Consequently, organisms do not have to expend valuable energy and materials to synthesize the "preformed" compounds supplied in the undefined medium. Microbiologists have also developed special media that can be used to select for the growth of certain organisms or differentiate between bacterial isolates based on physiological characteristics. These media are referred to as selective or differential media. Selective media contain at least one ingredient that inhibits the growth of unwanted organisms, but permits growth of the desired bacteria.
Exoenzymes can breakdown macromolecules ex: starch into smaller molecules that then can be taken through the cell's membrane and be used as individual glucose molecules for metabolic value. Mannitol provides the substrate for fermentation which makes it differential. Sodium chloride makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria.
Staphylocci thrive in this medium. Phenol red is the indicator. Used to isolate coliforms. Is selective for gram negative organisms because of the concentration of sugars and dyes. It is differential because acid produced by fermentation changes the color of dyes pH indicator.
The dyes are inhibitors and indicators. Is selective beacuse antibiotics colistin and nalidixic acid CNA interfere with DNA replication and affect membrane integrity of gram negative oranisms. Is differential because of the blood which allows hemolysis to occur. Sheep's blood provides the X factor heme and yeast provides B vitamins. Summarize the appropriate procedure for obtaining a pure culture from the growth of an isolated colony on a selective medium.
Remove several of what appears to be the same species using an inoculum loop. However, you cannot assume that there is only one species.
Therefore, you must do another streak of the inoculum to better isolate pure colonies. Identify the nutritional components, inhibitors, substrates, and indicators in the three media we tested. CNA: staphylococci, streptococci, enteroccocci are selected for Mannitol: Stahylococci spp. EMB: coliforms such as E. Distinguish the appearance of selected gram-negative bacterial colonies growing on EMB Agar, representing strong, moderate and non- lactose fermenters.
It contains two antibiotics, colistin and naladixic acid, which inhibit the growth of gram-negative bacteria, thus selecting for Gram-positive organisms. Provide the reasoning behind your choice and explain why this formulation is desirable.
Therefore, nalidixic acid inhibits DNA synthesis Gram negative bacteria are more sensitive to nalidixic acid than Gram positive bacteria. What did you use to establish what constituted "good growth? You do not know how the inhibitors may have affected the growth of the different organisms. So you can't compare growth on selective media. Even on TSA the growth of different organisms is likely different. One may grow really well and one less well thinner growth.
You might also inadvertently add different amounts of organism, but hopefully you are getting more consistent. If an organism can grow on both media, on which would you expect it grow better? There are defined media. Be sure to know the difference. I think all we used were undefined. What makes a medium undefined is the presence of an ingredient that is itself a mix of nutrients, growth factors and the like.
As Jane says, from one batch to another the amounts within those ingredients will change. They are really asking you to identify the undefined ingredients. Explain your answer. It would alter the medium's specificity because you could not select for the Staphylococcus.
Suppose a mistake is made in preparing a batch of MSA and the stating pH is 7. Sensitivity is getting false negatives. We are looking for a color change due to acid production. If you start at a higher pH, more alkaline, then it will take longer more acid to change the color.
With the diversity of microorganisms in the world, how can a single test such as MSA be used to confidently identify staphylococcus aures? Aures can ferminent mannitol, eliminating all other staph species. Suppose a mistake is made in preparing a batch of EMB and the stating pH is 7. We are looking for a color change due to acid production from lactose fermentation. Understand the necessity of performing a Gram stain and an oxidase test in conjunction with API20e testing.
The reasons are different. Perform the API20e study on an unknown organism. Know how to read the strip with the information sheet and determine the 7 or 9 digit code. In addition to the rate of assimilation of carbon sources, the nature of carbon source also affects the type and amount of the product. Methanol could be a very popular inexpensive carbon source for single-cell protein production, but being toxic to the cells even at low concentrations and low flash points, it can never be used in fermentation as media.
Hence, not only the cost even the dynamics of the carbon source must be considered whether it plays a role as a substrate in fermentation process or not. Like carbon, the selection of nitrogen source and its concentration in the media also play a crucial role in metabolite production.
Use of specific amino acids can increase the productivity in some cases and conversely, unsuitable amino acids may inhibit the synthesis of secondary metabolites Marwick et al.
