What Is Growth Medium?

Microbiological growth medium - also called Culture Medium, or Nutrient Broth, solution freed of all microorganisms by sterilization (usually in an autoclave, where it undergoes heating under pressure for a specific time) and containing the substances required for the growth of microorganisms such as bacteria, protozoans, algae, and fungi. The medium may be solidified by the addition of agar.

Growth medium - a source of nutrients in which a microorganism is placed to permit its growth, cause it to produce substances, or observe its activity under defined conditions; also called culture medium or growth medium . The medium is usually a solution of nutrients in water, or a similar solution solidified with gelatin or agar.

Growth medium (pl. growth media) - a synthetic medium which is filled with nutrients necessary to the growth of microorganisms or cells being cultured in the lab.

The majority of the cell is made of carbon, oxygen, hydrogen, nitrogen and phosphorous and these elements are necessary for forming membranes, proteins, nucleic acids and the other structures of the cell as described in Chapter 2. These nutrients are required in large amounts, consisting of more than 1% of the cells dry weight and are termed macronutrients (Table 6-1). A second tier of elements that are required in lower concentrations are calcium, potassium, magnesium, sulfur, iron and manganese and these are called micronutrients (Table 6-1). They make up between 0.1-1.0% of the cells dry weight micronutrients serve as less common parts of cellular structures or as necessary components for proteins. Although in smaller amounts, they too are absolutely required for the functioning of a living cell. Trace elements (Table 6-2) are compounds necessary in such small amounts that they are difficult to assign a requirement for. These are required in very small amounts (less than 0.1%) and their actual amount is difficult to measure. Growth factors (Table 6-3) are premade organic molecules that are necessary for growth, yet the microbe is unable to synthesize itself. Examples include vitamins, amino acids and nucleotides. These nutrients are the chemical substances used by the organism for biosynthesis and energy generation. Bacteria must acquire all these elements from their environment for growth.

We list here a few examples to give you an idea of the utility of nutritional information.

Bacterial nutrition began with studies directed at testing spontaneous generation. In these instances very simple medium containing chicken or beef broth were used for experimentation. Without these media it would have been impossible to test the validity of the hypothesis. Further work on bacteria led to the isolation of microbes and pure culture techniques. Again, without the ability to grow bacteria under controlled conditions, these experiments would be impossible.

Microorganisms are so small that in most cases large populations of them are needed to perform any experiment. To get these populations the scientist has to be able to grow large quantities of them and to do that, you have to know what they need nutritionally.

Knowing the nutritional requirements of a pathogen not only allows you to culture it in the laboratory for further study, but it can sometimes give you insight into its pathology. Pseudomonas aeruginosa, an important pathogen in burn patients and those with cystic fibrosis, will only express its virulence genes in medium low in iron. The low iron concentration serves as a signal to the microbe that it is inside a host. Understanding this facet of its pathology may help in the treatment of patients.

When trying to grow antibiotic-producing strains in industry, knowledge of the nutritional requirements of the microbe can enhance its growth and creating higher yields of the desired antibiotic. Knowledge of the nutritional needs of a microbe can also save money as just the right amount of each nutrient can be added to the growth medium.

As a final example, the ability of microbes to grow on different media can help to elucidate their metabolic capabilities. This is useful in identifying bacteria and can help us to understand their roles in the environment.

From these examples it should be clear that knowing the nutritional requirements of a microbe are extremely useful, but now the question becomes what are these requirements? One can get a reasonable estimate of this by examining the composition of the average bacterial cell. Bacterial cells contain a cell wall, membranes, ribosomes, proteins, RNA and DNA. If you break these structures down you end up with lipids, amino acids, sugars, nucleic acids and a few other compounds. By examining the chemical formulas of these compounds, it is possible to generate a reasonable estimate of the nutritional requirements of the cell. In this chapter we will look at what makes up the average bacterial cell, and give an insight into those requirements. We will also examine how these things are provided to a bacterium in the laboratory by examining the formulation and synthesis of culture medium.

Most often trace elements serve as cofactors in just a few enzymes, with only a small number of atoms being necessary per cell. Very careful experiments have shown requirements in many organisms for small amounts of zinc, copper, molybdenum and cobalt. These elements are normally in high enough concentrations as contaminants in water and the other ingredients added to the medium and therefore need not be added explicitly. Table 6-2 lists some trace elements and gives examples of their functions in bacterial cells. The assortment of micronutrients and trace elements required by an organism is dependent upon its metabolic life style and will vary from species to species.

Some species can use just a few simple compounds as their sources for all macronutrients, micronutrients and trace elements and from them synthesize all the complex molecules they need for growth. Other microbes do not have that broad a metabolic repertoire and their growth depends upon certain organic molecules that they are unable to synthesize. They therefore have to uptake these compounds from their environment. Some examples of growth factors include

Vitamins which are non-protein components of many enzymes

Amino acids for protein synthesis

Nucleic acids for DNA and RNA synthesis

The requirement or lack thereof for growth factors reflect the synthetic capability of a microbe and this in turn reflects the environment that they live in. If a compound necessary for growth is always present in the environment that a microbe lives in, it is very likely that the ability to synthesize that compound will be lost, since it can always be taken up from the outside. Bacteria vary greatly with regard to their growth requirements. Some bacteria, such as Anabaena variabilis, demand very little from their environment. They are able to grow in a medium consisting of a few salts, an atmosphere with CO2, N2 and light. Anabaena cells live in aquatic environments where nutrients can be scarce and sunshine is prevalent. They often have to be capable of synthesizing everything they need and generating energy from the sun. Others have very high growth requirements needing a number of vitamins, all twenty amino acids and all nucleotides. For example, Streptococcus is a resident of the mucous membranes and the intestines, with the host routinely providing many of its nutrients. It has therefore lost the ability to synthesize many compounds.

All living organisms need a source of carbon, energy and electrons to carry out their metabolic activities and bacteria have been classified based on the methods used to obtain these three important components. Carbon is one of the most important elements for living systems as it forms the backbone of all biological macromolecules. Microbes that obtain their carbon from carbon dioxide are termed autotrophs while those that rely on pre-made organic compounds in the environment are heterotrophs. Energy for carrying out cellular reactions comes either from the conversion of light energy or by the oxidation of chemicals. Phototrophic organisms utilize light as a source of energy, while chemotrophs obtain energy by the oxidation of either inorganic or organic compounds. For the cell to carry out many of its metabolic reactions a source of electrons is also necessary and again microbes can be classified by the methods they use to obtain them. Microbes that obtain their electrons from organic compounds are termed organotrophs while those that obtain electrons from inorganic compounds are called lithotrophs (literally rock eaters).

