Isolation and Screening of Microorganisms PDF

**Isolation and Screening of Microorganisms** Isolation and screening of microorganisms are fundamental techniques in microbiology, biotechnology, and environmental science. These processes help identify and obtain specific microorganisms from complex mixtures, enabling the study of their character...

**Isolation and Screening of Microorganisms** Isolation and screening of microorganisms are fundamental techniques in microbiology, biotechnology, and environmental science. These processes help identify and obtain specific microorganisms from complex mixtures, enabling the study of their characteristics and potential applications. Isolation of suitable industrial microorganism from the environment is by · Collecting samples of free living microorganism from anthropogenic or natural habitats. These isolates are then screened for desirable traits. · The alternative method is by sampling from specific sites where microorganisms with desired characteristics are considered to be likely components of the natural Microflora.  1. (2) (3)  (4) (5)  (6) (7)  (8) Example of Isolation of amylase producing microbes ================================================== After sampling of the organism the next step is of enrichment. **Enrichment** Enrichment (refers to the process of increasing the concentration of specific microorganisms or biomolecules in a culture or sample by providing selective conditions (such as specific nutrients or environmental factors)) in a defined growth media and cultivation conditions are performed to encourage the growth of the organism with desired trait. This will increase the quantity of the desired organism prior to isolation and screening. **Part 1**: **Isolation of Microorganisms** 1\. **Definition** **Isolation**: The process of separating a microorganism from a mixed population to obtain a pure culture. 1\. **Importance of Isolation** 1.1 **Research Applications** Pure Cultures: Essential for studying the characteristics, physiology, and genetics of specific organisms without interference from others. Pathogen Identification: Critical in clinical microbiology for diagnosing infections and determining appropriate treatments. 1.2 **Industrial Applications** Biotechnology: Isolated microorganisms can be used in fermentation, enzyme production, and the development of biofuels. Food Industry: Isolation of specific strains for fermentation processes in yogurt, cheese, and alcoholic beverages. 1.3 **Environmental Applications** Bioremediation: Identifying and isolating microbes capable of degrading environmental pollutants. Microbial Ecology: Understanding biodiversity and interactions in various ecosystems. 3\. **Methods of Isolation** **Streak Plate Method**: Involves spreading a diluted microbial sample across the surface of an agar plate to obtain isolated colonies. **Procedure**: 1. Sterilize an inoculating loop in a flame. 2. Dip the loop into the microbial sample. 3. Streak the loop across one sector of an agar plate. 4. Sterilize the loop again and streak into a second sector, dragging some cells from the first sector. 5. Repeat for additional sectors. Outcome: Individual colonies develop from single cells on the agar surface.  **Spread Plate Method**: Diluted samples are spread uniformly on the agar surface to create colonies. **Procedure**: 1. Prepare a dilution of the microbial sample. 2. Pipette a measured volume onto the surface of an agar plate. 3. Use a sterile spreader to uniformly distribute the sample over the surface. Outcome: Well-isolated colonies can form on the surface of the medium. **Pour Plate Method**: Microbial samples are mixed with molten agar and poured into Petri dishes, allowing growth within the agar. **Procedure**: 1. Prepare a dilution of the sample. 2. Mix an aliquot of the dilution with molten agar (around 45-50°C). 3. Pour the mixture into a Petri dish and allow it to solidify. Outcome: Colonies develop both on the surface and within the agar.  **Selective Media**: Use of media that favor the growth of specific microorganisms while inhibiting others (e.g., the use of Mannitol Salt Agar for isolating Staphylococcus species). 4\. **Considerations for Isolation** 4.1 Aseptic Technique Essential to prevent contamination and ensure the purity of isolated cultures. Key practices include: Sterilizing tools (loops, pipettes). Working near a flame or in a sterile environment. 4.2 Choice of Medium Selection of the appropriate growth medium is critical for successful isolation: Use of nutrient-rich media for fast-growing organisms. Selective media to isolate specific types of microorganisms. 4.3 Environmental Conditions Consider factors such as temperature, pH, and oxygen levels, which can influence the growth of different microbes. 4.4 Colony Morphology Observing colony characteristics (size, shape, color, texture) can provide initial clues about the identity of isolated microorganisms. **Part 2: Screening of Microorganisms** Screening of microorganisms is the systematic process of evaluating microbial strains to identify those with desirable traits for specific applications, such as biotechnology, pharmaceuticals, agriculture, and environmental management. It relies on our ability to isolate and culture them in the laboratory, on the design of culture conditions that allow maximum expression of their genetic potential, and on the development of sensitive methods for the detection and characterization of desired properties. Key Steps in Screening Microorganisms Sample Collection Collect samples from diverse environments (soil, water, compost) to maximize microbial diversity. **Functional Screening** Focus on identifying strains with specific functions or traits: Enzyme Activity: Screen for production of industrially relevant enzymes (e.g., proteases, cellulases). Antibiotic Production: Test for antimicrobial compounds using bioassays against various pathogens. Metabolic Profiling: Assess metabolic pathways to determine potential for bioconversion or bioremediation. **Data Collection and Analysis** Use statistical software to analyze screening data, identifying strains with significant activities. Validate results through repeat testing and confirmatory assays. **Selection of Promising Strains** Based on screening results, select strains that exhibit desired characteristics for further study or applications. Documentation Maintain detailed records of all procedures, observations, and results to ensure reproducibility and traceability. **Fermentation Media** Media used in the cultivation of microorganisms must contain all elements in a form suitable for the synthesis of cell substance and for the production of metabolic products. In laboratory research with microorganisms, pure defined chemicals may be used in the production of culture media, but in industrial fermentations, complex, almost undefinable (in terms of composition) substrates are frequently used for economic reasons. Depending on the particular process, from 25 to 70% of the total cost of the fermentation may be due to the carbohydrate source. In many cases, media ingredients are byproducts of other industries and are extremely varied in composition. General media requirements include a carbon source, which in virtually all industrial fermentations provides both energy and carbon units for biosynthesis, and sources of nitrogen, phosphorus and sulphur. Other minor and trace elements must also be supplied, and some microorganisms require added vitamins, such as biotin and riboflavin. The main factors that affect the final choice of individual raw materials are as follows. 