Singh et al. On the contrary, the same amino acid showed inhibitory effect in the production of candicidin from Streptomyces griseus Sanchez and Demain, Nevertheless, it is confirmed that nitrogen molecules have inhibitory effect on the metabolite production in some cases, whereas, some enhancer effects of nitrogen have also been reported Table 2.
Phosphate is another basic component which is required for the production of phospholipids present in the microbial cell membranes, and for the production of nucleic acids. The amount of phosphate which must be added in the fermentation medium depends upon the composition of the broth and the need of the organism, as well as according to the nature of the desired product.
For instance, some cultures will not produce secondary metabolites in the presence of phosphate, e. Sanchez and Demain reported that various secondary metabolites' production such as, actinorhodin, cephalosporin, clavulanic acid, streptomycin, tetracycline, vancomycin etc.
In most cases, lower concentration of phosphate is required for the initiation of the metabolite antibiotic production and beyond a certain concentration it suppresses the secondary metabolism and ultimately inhibits the production of primary or secondary metabolite.
High phosphate concentration was reported to inhibit the production of teicoplanin, a glycopeptide antibiotic Rokem et al. From the above description it is clear that changes in carbon or nitrogen sources of the production medium or variation from their optimum required concentration, may affect the nature of the end product or its productivity.
Therefore, the production medium with all the required components in appropriate concentration is required for the production of desired metabolite at large scale.
In order to standardize the production medium, the concept of medium optimization has emerged. Medium optimization studies are usually carried out in the chemical, food, and pharmaceutical industries, with respect to increase the yield and activity of the desired product. Currently, there is a very little knowledge available about the role of factors, their levels in controlling the metabolite e.
In order to enhance the productivity of the metabolites for e. The quantity and quality of nutrients available and the ability to assimilate successfully are the major determinants of microbial nature and its metabolic activity. Hence, during the medium optimization it must be considered that a minimal growth requirement of the microorganism must be fulfilled for obtaining maximum production of metabolite s. As the fermentation process progresses into lower-value, higher-volume chemicals, it becomes necessary to maximize the efficiency and minimize the production cost and waste by-products to compete effectively against the traditional methods.
During the medium designing and optimization, there are various strategies available which are frequently used to improve the efficiency of the production medium.
Figure 2 is a schematic representation of various techniques used in the medium optimization. Figure 2. Schematic representation of various techniques used in optimization studies. In the classical medium optimization technique, one-factor-at-a-time OFAT experiments, only one factor or variable is varied at a time while keeping other variables constant. The concentrations of the selected medium components were then changed over a desired range. Because of its ease and convenience, the OFAT has been the most preferred choice among the researchers for designing the medium composition and used in the initial stages in diverse fields Gonzalez et al.
This methodology is still in use even today, during the initial stages of medium formulation for the production of new metabolite or known compound from new source. Based upon the approach applied, OFAT is further sub-grouped into:. In this type of experiment, all the medium components are removed from the production medium one-by-one, and after proper incubation period, their effects on the production of secondary metabolite or the product of interest is observed in terms of suitable parameters.
Supplementation experiments are generally performed to evaluate the effects of various carbon and nitrogen supplements on metabolite production. Similarly, glycerol and peptone was found as a most suitable carbon and nitrogen sources for the production of antifungal and antibacterial metabolites from Streptomyces rimosus under submerged fermentation condition Singh and Rai, In addition to chemical and biological variables, several researchers used OFAT experiments to standardize the physical parameters such as pH, temperature, agitation and aeration requirements of the fermentation process Niwas et al.
Like any other technique, OFAT method of medium optimization has its own advantages and disadvantages. Vaidya et al. In this methodology, sometimes the optimum point may be missed completely, thus it requires a large number of experiments to determine the optimum level, which becomes laborious, time consuming, and uneconomical most of the time Gupte and Kulkarni, Nevertheless, OFAT technique can be a best screening tool when nothing about the media is known because of its ease and convenience.
The use of statistical method, i. Fisher proposed a basic theory of experimental design which shows that changing more than one component in the medium at a time can be more efficient over changing only one-factor-at-a-time Fisher, DOE is a series of experiments which are strategically planned and executed to obtain a larger amount of information about the effect of more than one parameter at a time on the output, i.