By combining the above sets of terms it is possible to describe the nutritional modes of an organisms. For example a chemoorganotrophic heterotroph obtains its energy from chemicals, its electrons from organic compounds and its carbon from premade organic compounds. In many cases a single organic compound can serve all these roles. A second example is a photoautotrophic lithotroph that will obtain energy from the sun, carbon from CO2, and electrons from inorganic compounds.

This method of classifying microbes, while useful in understanding the nutritional modes of microorganisms, is an artificial construct. There are microorganisms capable of more than one mode of growth and they will in fact cross categories depending upon environmental conditions. For example Rhodobacter sphaeroides is capable of growth on CO2, light and obtaining electrons from H2S, thus growing as a photoautotrophic lithotroph. However, the microbe is also capable of growing aerobically in the dark with an organic compound as its carbon source. In this case living as a chemoheterotrophic organotroph. Some microbes are even capable of growth using several modes at once. Beggiatoa species are capable of chemolithoautotropic growth on reduced sulfur compounds and CO2. However, in the presence of limiting amounts of reduced sulfur compounds they will supplement their metabolism by utilizing acetate (an organic compound) at the same time!

In this section we present some specific examples of microbes, their physiology and their habitats and relate this information to their nutritional classification. By examining examples of different types of microbes it will hopefully clarify what each type of nutritional category means. In addition this will begin to introduce you to the diversity of microbes living in the world. Later chapters will cover microbial diversity in much greater detail.

Interestingly many of the most studied microorganisms are chemoheterotrophs. One may get the mistaken sense that this means that chemoheterotrophs are the most common microbes in the environment and this is probably pretty far from the truth. Autotrophy and photosynthesis are very common metabolic activities and dominate a large number of habitats from the open ocean to deep in the earth. Here we present a representative of each of the four nutritional types to give you a sense of how these microorganisms differ in their metabolism.

One example of a photoautotrophic lithotroph would be the cyanobacteria Oscillatoria. This microbe has a photosynthetic system that is similar to that used by photosynthetic eukaryotes. In other words it generates its energy from the sun and uses carbon dioxide as its source of carbon. Due to their simple nutritional requirements, a few salts and sunlight, they are found in many places including damp soil, dripping rocks, fresh and seawater and in hot springs. Under the microscope they form filaments that are 5 µm wide and hundreds of µm long.

An example of a photoheterotrophic organotroph is the purple bacterium Rhodobacter sphaeroides. While this microbe is capable of growing photoautotrophically, it can also utilize simple carbon compounds as sources of electrons, yet still use light as its method of energy generation. These microbes are aquatic, often occurring in fresh water lakes in the zones several meters below the surface where light is still present, but oxygen is absent. In these anaerobic environments organic molecules are incompletely degraded to organic acids. R. sphaeroides has adapted its metabolism such that it can take advantage of these carbon sources when they are available.

An example of a chemoautotropic lithotroph is the ammonia oxidizer Nitrosomonas europaea. This microbe is found in many different habitats including soil, sewage, fresh water, the walls of buildings and on monuments; basically anywhere that ammonia is available. It is especially common in urban settings where high levels of nitrogen compounds are present due to pollution. N. europaea converts ammonia into nitrous acid and in the process consumes oxygen. The energy generated from this process is used to convert carbon dioxide into cellular material. It has a competitive advantage in places that contain oxygen and ammonia, but not much organic carbon. This explains its ability to grow in unusual places such as on the surface of bricks and monuments. This activity can be damaging since the metabolic activity of these microbes lowers the pH and slowly dissolves the stone. A second example of this class is Ignicoccus islandicus. This microbes was isolated from a submarine, ocean vent containing high temperature and high sulfur and hydrogen concentrations. hydrogen gas serves as the source of energy and electrons, while carbon dioxide is the source of carbon. Deep sea ocean vent communities are intriguing ecosystems containing many unique micro- and macroorganisms. These communities and their environment are described in more detail in Chapter 24.

There are a large number of chemoheterotrophic organotrophs and one example is Escherichia coli. Because of the notoriety of certain pathogenic E. coli strains, you may find it surprising that the vast majority of E. coli do not cause illness in humans, in fact they are common inhabitants of the intestines of many mammals. In the warm environment of the intestines they take advantage of the large amount of organic carbon compounds consumed by their host. These are degraded to generate energy and the same compounds are also used to synthesize cell material. Another example of a chemoheteroorganotroph is the brown rot fungus Gloeophyllum trabeum. This organism and its relatives are some of the major degraders of wood and its major polymer lignocellulose in terrestrial ecosystems. Wood serves as the source of energy, electrons and carbon for the fungus. This fungus and its relatives are essential for the efficient degradation of lignocellulose, a very recalcitrant polymer. G. trabeum uses a novel mechanism to attack the polymer by creating extracellular enzymes that produce hydroxide radicals (•OH) that rapidly attack the polymer. A final example of a chemoheterotrophic organotroph is Thermotoga maritima. This bacterium grows on a number of sugars, alcohols and organic acids in environments where oxygen is absent. T. maritima has the highest optimal growth temperature of any bacteria (although several archaea grow at higher temperatures) with a growth optimum near 90°C. It was originally isolated from heated ocean sediment near Volcana, Italy and has as its habitat areas of volcanic activity.

Before delving into the rules and recipes for growing microorganisms, it is important to point out that, at present, scientists cannot culture over 90% of the microbes in the environment. It appears that most bacteria are unculturable and the reasons for this are still being sorted out. In some cases these microbes may require very narrow gradients of certain nutrients. For example the microbe may only grow at oxygen concentrations between 4-6% and while these conditions are present in their natural environments, they are difficult to replicate in the laboratory. If conditions fall outside of these narrow ranges, the microbes may often become nonviable. In other cases the unculturables are out competed by culturable microbes that can grow faster given the ingredients and incubation conditions of the medium. Another factor is time. Some unculturable microbes are extremely slow growing, taking hundreds of days to accumulate to appreciable quantities. Chapter 24 examines unculturables microbes in greater detail. It is worth keeping in mind that there is much to learn about growing microbes and microbiologists are just beginning the process.

