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Is there a difference between Luria Bertani and Luria Broth? Or are they both the same thing? Is it necessary to autoclave LB medium after it has been made?
Although both names are quite common, both are wrong. In the original paper from 1951 Bertani was studying the lysogeny in E.coli, hence he called his media for this purpose "Lysogeny Broth" or LB in short. In the subsequent decades this name was transformed to "Luria Bertani" or "Luria Broth", which is incorrect. See the references 1 and 2 for more details.
For the second question: It is absolutely necessary to autoclave the media after preparation to avoid the contamination and growth of microorganisms which are present in our environment. Only making the media sterile makes sure that we can work with defined microorganisms.
- Studies on Lysogenesis I.
- The Limitations of LB Medium
What is LB medium used for?
Click to read full answer. In this regard, why do we use LB agar?
Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within.
Secondly, what is LB agar made of? LB Broth, also known as, LB medium, Lysogeny broth, Luria broth, or Luria-Bertani medium, is a commonly used nutritionally rich medium for culturing bacteria. First described in 1951 by Giuseppe Bertani, a 1-liter medium consists of 10 grams of tryptone, 5 grams of yeast extract, and 10 grams of sodium chloride.
In this regard, what is the difference between LB agar and LB broth?
Agar is a complex gelatinous carbohydrate, and is added to the LB broth, in order to form gel for bacteria to grow on as a microbial culture. LB is a rich medium containing peptone, yeast extract, NaCl and agar (for solid medium).
LB is the most common media used to grow recombinant Escherichia coli (E. coli). LB media was named by Giuseppe Bertani as “lysogeny broth” in due to the research he was conducting on lysogeny. LB is commonly incorrectly referred to Luria-Bertani media, Luria Broth or Lennox Broth. LB media is also used to culture a variety of other facultative organisms.
Components of LB Media
One reason LB is commonly used is because it is simple to make, with only a few ingredients, tryptone, yeast extract, and sodium chloride (NaCl). Tryptone, a mixture of peptides generated by digestion of casein with the pancreatic enzyme trypsin, provides nitrogen and carbon. Yeast extract provides vitamins (including B vitamins) and some trace elements. NaCl provides sodium ions for transport and osmotic balance. E. coli derived from the K-12 strain, one of the most commonly used parental strains of E. coli in use in molecular biology today are deficient in B vitamin production.
Historical Background of LB
LB is a nutritionally rich medium that has been used since the 1950’s to culture Enterobacteriaceae and for bacteriophage plaque assays. LB permits fast growth with good growth yields for many species. In 1951, Giuseppe Bertani developed LB to optimize plaque formation in a Shigella indicator strain of Enterobacteriaceae. Today, LB media is the most common media for growth of recombinant strains of E. coli. There are three common formulations of LB: LB Miller, LB Lennox, and LB Luria. All are sometimes referred to as LB, though the LB Miller formulation is the common or more standard formulation of LB. The alternate formulations of LB vary in the NaCl concentration (Table 1).
Table 1. Formulations of LB.
|Ingredients||%||Luria (g/L)||Lennox (g/L)||Miller (g/L)|
|Sodium Chloride (NaCl)||0.05, 0.5 or 1.0%||0.5||5||10|
The confusion about the name LB is understandable given the history of Bertani, Luria, and Lennox. Giuseppe Bertani was a member of the Luria lab at Indiana University when he formulated LB media. Ed Lennox was also a member of the Luria lab and worked with Bertani on some of the early lysogeny experiments utilizing Shigella. Salvador Luria published a paper in 1955 in which he copied the original formulation of Bertani and LB is sometimes incorrectly attributed to Luria due to his scientific stature, and so can also be incorrectly referred to as Luria Broth. The original Bertani formulation was 1.0% Bacto tryptone (10.0 g/L), 0.5% yeast extract (5.0 g/L), 1.0% NaCl (10.0 g/L), 0.1% glucose (1.0 g/L) pH adjusted to 7.0 with 1 N NaOH. Glucose was added after autoclaving. Over time, the addition of glucose has dropped out of all formulations of LB. The name Miller comes from the formulation in the book Experiments in Molecular Biology by Jeffery Miller, published in 1972, which does not contain glucose. The original and most common formulation of LB, Miller, contains 1.0% NaCl. In 1955, Ed Lennox was studying mechanisms of DNA synthesis utilizing strains of E. coli sensitive to osmotic stress developed a formulation of LB containing half the salt of the original formulation, i.e., 0.5% NaCl . This formulation is referred to as LB Lennox. Today, LB Lennox is used for cultivation of E. coli when using salt-sensitive antibiotics such as Blasticidin, Puromycin, and Zeocin. A third formulation of LB, containing the least amount of salt, is referred to as LB Luria. This formulation contains 0.05% NaCl. LB Luria is used to isolate marine organisms such as Vibrio cholerae.
Mechanisms of Growth in LB
LB media was originally designed for growth of bacteria at low densities. Exponential growth, a period of steady-state growth, is estimated to end when the OD600 (optical density at 600 nm) is between 0.6 and 1.0. It is known that growth of E. coli in LB usually stops when the OD600 reaches approximately 2.0 under normal growth conditions, corresponding to approximately 0.6 mg E. coli (dry weight) per mL. In 2007, D’Ari and colleagues undertook a comprehensive study of growth characteristics in LB, looking specifically at the physiology of E. coli K-12, one of the most common strains utilized in molecular biology today. They demonstrated that K-12 cells grown in the LB Miller with a final OD600 of 0.6 –1.0 are not always in the same physiological state. D’Ari and coworkers confirmed earlier observations of diauxic growth (growth in two phases) in LB and noted that exponential growth stopped at
OD600 0.3, much earlier than commonly thought. It is known that E. coli is known have poor growth when the pH exceeds 9.0, and it is not uncommon for LB media to change to a pH close to 9.0. However, when D’Ari and coworkers adjusted the pH of LB there was no effect on the growth curve, whereas, when glucose was added to the media, cultures could grow to OD600 6.49. This suggests that growth of E. coli in LB is carbon limited. As noted, the original formulation of LB by Bertani, contained 0.1% glucose. This research demonstrates that while LB is a good medium for routine growth, it should not to be used for physiological studies where reproducibility and a prolonged period of exponential growth is required.