1\. Cost and availability. 2\. Ease of handling in solid or liquid forms, along with associated transport and storage costs. 3\. Sterilization requirements and any potential denaturation problems. 4\. Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration and foaming during fermentation and downstream processing stages. 5\. The concentration of target product attained, its rate of formation and yield per gram of substrate utilized. 6\. The levels and range of impurities, and the potential for generating further undesired products during the process. 7\. Overall health and safety implications. Typically, the main elemental formula of microbial cells is approximately C~4~H~7~O~2~N, which on the basis of dry weight is 48% C, 7% H, 32%O and 14% N. Ideally, a knowledge of the complete elemental composition of the specific industrial microorganism allows further media refinement. **Some of the Frequently used Substrates in Industrial Fermentation.** **I. Carbon sources** A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance. Carbohydrates are traditional carbon and energy sources for microbial fermentations, although other sources may be used, such as alcohols, alkanes and organic acids. Animal fats and plant oils may also be incorporated into some media, often as supplements to the main carbon source. **1. Molasses** A dark colored viscous syrup containing 50--60% (w/v) carbohydrates, primarily sucrose, with 2% (w/v) nitrogenous substances, along with some vitamins and minerals. It is produced during the sugar extraction process from sugarcane or sugar beets. When sugarcane or sugar beets is crushed to extract juice, the juice is boiled to produce sugar crystals. The remaining syrup is molasses. **2. Malt extract** Aqueous extracts of malted barley can be concentrated to form syrups that are particularly useful carbon sources for the cultivation of filamentous fungi, yeasts and actinomycetes. **3. Starch and Dextrins** These polysaccharides are not as readily utilized as monosaccharides and disaccharides, but can be directly metabolized by amylase-producing microorganisms, particularly filamentous fungi. **4. Sulphite Waste Liquor** Sugar containing wastes derived from the paper pulping industry are primarily used for the cultivation of yeasts. **5. Cellulose** Cellulose is predominantly found as lignocellulose (is a complex biomass material ) in plant cell walls, which is composed of three polymers: cellulose (is a complex carbohydrate made up of long chains of glucose molecules), hemicellulose (any of a class of substances which occur as constituents of the cell walls of plants and are polysaccharides of simpler structure than cellulose, e.g. the dry seeds contain a substance called hemicellulose) and lignin (Lignin is a complex organic polymer found in the cell walls of many plants, particularly in woody plants). Relatively few microorganisms can utilize it directly, as it is difficult to hydrolyse. **6. Whey** Whey is an aqueous byproduct of the dairy industry. This disaccharide was formerly used extensively in penicillin fermentations and it is still employed for producing ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid. **7. Fats and Oils** Hard animal fats that are mostly composed of glycerides of palmitic and stearic acids are rarely used in fermentations. However, plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soya) and occasionally fish oil, may be used as the primary or supplementary carbon source, especially in antibiotic production. **8. Alkanes and Alcohols** *n*-Alkanes of chain length C~10~--C~20~ are readily metabolized by certain microorganisms. Methanol has high percent carbon content and is relatively cheap, although only a limited number of organisms will metabolize it. **II. Nitrogen Sources** Most industrial microbes can utilize both inorganic and organic nitrogen sources. Inorganic nitrogen may be supplied as ammonium salts, often ammonium sulphate and di-ammonium hydrogen phosphate, or ammonia. **1. Corn Steep Liquor** Corn steep liquor is a byproduct of starch extraction from maize and its first use in fermentations was for penicillin production. **2. Yeast Extracts** Yeast extracts may be produced from waste baker's and brewer's yeast from wood and paper processing. **3. Peptones** Peptones are usually too expensive for large-scale industrial fermentations. They are prepared by acid or enzyme hydrolysis of high protein materials: meat, casein, gelatin, keratin, peanuts, soy meal, cotton seeds, etc. **4. Soya Bean Meal** Residues remaining after soya beans have been processed to extract the bulk of their oil are composed of 50% protein, 8% non-protein nitrogenous compounds, 30% carbohydrates and 1% oil. **III. Water** All fermentation processes, except solid-substrate fermentations, require vast quantities of water. In many cases it also provides trace mineral elements and it is important for ancillary equipment and cleaning. **IV. Minerals** Normally, sufficient quantities of cobalt, copper, iron, manganese, molybdenum, and zinc are present in the water supplies, and as impurities in other media ingredients. **V. Vitamins and growth factors** Many bacteria can synthesize all necessary vitamins from basic elements. For other bacteria, filamentous fungi and yeasts, they must be added as supplements to the fermentation medium. **VI. Precursors** Some fermentation must be supplemented with specific precursors, notably for secondary metabolite production. **e.g.:** Phenylacetic acid, Phenylacetamide, D-threonine, Anthranillic acid **VII. Inducer** Inducers are often necessary in fermentations of genetically modified microorganisms (GMMs). **VIII. Inhibitors** Inhibitors are used to redirect metabolism towards the target product and reduce formation of other metabolic intermediates. **IX. Antifoams** Antifoams are necessary to reduce foam formation during fermentation. **e.g.: Natural antifoams include** i\. plant oils, ii\. deodorized fish oil, iii\. mineral oils and iv\. tallow. The synthetic antifoams are mostly i\. silicon oils, ii\. poly alcohols and iii\. alkylated glycols.