The goal of any culture medium is simply to provide an environment where the nutritional needs of the microbe are met and the microbe will multiply. In practical terms this means providing a source of carbon and electrons, a means for the microbe to generate energy, any necessary ions that contain the needed elements (Fe, Mg, S, P, etc). and finally any specific growth factors that the microbe may require. There can also be physical incubation parameters such as the appropriate temperature and the presence or absence of light that must be taken into consideration.

Media may be supplied to bacteria in liquid form (broths) where the cells grow suspended in an aqueous solution or in solid form (agars). Broth cultures are useful for growing large batches of microorganisms in a homogeneous environment. Solid media are useful in the isolation of pure cultures, enumeration of bacteria and for the selection of strains of bacteria with desired properties, among many other uses. Solid media contain a gelling agent that hardens when cooled and the most common gelling agent is agar.

Agar is an extract of red algae and is a long polysaccharide that has several unique properties useful in media preparation. Most bacteria cannot degrade agar, making it a stable gelling agent in most situations. Also, agar melts at 100 °C, but will not solidify until a temperature of 45 °C is reached. After gelling, it will not again melt until reheated to 100 °C. This is a very beneficial property since it makes it possible to prepare solid medium by first melting the agar during sterilization and then cooling it to just above 45 °C, when heat-sensitive compounds such as antibiotics or growth factors can be added aseptically (aseptically means in a fashion such that it does not get contaminated with microorganisms). It is also possible to mix most bacteria briefly with 45 °C agar if desirable and pour plates, a useful technique in the enumeration of bacteria and their viruses. Finally, solidified agar plates with bacteria on them can be incubated over a wide range of temperatures, making it ideal for the cultivation of a wide variety of microbes. Agar also has some problems that may make it unsuitable for the cultivation of certain bacteria. It is notorious for harboring impurities that may inhibit the growth of certain organisms. Also, a few microbes are capable of using agar as a carbon source. In these instances silica-based gelling agents can serve as a substitute.

The needs of a microbe can vary greatly and we now return to some of the above examples of nutritional types of bacteria to demonstrate this point.

To grow a cyanobacterium such as Oscillatoria (an example of a photoautotrophic lithotroph) a very simple medium is required. Since it forms its cellular material from carbon dioxide, nothing needs to be added to the culture as a carbon source. The medium need only be exposed to air, which contains a ready supply of carbon dioxide. Hydrogen and oxygen are obtained during the process of utilizing carbon dioxide. Since these microbes are also capable of using nitrogen gas as their sole source of nitrogen, air can also provide this nutrient. Phosphorous, sulfur, potassium, magnesium, calcium and iron are the only real additions to the medium. In other words, Oscillatoria, is metabolically very capable and only requires the presence of a few salts in order to grow. Incubation must occur in the presence of light and at the appropriate temperature. e, e. The presence of oxygen in the atmosphere is of no concern and in fact these microbes generate oxygen in the course of their photosynthesis.

CELL CULTURE APPLICATIONS (BD). Why Peptones in Cell Culture In the biopharmaceutical industry, concerns over using animal-derived components have prompted investigation into new forms of supplementation. Traditionally, cell culture media have been supplemented by the addition of serum or serum-derived components. While this helped to complete the diverse nutritional and growth requirements of the cells, the high product cost and variability between lots were difficult obstacles to overcome. New concerns over infectious agents such as viruses, mycoplasma, and prion diseases like BSE/TSE demonstrated the need for an alternative form of supplementation.1 For over 30 years peptones have been successfully used to replace serum in various cell culture applications.2 Peptones are more versatile and less expensive than serum products and have been shown to perform just as well (Figure 1). Additionally, some peptones have shown significant improvements in antibody production due to unique features such as anti-apoptosis properties. The wide variety of peptones available will help to meet the unique nutritional requirements of the cells, creating the opportunity for each step of the manufacturing process to be optimized for peak performance. Since they are comprised of small molecular weight peptides, fewer downstream purification steps are required. All of these factors combine to make peptones a viable supplementation option.

Peptone Selection Criteria Since every cell line is different, it is necessary to test an assortment of peptones at a variety of concentrations to optimize their performance. As shown in Figure 1, each peptone resulted in different antibody yields depending upon the concentration used. Peptones can also be used to optimize cell proliferation. As shown in Figure 2, the soy peptone produced better proliferation than the wheat, but the wheat exhibited a higher antibody yield. The right combination of peptones, as well as a consideration of feed strategy, is essential in order to optimize every portion of the process. Since lot-to-lot consistency is always a concern when using peptones, evaluations of multiple lots and a clear understanding of the product specifications is critical. Purification requirements are greatly reduced when peptones are used as a replacement for serum or any of its derived proteins. The gel in Figure 3 shows the differences in unpurified supernatants of hybridoma cells grown in variously supplemented media. Lane 4 shows the large amount of contaminating protein that is present when 10% serum is used as a supplement. While the samples supplemented with serum-derived components in Lanes 2 and 6 are cleaner, a great deal of purification is still required. The medium where a peptone was used as a replacement for serum (Lane 3) is just as clean as the protein-free medium used as a control (Lane 5).

The benefits achieved when peptones are used as a serum substitute will be enhanced if they are used with an optimized base medium. Figure 4 shows the differences in antibody yield when two different peptones are used in either IMDM or BD Cell™, a specialized hybridoma medium for high-level antibody yield. Previous work has shown that cells will not perform in IMDM without the proper supplementation. The level of antibody produced in IMDM with peptone supplementation was similar to the level achieved in BD Cell™ medium without peptone. The level in BD Cell™ was increased almost five-fold when peptones were added to the BD Cell™ medium.