LB and Selection
There are a range of additives that can be added to LB medium for identification or selection of a population of organisms. Antibiotics are often added to LB medium to select for cells that have been engineered to have resistance to one or more antibiotics. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) or Bluo-Gal (halogenated indolyl-β-galactoside) may be added to LB medium for blue-white screening for insertion of sequence into a multiple cloning site (MCS) in a plasmid containing the α fragment of the β-galactosidase gene. To induce expression of genes controlled by the lac promoter, the non-metabolizable lactose analog IPTG (isopropyl-beta-D-thiogalactopyranoside), is added to the medium. In bacteria where there is no sequence inserted into the plasmid MCS colonies will be blue since the X-Gal or Bluo-Gal will be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl. For plasmids where there is an insertion there will not be a functional β-galactosidase and the colonies will remain white.
Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems
Most of us doing research have a preferred material, a set of well-tried techniques, a standing list of unsolved problems, ways of looking at or of doing things, which we share to a large extent with colleagues in the same laboratory and others in the same area of specialization, be they friends, former associates, or competitors. All this and more is encompassed by the concept of 𠇎xperimental system” as introduced and used by Hans-Jörg Rheinberger (76, 77), a historian of science. His concept, rather flexible and rich in metaphors, may be easily adapted to the present narrative, which proceeds from a personal view rather than from a critical historical examination. A careful restriction of material, techniques, and nomenclature allows more constructive interactions between different laboratories and different generations of scientists in the same field. Of course, carried out to excess, this process will stifle developments in new areas and defer some discoveries. Max Delbr࿌k, who in the early forties had forcefully advocated the study of bacteriophage as the royal road to the secrets of replication and recombination, was quite outspoken about the necessity for workers on that road to use a common material (the T phages in Escherichia coli B) and precisely standardized techniques (1). Of course, the T phages are generally virulent, take-no-prisoner parasites of bacteria and thus could not instruct us about the more shadowy interactions between “weaker” bacteriophages and their bacterial hosts: about lysogeny, where infection meets heredity.
The term lysogenic—generating lysis—was applied very early after the discovery of bacteriophages and was used at first in broadly descriptive, uncritical ways. On the other hand, bacterial cultures that spontaneously (i.e., in the absence of obvious infection from the outside) produced bacteriophage, yet grew well, without gross evidence of lysis, were isolated as early as 1922. Varied interpretations were debated back and forth for several years, quite fiercely so in the francophone medical research community. While the development of the concept has been well summarized (68), it deserves a serious look from the point of view of the history and philosophy of science. The flavor of the debates is conveyed by one of the participants, Paul Flu (42 see also reference 84). Eugène Wollman (90, 91) of the Institut Pasteur was one of the few who saw at the time the genetic implications of concepts of lysogeny. Genetics was then rather underdeveloped in the Latin countries of Europe, immunology being the star of the day in medical quarters. Also, the debate only rarely focused on one type of experiment with well-defined and generally available material. In the early thirties a definition of lysogeny was reached (26) that is still valid, obviously without modern molecular implications. Anlage, a German word used in embryology, was applied by Burnet and McKie (27) to what we now call prophage. The concept of Anlage or prophage was based on the fact that no one had succeeded, using a variety of methods, in demonstrating the presence of infectious phage particles inside a lysogenic bacterium. World War II interrupted much of this work. The Wollmans died, and Flu barely survived, in Nazi concentration camps. Burnet took up more medically oriented work.
I encountered lysogeny in early 1949 while at Cold Spring Harbor as a research fellow in Milislav Demerec's laboratory. I was studying spontaneous and induced mutation in a streptomycin-dependent E. coli B strain. I had never worked with bacteria before. Puzzled once by a colony that looked sectorially nibbled, I showed it to Evelyn Witkin and to the late Gus Doermann, who, fortunately for me, had their laboratories just next to my room. They suggested the obvious explanation, phage contamination, but one of them added “There is also something called lysogeny…” (Actually, at that time the English word in use was lysogenesis or lysogenicity.) I had never heard of lysogeny before (although I had been exposed to Paul Buchner's  works on endosymbioses) and started digging for more information in the library.
At about the same time, unknown to me, two important events had happened. In Madison, Esther and Joshua Lederberg, in the course of their work on the K-12 strain of E. coli, then the only bacteria𠅊part from Pneumococcus—known to exchange genetic material, isolated a mutant that unexpectedly lysed when in contact with the parent strain. They came to the conclusion that the original K-12 strain was lysogenic for a previously unsuspected phage, which they named lambda, thinking it might be something like Tracy Sonneborn's kappa factor in Paramecium. This was briefly communicated in the first issue (January 1950) of Witkin's informal Microbial Genetics Bulletin actual publication came much later (58). In Paris, André Lwoff, a well known protozoologist and bacterial physiologist, took up the question of how phage particles are produced by lysogenic bacteria. The problem was bravely attacked by direct micromanipulation of individual, or small numbers of lysogenic bacteria, the oversized Bacillus megaterium, cultured in a droplet of broth under the microscope. At intervals, the liquid surrounding the bacteria was removed, replaced with fresh broth, and then tested for presence of bacteriophage. At the same time, the number of bacteria in the droplet was recorded, and the droplet was examined for bacteria that might be lysing. A strong result was that when the droplet contained phages, these were present in large numbers, as one might expect from the sudden lysis of one or more bacteria. Conversely, it was shown that the bacteria could grow and divide several times without producing any phage. However, the correlation of phage production with cell lysis could not be immediately demonstrated. This was later clarified by the finding that B. megaterium cells had the property of lysing spontaneously and nonproductively, leaving a visible cell ghost, while lysis linked to phage production was extremely rapid and left no microscopically visible traces of the bacterial bodies (69, 70).
In the fall of 1949 I joined Salvador Luria at Indiana University in Bloomington. As we were making work plans, I proposed that I study lysogeny. Luria was not too happy about it, as he had in mind other problems relating to the development of the virulent phage T2 (on which however I did work for several months). Perhaps—I am guessing—he had not mentioned lysogeny in his research grant proposal. . . . He was nevertheless very cooperative and promised to try to obtain some strains that I could use to study lysogeny. In fact, in January 1950, we received from the Lederbergs indicator strains that could be used with K-12 and also the classical Lisbonne strain, a lysogenic E. coli strain isolated in the early twenties by M. Lisbonne and L. Carrère, together with its Shigella indicator. I immediately started working on both sets of strains, looking at their cultural properties and trying to optimize growth and plaque formation. The obvious advantage with K-12 and lambda was that one could combine the study of phage with that of bacterial recombination, as the Lederbergs in fact had also begun to do. The alternative required the use of Shigella, officially a pathogen, which would call for more stringent lab safety precautions than the use of E. coli. Unfortunately, a few weeks later, Luria met Joshua Lederberg at some meeting and understood that he and Esther would have much preferred if I did not work on lambda. I was rather displeased at the time, although I recognized that the Lederbergs' request was within their rights, as their discovery of lambda had not yet been published in the open literature. Luria, Jim Watson (then in his last year of graduate school at Bloomington), and I were sharing a smallish laboratory, from which a corner had been cut out for Luria's desk. One late afternoon we had a serious discussion on how to proceed in view of the Lederbergs' request. I remember Jim declaring at the top of his voice that he would not want to be in a lab where one used routinely the presumably pathogenic Shigella. Nevertheless, Luria convinced me to leave lambda alone, at least for a time, and I accepted the challenge of using some extra care in handling Shigella. Looking back, however, it seems that this minor episode never let me develop the proper ling for the organism” (43) with respect to lambda.
