated. The MNP trchnique is used to quantifu the number of infectious viral units for hosts unable to grow on solid media. Serial dilutions of the virus are made in replicates. The last dilution in which the host is lysed is considered to contain one infectious viral particle. The number of infectious particles can then be calculated based on the number of replicates in each dilution where lysis has occurred. The PFU and MNP methods can also be used to capture new environmental viral isolates and to purify viral isolates in culture in order to obtain a clonal virus. I.4.1 Direct counts Direct counts of viruses from the environment provide the total number of VLPs without the need for culturing and often give estimates that are 100-1000 times higher than PFU counts. For TEM, viruses are harvested directly on to elecetron microscopy grids by centrifugation or are concentrated by ultrafiltration and then transferred to grids. Thereafter the viruses are stained with an electron dense material for example uracil acetate. This method can also be used to describe the VLPs according to their morphology a trait used in viral taxonomy. Tn this way TEM can be used to give information that enables the tentative taxonomic affiliation of the viruses to be established. In addition TEM can be used to visualize infected cells and counting, examining, and sorting microscopic particles that are suspended in a stream of gluid. FCM allows populations to be analyzed based on the physical and chemical characteristics of single cells. This accurate high throughput method also allows the quantification of subpopulations of fluorescent stained viruses that differ in their characteristics of gluorescence and light scattering. The mahor advantages of FCM are its ability to analyze a large number of cells rapidly and the provision of data suitable for statistical analysis.

II. BACTERIA
Bacteria are large domain of prokaryotic microorganisms. They are of a few micrometres in length bacteria have a wide range of shapes, ranging from spheres to rods and spirals. Bacteria are present in most habitats on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals, they provides good examples of mutualism in the digestive tracts of humans, termites and cockroaches. There were typically 40 million

bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water, there were approximately five nonillion bacteria on Earth forming a biomass that exceeds that of all plants and animals. Antonie van Leeuwenhoek in 1676 observed a bacteria using a single-lens microscope of his own design. He called them "animalcules" and published his observations in a series of letters to the Royal Society. The name Bacterium was introducedr by Christian Gottfried Ehrenberg in 1828. In fact Bacterium was a genus that contained non-spore-forming rod-shaped bacteria, as opposed to Bacillus, a genus of spore-forming rod-shaped bacteria defined by Ehrenberg in 1835.Louis Pasteur demonstrated in 1859 that the fermentation process is caused by the growth of microorganisms, and that this growth is not due to spontaneous generation. Ehrlich had been awarded a 1908 Nobel Prize for his work on immunology and the use of stains to detect and identify bacteria with his work being the basis of the Gram stain and the Ziehl-Neelsen stain. A major step forward in the study of bacteria was the recognition in 1977 by Carl Woese that archaea have a separate line of evolutionary descent from bacteria. This new phylogenetic taxonomy was based on the sequencing of 16S ribosomal RNA, and divided prokaryotes into two evolutionary domains, as part of the three-domain system. II.1 ISOLATION Isolated colonies of bacteria are the result of a single bacterium which has replicated many times and eventually formed a visable mass of genetically identical bacteria. The colony's shape, texture and colour can somtimes be helpful in identifying the species of bacteria. For example the collonies of Serratia marrceccens are typically pink, moist looking, round and small on nutrient agar. II.1.1 Isolation techniques In nature microbial cultures are mixed Identification relies upon isolating individual colonies Testing requires pure cultures As a result isolation technique provides an essential microbiological tool.

II.1.2 Streak plate isolation principle An original inoculum containing a mixture of bacteria is spread into 4 quadrants on solid media. The goal is to reduce the number of bacteria in each subsequent quadrant. Colonies are masses of offspring from an individual cell therefore streaking attempts to separate individual cells. Discrete colonies form as the individual cells are separated and then multiply to form isolated colonies in the later quadrants. An isolated colony, one that is not touching any other colonies, is assumed to be a pure culture.

Can an isolated colony be considered pure?

This is generally assumed, however. some colonies are very slow growers and may be too small to see.

some colonies may be growing under another colony selective media may be preventing reproduction of some bacteria so they may be present but not visible condensed water, capsules, slime, all represent areas where individual contaminant cells hide out.