BD Brand Peptones Meet Your Needs Throughout the years the successful use of meat-based peptones in cell culture applications has been widely accepted.3,4 In addition to their nutritional properties, meat-based peptones have exhibited many growth related properties such as the stimulation of both DNA synthesis and mitosis.5 BD Difco™ Proteose Peptone No. 3 is a porcine-based supplement that has shown great success as an alternative to serum in many systems. Since porcine-based products are in a lower BSE/TSE risk category than bovine-based products, Proteose Peptone No. 3 provides a way to reduce the risk of BSE/TSE while taking advantage of the complex properties of meat-based peptones.5 If it is necessary to completely eliminate all animal derived components, BD has a wide range of soy and yeast-based products to fit your needs. The various Yeast Extract and TC Yeastolate products are industry standards for use in insect cell cultures. j, c. In addition to the yeast offering, CHO and Hybridoma systems also benefit from the various soy products available. Whether your application need is to provide additional amino acid supplementation to your culture or to find a replacement for serum, there is a BD peptone available to help. While the concentration used should be optimized for each system, we recommend starting at a concentration of 1-10 g/L.

References 1. Heidemann, Zhang, Qi, Rule, Rozales, Park, Chuppa, Ray, Michaels, Konstantinov and Naveh. 2000. The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology 32:157-167. 2. Taylor, Dworkin, Pumper and Evans. 1972. Biological efficacy of several commercially available peptones for mammalian cells in culture. Exptl Cell Res 74:275-279. 3. Schlaeger. 1996 The protein hydrolysate, Primatone RL, is a cost effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties. J Immunol. Methods. 194:191-199. 4. Zhang, Zhou and Yu. 1994. Effects of peptone on hybridoma growth and monoclonal antibody formation. Cytotechnology. 16:147-150. 5. Jan, Jones, Emery and Al-Rubeai. 1994. Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture. Cytotechnology. 16:17-26.   Figure 5

FERMENTATION APPLICATIONS (BD)  Defined vs. Complex Media Fermentation media formulations are of two types: defined and complex. Defined media are made by the addition of chemically-defined ingredients to WFI (water for injection) or distilled water. Complex media are made with peptone digests or extracts of plant or animal origin (see “Hydrolysis to Hydrolysate”).1 The advantages of chemically-defined media can be greater reproducibility, cleaner downstream processing steps and simplicity in the analysis of the end product. The disadvantages can be lower yields and greater expense, especially if the list of media components include growth factors and vitamins.2 The advantages of complex media are that they are relatively inexpensive, support a wide variety of growth from a large group of microorganisms, promote growth of the more fastidious organisms that will not grow on chemically-defined media, stimulate toxin production and routinely produce higher yields than many defined media. k, j. The disadvantages of complex media are that the downstream processing may be more difficult and reproducibility can sometimes be compromised.

Selecting a Peptone This section will demonstrate a variety of uses for some of the BD peptones. When developing a new medium formulation, care should be taken in choosing the peptones for the new formulation. Individual experimentation with a variety of peptones is suggested to select the optimum peptone or combination of peptones. Figures 1 and 2 demonstrate such a preliminary screen for multiple peptones and two different organisms. The peptones are each 1% solutions with 0.4% glucose and buffering salts. Growth testing was performed using the Labsystems Bioscreen C Kinetic Optical Density Reader. Compare the growth support curves of Proteose Peptone No. 3: in Figure 1, the proteose curve demonstrates the least growth support; in Figure 2, proteose gave the second best growth support. Based on these results, one would likely create a medium formulation consisting of yeast and proteose to support the growth of Enterococcus faecalis and not use proteose in a Saccharomyces cerevisiae formulation.  

Moving From Animal to Non-Animal With the advent of the BSE/TSE crisis, a prime directive for the development of new fermentation products has been to either source the media’s raw materials from a country free from BSEs or reformulate the media using animal-free components.3 BD has paved the way by providing documentation on all raw material origins to ensure that biotechnology users are provided with all necessary paperwork. BD began reformulating media formulations from animal components to non-animal components in 1997. In 1998, we debuted our first Select APS™ (alternative protein source) products. Throughout the reformulation effort, it was noted that in each case, growth support was either enhanced or maintained. There was never any loss of growth support characteristics.  j, l, g, g, j, c. In the case of the LB Broth reformulation, the performance was increased (see Figure 3). The test organism was DH5α E. coli. The experiment was conducted in a shake incubator set at 250 rpm and 35°C. Another example where product yield was increased was when a medium (Todd Hewitt) was changed to a custom non-animal formulation (see Figure 4). E. faecalis was grown in side-by-side New Brunswick BioFlo 3 fermentors. In this case, the product yield is related to the mass or OD reading, which doubled when the medium formulation was changed to all non-animal components. Figure 5 demonstrates the variety of soy peptones available from BD. It also demonstrates the differing responses an organism may have to different peptones made with the same starting materials. For an E. coli with a plasmid, the Select Soytone provides better growth support than Trypticase™ Peptone. In this experiment the peptones were in 2% solutions with some buffering salts. The purpose of the experiment was to observe what type of growth support the individual peptones contributed to a multicomponent medium. Figure 6 demonstrates the rigorous quality control testing these media undergo. Three lots of Select APS™ Super Broth were growth tested using an E. coli strain containing a plasmid. The three growth curves are nearly identical in their growth support. Product consistency is demonstrated through the quality control testing of the final product. Figure 6