Using the Lisbonne strain (symbolized for brevity as Li) and Shigella, I set out to investigate essentially the same problem as Lwoff. Having suffered the fatigues of micromanipulation in the course of my thesis work (4), the idea of using his approach never crossed my mind. Besides, phage production in lysogenic B. megaterium is unusually high (e.g., one free phage particle for every two bacteria) as compared to other lysogenic strains (a few percent free phage), so that the direct microscopic approach would hardly be successful with most strains. I first isolated from Shigella a streptomycin-resistant mutant (later known as Sh/s or Sh-16) and showed that the phage produced by the Li lysogen was unaffected by streptomycin. Next I set up what I called a “modified single burst” experiment, in which exponentially growing lysogenic bacteria (washed to eliminate any free phage) were distributed among a set of tubes, and after further incubation the whole content of each tube was plated with the streptomycin-resistant indicator and a drop of streptomycin. This technique scores only the free phage present at the time of plating and not phage that the lysogenic bacteria would produce, in the absence of streptomycin, on the plates. If the phage were produced continuously during the lysogen growth, the plates would have randomly distributed plaques. If lysogenic cultures would produce phage in 𠇋ursts,” as when a phage-sensitive cell is infected by a virulent phage, most plates would have no plaques and a few would have a large number of plaques, that is, presumably, all the phage progeny of one bacterium. Once the parameters were adjusted, the experiment worked beautifully (5). It confirmed that phage production by a lysogen was discontinuous, involving rare, large bursts of phage. A day's work thus allowed one to measure the frequency of spontaneous phage production, even down to very low levels (one burst per 45,000 cell generations for strain Li), and the average burst size: more than one could obtain in months of micromanipulation. But there was a surprise in store: while the phage recovered from cultures of strain Li was known to be rather heterogeneous as to plaque size, in my experiment the plaques looked different in different bursts. Further investigation showed that strain Li did indeed produce three immunologically distinct types of phages, which I named P1, P2, and P3, all three able independently to establish lysogeny in the Shigella strain and that as a rule they were produced in homogeneous bursts, independently of each other (5). I continued working with the phages from strain Li, giving particular attention to P2, in which it was relatively easy to recognize various plaque-type mutants.
Meanwhile, at the Pasteur, Lwoff and several collaborators had been looking for conditions that might affect the ision” of a lysogenic cell to shift from normal growth to the suicidal production of a burst of phage. After the failure of various chemical treatments, they obtained a dramatic success: exposure to a very small dose of UV light caused nearly all the bacteria in their lysogenic B. megaterium cultures to lyse and produce a burst of phage (71). The discovery of UV induction, as the new phenomenon was called, attracted much attention to lysogeny. Even Delbr࿌k, who only a few years earlier had expressed doubts about it (39), embraced lysogeny, mostly, one should add, through the enthusiasm of his colleague, Jean Weigle, professor of physics at the University of Geneva, who had just then taken early retirement and committed himself to research on phage (83). It was soon found that also a phage in Pseudomonas (51) and lambda in E. coli K-12 (89) could be induced by UV light. On the other hand, my attempts to induce strains Li or Sh(P2) (i.e., Shigella isolates made lysogenic for P2) were totally negative. This was disappointing but was also the first indication that there might be basic biological differences between lysogenic systems.
Several interesting questions about lysogeny were open. In an established lysogenic bacterium would there be one prophage copy or many copies? If just one, how would it segregate regularly at cell division? And what was the modus operandi of immunity to superinfection? Was immunity due to a diffusible, prophage-specific product (“immunity substance”, “repressor”), which would block the development of a superinfecting phage, or was it the result of some necessary interaction with a special bacterial site? Preliminary bacterial crosses by Esther Lederberg, mentioned in her 1950 report, had indicated some kind of linkage, albeit complex, between lambda lysogeny and certain genetic markers in K-12. The possibility of chromosomal control of a population of cytoplasmic elements (as in the case of kappa in Paramecium) was not excluded. I used P2 plaque-type mutations as markers in superinfection of Sh(P2) lysogenic cells, trying to follow the fate of the superinfecting phage. The latter, it turned out, was not degraded or rejected by the immunity system of the lysogen it was only blocked in its replication (as “superinfection preprophage”) and distributed randomly to the daughter cells as the lysogen continued to grow and divide. When a cell that carried it happened to lyse, the superinfecting phage would participate in the burst essentially on equal footing with the prophage. On rare occasions, the resident prophage (or some of its genes) would be replaced by the superinfecting type. Still more rarely a stable doubly lysogenic strain would be established. A P2 mutant (the clear plaque type “weak virulent”) that had lost the ability to lysogenize could be carried in the blocked state for several cell generations and, rarely, even behave like a second prophage, i.e., be inherited by all progeny cells, as long as the original prophage (turbid plaque type) was present it behaved as expected of a mutant with a recessive mutation affecting immunity. These results (6, 7, 8, 9, 10), while open to more complex interpretations, supported well both the idea of one prophage, or exceptionally two, per bacterial cell (more precisely, per nucleoid) and the concept of a specific prophage product interacting with superinfecting phage. Meanwhile, a new strain of E. coli, later called C (16), appeared on the scene, indirectly the result of a finding by Cavalli and Heslot (35). Strain C was sensitive to lambda and to all phages from strain Li, gave decent plaques with P2, and furthermore (35) would cross with K-12 strains. Gradually, I shifted from Shigella to strain C for most of the work with P2, and then bacterial crosses to establish the chromosomal location of prophage could be performed (10, 14). While I here emphasize work with P2, most readers will know that during that time (1951 to 1957) rapid progress was made in the understanding of the genetics of K-12 and lambda, as then reviewed (53) by two of its main contributors. It became quite clear that the lambda prophage itself was actually anchored at a specific site, thought at first to be the only one possible, on the bacterial chromosome. A detailed linkage analysis (28) and the then new notions of chromosome circularity led to Allan Campbell's proposal of his well-known integration model.
