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I would like to grow E coli cells on cellophane on top of LB plate, and then grow yeast cells on the same LB plate. Does (or how well) yeast grow on LB plates? Is there a composition media that can be good for both E. coli and yeast? Has anyone done this experiment?
Not well, they need dextrose. Use YBT:
20 g Casein Peptone Tpe-M 10 g Yeast Extract 20 g Dextrose 17 g Agar
q.s. to 975 mls in di-water.
pH to 6.2 w 5M NaOH
q.s. to 1L with di-water.
Autoclave for 45 min at 121C
Aseptically dispense in Petri dishes.
Store at 4C for up to 12 weeks.
If you want to grow Co-culture do so in LB supplemented with 20g/L Dex.
4.2: Yeast growth media
- Contributed by Clare M. O&rsquoConnor
- Associate Professor Emeritus (Biology) at Boston College
For routine culture, scientists usually use rich media that supply all the nutrients that cells need to grow. The individual components of rich media are often undefined.For example, yeast are commonly grown in a medium known as YPD, which is simple and inexpensive to prepare. The &ldquoY&rdquo in YPD refers to a yeast extract, which contains the water-soluble compounds generated when yeast are forced to self-digest. (Those of you who have visited Australia may have encoun- tered yeast extract in the popular spread, Marmite.) The &ldquoP&rdquo refers to peptone, a mixture of peptides and amino acids prepared by digesting animal protein with proteases. The &ldquoD&rdquo refers to dextrose, or glucose, which is the favored carbon source of yeast (Sherman, 2002).
Because YPD is composed largely of crude extracts, its composition may show significant batch-to-batch variation. This variation is rarely a problem, because YPD contains more than enough essential nutrients to satisfy the metabolic requirements of cells. Many experiments, however, require media with a defined composition. To meet this need, the yeast community has developed a variety of synthetic media. Individual components of the synthetic media may be manipulated to suit the needs of an experiment. (Later in the semester, we will use defined media to select for particular genotypes.)
Yeast can be grown in liquid cultures or on the surface of plates containing solid media. Agar is usually used to solidify liquid growth media when preparing plates. Strains are typically maintained on agar plates for routine use. Cells grow in colonies on plates. The cells in a colony are genetically very similar, if not identical, because they are derived from the same progenitor cell. Most yeast strains can be stored on plates in the refrigerator for several months with minimal loss of viability.
How to make LB agar plates
- Weigh out 7.5 g agar, 5 g tryptone, 5 g sodium chloride (NaCl) and 2.5 g yeast extract and add to a 1 L Duran bottle.
- Measure out approximately 400 mL of distilled water and add to the Duran bottle.
- Shake the bottle to dissolve the reagents.
- Once the reagents have fully dissolved, adjust the pH to 7.0 by using sodium hydroxide (NaOH) solution (1 N).
- Once the pH is adjusted, top up the solution to 500 mL by using distilled water.Optional: set a water bath to 55 o C.
- To sterilise, autoclave the solution on a liquid cycle (20 min at 15 psi).
- Leave the solution to cool to approximately 55 o C, or warm enough to be held in your hand without burning it.If you do not have enough time to keep an eye on the temperature, put the bottle into the water bath. This will prevent the solution from setting prematurely.
- When cooled, add the appropriate antibiotic into the solution and swirl to mix.
- Prepare Petri dishes in a sterile environment (surfaces cleaned with 70% ethanol etc.). Carefully pour a thin layer of solution into the Petri dishes to cover the bottom of the plate (approximately 10 – 20 mL per plate). Try to avoid transferring or creating any bubbles.
- Leave the plates to set before storing them in the fridge (see below).
Does yeast grow on LB agar plates? - Biology
Yeast have simple nutritional needs. Unable to carry out photosynthesis, they require a reduced carbon source which can be as simple a compound as acetate. In addition, they also require a nitrogen source such as ammonium sulfate. Yeasts can use a variety of organic nitrogen compounds, including urea and various amino acids. The only other complex compound that they require is the vitamin, biotin. Of course, they also require a variety of salts and trace elements.
Another characteristic of most yeast, including S. cerevisiae, is that they divide by budding, rather than by binary fission (Byers 1981). A small bud emerges from the surface of the parent cell and enlarges until it is almost the size of the parent.