MEAT PEPTONES AND MEDIA Meat peptones are proteins from animal sources that have been hydrolyzed, or broken down into amino acids and peptides, to provide nitrogen for microorganisms. Meat peptones can be tailored to specific nutritive needs of microorganisms by controlling the quality and origin of the protein, the quality and source of the enzyme used to digest the protein, and the methods used for hydrolysis, concentration and drying the peptone. Sources of animal protein include meat from muscle tissue or offal and gelatin. Muscle tissue and offal (waste parts, entrails) are utilized fresh, frozen or dried, but offal is often used fresh. Gelatin is extracted by boiling collagen, the fibrous protein found in connective tissue, bone and cartilage. A variety of proteolytic enzymes, or proteases, may be used to accomplish enzymatic hydrolysis of animal protein. Pepsin and trypsin are widely used for animal peptone manufacture. Pepsin is isolated from porcine or other animal stomach. Trypsin, along with chymotrypsin, carboxypeptidase A, carboxypeptidase B, and elastase, are enzymes isolated from animal pancreas. Peptone manufacture includes the following steps: hydrolysis/digestion, centrifugation, filtration, concentration and drying. Animal tissues are chopped in order to prepare for digestion, and demineralized water is added to the starting constituent(s) to form a thick suspension of protein material. The material is placed in large-capacity digestion vessels, which are stirred continuously. Base or acid is added to bring the pH of the protein suspension to the optimum for the specific enzyme being used. For example, pepsin is most effective at pH 2.0 and trypsin shows maximum activity at pH 8.5.1 When the pH and temperature are optimal, the enzyme is added. The amount of enzyme necessary, time for digestion, and control of pH and temperature are dependent on the degree of hydrolysis intended. Once protein digestion is complete, the suspension may be heated to inactivate the enzymes. The protein/enzyme slurry is then centrifuged and/or filtered to remove the insoluble materials and to clarify and concentrate the material. The peptone solution may be vacuum-evaporated to rapidly concentrate the peptone. The peptone syrup, which contains approximately 67% solids, may undergo further processing for pH adjustment or filtration. The final drying step of the process further concentrates the protein by spray-drying or by pan-drying in vacuum ovens, which readies the material for packaging.   Product Description Beef Extract is derived from infusion of beef and provides an undefined source of nutrients. Beef Extract is not exposed to the harsh treatment used for protein hydrolysis, so it can provide some of the nutrients lost during peptone manufacture.1 Beef Extract is a mixture of peptides and amino acids, nucleotide fractions, organic acids, minerals and some vitamins. “Its function can therefore be described as complementing the nutritive properties of peptone by contributing minerals, phosphates, energy sources and those essential factors missing from peptone.”2

Applications Beef Extract is intended to replace aqueous infusion of meat in microbiological culture media. Beef Extract is frequently used at a concentration of 0.3 to 1.0% in culture media, although concentrations may vary depending on the nutritional requirements for the medium formulation. Beef Extract was used in media for early studies of non-sporulating anaerobes of the intestinal tract and as a stock broth in the study of nutritional needs of streptococci. Prokofeva et al.3 used Beef Extract for growing thermoacidophilic organisms newly isolated from hot springs in Kamchatka, Russia. Kataoka and Tokiwa4 used Beef Extract as a nitrogen source in studies of mannose production by Clostridium tertium strains isolated from soil and methanogenic sludge. In addition, Beef Extract is a nutritive ingredient in many classical culture media, including Antibiotic Assay media described in The United States Pharmacopeia,5 and several media recommended for standard methods applications

BEEF EXTRACT POWDER BACTO™ BEEF EXTRACT, DESICCATED   Physical Characteristics Beef Extract Powder is a light to medium brown, free-flowing, homogeneous powder. Bacto™ Beef Extract, Desiccated is the dry form of Beef Extract paste. It is a medium to dark brown, free-flowing, homogeneous powder.   Availability Beef Extract Powder 212303, 500 g Bacto™ Beef Extract, Desiccated 211520, 500 g   References 1. Cote. 1999. Media composition, microbial, laboratory scale. In Flickinger and Drew (ed.), Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation, p.1652. John Wiley & Sons, Inc., New York. 2. Bridson and Brecker. 1970. Design and formulation of microbial culture media. In Norris and Ribbons (ed.), Methods in microbiology, vol. 3A, p. 250. Academic Press, New York. 3. Prokofeva, Miroshnichenko, Kostrikina, Chernyh, Kuznetsov, Tourova and Bonch-Osmolovskaya. 2000. Acidilobus aceticus gen. nov., sp. nov., a novel anaerobic thermoacidophilic archaeon from continental hot vents in Kamchatka. Int. J. Syst. Evol. Microbiol. 50: Pt 6:2001-2008. 4. Kataoka and Tokiwa. 1998. Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertium KT-5A, from lotus soil. J. Appl. Microbiol. 84:357-367. 5. United States Pharmacopeial Convention. 2001. The United States pharmacopeia 25/The national formulary 20—2002. United States Pharmacopeial Convention, Inc., Rockville, Md. 6. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, DC. 7. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC International, Gaithersburg, Md. 8. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, DC.   Product Description Bacto™ Brain Heart Infusion (BHI) is a microbiological culture medium used for cultivating fastidious microorganisms, including streptococci, pneumococci and meningococci. In 1919, Rosenow1 devised an excellent medium for culturing streptococci by supplementing dextrose broth with brain tissue. Hayden2 revised Rosenow’s procedure by adding crushed marble to the medium and reported favorable growth of dental pathogens. Brain Heart Infusion is a modification of the media described by Rosenow1 and Hayden2 in which infusion from calf brains has replaced the brain tissue, and disodium phosphate has replaced the calcium carbonate. Infusion from beef heart, calf brains and Proteose Peptone provide nitrogen, carbon, sulfur and vitamins in Brain Heart Infusion media. Dextrose is a carbon energy source, sodium chloride maintains osmotic balance in the medium, and disodium phosphate is a buffering agent. Bacto™ Brain Heart Infusion, Porcine was developed as an alternative to the classical Brain Heart Infusion formula, and replaces calf brains and beef heart with pork brains and heart. BHI, Porcine was formulated with no bovine components to minimize Bovine Spongiform Encephalopathy (BSE) risk. Infusion from pork brains, infusion from pork heart, and Pork Peptone No. 2 provide nitrogen, carbon, sulfur and vitamins in Brain Heart Infusion, Porcine. Dextrose is a carbon energy source, sodium chloride maintains osmotic balance in the medium, and disodium phosphate is a buffering agent.

Applications Brain Heart Infusion media are specified in several standard methods references for food testing.3-5 Standard Methods for the Examination of Water and Wastewater recommends Brain Heart Infusion media in tests for the verification of fecal streptococci.6 Brain Heart Infusion is listed by the National Committee for Clinical Laboratory Standards (NCCLS) as a medium for use in the preparation of microdilution trays for antimicrobial susceptibility testing by the broth microdilution procedure.7 BHI broth has been used in various microbiological studies. e, h, h, b, l. BHI is recommended for growth of most ATCC™ strains of Pasteurella multocida and has been cited in many studies on P. multocida fowl cholera vaccines. Duffy et al.8 used BHI as a fermentation medium for pH studies on Escherichia coli O157:H7. Tan et al.9 utilized BHI to grow Fusobacterium necrophorum for leukotoxin production studies. Likewise, Van Tassell et al.10 made enterotoxin preparations by growing Bacteroides fragilis in BHI. BHI, Porcine was developed for pharmaceutical and vaccine production and can replace the traditional BHI depending on organism and production application.