Outside of lysogeny, P1 and P2 also contributed more generally to the early progress in bacterial genetics. (Little was ever done with P3.) An example is the discovery of “host-controlled variation”, now more commonly called “restriction and modification.” I noticed it in P2 (using strain B as the restricting host, Shigella being the standard host) and did not know what to make of it. Jean Weigle, with whom I often corresponded, noticed it in lambda (using strain C as the permissive host, K-12 being the standard host). Being aware of my results, he immediately recognized the similarities of the two findings. Shortly before that, a minor laboratory accident, as told by Luria (66), had led to the discovery of another, albeit more complex case of host-controlled variation (67). Although no satisfactory mechanistic explanation was in sight at the time, Jean and I were encouraged by the parallelism between our two, totally independent “systems” and decided to publish our findings together (16). It rarely happens that a new phenomenon, observed in two different systems, in different laboratories, is described in the same paper, in a comparative manner. This strengthened the evidence and hinted at the generality of the phenomenon, while scoring a point for cooperation versus competition in science and human affairs. (A similar case, several years later, was that of a paper by René Thomas and Elizabeth Bertani , which reported parallel experiments with lambda and with P2 to more precisely define the mode of action of the immunity repressor.) Growing P2 in E. coli B led to another unexpected finding: the presence of a defective prophage (related to P2 but with a different immunity specificity) in this most traditional phage host strain (37). Today defective prophages are an almost daily finding in genomic analyses of bacteria.
P1 also gave some surprises. Its establishment of lysogeny in Shigella was almost absolutely controlled by temperature (17): very high frequencies of lysogenization and no immediate phage production when the infected bacteria were kept at 20ଌ after infection, 100% lysis with phage production at 40ଌ. This property was apparently lost as P1 was pted,” through at least two mutational steps, k and c (60, 88), to grow more efficiently on K-12, and no one has returned to the study of the original wild type in Shigella. The reason for this neglect of course is the fact that P1 was found capable of transduction and began to be used almost exclusively in K-12 derivatives. Transduction, discovered in 1951 by Zinder and Lederberg in Salmonella (94), and correlated to phage P22, allowed genetic analysis of closely linked mutations but no gross mapping. On the other hand, fine-structure mapping was still impracticable with E. coli K-12. The credit for discovering this capacity of P1 goes to Ed Lennox, a physicist then in the process of converting to biology, who one morning in early 1954 entered our laboratory proclaiming: “Joe, let's try and see if your phages can transduce!” I had by then some auxotrophic mutants of strain C that could be used to test the idea. We did the experiment the following day, and it turned out that indeed P1 (but not P2) was a very efficient transducer. I still have copy of a letter to Jean Weigle, dated 16 April, where I overenthusiastically wrote: “We spent a week of great excitement (i.e., Ed Lennox and I). My phage P1 can transduce. P1 grown on Shiga can transduce the Arginine character in coli C, a Galactose marker also in coli C, and a Streptomycin marker in coli B. Enough?…It will be possible to study transduction and genetic recombination in the same organism compare the temperature effect on lysogenization and perhaps on transduction perhaps succeed in transducing prophage P2 through phage P1 et cetera et cetera.” The finding was reported at the Oak Ridge meeting on genetic recombination that same spring (59) and was followed by the very comprehensive paper of Lennox (60). Both Lennox (60) and Jacob (52) demonstrated cotransducibility of lambda prophage with bacterial markers. Later, P2 prophages at three different sites were cotransduced and oriented by means of P1 (31).
While older geneticists were infecting and crossing, the methods of what is now molecular biology came on the scene, on the heels of electron microscopy and ultracentrifugation. Over a few decades the enthusiasm for lambda as an experimental system made lambda the paradigm for lysogeny (47, 48, 75, 82). It is now so well known down to the molecular level that it is usefully modeled in silico (2). P1 also attracted numerous followers, at first as a tool in transduction and later in its own right (93), because of its unusual chromosomal behavior (50). A few, including myself, continued working on P2, although at times with a feeling of isolation and the worry that perhaps its differences from lambda were not sufficiently important to justify the effort. This, I believe, turned out not to be the case (12).
Particles of P2 and lambda differ structurally. Unlike lambda, P2 DNA replication follows a typical rolling-circle model throughout its reproductive cycle (54, 64, 73, 79). This had been suggested by two striking findings, the strict requirements of P2 DNA replication for the host cell Rep function (32) and for a cis-acting phage protein (62), as is the case for the very different, virulent phage, φX174. Encapsidation occurs directly from monomeric circles (74). Several P2-specific attachment sites, different from that of lambda, exist on the bacterial chromosome (3, 14, 33, 55). Genetic recombination in P2 mixed infections occurs with extremely low frequency (11, 15): the P2 genetic map (13, 61) had to be obtained with the help of UV irradiation to stimulate recombination. The P2 regulatory circuit, lysis versus lysogeny, is simpler (78) than that of lambda. The P2 prophage integration mechanism, while a typical site-specific recombination, has special features (20, 30, 46), in part responsible for blocking detachment of the prophage on derepression. No case of specialized transduction is known in P2 on the other hand, its complementary eventuction of a bacterial marker—has been described (56). A great boost for P2 studies was the discovery in 1966 by Erich Six (81) of the remarkable satellite phage P4 and its complex interactions (“transactivation”) with P2 and P2-like phages (22, 29, 36, 65). A number of very intriguing observations, peculiar to P2, remain incompletely understood and deserve further study: striking metabolic effects on the frequency of lysogenization (18, 19) cell sensitization to a small molecular product (21) the complex interactions between P2 nonessential gene old, the host cell, and a coinfecting phage lambda (24, 44, 63, 72, 80) and others. Based on the results of screens of coliforms in Paris and Los Angeles (23, 53) and the Dhillons' extensive work in Hong Kong (40), it became clear that—to the extent that modern notions of segmental evolution (57) allow it— P2 and lambda are representative of two main groups of temperate phages for such bacteria. Within the P2-like group, phage 186 was studied in great genetic and molecular detail by Barry Egan and his students (41, 92) phage 299 was studied to a lesser extent by E. I. Golub (45). More recently, other phages obviously related to P2 have been encountered in several other species besides E. coli and as defective prophages in DNA sequencing studies (34).
