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What is the purpose of pH adjustment & autoclave during medium preparation?

The optimal pH is required for microbial growth  in the culture media. Different microorganisms prefer different pH of culture media that suitable for the growth. Mostly microogranism prefer to grow in the culture media of pH 6.5 -7.0. meanwhile, most animal cells grow in pH 7.2-7.4. To maintain the best optimal pH of media, the buffer is added as some microorganisms tend to release acidic products. The acidic product will interfere the growth of microorganisms. the adjustment of the pH of the dehydrated media will fix the final specific pH of the finished culture media conforms the label specification when the medium cooled to 25°C. Moreover, pH adjustment is crucial for the nutrients absorption. Normally, optimized environment for minor and major minerals and also agar activity is pH 5.8. In this condition, nutrients will be absorbed as best as possible.

Autoclave is a machine for sterilization the lab apparatus, waste and media at high temperature in specific period of time. The autoclaving of most culture media for final sterilization at 121°C for 15 minutes. During preparing the media, the media contaminate with surrounding microbial. By autoclaving the media, it will sterilize it and kill microbial. It is crucial to autoclave at specific temperature and time because nutrients in the media will denature. Some media also do no need to autoclave, for example during fermentation of Salmonella spp, Xylose Lysine Deoxycholate agar (XLD) will be used and this selective growth medium do not need to autoclave it.

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What is synthetic medium and how it differs from natural medium?

👉 Synthetic medium is a chemically defined medium made from purified materials whose exact composition can be determined. A chemically defined medium provides the purest and most consistent cell growth environment since it is free of animal-derived components.

♦️ It is an artificially prepared composition of chemically known organic and inorganic components. Synthetic media comprised of serum-containing media, serum free media, chemically defined media, protein free, media, and balanced salt solution (BSS). The advantages of serum media is that the media are supplemented with growth factor and hormones that can support cell attachment.

👉 While natural medium is composed of natural biological components which are chemically undefined and comprises of natural biological fluids such as tissue extracts, plasma clots and serum.

♦️ usually used in animal tissue cultures and has poor reproducibility due to lack of knowledge on its exact composition.

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What is the purpose of isolation of microorganisms from their natural environment?

To investigate the specific role of a specific microorganism in its environment, it must be isolated in pure culture. Thus, the purpose of the isolation is to cultivate microorganisms outside of their natural environment so that it can identify more about the morphology of the enzyme function. The enzymes produced are useful so that the chemical reaction aiming the microorganisms can take place and ensure their living.

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What is medium formulation and its importance ?

The medium formulation adds improvement of adequate components amount needed by the cells in a bioreactor for bioprocess development to satisfy all the nutritional and chemical environment requirements of the cells and fulfil the technical objectives of the bioprocess development. The importance of medium formulation is to provide a production medium that allows optimal growth of the microorganism for the target product such as biomass or primary metabolites and provides an initial period of cell growth, followed by conditions optimised for secondary metabolite production. At this point, the supply of one or more nutrients may be limited, and rapid growth ceases. Therefore, fermenter control, nutrients availability and fermentation efficiency are important factors in medium formulation for the effectiveness of the microbe activities.

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The use of biological, biochemical, molecular, or chemical methods for the detection, identification, or enumeration of microorganisms in a material. One of the importance’s of microbiological is rDNA (Recombinant DNA) technique involving E.coli bacteria was adopted to produce large amounts of human insulin for the individuals with diabetes. On top of that, Microbiological water analysis is a method of analyzing water to estimate the numbers of bacteria present and to allow for the recovery of microorganisms to identify them. The method of examination is the plate count. Besides these, Food and water safety are important to our health. Food testing assists manufacturers in avoiding the distribution of contaminated food or beverages to consumers. The FDA routinely tests for harmful germs such as E.coli, salmonella, and listeria. Food safety standards violations may be costly for food manufacturers, restaurants, hotels, and school cafeterias. Any food-selling establishment is subject to health inspections and failing such an examination has significant financial repercussions.