While this is happening the chromosomes of the parent replicate. At mitosis, when the nucleus divides, one of the nuclei is transferred to the bud, and then the two cells separate.
Schizosaccharomyces pombe, another popular yeast, is remarkably similar to S. cerevisiae, except that it grows as cylindrical cells that elongate and split in half by binary fission, much like bacteria. Its cell division is so convenient to observe that this yeast also has potential for use in teaching biology.
The yeast life cycle, like that of all higher organisms, includes a step known as meiosis, where pairs of chromosomes separate to give new combinations of genetic traits.
Ascomycetes, such as baker's yeast, are popular for genetics research because the ascospores they produce in each ascus are the products of meiosis. When yeast are nutritionally stressed, for example by deprivation of either a carbon source or a nitrogen source, diploid yeast will sporulate. The diploid nucleus goes through meiosis, producing four haploid nuclei which are then incorporated into four stress-resistant ascospores, encapsulated in the ascus(see Figure 1). This packaging of the four meiotic products makes genetic analysis particularly simple.
The ease with which baker's yeast can be maintained as either haploid cells or diploid cells is another characteristic that makes them attractive to geneticists.
What Are Yeast Good For?
People have used yeast, undoubtedly one of the earliest domesticated organisms, for controlled fermentation of food and drink and for leavening in baking throughout recorded history. Today, they are also used in a variety of commercial fermentation and biomass conversion processes. Their usefulness is based on their ability to convert sugars and other carbon sources into ethanol in the absence of air (anaerobic), and into carbon dioxide and water in the presence of air (aerobic). Ethanol is a valuable alternative to petroleum as a fuel and as a raw material for the manufacturing of many important commercial chemicals.
Yeast is also good food. It is rich in protein and is an uncommonly good source of the B vitamins. It provides a valuable source of nutrients that are important in low-meat or vegetarian diets. But while few emanations from the kitchen are quite as tantalizing as the yeasty aroma of baking bread, most people agree that pure yeast tastes pretty bad.
Other genera of yeast also have practical uses. Some can use hydrocarbons, such as petroleum, as a carbon source. These organisms can literally convert petroleum into protein. They are being used to remove petroleum as a pollutant from the environment and to convert low-grade hydrocarbons into protein for consumption by animals.
Is There Anything Bad About Yeast?
Yeast can indeed be germs. In fact, one extremely common pathogenic yeast, Candida albicans, is carried by most people in a benign form (Fincham & Day 1971). While in normally healthy people it is harmless, in those whose immune response is weakened it can become infectious and turns into a serious pathogen. These infections are particularly hard to control in humans because yeast metabolism is so similar to ours. Drugs that are toxic to the yeast are also toxic to people. Another particularly nasty pathogenic yeast, Cryptococcus neoformans, produces a life-threatening meningitis. These yeasts are known as "opportunistic pathogens" because they are a serious threat only to people with impaired immune responses like those who have AIDS.
How Do Yeast Grow?
Both haploid and diploid yeast cells divide by budding (see Figure 2). The cell division cycle begins with a single, unbudded cell (Pringle & Hartwell 1981 Byers 1981). This cell buds, the bud grows to nearly the size of the parent cell, the nucleus divides, and the two cells separate into two unbudded cells. The cycle then begins again for both of the cells. The result is an exponential increase in the number of cells with a doubling time equal to the mean cell-division-cycle time. This varies with the strain, the growth medium, and the temperature, but can be as short as one hour. At this rate, a single cell can grow into a barely visible colony in one day.
The growth behavior of yeast cultures is similar to that of bacteria. When a growth medium is inoculated, the cells require a period of preparation before they start dividing. Following this lag period which may be up to several hours they rapidly enter the exponential phase during which their number and mass double at equal time intervals. After a period of growth at a relatively constant exponential rate, some environmental condition becomes growth limiting so that the rate of increase diminishes and growth eventually stops. The population and mass become constant. The culture remains stationary and the cells remain viable for several hours if the culture is refrigerated the cells remain viable for months. Eventually the cells die, and at room temperature or warmer they will undergo autolysis: their own digestive enzymes become active and they literally digest themselves, reducing their proteins and nucleic acids to their simpler components they produce a particularly unpleasant stench in the process.