The above recollections seem to indicate that at about the middle of the last century, starting before the formulation of the DNA structure and independently of the introduction and diffusion of “molecular” methods of analysis, there was a confluence of the rigorous approach of phage work with more traditional bacteriological perspectives, energized by the new remarkable findings about gene transfer. Studies of lysogeny were rather central in this process of broadening interest with respect to problems and materials. As a result, several new 𠇎xperimental systems” à la Rheinberger were developed in the fifties and sixties, lambda being the most successful. One is tempted to generalize these observations and suggest that it is the way of scientific progress to alternate between periods of broad and somewhat haphazard exploration and periods of highly focused in-depth analysis of particular problems or materials. On the one hand, as Francis Crick once wrote (38), w molecular biologists would care to be caught studying the colour of butterflies' wings… .” The tendency in molecular biology (as it was, mutatis mutandis, in classical genetics) is for one to analyze the experimental material to the lowest possible structural level and thus invest heavily in one's system. On the other hand, the naturalist looks open mindedly for what may happen to be there and how it might be related to what has already been seen: in a way, he scouts for new experimental systems.
A comment may be made concerning induction: not lambda's, that of philosophers. It is hard to see how our intelligent species would have ever evolved without trusting induction, yet there are everyday examples of the risks of excessive reliance upon inductive predictions. After the B. megaterium phage, a phage in Pseudomonas, and lambda in K-12 were found to be inducible by UV light, it was hard to believe that P2 was not. How do you prove a negative? Similarly, after seeing that both lambda and P2 prophages were present in single copies, physically integrated in the bacterial chromosome at specific sites, it was very surprising when P1 was found to be present as one unattached copy and yet be regularly transmitted to the bacterial progeny without losses (50) or Mu to be capable of inserting itself anywhere in the chromosome (85, 86). Perhaps most surprising, after the early efforts had convinced everyone that lysogens produce phage through the lysis of individual cells, was the discovery that filamentous phages are indeed continuously secreted by growing cells, without lysis (49).
Postscript. My first paper on lysogeny (5), describing the modified single-burst experiment and the isolation of P1, P2, and P3, also contained the formula of the LB medium which I had concocted in order to optimize Shigella growth and plaque formation. Its use has since become very popular. The acronym has been variously interpreted, perhaps flatteringly, but incorrectly, as Luria broth, Lennox broth, or Luria-Bertani medium. For the historical record, the abbreviation LB was intended to stand for “lysogeny broth.”
PDusk and pDawn
The pDusk and pDawn plasmids are engineered DNA systems that allow someone to control gene expression using light. Both pDusk and pDawn make R e d Fluorescent Protein (RFP), wh ich makes the colonies reddish in color. The difference is that pDawn turns on the RFP gene when exposed to light, while pDusk will only make RFP when kept in the dark . The pDawn system allows you to control the expression of RFP in the light. This kit teaches you many basic molecular biology techniques, while also giving you the ability to perform cool experiments using light to control gene expression.
The pDusk and pDawn systems are activated by blue light but since most white light contains blue light any sort of ambient light should work.
- 1 hour Make plates (set aside more time if it's your first time making plates)
- streak out bacteria onto an LB Agar plate (takes
Day of experiment overview
- Mix bacteria t ogether with the plasmids, and transformation mix (takes
Incubate and wait for growth
1 hour, but leave more time if it&rsquos your first time)
Step by step walk-through of making plates with photos at: https://goo.gl/7yzpA1
Agar plates provide a solid media nutrient source for bacteri a to grow on. The standard media that is used is LB (Luria Broth, Lysogeny Broth, or Luria Bertani Broth). This contains a carbon source, a nitrogen source, and salt (many strains of bacteria like salt!).
The top of the plate is the larger part.
- Find the tube labelled &ldquoLB Agar M edia &rdquo dump its contents into the 250mL glass bottle. (You will need to make plates out of each kind of media . Rinse bottle between media. )
- Using the 50mL conical tube labelled &ldquo Tube for Measuring &rdquo, measure and add 150mL of water to the glass bottle.
- Making agar is like making jello-- heat the agar to dissolve it, then it will solidify when it cools. Heat the bottle in the microwave for 30 seconds at a time, being careful not to let the bottle boil over. DO NOT SCREW THE LID DOWN TIGHT! (just place it on top and give it a slight turn).
- The media is completely dissolved when the liquid looks yel low . This should take about 2 -3 minutes total of microwaving. Take the bottle out , (caution contents hot), and let it cool until you are able to touch it without much discomfort. This will take 20-30 minutes.
- While the bottle remains somewhat warm, pour the plates. One at a time, remove the lid of 7 plates and pour just enough of the LB agar from the bottle to cover the bottom half of the plate. Put the lid back on.
Making Competent Bacterial Cells for Transformation
&lsquoCompetent&rsquo means the bacterial cells are able to intake foreign DNA. The cells&rsquo walls normally prevent things from entering , but mixing the bacteria with chemicals and salts that change the cells&rsquo walls, allow the cells take up DNA from the environment. This process is called a &lsquotransformation&rsquo. We put all the components into synthetic DNA to trick the bacteria into thinking that our DNA is its own DNA , so they make the RFP.
The bacterial transformation mix contains:
10% Polyethylene Glycol(PEG) 8000
PEG 8000 is thought to play several different roles in transformation, though nobody really knows for certain. Since both DNA and cell walls are negatively charged, they reject each other. PEG 8000 is thought to function by shielding the charge of the DNA, thereby making it easier to permeate the cell wall. PEG 8000 is also thought to help transport the DNA into the cell, as well as make the cell membrane itself more porous.
5% Dimethyl Sulfoxide (DMSO)
DMSO is sometimes used to treat ailments in humans. In a transformation it is thought to permeabilize the cell wall. Also, sometimes DNA folds into complex structures that make it more difficult to pass through the cell wall. DMSO also might help to break these DNA structures down.
25mM Calcium Chloride(CaCl 2 )
Similarly to PEG 8000, CaCl 2 is thought to shield and neutralize the negative charge of DNA, thereby making it more likely to enter into the cell.
- Take one of the tubes of dried E. coli BL21, add water to the top and shake till it is all dissolved. Next, using your pipette put 100uL of the bacteria solution onto a new LB plate you made and using an inoculation loop gently spread or &ldquostreak&rdquo the bacteria. Let the plate grow overnight
- Use a syringe to transfer 0. 1 mL of Transformation mix to a microcentrifuge tube with pDawn . Make sure you mix the transformation mix with the tiny droplet of pDawn DNA in the microcentrifuge tube. Repeat with with pDusk DNA using another syringe. (Syringes can be washed with soap and water for reuse!!)