Cultivation of a photoheterotrophic organotroph such as Rhodobacter sphaeroides is somewhat similar to that of Oscillatoria since it also grows photosynthetically. However, this microbe will only photosynthesize if oxygen is absent and therefore the culture medium has to be devoid of oxygen. While this may appear at first to be a daunting task since air contains over 20% oxygen, it is in fact quite simple for broth cultures. A narrow neck bottle is filled with medium and a cap applied. R. sphaeroides will readily consume the oxygen remaining in the medium and very little can diffuse back into the capped bottle.

The medium has similar salts as that described for Oscillatoria, but often contains an organic compound such as succinate or malate. R. sphaeroides uses this as a carbon and electron source and the presence of these organic acids increases the growth rate of growth of the microbe. R. sphaeroides requires the addition of a few growth factors in the form of vitamins. Optimal incubation temperatures approach those of a warm lake in the summer (28°C), a common natural habitat for the organism. i, l, k, e, f. A chemoautrophic lithotroph also has to be metabolically capable, utilizing carbon dioxide as its source of carbon and does not require the addition of an organic carbon source to the medium. In contrast to the photosynthetic bacteria discussed above, it requires the addition of a chemical that can serve as a source of energy and electrons. In the case of Nitrosomonas europaea that energy source is ammonia and a relatively large amount of ammonia must be added to get adequate growth. Oxygen is required in order for the microbe to be able to utilize ammonia as an energy source, so the culture must be exposed to air. Metabolism of the ammonia produces nitrous acid and a strong buffer must be added to the medium to prevent the pH dropping too low, since ironically these microbes grow best near neutral pH. The only other requirement is the addition of ions similar to those described above.

Cultivation of a chemoheterotrophic organotroph requires the addition of organic carbon compounds to serve as sources of carbon, electrons and energy. In many cases a single compound can serve all these roles. In the case of E. coli, it is capable of utilizing glucose as its sole source of carbon, electrons and energy, being able to synthesize all other required organic compounds from it. A nitrogen source is provided, typically in the form of ammonia, but several nitrogen-containing organic compounds can also be utilized. The only other requirement is the addition of ions as with all the other media described above. The intestine contains little oxygen and E. coli is therefore capable of growth in its absence, but can also grow in its presence. Incubation can therefore occur in the presence or absence of oxygen and the microbe grows best near body temperature (37°C).

Due to the large number of bacterial types studied in laboratories throughout the world and the diverse purposes scientists have for growing them, there are many different types of media available for the cultivation of microbes. Culture media fall into two broad types, chemically defined media where the exact components of the medium and their amounts are known, and complex media where chemically complex ingredients such as an extract of yeast cells is added.

In contrast, complex media will use extracts of a variety of things, including left-over animal parts (cow brains and hearts), yeast (from brewing) or digests of plants or animal slurries (peptones are one example of this category). The exact composition of these extracts is often unknown. The sources of these extracts often take advantage of waste products from other industries to save money.

Minimal media supply only the minimal number of nutrients required for the growth of a microorganism. By definition, all minimal media are chemically defined. However, chemically defined media can provide more than the minimum needed for a microbe so not all chemically defined media are minimal media. Minimal media are useful for the study of nutritional requirements of microbes and in genetic analyses.

Rich media supply a large assortment of nutrients, usually from extracts, and support the growth of a wide variety of microorganisms. Rich media are useful for the cultivation of pathogens, analysis of unknown samples and also in genetic analyses.

Media can also be classified by its interaction with the microorganism. Selective media is formulated to inhibit the growth of undesirable species while encouraging the growth of desired organisms. This can be achieved by adding compounds to the medium to encourage the growth of some bacteria or inhibit the growth of undesirables. Omitting compounds that unwanted microbes need is another strategy to make a medium selective. Selective medium is especially useful in the selection of bacterial strains with desired properties and finds great utility in genetic experiments with microorganisms.

An enrichment medium is a particular type of selective medium. Its goal is to encourage the growth of a particular class of microbes from an environment in the hope of increasing their number to the point that they may be isolated in pure culture. These media will omit a nutrient needed by most undesired bacteria or include something that only the desired class of microbes can use. Selective agents that inhibit competitors may also be present. These media are typically formulated to mimic the environment of the desired class or will take advantage of a known metabolic property of the desired class. The distinction between selective and enrichment media is somewhat arbitrary since any selective medium could be used to enrich for isolates from the environment. The name given to a medium (selective vs. enrichment) has more to do with its use than its formulation. Enrichment medium are normally a first step when isolating microbes from the environment, with the goal to increase the population of the desired microbes.

Differential media does not inhibit the growth of any microbe, but causes bacteria that grow up on it to have a different appearance due the formulation of the medium. Table 6-15 shows the formulation of Lactose fermentation broth. The medium is differential because of the presence of lactose as a carbon source and bromcresol purple, an indicator dye. Organisms capable of fermenting the lactose to acid change the pH of the medium, which causes the bromcresol purple in the medium to turn from purple to yellow. Those bacteria that are unable to ferment lactose will still grow, if another carbon and energy source is provided that they can use, but no color change will be observed in the broth.

In almost all cases, once the ingredients of a medium are dissolved in solution, they must be treated to eliminate any microorganisms that might have entered from container surfaces, media ingredients, weighing papers, or other surfaces that have contacted the ingredients. If this is not performed correctly, contaminating microbes might grow and make the desired microbiological investigations impossible. Sterilization is defined as the inactivation (or removal) of all life forms and also viruses in a specific area or volume. Culture media must be made sterile without inactivating nutrients necessary for growth of the microorganism.

Once a medium is made and sterilized, it is ready to be inoculated with a source of microorganisms and incubated under appropriate physical conditions to encourage their growth. A final consideration is protecting the inoculated medium from contamination by microorganisms that may enter from the air. For broth cultures, the medium is stored in containers with narrow necks and the opening is plugged with foam or cotton. Most commonly an Erlenmeyer flask is used for broth media in the lab. In the case of solid medium, petri dishes are used, which are dishes with covers.