Normal yeast can grow either aerobically, in the presence of oxygen or anaerobically, in the absence of oxygen. Under aerobic growth conditions they can support growth by oxidizing simple carbon sources, such as ethanol, acetate or glycerol. If they have adequate oxygen, they will completely oxidize their carbon sources, usually sugars, to carbon dioxide and water. However, under anaerobic conditions, deprived of oxygen, yeast can convert sugars only to carbon dioxide and ethanol, recovering less of the energy. In either case, growth will be limited by some essential nutrient or the accumulation of the toxin.
Yeast grow equally well in liquid media or on a nutrient surface such as an agar plate or an exposed surface of some kind of food. In liquid they must be stirred or shaken if they are to remain aerobic otherwise, they settle to the bottom of the container, consume the dissolved oxygen, and grow anaerobically. On a nutrient surface in a ventilated container, they grow aerobically with each cell forming a visible colony of up to 100 million cells within 2 or 3 days.
The Yeast Life Cycle: A Complete Sexual Cycle
In sexual organisms, the life cycle (see Figure 3) is composed of a series of events that alternate between a haploid phase and a diploid phase (Fincham & Day 1971). The transition from the diploid to the haploid phase is the consequence of a specialized cell division -- meiosis -- in which the nucleus divides twice following a single replication of the chromosomes. Meiosis yields four haploid nuclei. During meiosis, paired homologous chromosomes in the diploid nucleus interchange parts and are distributed into the haploid nuclei yielding new combinations of genetic traits. In higher organisms a germ-cell line is differentiated from the body (somatic) cells. The germ-cell line produces the reproductive cells that form the gametes. The gametes are the cells which carry the genetic information to the next generation while the somatic cells form the rest of the organism and play no direct role in heredity.
The transition from the haploid to the diploid phase results from mating (sexual conjugation) between gametes. This leads to the formation of a zygote in which two parental haploid nuclei fuse to form a diploid nucleus. In higher organisms, the gametes are differentiated germ line cells. In higher animals, different gametes are produced by individuals of opposite sex. However, in higher plants, they are produced by different structures, either on the same plant or on different plants.
In yeast, as in other sexual microorganisms, the complete sexual cycle is present but is much simpler than in higher plants and animals. Permanent differentiation between germ line and somatic cells does not occur. Instead, transient differentiations occur under appropriate conditions to facilitate the essential events. The simplicity of the sexual cycle in yeast and the ease with which all the events can be manipulated and observed provides a unique opportunity for teaching this normally obscure and abstract process through simple, direct, concrete observations.
Mating in yeast which is mediated by diffusible molecules -- pheromones -- can be readily demonstrated (Manney, Duntze & Betz 1981). When cells of opposite mating type are mixed on the surface of agar growth medium in a petri plate, changes become apparent within two to three hours. As each type of cell secretes its pheromone into the medium, it responds to the one produced by the opposite type (MacKay & Manney 1974). They each respond by differentiating into a specialized functional form, a gamete. The cells stop dividing and change their shape (see Figure 1). They elongate and become pear-shaped. These distinctive cells have been termed "shmoos" because of their resemblance to the mythical but lovable animals in the "Li'l Abner" comic strip. Cells of opposite mating types that are in contact or close proximity join at the surface and fuse together forming a characteristic "peanut" shape with a central constriction -- two shmoos fused at their small ends. The two haploid nuclei within each joined pair fuse into a diploid nucleus, forming a true zygote. The diploid promptly buds at the constriction, forming a characteristic "clover leaf" figure. One can easily observe all of these stages under the microscope.
The mating pheromones that are secreted by haploid cells are small peptide molecules that diffuse through agar (Betz, Manney & Duntze 1981). Consequently, their existence and their effects on cells of the opposite mating types are easy to demonstrate. If cells of the mating type are grown overnight on agar medium, a high concentration of the pheromone accumulates in the agar surrounding the growth. Then if cells of the mating type a are placed on this agar, they begin to undergo the "shmoo" transformation within a couple of hours. The effect is quite striking. One can demonstrate the same effect in a liquid medium in which mating type cells have been grown. In fact, when yeast were first studied yeast biologists used these simple methods to isolate and characterize the pheromones.
