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Tuesday, March 5, 2013

Yeast Culture - cell number, Cell Size - Methods and Calculations

Growth and culturing yeast 

Humans have cultured the yeast S. cerevisiae for thousands of years. It is at or near the top of the “first domesticated organism” list. Beer and wine are considered by many to be the pinnacle of human achievement. The ancients in their wisdom fermented sugary foods they could gather as a means to preserve the nutritional value of fruits. The additional recreational value in the consumption of such fermented preparations did not go unnoticed. Small amounts of fermented material were used to inoculate foods in order to obtain reliable preparations.

Yeast can be grown on agar plates or in liquid using defined or complex media.






Feeding Yeast

Yeast can be grown in many kinds of media, in liquid cultures or on agar surfaces. Wild yeast need only a carbon source, ammonium as a nitrogen source and a few vitamins and off they go. Yeast can grow on many different carbon sources from sugars such as glucose (aka dextrose, which they like to ferment to ethanol), galactose, sucrose, raffinose, to other carbon compounds like glycerol and ethanol. Our lab strains havemutations that render them unable to grow on minimal medium (YMD, the Y stands for Yeast, the M for minimal and the D for dextrose). In such cases we may add nutrients they need and cannot make from scratch, for example the base uracil or the amino acid leucine. This method is called the "drop in" method where a supplement is added to minimal medium, eg YMD + leu. So the leucine requiring-strain will not grow on YMD but will on YMD + leu.

Some researchers use an alternative method in which a complete medium with everything even the most debilitated yeast strain needs is present in a mix of purified vitamins, nutrients and minerals. This is called "synthetic complete medium" (SC) and when it has dextrose it is called SCD. To identify a leucine requiring mutant we would "drop out" the leucine and make SCD – leu. In this case the leucine requiring strain would not grow on the SCD – leu, but would on SCD. I know this seems confusing, but we will do both in this class, it is good for budding yeast geneticists (ie geneticist of budding yeasts, and budding geneticists) to learn both ways. Really they are very much the same, but one needs to know what is in or missing from the medium to know the phenotype. So this extra challenge will serve to keep us all on our toes.

Rich media are also called undefined media or complex media. These do not contain known amounts of nutrients but are extracts of different things that are rich and contain all the goodies in various forms to support growth. They are not good for defining auxotrophic phenotypes because most auxotrophs grow readily on rich media.

Objective: Setting up cultures

Tools- sterile petridish, toothpicks, loop, YPD plates, yeast culture (bottle of red tail ale or a yeast culture provided)

Directions: Open culture (5-10 ml).

(If using beer with live yeast, leave bottom 10 ml and decant the rest in a glass. Save decanted beer (to drink later). Swirl bottom of bottle and pour into sterile Petri dish).

Streaking a plate: Flame the loop until it is glowing red. Let it cool in the air. Touch the side of the agar to make sure it is cool (hissing and spitting indicate that it is still hot). Take a loopful of liquid, or just a touch of a colony from the culture to be grown. Patch this in a small area of the agar plate near the side. Flame the loop again and let it cool. Streak through the patch of cells and out onto the agar surface. Repeat each time streaking through the last streak, in order to “dilute” the streak. The idea is to obtain isolated colonies that have arisen from a single cell.

For simple transfers, a sterile toothpick can be used to pick up a small amount of cells and make a small patch on a new plate.

Setting up a liquid culture: From a plate, take a sterile loop and pick up cells from a colony. Disperse into the liquid.

From a liquid culture; make sure the cells are well suspended. Measure a known volume using a sterile pipette (or with a sterile-tipped pipetter). If you wish to start the culture at a known cell concentration, you will need to know the cell number of the innoculum.

Yeast will grow at different rates depending on the temperature. Yeast will grow relatively quickly at temperatures from 37C down to 18C and more slowly at temperatures down to 5C.

Most Saccharomyces cannot survive temperatures in excess of 42C.

How to mate yeast:

Mix two strains of yeast on a plate on which they can both grow (YPD). Incubate at least 6-8 hours, better overnight or 24 hours. Patch or streak to selective medium on which only the diploid and not either haploid, can grow. Test that the strain is diploid by confirming that it sporulates. Multiple crosses can be done simultaneously by crossreplica plating.



YP-Media/ Plates

            Ingredients                                                                 Amount

            Yeast extract                                                                10 g
            Peptone                                                                       20 g
            Bacto-Agar                                                                   20 g
           
            Water                                                                          900 ml

Place a magnetic stir bar before autoclaving.
2          Separately sterilize
            Glucose                                                                       20 g
            Water                                                                          100 ml
Autoclave solutions 1 and 2 separately Mix the two thoroughly and then pour plates.

YPD liquid media is made as above except that solution 1 does not contain Agar. Store solution 1 (liquid media) in 90 ml aliquots and solution 2 in 100 ml aliquots.

YM Plates/media

            Ingredients                                                                 Amount
           YM Media
1          Yeast Nitrogen Base                                                      6.7 g
            (without amino acids)
            Bacto-Agar                                                                  20 g
            Water                                                                         900 ml
2          Separately sterilize
            Glucose                                                                      20 g
            Water                                                                         100 ml

Autoclave solutions 1 and 2 separately and Mix the two thoroughly and then pour plates.