Bacteria can be cultivated in either closed systems (also termed batch cultures),where nothing is added or removed during incubation, or in open systems where fresh medium is added and spent medium and cells are removed. Closed systems involve a single vessel and are usually started from a small inoculum. Examples of closed systems include test tubes, flasks and any other container that is then filled with a discrete amount of medium. Open systems are more complex due to the need to aseptically add and remove medium.

Continuous cultures can be set up using two methods. In a chemostat, sterile medium is fed into the culture at the same rate that medium containing microbes are removed. One essential nutrient in the medium is limiting and controls the total yield (cell number) present in the chemostat. A key element of the chemostat is that the growth rate and total yield can be controlled independently. In batch culture, growth rate and total yield are both tied to the medium used and physical incubation conditions. In the chemostat, growth rate can be regulated over a wide range of values by the flow rate of medium through the culture while total yield depends solely on the concentration of the limiting nutrient in the medium.

The second type of continuous culture is a turbidostat. This method uses the same basic set up as a chemostat except that a photocell monitors the optical density of the culture. The flow rate of medium coming into the culture is adjusted to automatically maintain a predetermined turbidity. Chemostats are useful in studying the physiology of a microbe since it is possible to limit just one nutrient and see the effect of the microbe. With such studies it is possible to determine how efficiently the microbe uses nutrients. Turbidostats find utility when a constant supply of metabolically identical cells are desirable.

In water, the hydrogen ion concentration will range from 1 x 10-14 M (a pH of 14) to 1 M (a pH of 0). Microbes are found at almost any conceivable pH, with most common bacteria growing at or near neutral pH (7). It is possible to classify bacteria based upon the pH at which they grow. Acidophiles have pH optima in the range of 1-5.5, and include many fungi and some obligate acidophilic bacteria such as Thiobacillus, Sulfolobus and Thermoplasma. Neutrophiles prefer a pH in the range of 5.5-8.0 and most organisms fall into this category. Pathogens of mammals usually have extraordinarily narrow pH ranges very close to 7.0 where they will grow well. Alkalophiles prefer a pH in the range of 8.0-11 and are found in highly basic soda lakes and high carbonate soils. e, l, b, b, c, f, a. Water is the solvent that almost all molecules of life dissolve in, and thus all organisms require water to grow. The removal of water from food has been a favorite method of preservation for many centuries for this very reason. Water availability (water activity) is a measurement of how much free water is available to carry out necessary reactions in a cell. Pure water has a water activity of 100% and the activity decreases as solutes are added to the solution.

Water can be made less available by evaporation or, in the case we will be considering, by being bound up by solutes in the solution. As the concentration of solutes in a solution increases, it causes two problems for the cell. The first relates to osmosis. In the presence of a semi-permeable membrane, water will try to flow in a direction that will balance the amount of solute on each side of the membrane. In dilute environments, this flow of water can cause the cell to swell, as water pushes into the cell and most bacteria contain a cell wall to prevent this type of damage. However, in environments high in solute concentration, the water will flow out of a cell causing dehydration and damaging the cell membrane.

The second problem occurs only in high-solute environments and relates to their enzymes and the reactions they carry out. In solution with high levels of solute, the water is bound by the solutes and cannot interact with the enzymes of the cell. Enzymes need water bound to them to remain in solution and often to carry out their reactions. This can cause the proteins to fall out of solution, and will also inhibit their activity.

Microbes that require high concentrations of salt in their environment are called halophiles. Mild halophiles require 1-6% salt, moderate halophiles require 6-15% salt, and extreme halophiles require 15-30% NaCl for growth. Halotolerant bacteria can endure moderate salt concentrations, but grow best in its absence. Many of the halophiles encounter NaCl or KCl in their natural environment and thus much of the work with halophiles uses these salts as the solutes. Even though halophiles are osmophiles (and halotolerant organisms are osmotolerant) the term osmophiles is usually reserved for organisms that are able to live in environments high in sugar. Most of these microbes tend to be yeast strains. Organisms that prosper in dry environments are called xerophiles.

As the concentration of solutes in a solution increases, the growth rate of organisms changes. For non-halotolerant bacteria such as E. coli or Pseudomonas, growth is at a maximum in dilute solution and decreases as the water activity decreases (Figure 7-12). Halotolerant bacteria still grow best at low solute concentrations, but they show a much shallower slope in a graph of growth rate versus water activity. Staphyloccocus aureus, a pathogen of humans, is a halotolerant microbe and this is used to help identify it. Halophiles show a peak growth rate at an optimum salt concentration, growing less well at lower and higher water activities. Halobacterium, the predominant species of the Great Salt Lake in Utah, is a halophile that grows well even in saturated salt solutions.

Microorganisms are found in almost every environment on earth. They can survive in a great many environments because they are small and easily dispersed, occupy very little space, need small quantities of nutrients, and are remarkably diverse in their nutritional requirements. They also have a great capacity for adapting to environmental changes.

Organisms that interest people in the health.sciences account for only a small portion of all microorganisms. These organisms are ones which have adapted to the conditions found in or on the human body.

Different organisms can grow in a wide range of environments-- from highly acidic to very alkaline, from subfreezing temperatures to volcanic eruptions, and with or without oxygen. Growth can be influenced by a variety of physical factors including: pH, temperature, oxygen concentration, and moisture.

Most species of bacteria grow best in a medium with a pH of 7.0. (Note: a pH of 7.0 is considered neutral.) This means that the number of acidic ions (protons, or H+) is equal to the number of basic ions (hydroxyl, or OH-). However, many bacteria can live and multiply as a pH 5.0 (acidic) to a pH 8.0 (basic).

As bacteria grow in their natural habitat or in the laboratory they produce acid as a by produce of their matebolism. This acid by-product often inhibits their growth by changing the surrounding environment.

To help prevent this in the laboratory buffers are used. Buffers are usually a mixture of monohydrogen and dihydrogen phosphates (K2HPO4 or KH2PO4). These salts limit the pH changes because they can combine chemically with hydrogen ions of strong acids and the hydroxyl ions of strong bases to produce neutral compounds. In other words the buffers resist radical changes.