Shmoos then, are the gametes in yeast. They differentiate from normal vegetative haploid cells only when a cell of the opposite mating type is present. In a like manner, any diploid cell can go through meiosis forming haploids which have the potential to become gametes (Esposito & Klapholz 1981 Fowell 1969b). Meiosis is part of the process of sporulation which is initiated when diploid cells are transferred to a nutritionally unbalanced medium. But the changes become apparent under the microscope only after three to five days when the asci become quite distinctive. Theoretically, all asci should contain four spores but in practice, many contain only two or three. The ascus has a lumpy shape, much like oranges inside a cloth bag. Treating the sporulation mixture with a readily available crude preparation of digestive enzymes from garden snails will remove the wall of the ascus, liberating the spores. When the spores, either within the ascus or after being liberated, are returned to a nutritionally adequate environment, they germinate and undergo vegetative growth in a stable haploid phase. Haploid strains occur in two mating types, called a and (alpha). Within each ascus, two spores are normally mating-type a and the other two are . When a cell of one mating type encounters one of the other mating type, they initiate a series of events that leads to conjugation. The result is a diploid cell, which grows by mitotic cell division in a stable diploid phase. If one merely transfers a sporulated cell culture to growth medium the result is a mixed population of haploid strains and new diploid strains which are analogous to the progeny from a cross between diploid higher organisms.
Normally, yeast geneticists isolate the spores, either randomly or by micromanipulation, to prevent the haploid strains from mating and forming the next generation. This degree of control and the ability to observe the genetic traits in the haploid phase makes genetic analysis in yeast powerful and efficient. The analysis of random populations of spores, while time-consuming, is simple enough for high school students, and is particularly well-suited to individual projects for advanced students.
DIYeast: Creating Plates
So you’ve acquired some wild yeast and bugs from your backyard, fruit or barrel room. Maybe you’ve brewed with it and now have your own unique house culture. That’s awesome. You are a rock star.
But what do you really have? Is it wild yeast? Is it something funky, like Brett? Is it all of the above? What’s causing that sour fermentation, if at all? Mixed wild cultures are wonderful, and are as close as we’ll ever get to replicating ancient fermentation practices.
It’s not until you isolate individual pure strains that you’ll be able to get some kind of control over your microbes and be able to accurately reproduce similar batches. You’ll also learn what makes each strain tick, and what beer styles and ingredients they meld with best. Plus, once you isolate your own yeast strain, everyone will think you’re a whiz kid.
Before proceeding, checkout Bootleg Biology’s Backyard Yeast Wrangling Tool Kit. Everything the experimental homebrewer needs to capture wild bugs, create agar plates, and isolate wild yeast.
Star San (put some in a cheap plastic spray bottle to clean your work surface)
Plastic Sterile Petri Dishes with Lids (glass dishes are reusable but more expensive)
Parafilm (stretchable plastic wrap is a good substitute)
Bunsen or portable burner (using a gas stove in your kitchen is cheaper and almost as effective)
- Dark/warm area to incubate plates (the warmer it is, the quicker you’ll know if your plate is contaminated)
Antimicrobial UV Light (not required) 2
Almost all of these items are already in your kitchen or your homebrew toolkit…with the exception of petri dishes. Plastic petri dishes are inexpensive and easily found on Amazon. They typically come in 60mm or 100mm diameters. If you’re trying to isolate microbes from a mixed culture like a Wild Yeast Starter, it’s probably best to go with the larger size dish to increase the likelihood of isolating a strain.
Agar is a gelatinous-like substance made from seaweed that when boiled with wort causes the mixture to set quickly into a solid, jelly-like medium, that individual microbe colonies can grow and feed on.
Creating a Simple Agar Plate: 3
- 300 mL of 1.040 (or lower) wort (If using extract, boil water and DME mixture before adding other recipe ingredients)
- 5 grams of agar agar powder (Add more if liquid fails to set)
- Dab of yeast nutrient (other sources of Amino Acid can be added due to their benefit to yeast metabolism, but aren’t required)
- Of course, you can also skip the recipe and grab Bootleg Biology’s Wild Yeast Agar Blend
- Heat wort in a small pot up to near boiling and stir in agar.
- Bring to a boil. Agar and wort won’t successfully bind if not brought up to a boil.
- Let cool for several minutes until temperature is in the low-100’s Fahrenheit or approximately 5-10 minutes. Watch your pot closely, since agar can set very quickly the medium still needs to be a thick liquid when poured into the petri dish.