Use of a microscope (Bright field and phase contrast)

Our microscopes can be used for either conventional bright field or phase contrast microscopy. If a specimen is pigmented or stained, use the bright field adjustment. If a specimen is unstained, as most of our yeast preparations will be, use a phase contrast adjustment. Phase contrast is a way to get image contrast using the differing refractive indexes of the different materials in the biological sample. It uses constructive and destructive interference to make different parts of the sample look brighter or darker, respectively.

To make this work, the scope needs to be properly adjusted! YOU must learn (or perhaps re-learn) your way around the microscope. It isn't that hard, and unless you are a brute and force parts that obviously won't move, you won't break it. Two simple rules are "don't force it" and "keep your mitts off the glass parts" (use lens paper).

To use the microscope, please pay close attention to the following steps:

Illumination: A small unit called a transformer converts the 120 volt from the standard AC outlet into smaller voltages (2.5V, 3.5V, 4.5V, 5.5V and 8V) depending on how you insert the plug into the transformer. See the diagram.

The different voltages correspond to illumination brightness- the higher the voltage the brighter the light in the microscope. As you change from low to high power (10x to 100x), you may need to use brighter light. Therefore use the lower voltages for the lower magnification objectives (10x) and use the higher voltages for the 100x objectives.

To start out, insert the microscope plug into one of the lower voltages (on the transformer) since you will be using low magnifications first. Then plug the transformer into the main power outlet and turn the switch on- the lamp should go on.

Carefully prepared wet mounts will be a aid to you as you familiarize yourself with this instrument. Slides and coverslips should be clean. An air bubble under the coverslip will make it easy to focus on the specimen plane. Excess liquid should be blotted with a wipe.

Begin observing cells with the 10x objective and increase magnification as needed.

Typical sequence of action for alignment is:

Clamp slide onto microscope stage and position slide using the mechanical stage knobs so that the specimen appears to be in the optical path

Focus objective on an air bubble or specimen.


Use a culture of E. coli and a culture of yeast

Yeast cell number determination: How many yeast do I have??

Goals

1. Learn to determine yeast cell number by at least two methods:

Petrof-Hauser counting chamber

Optical density at 600 nm (light scattering)

2. Determine over what range of optical density measurements is cell number proportional to optical density at 600nm

3. Determine cell number represented by OD600=1

Objective: count yeast cells in culture. Determine the relationship between cell number and OD600

We will use two different methods of determining cell number: optical density at 600 nm and direct microscopic cell counts using counting chambers.

We will compare these methods and calibrate them to each other.

Stationary cultures of yeast will be available. Medium for diluting yeast to different cell concentrations will also be available.

Method1- optical density measurements:

1 Turn on the spectrophotometer and set the wavelength to 600 nm

2 Using the stationary culture, make the following dilutions: 1:5. Then use the 1:5 dilution and further dilute it two-fold each time till you obtain a 1:160 dilution.

3 Zero the spectrophotometer using the dilution medium (diluent) and the undiluted culture as well.

4 Measure the OD of each dilution and note the values.

Method 2 (Optional): Viable cell count

After measuring the OD of the culture, plate an appropriate volume of diluted cell suspension so that 50-300 cells are spread on the plate. Use the following factor to estimate this: 1 OD = 3x107 cells/ml

To be sure you are in the right range, plate 5 times more and 5 times less than your best estimate.

After the plates dry, incubate at 30C for 2-3 days. Count the colonies

Method3- Petrov-Hauser counting chamber

1 Place the cover slip on the chambers (the long way) so that it covers as much of both of the mirrored chambers as possible.

2 charge the chamber with a minimum volume of cell suspension derived from the dilution series of the culture (usually 10-12 ul) by carefully placing the pippetteman tip in the groove just below the coverslip and pushing the fluid in slowly until the chamber is full (but not more).

3 Place the chamber on the microscope. You should see the grid like this if you are under 320-400X magnification (the 40X objective with either 8X or 10X eyepieces:

The entire area of the grid is laid out like this



4 Count the cells in an appropriate grided area. If there are few cells, count all 25 of the  



squares bounded by triple lines. If there are more cells, count only 5 squares (the center and four corner). If there are many cells, count only the center square. Shoot to count about 100 cells per sample no matter how many squares you are counting (Why?).

5 multiply the cell number by the volume factor to determine cells per milliliter.

The chamber grid is 1 mm by 1 mm and the volume above the grid is 0.1 ul. Don’t forget to multiply if you counted only 1 (X25) or 5 (x5) squares. If you have 137 cells in 25 squares, how many cells per ml do you have in your culture? Remember that a ml is equal to 1 cubed centimeter. The volume you are sampling here is 0.1 cubed mm. Can you figure the conversion factor of this volume to a ml?


Cell depth is 0.1 mm

Volume is 0.1 ul

Ruling pattern is 1/400 sq mm

Rulings cover 9 sq mm. Boundary lines of the Neubauer are the center lines of the groups of three. The central sq mm is rules into 25 groups of 16 small squares, each group separated by triple lines, the middle on of which is the boundary. The ruled surface is 0.1 mm below the coverglass, so that the volume of the 16 small squares is 0.0025 cubic mm.

The number of cells per cubic mm = number of cells counted per sq mm x dilution x 10

The number of cells per ml = number of cells counted per sq mm x dilution x 10,000

1 milliliter (ml) = 1000 cubic millimeters
1 microliter (ul)= 1 cu mm

Cell size determination: How big is yeast??

Look at yeast under the microscope.

There are three standard cultures of yeast- a haploid with mating type "a", another haploid with mating type "" (alpha), and a diploid.

Source
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