Some of growth media manufacturers are listed here: ABI/Advanced Biotechnologies Inc.,  Becton Dickinson & Co., Binding Site Inc., BIO 101 Inc., BioWhittaker Inc., Digene Diagnostics Inc., Fisher Scientific Co., Gibco BRL Div. of Life Technologies Inc., ICN Biochemicals Inc., Intermountain Scientific/BioExpress, Merck KGaA, Midwest Scientific, Quality Biological Inc., Sigma Chemical Co., Thomas Scientific, Wako BioProducts.

Selective media are supplemented with ingredients which enhance the growth of certain bacteria and suppresses the growth of others. For example the addition of certain dyes such as eosin methylene blue is used to detect preferentially enteric bacteria in water supplies. Differential media help to distinguish between different groups of related microorganisms. Blood agar plates for example will distinguish between hemolytic and non-hemolytic strains of Streptococci. Additional growth parameters include temperature and oxygen availability

Microorganisms can be cultivated either in liquid or solid growth media. Liquid media cultivation is done either in small Universals (up to 20 ml), in Erlenmeyer flasks (up to 1 l) and fermenters (from 1 l onwards up to large industrial scale vessels with a capacity in excess of 100000 l).

One differentiates two types of liquid cultivation: batch cultivation and continuous cultivation. Most cultivations on a laboratory scale are carried out using batch culture techniques. This employs a closed system such as a flask or a fermenter in which the nutrient supply is not replenished and therefore leads to the observation of above growth curve.

Alternatively cells can also be grown in continuous culture using a chemostat. In this system fresh nutrients are continuously fed into the culture vessel and spent broth/biomass is removed at volumetrically equivalent levels; therefore the culture volume of the vessel remains contant. A continuous culture vessel, once set up and running can be operated for weeks or even months. The microbial cells inside are in exponential phase and divide at a constant growth rate, which at optimal growth conditions can be equal to the maximum growth rate. Continuous cultivation are sometimes used on an industrial scale for example the production of single cell protein, based on certain bacteria and fungi.

Downstream processing. This expression refers to the techniques which need to be carried out after growth. At this stage the biomass has to be separated from the culture medium for further processing. This is achieved by various techniques such as centrifugation (high g-forces will pellet cells towards the bottom of a container), filtration or flocculation of the cells. Growth of a culture in liquid is assessed by establishing a socalled growth curve in which at regular intervals samples are withdrawn and analysed for the number of cells present in the sample. The quickest way to obtain an idea of cell numbers is by turbidity measurements at a particular wavelength (usually 578 nm); It is based on the fact that cell in suspension have a light scattering effect the extent of which is directly proportional to the cell density.

The plotting of the logarithm of cell numbers over absorbance will give a straight line; hence this procedure enables the microbiologist to determine the cell number by measuring the absorbance of light at visible wavelength. Also these standard curves are species specific and therefore will have to be done with each separate microorganism. It will also give only satisfactory readings for cultures at a cell density of 105 to 5x107 cells per ml.

In order to obtain a more accurate figure as to the actual cell numbers in the liquid culture one has to withdraw at regular intervals aliquots from the culture and count the cell number on a counting chamber called Haemocytometer. g, f, f, g, k, h. This gives the socalled total cell count (TCC). Alternatively one can subject the aliquot to serial dilution and plate the culture out on a suitable growth medium on a socalled spread plate, pour plate or by using the Miles-Misra drop count technique.

The number of colonies one counts the next day is known as the number of colony forming units (cfus) a number which is reflecting the number of dividing cells present in the culture. This gives the socalled viable cell count. The main disadvantage of this technique lies in the fact that one has to wait at least 24 hours until one can determine the cell number. The advantage is that any contamination which may accidentally may have entered the liquid culture may readily be identified.

Any medium for the cultivation of bacteria must provide certain basic nutritional requirements, which include (1) a carbon source that may also serve as an energy source; (2) water; (3) a nitrogen source; (4) a phosphate source; and (5) various mineral nutrients, such as iron and magnesium. Some bacteria are capable of growth on a medium consisting of a single carbon source, such as the carbohydrate glucose; a simple nitrogen source, such as ammonium salts; and inorganic salts, such as phosphates. This kind of medium is termed defined or synthetic because its exact chemical composition is known. For routine laboratory work, however, complex media are employed where the basic nutrients are provided by complex nutrients, such as plant and animal extracts in which the exact composition is not known. For example, beef extract and peptones (hydrolyzed protein) are the basic ingredients of nutrient agar. These materials supply a variety of carbon sources, nitrogen compounds in the form of amino acids, and a mixture of cofactors, such as vitamins. This basic medium can be further enriched to support the growth of more fastidious types of bacteria by the addition of carbohydrate sources, yeast extract, and materials such as plasma or blood, which provide a variety of complex nutritional factors. A broth medium is one in which the components are simply dissolved in water. The addition of agar-agar (a complex carbohydrate extracted from seaweed) results in a solid medium. Agar is an ideal solidifying agent for microbiological media because of its melting properties and because it has no nutritive value for the vast majority of bacteria. Solid agar melts at about100°C; liquid agar solidifies at about 42°C.

Because microbes are ubiquitously distributed in the environment, during the preparation of any culture medium, bacteria are introduced from many sources such as glassware, dry medium components, air, and so on. These bacteria would eventually grow and flourish if the medium were not sterilized, that is, if these unwanted microbes were not destroyed.

Sterilization procedures eliminate all viable microorganisms from a specified region. Culture dishes, test tubes, flasks, pipettes, transfer loops, and media must be free of viable microorganisms before they can be used for establishing pure cultures of microorganisms. The culture vessels must be sealed or capped with sterile plugs to prevent contamination. There are various ways of sterilizing the liquids, containers, and instruments used in pure culture procedures; these include exposure to elevated temperatures or radiation levels to kill microorganisms and filtration to remove microorganisms from solution.

Media preparation for the microbiology laboratory involves the use of an autoclave for sterilization, which permits exposure to high temperatures for a specified period of time. Generally, a temperature of 121°C (achieved by using steam at 15 lb/sq in) for 15 minutes is used to heat-sterilize bacteriological media. Much of the time spent in preparation for the bacteriology laboratory involves preparing the media for growing bacteria; that is, mixing and sterilizing the growth media in suitable sterile culture vessels.

Culture medium - a liquid or gelatinous substance containing nutrients in which microorganisms or tissues are cultivated for scientific purposes.

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