- Pour a small layer of liquid on to the petri dish and move to your draft-free storage area. It’s important to let the liquid cool before covering with petri dish lid to prevent condensation from forming on the lid, which helps reduce the likelihood of mold growth.
- If a good agar/wort ratio was used, the agar plates should set within a few minutes. If you find your plates aren’t setting, you may need to reboil your mixture with more agar added.
- Wrap the edges of the plate lid with stretchable plastic wrap or stretched 1/2 inch strips of Parafilm. This will help reduce agar plate contaminants and prevent the plate from drying out if you don’t use the plate shortly afterward.
- Leave your plates upside down (prevents condensation from falling on your agar medium) for a couple days in a warm, dark place to incubate. This will help determine which plates are contaminated with mold spores or other uninvited guests, making them unusable 2 . When you’re done, store upside down in a resealable plastic bag and refrigerate.
Remember, you’re not working in a clean room with a hood, you will get contaminated plates. So don’t be sad when some of the plates you’ve created look like this after incubation:
Now that you’ve made your own agar plate, the last thing to do is to Isolate Your Yeast.
I. Making culture media requires patience and attention to detail. Watch Video 1: Making Microbiological Media
Watch Video 1: Making Microbiological Media by Bio-Rad. URL:https://youtu.be/BH4ESgWU_Eo
II. Pouring Agar plates. Watch Video 2: Solid Media Preparation, video was filmed at NC State Microbiology labs.
Watch Video 1: Solid media preparation, video was filmed at NC State Microbiology labs. URL:https://youtu.be/P7M_MCXbZjc
LB Agar Lennox ( MB38901 )
Description: Recommended medium for the test of Escherichia coli in molecular genetics studies. LB Agar Lennox is nutritionally rich medium developed by Lennox for the growth and maintenance of pure culture of recombinant strains of E. coli. Tryptone provides nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is source of vitamins, particularly the B-group. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Bacteriological agar is the solidifying agent. If desired, antibiotics can also be added.
Directions for use: Suspend 35 grams of medium in 1 L distilled water. Mix well and dissolve by heating and stirring. Boil for one minute until complete dissolution. Sterilize in autoclave at 121ºC for 15 minutes. Cool to 45-50ºC, mix well and dispense into plates.
– Tryptone: 10 g/L
– Sodium Chloride: 5 g/L
– Yeast Extract: 5 g/L
– Bacteriological Agar: 15 g/L
(Final pH 7.0± 0.2 at 25 ºC)
Soluble in: water
Storage: Store at 2-25 ºC. Once opened keep powdered medium closed to avoid hydration. Protect form light and moist
An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics. 
|Related items||Petri dish|
Individual microorganisms placed on the plate will grow into individual colonies, each a clone genetically identical to the individual ancestor organism (except for the low, unavoidable rate of mutation). Thus, the plate can be used either to estimate the concentration of organisms in a liquid culture or a suitable dilution of that culture using a colony counter, or to generate genetically pure cultures from a mixed culture of genetically different organisms.
Several methods are available to plate out cells. One technique is known as "streaking". In this technique, a drop of the culture on the end of a thin, sterile loop of wire, sometimes known as an inoculator, is streaked across the surface of the agar leaving organisms behind, a higher number at the beginning of the streak and a lower number at the end. At some point during a successful "streak", the number of organisms deposited will be such that distinct individual colonies will grow in that area which may be removed for further culturing, using another sterile loop. It is crucial to work sterile to prevent contamination on the plates. 
Another way of plating organisms, next to streaking, on agar plates is the spot analysis. This type of analysis is often used to check the viability of cells and performed with pinners (often also called froggers). A third used technique is the use of sterile glass beads to plate out cells. In this technique cells are grown in a liquid culture of which a small volume is pipetted on the agar plate and then spread out with the beads. Replica plating is another technique in order to plate out cells on agar plates. These four techniques are the most common, but others are also possible. It is crucial to work in a sterile manner in order to prevent contamination on the agar plates. Plating is thus often done in a laminar flow cabinet or on the working bench next to a bunsen burner. 
Making agar stabs for storage and transport of bacterial strains.
|5 g||Yeast extract|
|10 g||Sodium chloride|
Autoclave. Transfer to cryovials before media cools. Can store the vials at 4 C for a few months.