Some special considerations

Different species of microbes represent challenges. Encapsulated bacteria are sticky and dont separate well. Some species are motile and do not stay where you streak them spreading across the plate. Fungal spores easily contaminate cultures within a plate. Organisms can gain entrance to a Petri dish through water or the edges, or from the air currents while you are streaking.

Isolation Requires Aseptic Technique

Aseptic technique is the process of:

Preventing contamination of a culture with environmental microbes. Preventing contamination of yourself or the environment with the organism in the culture Remember everything is contaminated with a variety of environmental microbes. Remember microbes are invisible, you must see with your minds eye during these procedures.

II.2 CULTIVATION Protocols for use of cultivation of bacteria, use of general growth, enriched, selective and differential media, plate pouring, determination of temperature range for growth and determination of salt tolerance capacity of bacteria. In nature, bacteria exist as mixed populations. In the laboratory these populations must beseparated so that characteristics of individual species may be observed. A number of basictechniques are used in microbiology.

First, microorganisms must be removed from natural environments and cultured in thelaboratory. This requires artificial media and surfaces on which bacteria may grow. Thisalso requires knowledge of nutritional requirements and environmental requirements such as temperature of incubation and the requirement of oxygen. Second, bacteria of interest must be separated from all other bacteria in theenvironmental sample. This requires separation techniques that allow isolation of a pureculture of one type of bacteria.

Third, once a pure culture is achieved, no contaminating bacteria can be introduced fromthe environment. This requires that all media and lab supplies be sterile that is containno bacteria that may contaminate the culture of interest. Fourth, techniques are needed that facilitate working with pure cultures. This requiresaseptic technique and techniques of storage for pure cultures.

II.2.1 Culture media The fundamental function of bacterial media is to provide nutrients for the growth of microbes in the lab. Media designed for growth are defined below. 1. Nutrient material prepared for the growth of microorganisms. 2. Can be used to: Enrich the numbers of bacteria. Select for certain bacteria and suppress others. Differentiate among different kinds of bacteria.

II.2.2 Characters of media 1. Must contain basic nutrients (C, H, N, O, P, S) & growth factors. 2. Must have suitable moisture content, pH and salt concentration. 3. Must be sterile. II.2.3 The media may be: 1. Liquid medium: Components are dissolved in water and sterilized. The simple liquid media is nutrient broth. 2. Semisolid medium: A medium to which has been added a gelling agent. The most commonly used is agar. Gelatin. Silica gel used when a non-organic gelling agent is required. II.2.4 Types of culture media

1. Complex media: Often consist of plant or animal extracts, such as soybean, meal, milk protein ... etc. Include most routine laboratory media. e.g. tryptic soy broth. Basal media:

Aqueous extract of meat. e.g. nutrient broth/agar. Enriched media: Complex media to which additional nutrients are added (As blood, egg or serum) which are required by many pathogens. e.g. Blood agar. Contains mammalian blood (usually sheep or horse), typicallyat a concentration of 510%. Use: isolate pathogenic organisms & detect hemolytic activity.

e.g. Chocolate agar.

Type of blood agar where blood cells lysed by heating to 56 C. Growing fastidious respiratory bacteria "Haemophilus influenzae".

Differential media:

Contain indicators that react differently with different organisms. For example, producing colonies with different colors. Used in identifying specific organisms.

Selective media:

Contain agents that inhibit the growth of certain bacteria while permitting the growth of others.

e.g. tetrathionate bile/brilliant green.

Use: Isolate specific organisms from a large population of contaminants.

e.g. Mannitol Salt Agar (MSA), Selective for Staph. aureus. 2. Synthetic media: Chemically defined media contain pure chemical compounds. Ingredient must be of analytical quality. Water must be distilled. e.g. media used in bacterial genetics experiments.