Most DOE procedures allow the preliminary screening of 2—10 medium factors in a limited number of experiments. In this method, several medium factors or components are compared simultaneously and the effects are observed and ranked based on the results. Once the response variables are determined and ranked, statistical performance parameters are generated from the subsequent analysis. The interaction between the factors can be estimated systematically in DOE Haaland, After getting the basic idea about the fermentation production process from the literature or from the classical experiments, designing of the experiments are more effective to determine the impact of two or more factors on a response than OFAT.
With the advancement of statistical techniques, medium optimization has found new dimensions, as these techniques improve the efficiency of the process, reduces the time required in the process and labor cost etc. Being, biological in nature, the microbial processes contain relatively large amount of natural variations. The networks associated with the microbial reactions are complex, and several factors affect different parts of the networks.
Rational experimental design and statistical evaluation of the results increase the knowledge about the reliability of the information obtained during the experiments. By using experimental design, the amount of experiments required to obtain a for reliable process optimization can be reduced Elibol, For example, during the study of rate of methane and carbon dioxide gas production from Methanosarcina barkeri bacterium growing on methanol, medium optimized through experimental design was found to give 1.
Given this, it is widely accepted that in order to have an improved media by employing the experimental design approach; we require both a design as well as the optimization technique.
The DOE defines the medium variants to be tested such as, number of replicates and the arrangement of the tests in a harmonized pattern etc. Based upon the obtained experimental data, optimization technique is used to predict a mathematical model and improve the medium composition.
Experimental design is a study plan to get defined goals or objectives. Modern statistical techniques provide us powerful tools for the evaluation of the components or variables effects based on the experimental results. Hence, the experiments must be planned properly with the sufficient sample size to obtain adequate data which is essential to answer the objective as efficiently as possible. Such types of techniques are commonly called as DOE.
In a full factorial design, all the combinations of the factors, e. In contrast, in a partial factorial analysis, only few well reported combinations are picked-up and tested. Usually, partial factorial analysis is done, when the full factorial design is not possible and some or little knowledge about the interactions of the medium components for a particular strain is available. All the components present in the medium do not contribute in the metabolite production. Hence, it is utmost important that the non-contributing factors, should be eliminated from the study as early as possible.
In , R. Plackett and J. Plakett Burman Design PBD , is a two-level design, which is very useful for economically detecting the main effects and assuming all the other interactions are negligible when comparing the some important major effects, i. An example of PBD has been given in Table 3. In this design, there are two types of variables, i. Each variable is represented in two levels, i. Each horizontal row represents a trial and each vertical column represents the either of two levels high or low of each independent and dummy variables in all the trials.
Usually, the classical experiments help in the selection of independent and dummy variables. The effect of each variable is determined by the following equation:.
PBD is an authentic method to evaluate the relative importance of various variables or medium components for specific output, for e. Use of PBD decreases the total number of experiments, tremendously Adinarayana and Ellaiah, , as the interaction effects of the variables not consider and only those variables that actually affect the production of desired metabolite are screened.
Likewise, during the initial studies of medium optimization for antibacterial metabolite production from Streptomyces sp, we have used PBD to identify the most effective components in the media and reported soybean meal, calcium carbonate, and potassium phosphate can significantly increase the antibiotic production Banga et al.
Even though PBD is a good method to identify the important components, but there are some drawbacks associated with its efficiency. PBD should be used only when the factors have no interactions, or have only additive effects on the output, otherwise the results of the factor analyzed will be enhanced or masked by other factors as it fails to interpret if the effect of one factor depends on another factor.
Nevertheless, in the DOE, PBD is a starting point and one should use it to determine the follow-up experimentation list. In order to overcome the problems associated with the PBD method, Dr. Taguchi technique offers three-stages of off-line quality control features, like system strategy, parameter designing and tolerance design phase Pignatiello, The system strategy helps in finding the experimental levels of design features while parameter designing shows the factor level and provides the paramount effects of the process, whereas the tolerance design phase improves the elemental tolerance that considerably effect the product formation Muhammad et al.
This design helps in determining the factors affecting the product significantly with a minimum number of experiments, thus saving time and resources. At the end of the experiment, the factors are assigned to appropriate columns. Unlike PBD, it analyses the main effect and two factor interactions. However, higher order interactions are assumed as negligible. Noise, i. Uncontrolled variables noises generally cause the loss of the quality. This effect of noise can be removed by employing the Taguchi methodology Aggarwal and Singh, The Taguchi method becomes very helpful in measuring the quality by the deviation of a functional characteristic from its target value.
