How to isolate yeast and what is the pure culture of yeast?

Potato dextrose agar medium.

Cultivation and isolation of yeast.

The objective of the experiment was to learn the techniques and methods of cultivating and isolating yeast.

Experimental principle] Most yeasts are saprophytic, and their optimal pH is 16, which is commonly found in environments with high sugar content, such as garden soil, vegetable soil and fruit peel and other plant surfaces. Yeast grows quickly and is easy to isolate and culture, and in liquid media, yeast grows faster than mold.

Taking advantage of the fact that yeast prefers an acidic environment, the culture medium of yeast is obtained by using acidic liquid medium (the advantage of this is that acidic culture conditions can inhibit the growth of bacteria), and then separated by scribing method on solid medium.

Experimental materials and utensils].

1. Peels of sugarcane, ripe grapes or apples, indigo dyeing solution, 1ml sterile straws, sterile petri dishes, etc.

2. Potato glucose agar medium:

Ingredients: potato (200 g), glucose (20 g), agar (15-20 g), distilled water (1000ml).

Preparation method: 1) Peel the potatoes first, slice them, weigh 200 grams and add 1000ml of distilled water, boil for half an hour, filter them with gauze, and make up the amount of distilled water to 1000ml

Make 20% potato juice.

2) Add agar to 20% potato juice, boil to dissolve, replenish with water and autoclave at 115 degrees Celsius for 20 minutes.

3) Add glucose to make potato glucose agar medium for culturing yeast.

3. Lactic acid potato glucose culture medium: the formula is the same as above, but lactic acid is added without agar, and the amount of lactic acid containing 5ml per 1000ml of culture medium is added, and the test tubes are divided into separate tubes.

Experimental procedure] l. Inoculation: Take a small piece of peel, do not need to rinse, directly connect it into the lactic acid potato glucose culture liquid tube, put it for 28-30, and culture for 24 hours, it can be seen that the culture medium becomes turbid.

2. Culture, take the above culture medium with a sterile pipette, inject it into another tube of lactic acid potato glucose culture medium, and place it for 28-30 and then culture for 24 hours or slightly longer (if it is too long, mold will grow).

3. Observation: Use the aseptic operation method to take a little bacterial solution and put it in the blue staining solution of the glass slide**, mix it well, add a coverslip to make a water-immersed piece, and first use a low magnification lens and then change to a high magnification lens to observe the morphology and budding reproduction of yeast.

Live yeast can reduce the blue so that the bacteria are not colored, and this method can be used to determine whether the yeast is alive or dead.

4. Separation: Potato glucose agar medium is dissolved and made into plates. The yeast culture broth was separated by the streak method to obtain a single colony. A single colony was picked and repeatedly re-streaked for separation and purification, and finally a pure culture was obtained.

Yeast MethodomycesThe pure culture is the progeny obtained from the culture and propagation of a cell.

A method of purifying and separating microorganisms by zoning and marking on the surface of the plate medium with an inoculation loop is to dilute the microbial sample on the surface of the solid medium for many times from point to line to achieve the purpose of separation.

Introduction to yeast knowledge:

Yeast is the earliest application of human beings, and it is also the most widely used microorganisms, and people often use its fermentation effect to make various leavened foods and wine. Yeast is a live yeast cultivated from a purebred culture in a factory, and the yeast containing about 70% water is called pressed yeast, and the one containing about 10% water is called dry yeast.

Yeast powder. It is mainly used for bread making or steamed buns to accompany medium flour.

The main function is to expand gluten and increase the volume of dough, so that the finished product has a tougher taste.

SummaryThe key to the success of yeast and bacteria isolation, purification and preservation is low temperature, drying, and vacuum.

Microbial culture preservation method:

1. Passage preservation method.

Some microorganisms die very quickly when subjected to treatments such as freezing or drying, so in this case, the only resort is to subculture preservation methods. Subculture is to regularly transfer and preserve strains after culture, which is the most basic microbial preservation method, such as the preservation of commonly used production strains such as yogurt. The shelf life temperature of most strains is 5.

Although the subculture preservation method is simple, its disadvantages are also obvious, such as: the tampon of the culture tube is often easy to mold; The genetic traits of the strain are prone to mutation; During repeated passages, the strain's pathological purity and ability to form physiologically active substances were reduced.

2. Liquid paraffin cover preservation method.

Compared with the previous method, this method preserves the strains for a longer time and is suitable for the preservation of molds, yeast trouser leakage, actinomycetes and aerobic bacteria. This method prevents dryness and weakens microbial metabolism by restricting the supply of oxygen.