- Using an inoculation loop, gently scrape an area of the size of a pencil eraser of bacteria off of your fresh plate and mix it into the transformation p Dawn mix. Mix until any big clumps have disappeared. Twirling the inoculation loop can work, but avoid making bubbles. Repeat with pDusk using a clean inoculation loop.
- Make a warm water bath for the next step. The water should be 42ºC (108ºF) water. You can approximate this temperature by using water that is warm, but comfortable enough such that you can still keep you hand in it.
- Add 1.5 mL of room temperature water (or fill to the top) to one of the LB media microcentrifuge tubes and shake to dissolve the LB.
- Using a clean syringe , add 0.5 mL of LB media to your competent cell mixture containing your DNA.
- Incubate the tube at 37 º C (99 º F) for 2 hour or 4 hours at room temperature. This step allows to bacteria to recover and replicate the DNA and perform the engineering process. Take an LB/ Kan plate out of the fridge and bring them to room temperature
- Pour half of the LB/competent cell mix to your LB/Kan plates and gently spread the bacteria around the plate with an inoculation loop. L et dry for 10 minutes before putting the lid back on.
- Flip the plate upside down to prevent condensation from forming and dripping onto your bacteria.
- Incubate the plate at room temperature for 24-48 hours. Keep pDusk in a dark place or wrap the plate with tin foil.
- If your transformation was successful, you should have a plate that looks like the one below. If you kept it in the dark the pDusk plate should look a little red. If you kept it in the light the pDawn plate should look red. If not, give it another shot, Science doesn&rsquot always work on the first try. Also, feel free to contact us at [email protected] and we will help you troubleshoot.
Now that you have colonies growing, you can do a couple of tests to see how pDusk and pDawn behave differently in light and in dark conditions.
The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or lysogeny broth medium. Liquid media are often mixed with agar and poured via a sterile media dispenser into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar (the exception being some species in the genera: Cytophaga, Flavobacterium, Bacillus, Pseudomonas, and Alcaligenes). Bacteria grown in liquid cultures often form colloidal suspensions.  
The difference between growth media used for cell culture and those used for microbiological culture is that cells derived from whole organisms and grown in culture often cannot grow without the addition of, for instance, hormones or growth factors which usually occur in vivo.  In the case of animal cells, this difficulty is often addressed by the addition of blood serum or a synthetic serum replacement to the medium. In the case of microorganisms, no such limitations exist, as they are often unicellular organisms. One other major difference is that animal cells in culture are often grown on a flat surface to which they attach, and the medium is provided in a liquid form, which covers the cells. In contrast, bacteria such as Escherichia coli may be grown on solid or in liquid media.
An important distinction between growth media types is that of defined versus undefined media.  A defined medium will have known quantities of all ingredients. For microorganisms, they consist of providing trace elements and vitamins required by the microbe and especially defined carbon and nitrogen sources. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.
A good example of a growth medium is the wort used to make beer. The wort contains all the nutrients required for yeast growth, and under anaerobic conditions, alcohol is produced. When the fermentation process is complete, the combination of medium and dormant microbes, now beer, is ready for consumption. The main types are
- cultural media
- minimal media
- selective media
- differential media
- transport media
- indicator media
Culture media Edit
Culture media contain all the elements that most bacteria need for growth and are not selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.
An undefined medium (also known as a basal or complex medium) contains:
- a carbon source such as glucose
- various salts
- a source of amino acids and nitrogen (e.g. beef, yeast extract)
This is an undefined medium because the amino-acid source contains a variety of compounds the exact composition is unknown.
A defined medium (also known as chemically defined medium or synthetic medium) is a medium in which
Examples of nutrient media:
Minimal media Edit
A defined medium that has just enough ingredients to support growth is called a "minimal medium". The number of ingredients that must be added to a minimal medium varies enormously depending on which microorganism is being grown.  Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow "wild-type" microorganisms. Minimal media can also be used to select for or against recombinants or exconjugants.
Minimal medium typically contains:
- a carbon source, which may be a sugar such as glucose, or a less energy-rich source such as succinate
- various salts, which may vary among bacteria species and growing conditions these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acids
Supplementary minimal media are minimal media that also contains a single selected agent, usually an amino acid or a sugar. This supplementation allows for the culturing of specific lines of auxotrophic recombinants.
Examples of lysogeny broth in the following topics:
- The most common growth media nutrient broths (liquid nutrient medium) or LB medium (LysogenyBroth) are liquid.
Plasmids and Lysogeny
- Both plasmids and lysogeny are used by bacteria and viruses to ensure transfer of genes and nucleic acids for viral reproduction.
- Lysogeny is the process by which a bacteriophageintegrates its nucleic acids into a host bacterium's genome.
- Lysogeny is utilized by viruses to ensure the maintenance of viral nucleic acids within the genome of the bacterium host.
- Lysogeny is one of two major methods of viral reproduction utilized by viruses.
- An example of a virus which can promote the transformation of bacterium from a nontoxic to toxic strain via lysogeny is the CTXφ virus.
Temperate Bacteriophages: Lambda and P1
- With phage the term virulent is often used as an antonym to temperate, but more strictly a virulent phage is one that has lost its ability to display lysogeny through mutation, rather than a phage lineage with no genetic potential to ever display lysogeny (which more properly would be described as an obligately lytic phage).
- In lysogeny, P1 can exist within a bacterial cell as a circular DNA, in that it exists by replicating as if it were a plasmid and does not cause cell death.
- During lysogeny, new phage particles are not produced.
- A unique feature of phage P1 is that during lysogeny its genome is not incorporated into the bacterial chromosome, as is commonly observed during lysogeny of other bacteriophage.
- This virus is temperate and may reside within the genome of its host through lysogeny.
History of Microbiology: Hooke, van Leeuwenhoek, and Cohn
- Lazzaro Spallanzani (1729–1799) found that boiling broth would sterilise it and kill any microorganisms in it.
- He also found that new microorganisms could settle only in a broth if the broth was exposed to the air.
- By boiling the broth beforehand, Pasteur ensured that no microorganisms survived within the broths at the beginning of his experiment.
- Nothing grew in the broths in the course of Pasteur's experiment.
- This meant that the living organisms that grew in such broths came from outside, as spores on dust, rather than spontaneously generated within the broth.
Pasteur and Spontaneous Generation
- In summary, Pasteur boiled a meat broth in a flask that had a long neck that curved downward, like a goose.
- The idea was that the bend in the neck prevented falling particles from reaching the broth, while still allowing the free flow of air.
- When the flask was turned so that particles could fall down the bends, the broth quickly became clouded .