The Taguchi approach is a fully developed method having advantage of saving experimental time, product cost and improving the quality as well which is a basic requirement for the optimization of any fermentation process Chanin et al. Recently, Muhammad et al. As PBD considers only main effects and ignores the interactions among the factors, therefore, a new design is required. Nowadays it is widely used in response surface methodology RSM for building a second order quadratic model for the response variable without using a complete three-level factorial experiment.
This point is often replicated in order to improve the precision of the experiment; star points , experimental runs identical to the center points except for one factor, which will take on values both below and above the median of the two factorial levels. The numbers of star points are double the number of factors used in the design.
Table 4. Table 5. A summary of designs and optimization techniques used for the improvement of production media in some of the published studies. The Box-Behnken design is an alternate to CCD, it is independent of quadratic design, which does not contain an embedded factorial or fractional factorial design Ferreira et al. In this design, the treatment combinations are at the mid-points of the edges of the process space and at the center.
These designs are rotatable or near rotatable and require 3 levels of each factor. The designs have limited capability for orthogonal blocking compared to the central composite designs. Through site-directed mutagenesis or customized primers, individual mutations in DNA can be made. By changing the amino acids transcribed from DNA through individual mutations, the importance of those amino acids with respect to gene function can be analyzed.
However, this process can be difficult, particularly when genes act in concert with varying expression with respect to gene activity. The length of time it takes to run a successful PCR and perform other techniques before additional studies can be done protein expression, isolation, and purification, for example , makes biochemical research time-consuming and difficult.
However, PCR, coupled with other biochemical techniques, allows us to analyze the very core of organisms and the processes by which they function. Common PCR protocols in labs today include knockout genotyping, fluorescence genotyping and mutant genotyping. Researchers can use PCR as a method of searching for genes by using primers that flank the target sequence of the gene along with all other necessary components for PCR.
If the gene is present, the primers will bind and amplify the DNA, giving a band of amplified DNA on the agarose gel that will be run. If the gene is not present, the primers will not anneal and no amplification will occur. The ability to identify specific genes to specific organisms has increased the use of PCR and has allowed it to be more specific and eliminate the possibility of cross contaminants.
The identification of specific genes to specific organisms has important medical diagnostic value. PCR is a reliable method to detect the presence of unwanted genetic materials, such as infections and bacteria in the clinical setting. It can even allow identification of an infectious agent without culturing. PCR can reveal the presence of HIV in people who have not mounted an immune response to this pathogen, which may otherwise be missed with an antibody assay.
Additionally, PCR is used for identifying bacterial species, such as Mycobacterium tuberculosis in tissue specimens. With the use of PCR, as few as 10 bacilli per million human cells can be readily detected. The bacilli are identified by using Mycobacterium tuberculosis specific genes.
Privacy Policy. Skip to main content. Culturing Microorganisms. Search for:. Microbial Culture Methods. Enrichment and Isolation Understanding the nutritional requirements of bacteria can aid their enrichment and isolation. Learning Objectives List the growth phases of microrganisms and the different types of growth media available to culture them. Key Takeaways Key Points The most common growth media for microorganisms are nutrient broths and agar plates.
Open cultures allow for a replenishment of nutrients and a reduction of waste buildup in the media. Differential media or indicator media distinguish one microorganism type from another growing on the same media. Key Terms closed culture : A closed culture has no additional nutrients added to the system, and waste products are not removed.
Cultures in a closed system will follow a predicted growth curve. Enriched media : Contains nutrients required to support the growth of a wide variety of organisms. Pure Culture A pure culture is a population of cells or multicellular organisms growing in the absence of other species or types. Learning Objectives Describe how pure microbial cultures can be grown in agar-based growth medium. Key Takeaways Key Points A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.
The most common form of microbial cultures are liquid or solid agar. Key Terms agar : A gelatinous material obtained from the marine algae, used as a bacterial culture medium, in electrophoresis and as a food additive. Preserving Bacterial Cultures Bacteria can be stored for months or years if they are stored at C and in a high percentage of glycerol.
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