3. Carrier preservation method.

That is, the method of adsorbing microorganisms on an appropriate carrier for dry storage. Commonly used methods are:

Soil Conservation Method.

Sand conservation method.

Silica gel preservation method, magnetic bead preservation method, bran preservation method, paper disc judgment (filter paper) preservation method.

1.Colony morphology observation was carried out according to the general microbial experiment guide, and the characteristics of the isolated fungal colonies were observed.

2.Observation of cell morphology.

According to the simple preparation method of the general microbial experiment guide, the low magnification lens was observed and drawn first, and then the high magnification was observed.

Expansion: What is the difference between a yeast colony and a filamentous fungus:

1. Yeast is a single-celled eukaryotic microorganism, and its morphology is usually spherical, oval, sausage-shaped, oval-shaped, lemon-shaped or lotus-shaped, etc., which is much larger than the single-celled individual of bacteria, generally 1 5 or 5 20 microns. Yeasts are flagellated and cannot swim. Yeast has a typical eukaryotic cell structure, with cell wall, cell membrane, nucleus, cytoplasm, vacuole, mitochondria, etc., and some also have microsomes.

The difference between colonies is very clear. The hairy surface is the colony of the filamentous fungus; If there is no hairiness on the surface, it is a yeast colony.

2. In more detail, the colony characteristics of most yeasts are similar to those of bacteria, but they are larger and thicker than bacterial colonies, the surface of the colonies is smooth, moist, viscous, easy to provoke, the texture of the colonies is uniform, the color of the front and back sides and edges, and the first part are very uniform, and the colonies are mostly milky white, a few are red, and some are black.

Colonies of filamentous fungi (mucor, penicillium, aspergillus, etc.) usually grow rapidly and have a cotton-like surface, or velvety, flocculent, rope-like, bundle-like; Most are white at first, then turn gray to brownish-gray. When it matures, a large number of spores will be produced on the surface, and smoke spore powder will scatter when touched.

Don't you really understand that you mean a mixture of two bacteria? The steps are as follows:

1. Prepare LB medium plates and YPD medium plates.

LB Formula: Tryptone 1%, Yeast Extract, NaCl 1%, Agar Powder 2%.

YPD formula: yeast paste 1%, peptone 2%, glucose 2%, agar powder 2%.

2. Use the inoculation ring to dip the bacterial solution to scribble on two plates.

3. Culture: LB plate 37 culture, YPD plate 28 culture.

4. After 18 hours of LB plate culture, obvious colonies can be seen with the naked eye. The colonies were smeared and microscopic, the E. coli colonies were selected, and the LB liquid medium was continued to be cultured, and then the single colonies were picked by scribing microscopy on the LB plate again, and the pure E. coli colonies were basically obtained.

5. After 24 hours of YPD plate culture, YPD plate 28 was cultured.

4. After 18 hours of LB plate culture, the colonies that are more obvious than Yuxiao can be seen with the naked eye. The smear microscopic examination of each colony, the yeast colony was selected, and the yeast colony was connected to the YPD liquid medium to continue the culture, and then the single colony was picked by scribing microscopy on the YPD plate again, and the pure yeast colony was basically obtained.

Brewing microbiology research has achieved the isolation and preservation of more than 5,000 yeast resources.

There are two main methods of traditional yeast preservation: first, the yeast is stored in a medium containing a certain proportion of glycerol in the shed, and stored at low temperature or liquid nitrogen; Second, add protective agents such as skimmed milk powder to the yeast, and freeze it and dry it for preservation. The traditional method can make the yeast successfully resuscitate and culture, and gradually scale up the production to achieve the expression and harvest of the target protein.

However, the traditional preservation method will cause a large number of yeast death, especially with the increase of storage time, the viability rate of yeast shows a significant downward trend, which affects the expanded yeast culture after recovery.

Background technology:

Yeast is a single-celled fungus, which is a common name for several families of single-celled fungi such as ascomycetes and basidiomycetes. Yeast is harmless, easy to grow, and can break down macromolecular substances into small molecule substances that are easy to use for cell metabolism, and belong to heterotrophs. Yeast is the earliest microorganism used in the history of human civilization, which can be used not only for brewing and production, but also as a model organism for genetic engineering and cell cycle research, and is favored by the majority of scientific researchers.

Saccharomyces cerevisiae was the earliest to be recognized in molecular genetics, and it was also the first yeast host for exogenous gene expression, and the exogenous gene expression system of Pichia chain plum as the host has developed rapidly in recent years and is also the most widely used.