- In detail, Pasteur exposed boiled broths to air in vessels that contained a filter to prevent all particles from passing through to the growth medium, and even in vessels with no filter at all, with air being admitted via a long tortuous tube that would not allow dust particles to pass.
- Nothing grew in the broths unless the flasks were broken open, showing that the living organisms that grew in such broths came from outside, as spores on dust, rather than spontaneously generated within the broth.
The Lytic and Lysogenic Cycles of Bacteriophages
- Those phages able to undergo lysogeny are known as temperate phages.
- Even though there are similarities between lysogeny and latency, the term lysogenic cycle is usually reserved to describe bacteriophages.
- Another method of bacterial culture is liquid culture, in which the desired bacteria are suspended in liquid broth, a nutrient medium.
- The experimenter would inoculate liquid broth with bacteria and let it grow overnight (they may use a shaker for uniform growth).
Tissue Culture of Animal Viruses
- Viruses cannot be grown in standard microbiological broths or on agar plates, instead they have be to cultured inside suitable host cells.
Complex and Synthetic Media
- Luria Broth as shown here is made with yeast extract, as yeast extract is not completely chemically defined Luria Broth is therefore an undefined media.By Lilly_M [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0-2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons
Minimal Inhibitory Concentration (MIC)
- MICs can be determined on plates of solid growth medium (called agar, shown in the "Kirby-Bauer Disk Susceptibility Test" atom) or broth dilution methods (in liquid growth media, shown in ) after a pure culture is isolated.
- For example, to identify the MIC via broth dilution, identical doses of bacteria are cultured in wells of liquid media containing progressively lower concentrations of the drug.
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VWR Life Science Premixed LB Broth
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Is there a difference between Luria Broth and Lysogeny Broth? - Biology
Rich media used for routine culture of E. coli and other bacteria at high cell densities.
|10 g||50 g||Tryptone|
|5 g||25 g||Yeast Extract|
|10 g||50 g||NaCl|
Add dH2O to final volume. Autoclave. If mixing up large batches and aliquoting, be sure to add exact volumes to final media bottles so that they will be ready for addition of antibiotic to known concentrations.
This recipe is from Miller JH. (1992) A Short Course in Bacterial Genetics. CSHL Press. Handbook Unit 25.5.
Note: Be aware that (1) there are several other formulations that may be called LB but vary the amount of NaCl. Using the wrong one can cause large changes in growth. (2) LB was originally supposed to stand for "Lysogeny Broth" and you may also see it called "Luria Broth" (more about this).
Expected results: E. coli strains grow to 5×10 9 cells/ml final density in LB.
Variant: 0.1×LB is made with 1 g Tryptone, 0.5 g Yeast Extract, and 10 g NaCl per liter. (It is 0.1× in the nutrients, but not the salt!)
Is there a difference between Luria Broth and Lysogeny Broth? - Biology
When it comes to growing bacterial colonies, LB-agar steps up to the plate &ndash but first you have to get it into a gel state (a situation to which its friend agarose can surely relate!) &ldquo-ose&rdquo is an ending that&rsquos usually used to indicate something&rsquos a sugar &ndash and agarose is a sugar &ndash but so is agar! So what&rsquos the difference? They&rsquore related, but they differ by more than just a few letters and those differences make them useful for making gel matrices for different tasks &ndash agarose gels for separating DNA fragments by size and agar gel plates for growing bacteria)
A gar is a fish-like thing and AGAR (aka agar-agar (seriously!)) is a mixture of 2 sugars &ndash agaropectin & the one we&rsquore more familiar with, agarose. So you get agarose by purifying agar. And to understand why you&rsquod go through that trouble sometimes, but not other times, it helps to know a bit more about what these sugars are.
Agarose is a polysaccharide (&ldquopoly&rdquo means many & saccharide&rsquos sugar, so a polysaccharide is a long chain of repeating sugar subunits joined together). It&rsquos an example of a polymer. Polymersare long chains of repeating subunits. Different polymers have subunits of different types (e.g. nucleotide subunits link up to give you the polymers we call DNA or RNA amino acids join to form proteins and monosaccharide sugars chain-ify to give make complex carbs (like agarose!))
Sugars have lots of hydroxyl (-OH) groups (which water happily sticks to, helping you form a gel &ndash an &ldquoinfinitely&rdquo interconnected (like 7° of Kevin bacon) polymer mesh containing water. (more to come)). Individual sugar units (monosaccharides) often adopt ring structures (as is the case in agarose) in which the -OH groups stick out like legs. Sugars can have the same &ldquolinkage&rdquo but have the linked groups sticking out in different ways & we use &ldquoL&rdquo and &ldquoD&rdquo to refer to which direction in space the legs point. More on such stereochemistry here: bit.ly/2Q8Dnax
Monosaccharides can use their -OH&rsquos to link together. Linking 2 gives you a disaccharide . Add a few more and you get oligosaccharides (oligo means few). Link lots and you get a polysaccharide (poly meaning many).
And speaking of many, the multiple -OHs mean there are multiple potential linkage sites (which we specify by which number &ldquoaddresses&rdquo on the sugar rings are joined). You can get &ldquobranching,&rdquo but in agarose, you have linear chains (of about 400 subunits). (Although branches can be introduced using &ldquocrosslinkers&rdquo to strengthen agarose so that it can do things like make size exclusion chromatography (&ldquogel filtration&rdquo) resin which can withstand the high pressures generated in FPLC (Fast performance liquid chromatography).
In agarose, the repeating subunit is a sugar duo (disaccharide) of galactose (D-galactose to be precise) linked to a modified galactose monosaccharide, 3,6-anhydro-L-galactose. We call this duo AGAROBIOSE (Linking a galactose to a glucose gives you lactose, a disaccharide you might be more familiar with).
In galactose, the rings have 6 sides with 4 -OH legs, 5 -H legs, & 1 methylhydroxyl (-CH₂OH) leg. We often don&rsquot draw the &ldquo-H&rdquo legs because they hide the more interesting legs that are capable of reacting. In agarose&rsquos modified galactose, 2 of the -OH groups have linked up & kicked out a hydrogen (&ldquoanhydro&rdquo) so that the methyl hydroxyl arm is bridged to an -OH arm forming a &ldquobridge&rdquo over the ring.
About 2/3 of agar is agarose but, agar also contains AGAROPECTIN. It&rsquos really similar (it has alternating D & L galactoses) BUT many of those galactoses have modifications. There are several different modifications including adding sulfate(s), pyruvic acid, or methyl groups, or sneaking in one of those linked-leg versions like&rsquos in agarose.