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Medium optimisation is an essential process done before media preparation. It includes independent factors like pH, temperature or nutrient being studied for maximum growth of desired organism.

Different combinations and sequences need to be investigated to produce biomass with the best physiological state, constituted for product formation. Other than that, undesired materials production can be minimised while target product can produce maximum yield. Next, it is also important to ensure products are readily available to fulfil the demand from the market.

However, when considering the biomass growth phase in isolation, it must be recognised that efficiently grown biomass produced by optimised high productivity growth phase is not necessarily best suited for its ultimate purpose.

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Glucose is a fundamental six-carbon sugar that is one of the most important compounds in biochemicals. It is the most prevalent monosaccharide on the globe, and its size is equivalent to heavier amino acids. Glucose is the major energy resource in cell culture because it may penetrate the plasma membrane via facilitated diffusion and transporting proteins. The energy contained in the high-energy phosphate bonds of adenosine triphosphate (ATP) is used to manufacture it in an interconnected and unrestricted manner. ATP is significant since it transfers power inside the cell and is frequently referred to as the cell's energy fuel. ATP is utilized by all known living species and is necessary in signaling pathways for intracellular signaling, in addition to acting as an energy source. Furthermore, glucose supplies the reducing power required to counteract reactive species (oxidative stress) that develop in vivo and in vitro. Therefore, optimal glucose concentrations in microbiological medium are also critical for healthy cell development and effective product yield.

Peptone is primarily used in culture mediums to provide nitrogen which is a protein-amino acid combination made from the breakdown of natural sources such as animal tissues, milk, and plants. The amino acids and other simpler nitrogenous substances found in peptone can be used by most organisms. Generally, peptones are frequently used as serum-free culture media additives. Peptones' growth-enhancing action in batch cultures may have a bidirectional impact, while it may encourage fast cell development in the early days of culture. In many circumstances, the complex infusion media can be substituted with simpler media made with the appropriate peptones instead of the meat infusions. As peptone is known as a protein and amino acid combination derived from natural sources such as animal tissues, milk, and plants, the role of it in nutrition agar is to supply a protein source for microorganisms to feed on.

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Media that also known as bacterial culture media is a growth medium to grow bacteria. It simply functioning as food to microorganism. It contains everything bacteria require to grow outside the body and in the laboratory. Different types of media are used to grow various types of cells. Cell culture media, which employ cell types originating from plants or animals, and microbiological culture media, which are used to grow microorganisms such as bacteria or fungus, are the two primary types of growth media. Culture medium is a rich source of carbon and nitrogen. It also contains small components such as inorganic salt, vitamins, growth factors, and a buffer. Colloidal suspensions are frequently formed by bacteria cultivated in liquid cultures.

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To obtain an effective medium, the medium must contain known ingredients important for the microbial growth, such as a defined carbon and nitrogen source, trace elements as well as  important vitamins required by the microbe. Choosing a medium with ingredients which fit the microbial needs is vital to ensure the effectiveness of the medium
To know more about the effectiveness of the medium fed to the bacteria, the growth of the cells can be calculated since the relationship between them determine how the media work or not. Two major approaches are used to measure cell growth. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells.

The direct methods contain techniques that can help in measuring the microbial growth such as, the measurement of dry cell weight by using either membrane separation or centrifugation, the counting of total cell number by using a haemocytometer slides and the counting of viable cell number by multiplying the number of colonies present with the dilution factor. Other than that, microbial growth in medium can also be assessed by determining the turbidity of the media culture. For microorganisms with high sedimentation coefficients, the microbial growth can be assessed by using the packed cell volume technique.

For indirect methods, it includes a few types of measurement which are through the measurement of cellular components such as the microbial DNA, RNA, and protein, as well as the measurement of the biomass concentration of substrate consumption or product formation such as ethanol, acetic, and lactic acid. Apart from that, indirect methods also involve observing the changes in viscosity of the media.