Microbiology is a branch of science dealing with the study of organisms which are too small to be seen clearly by unaided eye. Roughly the organisms with a diameter of 0.1mm or less, which can not be seen by the human eyes unaided, come under the domain of microorganisms. Thus, the laboratory work dealing with the microorganisms needs special care to avoid infection and infusion for maintaining good health. In fact all microorganisms should be treated as potential pathogens. Thus, for working with microorganisms aseptic techniques must be followed by students as well as by the laboratory assistants and teachers.

Microorganisms, being ubiquitous, are found in soil, air, water, food, sewage, on body surfaces as well as on degrading and decomposing substances. Their survival and continued growth mostly depend on an adequate supply of nutrients and favourable growth environments. The culture media (nutrients) consisting of chemicals support the growth of microorganisms in a given condition. They are devised in such a way that the organism should get all the nutritional requirements. These are usually prepared in laboratory by weighing and dispensing the individual ingredients. Generally, the common media contain both inorganic and organic nutrients; however, for the cultivation of large number of microorganisms, specialized media are required. Basically the culture media are of three types: (1) Natural, (2) Semi synthetic and (3) Synthetic.

Under natural habitats microorganisms usually grow in mixed populations containing several species. This is really a problem for microbiologists because a single type of microorganism can not be studied adequately in a mixed culture. In laboratories these populations can be separated into pure cultures. The pure cultures contain only one type of organism and are thus suitable for studying the cultural, morphological and biochemical characters of an individual species. In the present days there are several ways of developing pure cultures in laboratories. Some of which are as follows:

4.1 From Soil Soil contains varied group of microorganisms such as fungi, bacteria, actinomycetes, algae and protozoa. The type of nutrients available, moisture, temperature and pH of the soil determine the nature of microbial population in soil. Different laboratory techniques for isolation of microorganisms have been detailed in chapter-III. These are used in particular for the isolation of specific type of microorganisms. All the techniques adopted are just to segregate the pathogens/ microorganisms from the mixture of microbial population present in nature. (a) Isolation of Fungi Soil contains large number of fungi and constitutes a major place of microbial population. They can be isolated in pure form by following the methods given below:

Generally microorganisms are so small and colourless in their natural state that it becomes difficult to observe them clearly under microscope. Stains and dyes, which are colouring agents, are used by biologist for better understanding of the morphological and anatomical structures of the organisms under study. Stains are prepared with great care and specification to get satisfactory result, while dyes are not as exacting in their characteristics. Dyes are of two types, natural and synthetic. The natural dyes are used for histological purposes, whereas the synthetic dyes for bacterial stain. Usually dyes which are derivatives of cyclic compound benzene of benzole, are prepared from one or more substances found in coal-tar, and so are called coal-tar dyes. Chemically dyes are organic compounds containing both chromophore and auxochrome groups linked to benzene rings. Chromophore group provides the compounds the property of colour and thus benzene containing chromophores are called chromogens. It has no affinity to combine with fibres or tissue and as such not called dye. For being a dye, compound should contain an auxillary group which provides the compounds the properties of electrolytic dissociation. The dyes may be acidic, basic or neutral. The acidic dyes are eosin, acid fuchsine and picric acid whereas the basic dyes are safranin, methylene blue, crystal violet etc. The acidic dyes ionize to give the dye portion of molecule a negative electrical charge. They are salts of sodium, potassium, calcium, magnesium etc. The basic dyes ionize to give the dye portion of molecule a positive electrical charge and are salts of chlorides, sulphates or acetates.

The science dealing with the measurement of size of any microscopically visible microorganism is known as micrometry which is based on the principle of physics. Before measuring a cell, the diameter of the microscopic field must be established by means of optic devices, namely an ocular micrometer and a stage micrometer. a)Ocular micrometer : It is a circular disc that is placed in the eyepiece. The disc is graduated in to several small divisions marked from 0 to 100.It is placed inside the eyepiece of the microscope. The distance varies according to the objective of the microscope which can be easily determined by using a stage micrometer. b)Stage micrometer : It is a glass slide graduated in 1 mm. Again 1mm is divided into 10 large divisions and 100 small divisions (10 x 10 = 100 divisions). These divisions are equally placed therefore 1 small division is equal to 0.01 mm or 10 μm.