II.3 CHARACTERIZATION Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentially to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3

signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates. II.4 ENUMERATION In the study of microbiology, there are numerous occasions when it is necessary to either estimate or determine the number of bacterial cells in a broth culture or liquid medium. Determination of cell numbers can be accomplished by a number of direct or indirect methods. The methods include standard plate counts, turbidimetric measurements, visual comparison of turbidity with a known standard, direct microscopic counts, cell mass determination, and measurement of cellular activity. In this exercise, you will compare three methods of bacterial enumeration: the standard plate count, turbidimetric measurement and direct microscopic counts. II.4.1 Standard Plate Count (Viable Counts) A viable cell is defined as a cell which is able to divide and form a population (or colony). A viable cell count is usually done by diluting the original sample, plating aliquots of the dilutions onto an appropriate culture medium, then incubating the plates under proper conditions so that colonies are formed. After incubation, the colonies are counted and, from a knowledge of the dilution used, the original number of viable cells can be calculated. For accurate determination of the total number of viable cells, it is critical that each colony comes from only one cell, so chains and clumps of cells must be broken apart. However, since one is never sure that all such groups have been broken apart, the total number of viable cells is usually reported as colony-forming units (CFUs) rather than cell numbers. This method of enumeration is relatively easy to perform and is much more sensitive than turbidimetric measurement. A major disadvantage, however, is the time necessary for dilutions, platings and incubations, as well as the time needed for media preparation. II.4.2 Turbidimetric Measurement A quick and efficient method of estimating the number of bacteria in a liquid medium is to measure the turbidity or cloudiness of a culture and translate this measurement into cell numbers. This method of enumeration is fast and is usually preferred when a large number of cultures are to be counted. Although measuring turbidity is much faster than the standard plate count, the measurements must be correlated initially with cell number. This is achieved by determining the turbidity of different concentrations of a given species of microorganism in a particular medium and then utilizing the standard plate count to determine the number of viable organisms per milliliter of sample. A standard curve can then be drawn (e.g., this lab protocol section), in which a specific turbidity or optical density reading is matched to a specific number of viable organisms. Subsequently, only turbidity needs to be measured. The number of viable organisms may be read directly from the standard curve, without necessitating time-consuming standard counts. Turbidity can be measured by an instrument such as a colorimeter or spectrophotometer. These instruments contain a light source and a light detector (photocell) separated by the sample compartment. Turbid solutions such as cell cultures interfere with light passage through the sample, so that less light hits the photocell than would if the cells were not there. Turbidimetric methods can be used as long as each individual cell blocks or intercepts light; as soon as the mass of cells becomes so large that some cells effectively shield other cells from the light, the measurement is no longer accurate. Before turbidimetric measurements can be made, the spectrophotometer must be adjusted to 100% transmittance (0% absorbance). This is done using a sample of uninoculated medium. Percent transmittance of various dilutions of the bacterial culture is then measured and the values converted to optical density, based on the formula: Absorbance (O.D.) = 2 - log % Transmittance. A wavelength of 420 nm is used when the solution is clear, 540 nm when the solution is light yellow, and 600-625 nm is used for yellow to brown solutions.

II.4.2 Direct Microscopic Count Petroff-Hausser counting chambers can be used as a direct method to determine the number of bacterial cells in a culture or liquid medium. In this procedure, the number of cells in a given volume of culture liquid is counted directly in 10-20 microscope fields. The average number of cells per field is calculated and the number of bacterial cells ml-1 of original sample can then be computed. A major advantage of direct counts is the speed at which results are obtained. However, since it is often not possible to distinguish living from dead cells, the direct microscopic count method is not very useful for determining the number of viable cells in a culture.

III. FUNGI
A fungus is a member of a large group of eukaryotic organisms which includes microorganisms such as yeasts and molds as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria. One major difference is that the fungal cells have cell walls that contain chitin, in otherhand the cell walls of plant which contains cellulose. The branch of biology which devoted to the study of fungi is known as mycology, which is often regarded as a branch of botany, even though genetic studies have shown that fungi are more closely related to the animals than to the plants. Most of the fungi are grow as hyphae, which are cylindrical, thread-like structures of about 210 m in diameter and up to several centimeters in length. Fungi have a worldwide distribution which grows in a wide range of habitats, including extreme environments such as deserts or areas with high salt concentrations or ionizing radiation as well as in the deep sea sediments. Some of them can survive the high UV rays and cosmic radiation occuring during the space travel. Most commonly they grow in terrestrial environments, though several species live partly or solely in aquatic habitats. III.1 ISOLATION