Unlike the consistent repeating nature of AGAROSE, it&rsquos only the alternating D- & L- galactose &ldquoskeleton&rdquo that&rsquos consistent in agaropectin. Its modifications are scattered throughout the chain (for instance,
every 10th is attached to a sulfate through an -O-sulfate linkage, but that&rsquos just on average, and it&rsquos not likely they&rsquore precisely, evenly, distributed). And, while the chains tend to be shorter than the agarose chains, their length is also variable, so, agaropectin&rsquos really quite a mix & you never know quite what you&rsquore gonna get!
Why use one over the other?
DNA has a lot of negatively-charged phosphate groups (phosphorus surrounded by 4 oxygens). This will serve the basis for them moving through the gel towards the positive end. So we need the gel to be neutral.
Agar has a lot of sulphate groups (sulfur surrounded by oxygens). These are also negatively-charged, so they can interfere with how the DNA moves through the gel. So it would make a bad matrix for electrophoresis.
BUT agarose is neutral, making a good matrix for electrophoresis.
BUT agarose is also more expensive because it has to be purified. So if you don&rsquot need to worry about the physical charge issue, might as well use something that has a lower *monetary* charge! Because agar requires less processing, its cheaper & perfect for use as a matrix to hold bacteria food!.
In agar plates, it&rsquos not the agar itself that&rsquos providing nutrients. That&rsquos one of the great things about agar &ndash *most* bacteria can&rsquot eat it. Instead, the agar just provides &ldquoscaffolding&rdquo to house the nutrients the bacteria need. And often those nutrients are provided through a liquid bacterial food &ldquogrowth media&rdquo called LB, which, no matter what your textbooks might say, (originally at least) stands for Lysogeny Broth. Sometimes initials for Luria, Lennox, or Luria & Bertani get credit for the name, but it really stands for LYSOGENY BROTH and its recipe was first published (by Giuseppe Bertani) in 1951. He was using it when studying lysogeny (a process where a bacteria-infecting virus called a bacteriophage (&ldquophage&rdquo) inserts its own DNA into a bacteria&rsquos DNA & bides its time until conditions are right for entering the lytic phage where it cuts itself out, makes lots of copies and bursts open the cell) http://bit.ly/2HLuB1S
The point of LB is basically to give the bacteria what they need to grow, divide, and do what we want (like make copies of DNA we put in them and/or make copies of proteins using instructions from DNA we put in them). And to do it cheaply.
Note: For the protein-making and large-scale DNA-making, we grow bacteria in straight-up LB liquid &ndash &ldquoin suspension&rdquo with shaking &ndash but when we need to isolate specific groups of bacteria that are all derived from the same &ldquoparent cell&rdquo and thus genetically-identical, we embed that LB into a gel so that different genetically-distinct &ldquocolonies&rdquo don&rsquot intermingle, but instead grow as gloopy dots. If you want to learn more about various media for the suspension stuff check out this post http://bit.ly/bacterialmedia but today I&rsquom going to focus on the gel-trapped form.
We don&rsquot give the bacteria 5-star cuisine. Instead, we want to spend as little money as possible while still giving them the nutrients they need. At a minimum, we need to give them a source of energy, something they can break down (catabolize) to make ATP &ndash such things can be sugars, proteins, fats. In addition to breaking things down, they need to be able to make things like proteins and DNA (do the anabolic part of metabolism). This requires nutrients that provide the elements needed like carbon and nitrogen, which thankfully you can get with a simple recipe that&rsquos sufficient for lots of bacteria.
There are 3 main components (though 2 of those components themselves have a lot of components.
- TRYPTONE -> this is a mix of peptides formed by the digesting a protein called casein with pancreatic enzyme -> this provides amino acids the bacteria can use to make new proteins
- YEAST EXTRACT -> this &ldquoautolysate&rdquo of yeast is basically just whatever happened to be in yeast (organic compounds including vitamins, trace elements, etc.) &ndash and if it was good enough for the yeast&hellip
- SODIUM CHLORIDE (NaCl)(table salt) -> allows for osmotic balance, transport, etc.
A few of the major LB formulations are the &ldquoMiller,&rdquo &ldquoLennox,&rdquo & &ldquoLuria&rdquo versions & they differ in the amount of salt they have. Miller & Bertani drown the bacteria in NaCl (10g/L) whereas Lennox just uses 5g/L and Luria just 0.5g/L -> such low salt recipes are good if you&rsquore using a salt-sensitive antibiotic. in the original paper, Bertani also added glucose, but most later recipes leave it out.
And speaking of leaving things out, we need to make sure we &ldquoleave out&rdquo bacteria we don&rsquot want, which we can do by &ldquoselecting for&rdquo the bacteria we do want using selection media, which contain things like antibiotics etc. that suppress the growth of things you don&rsquot want to grow. For example, when we put genes into bacteria, we normally do it in the form of circular pieces of DNA called plasmids. We design those plasmids to also have an antibiotic resistance gene, so we can spike the food with that antibiotic and it can still grow, but other stuff can&rsquot http://bit.ly/2tcW4ky
There&rsquos also differential media &ndash this allows for &ldquoscreening&rdquo as opposed to &ldquoselection&rdquo &ndash you don&rsquot keep things from growing, but you change how they appear &ndash for example, we use X-gal for blue-white screening http://bit.ly/2MxNPs2
After I put a plasmid with my gene into bacteria and get colonies, I pick a few of those colonies and put them in liquid LB (with antibiotic) to grow overnight to make lots of copies of the plasmid, then I can purify out those copies and send them for sequencing to check for typos before l enter the &ldquoexpression prep&rdquo part.
Regardless of what media you use, you need it to be sterile. So you autoclave it (stick it in a really hot, high pressure dishwasher) -> make sure the bottles aren&rsquot sealed tight or they&rsquoll explode (thankfully I haven&rsquot made this mistake) &ndash and don&rsquot re-tighten the lids until the bottles have cooled of or the lids will get stuck (I *have* made this one). Another mistake not to make -> don&rsquot add antibiotics before autoclaving, or you&rsquoll inactivate them. We usually don&rsquot add it until right before we&rsquore ready to use it.
In undergrad, I made all my own media, but here, we use so much of it, we have a &ldquomedia-maker&rdquo lab technician who&rsquos amazing and makes & sterilizes our bacterial growth media. You know something&rsquos been through an autoclave if the lines on its autoclave tape are black &ndash the tape is temperature-sensitive so it color-changes when it gets really hot. I really want to design a line of joke and/or trivia messaged autoclave tape &ndash so if the whole teaching thing doesn&rsquot work out, I guess I have a back up!