The term growth for bacteria and other microorganism usually refers to changes in the total population rather than an increase in size or mass of an individual organism. As the mechanism of conservation and dissipation of heat generated during metabolism lacks in bacteria, the enzyme system in bacteria gets directly influenced by temperature. The purpose of studying the bacterial growth curve is a) to become familiar with the population growth dynamics of bacterial culture and b) to plot a growth curve and determine the generation time of bacterial culture. A number of methods are now available for measuring bacterial growth. Some important ones are 1)By colony counting or cell counting 2)By weighing the cells 3)By cell activity (turbidity method)

Our exposure to wide variety of chemicals has increased markedly in the recent past. Most of these compounds are mutagenic as well as potential carcinogens. Emphasis is being laid to develop method for detection of environmental carcinogens so that their use can be avoided. Despite the fact that mammalian cell structure and human enzymatic pathways differ from those in bacteria, the chemical nature of DNA is common to all organisms; this permits the use of bacterial test systems for rapid detection of possible mutagens. The Ames test which was developed by Bruce Ames in 1970, at the University of California, U.S.A. is a mutational reversion assay employing several special strains of Salmonella typhimurium, each of which has a different mutation in the amino acid histidine biosynthesis operon. S. typhimurium, the indicator organism, used in this test is the mutant strain that cannot synthesize histidine (His-), lack the enzyme to repair DNA so that mutation shows up readily and has leaky cell wall that permit the rapid entry of chemicals. In addition, this S. typhimurium strain carries mutation enhanced plasmid, the combined effect of these characters increase the sensitivity of the organism to mutagenic treatments.

Antibiotics are produced by some true bacteria, actinomycetes and fungi that destroy or inhibit the growth of other microorganisms. Some antibiotics are synthesized or modified in the laboratory; however, their origins are always in living cells. 9.1 Assay of Antibiotics Soil serves as the major repository of microorganisms some of which produces antibiotics. Four groups of soil microorganisms viz; Streptomyces, Bacillus, Penicillium and Cephalosporium represent three microbial types, namely, actinomycetes, true bacteria and molds respectively. Following procedure may be applied to isolate antibiotic-producing microorganisms from soil:

10.1 Transformation of E. coli by electroporation Principle Preparation of electro-competent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid –log phase, chilled, centrifuged, washed extensively with ice cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high voltage electrical discharge. Alternatively, the cell suspension may be snap frozen and stored at-70oC for 6 months before electroporation, without loss of transforming efficiency. Since E.coli cells are small, they require very high strengths (12.5- 15KV cm-1) for electroporation as compared to those used to introduce DNA into eukaryotic cells. Electroporation is temperature dependent and is best carried out at 0-4°C. The highest efficiency of transformation is obtained when the concentration of input DNA is high (1-10 μg/ml) and when the length and intensity of the electrical pulse are such that only 30-50% of the cells survive in the procedure. Under these conditions, as many as 80% of the surviving cells may be transformed. Higher frequencies of transformation (colonies/ molecules input DNA) are obtained when the DNA concentration is low (?10pg/ml). The method outlined in this protocol is followed with most strains of E.coli and with plasmids <15kb in size. However, substantial variation in the efficiency of transformation between the strains of E. coli. has been reported. As in case with chemical transformation, linear plasmid DNAs introduced into E.coli by a pulsed electrical discharge transform very efficiently from 10 to 1000 fold but less efficiently than the corresponding closed circular DNA – perhaps because the exposed terminal of linear DNA are susceptible to attack by intracellular nucleases.

Identification of enteric bacilli is of prime importance for controlling intestinal infections. This can be achieved by preventing contamination of food and water by a group of bacteria belonging to family – Enterobacteriaceae which are found in the intestinal tract of human beings and lower mammals. The microorganisms of this family are short, gram negative, non spore forming bacilli. Such common enteric bacteria are a) pathogens such as species of Salmonella and Shigella b) occasional pathogens like species of Proteus and Klebsiella and c) normal intestinal flora like species of Escherichia and Enterobactor.

Viruses are smaller than bacteria and require living cell for their growth. They can be detected through the bacteria to which they parasitise. Viruses are associated with both eukaryotes as well as prokaryotes and contain either DNA or RNA; only few have both DNA and RNA. Viruses that infect bacteria are called bacteriophage. They consist of head or capsid and a tail. The head may be round, oval, polyhedral and is made up of proteins while tail is hollow and provides an exit for nucleic acid to enter in bacterial cell. The tail also helps in attachment to the cell wall surface of bacteria. After injection of the nucleic acid into bacteria they multiply and form large number of bacteriophage particles which cause lysis of the bacterial cell wall releasing many bacteriophages. Phages that cause lysis are known as lytic phages and that do not cause lysis are called temperate phage. The most common lytic phage is E.coli – a coliphage.

With increasing industrialization, water sources available for consumption as potable water are being adulterated with industrial, animal and human wastes. As a result water has become a formidable factor in disease transmission. Polluted water usually contains large amount of organic matter which forms nutritional base for growth and multiplication of microorganisms. Usually the water with non-pathogenic organisms, like autotrophs and saprophytic heterotrophs, are not of major concern, but presence of intestinal contaminants of fecal origin are of great concern. These pathogens are responsible for intestinal infection such as bacillary dysentery, typhoid fever, cholera and paratyphoid fever. The detection of all the intestinal pathogens on a routine basis would be a tedious and difficult task, however, it would be easy to demonstrate the presence of some of the nonpathogenic intestinal organisms like Escherichia coli and Streptococcus faecalis. These are always found in intestine but their detection confirms the contamination of water with fecal material. Such organisms are also called pollution indicator.

14.1 Culture from Urine Infection of urinary tract due to microorganisms affect patient of all age group and both sex., and they vary in severity from an unsuspected infection to a condition of severe systemic disease. Urine is a ordinarily excellent culture medium for the multiplication of common pathogen of urinary tract. Most bacteria found in the urine are due to contamination of skin flora during passage and they tend to increase greatly, exceeding 106/ml. When the bacterial count exceeds 105 per ml of urine it indicates significant bacteriuria and is indicative of a urinary tract infection. Infection of the urinary tract may be limited to a single tissue/ organ or they may spread upward and involve the entire urinary system. Escherichia coli is the most common urinary tract pathogen. Pseudomonas aeroginosa causes another important urinary tract infection, which is characterized by blue green pus. Cystitis (inflammation of urinary bladder), Pyelonephritis (inflammation of the kidney) are permanent diseases of urinary tract. Urinary tract infections are generally characterized by bacteriuria, bacteria in the urine. Quantitative microbiological assay of urine can identify the probable pathogen. For this purpose, calibrated loop- direct streak method and pour plate method are routinely used. Now-a-days commercially prepared special plastic device called the urine dip slide and diagnostic urine–culture called Bacturcult is being used for isolation and identification of microorganisms from genitourinary tract infections. The Bacturcult is a sterile disposable plastic tube coated with a special medium on its inner side for detection and identification of bacteriuria.

Fermentation The term fermentation is loosely used to denote any process in which microorganisms are involved, regards of whether or not the organism is growing in the presence of molecular oxygen. Thus, it can be defined as 'an ATP generating metabolic process in which organic compounds serve both as electron donor (becoming oxidized) and electron acceptor(becoming reduced). Energetically, fermentations are poor energy yielding process and products have lower energy content then the substrates. Since there is no net oxidation in fermentation, the number of molecules of C, H and O remain the same in products as in the substrate.

Appendices [Appendix – 1] Microbiological Culture Media A large number of culture media are mentioned in this manual to carryout different laboratory experiments. The composition of media as detailed in the manual are listed below alphabetically alongwith the page number of the text.