Microorganism culture

Microorganism cultures. They have an increasing use in Biotechnology and environmental protection, so there is a need to maintain them so that the properties that make them important remain stable.

Table of Contents

Summary

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1 Microbial culture
2 Culture medium
- 1 Classification of culture media
  - 1.1 Depending on its consistency
  - 1.2 Depending on its composition
  - 1.3 Depending on the use that is given
- 3 Storage conditions of the culture media
- 4 Preparation of a culture medium
- 5 Bacterial growth
- 6 Cultivation of strict aerobic microorganisms
- 7 Culture of strict anaerobic microorganisms
- 8 Conservation of crops
- 9 Sources

Microbial culture

A culture is the growth of microbial populations in a controlled manner in an artificial medium.

Culture medium

Support that will contain the necessary nutrients, as well as optimal pH and humidity conditions that allow the development and growth of microorganisms.

Culture media classification

Depending on its consistency

Solids: A solidifying or gelling agent is added to the solid culture medium. They are prepared in an erlenmeyer flask, allowed to cool, and can then be transferred to either a plate or a tube.

The most common gelling agents are:

Agar Agar: It is an algae that is characterized by being inert against microorganisms and because it is liquid at boiling temperature and solidifies between 45-50ºC.
Liquids or broths: it contains the nutrients and, instead of adding the agar, they have the water that is used to dilute the culture medium. They are prepared in a tube.
Semisolids: contain agar (<5-10%). They are used when we want to observe bacterial mobility. It is done in a tube and the sowing is used with a straight handle.

Depending on its composition

Empirical: those in which its composition is not exactly known. They are no longer used in Laboratories.
Chemically defined: the composition and concentration of each of the nutrients that make up the culture medium are known exactly.
Complex or indefinite: They contain highly nutritious substances, but of indefinite composition. They usually carry substances such as meat extracts , peptone, yeast extracts , bovril … The advantage of this culture medium is that it contains many growth factors, so we can grow a large number of microorganisms in it. The downside is that we do not know what the microorganism is consuming. Examples:

Nutrient broth: meat extract + peptone + water

Nutritive agar: meat extract + peptone + water + agar.

Depending on the use that is given

Enriched: they are those means in which to a base medium certain nutritional substances are added that are necessary for the growth of a demanding microorganism. Example: blood agar.
Enrichment: used only when the microorganism in the sample is suspected to be in a very small number. It is provided with nutrients and is expected to grow in 6 to 8 hours. This is used, for example, when food contamination by Salmonella is suspected.
Differentials: it incorporates certain substances that allow us to differentiate between two types of microorganisms. Eg salt and mannitol agar: allows us to differentiate in the case of staphylococci, those that use mannitol in their metabolism from those that do not. If they use it, they produce acids, so the pH decreases and the indicator changes color.
Selective: they have certain substances incorporated that inhibit the growth of a microorganism and facilitate the growth of the microorganism that interests us. Eg salt and mannitol agar: NaCl (5%) inhibits the growth of certain mo and facilitates the development of others that grow better in that medium.
Transport and maintenance: they are used when the sample cannot be processed immediately after taking it.

Storage conditions of the culture media

Most of the culture media are dehydrated and these should be preserved as follows:

Fresh, dry, without sunlight and some in the fridge (eg Salmonella).
The least possible openings and closings so that the products do not catch humidity.

When the media are prepared both by companies and by the Laboratory itself, they must be stored taking into account:

The temperature must be between 2-8ºC. They will be kept for 4-6 weeks.
If it is necessary to recast, it will be done in a water bath or in an autoclave for 5 min.

Preparation of a culture medium

Since most of the culture media are dehydrated, the preparation will require the addition of distilled or demineralized water with a pH close to 7. To prepare the culture medium, use a clean erlenmeyer flask and rinse with distilled or demineralized water. . The volume of the erlenmeyer should be double the volume of the culture medium that we want to prepare.

Steps to take:

Depending on the volume of culture medium that we are going to prepare, weigh in a watch glass the necessary amount of the dehydrated culture medium.
Measure the quantity of distilled water required in a test tube .
Add part of the water to an erlenmeyer suitable for the volume that we are going to prepare.
Add the culture medium to the erlenmeyer flask.
Shake until partial dissolution and heat a little.
Add the rest of the water that will serve to clean the surface of the object used to weigh.
We place the erlenmeyer on the lighter, supported on a grid that allows the heat to diffuse and heat for a period of time that will be determined in the product package.
Finally you must go through the sterilization process.

All culture media are prepared in Erlenmeyer, regardless of whether it is solid or liquid. What differentiates its preparation is the way to sterilize:

Solid plate culture media: Prepared and sterilized in the Erlenmeyer flask. For sterilization, a cotton plug wrapped in gauze is placed, covered with paper and sealed with an adhesive tape.
Solid or liquid culture media in tube: they are prepared in the erlenmeyer and sterilized in the tube. The tubes are filled with a volume ranging from 5-10 mL (we use a pipette), they are covered with a metal cap and covered with paper and adhesive tape. They are sterilized placed in a rack, although to increase the surface they are usually placed at an angle.

Bacterial growth

Bacterial growth requires a series of substances and environments that facilitate its development. These characteristics can be: PHYSICAL Temperature: taking into account the temperature, we can classify the microorganisms in:

Mesophiles: they are capable of developing at temperatures that can range between 20-45ºC. Optimum temperature = 35-37ºC.
Thermophiles: they are capable of developing at temperatures above 45ºC.
Sicrófilos: they are able to develop at temperatures ranging from 0-20ºC.

PH: the optimal pH in which most microorganisms develop is neutral, ranging from 6.5-7.5. Mo that develop at pH = 4 are called “acidophiles”. Sometimes, as a consequence of bacterial metabolism, residues are produced that acidify the medium. Buffers are used to keep the medium neutral. The most widely used is that of phosphate salts, which also serve as food for microorganisms. Osmotic Pressure: it is the pressure that originates by diffusion or exchange of solutions of different concentration through a membrane. Most microorganisms inhabit hypotonic media. Its membrane must be selectively semipermeable for this phenomenon to occur. CHEMICALS Oxygen O2: Despite being a fundamental element for most organisms, it is quite toxic and has corrosive effects.

Carbon: it is an element that is part of the basic structure of living matter. The sources from which mo obtain this element are: Proteins (in the form of peptones. It serves to differentiate mo, for example, when we have a culture medium with lactose and peptones). Carbohydrates (mainly glucose and lactose. It is used when we do not care about the type of mine that grows in that crop). Lipids.

Nitrogen, Phosphorus and Sulfur: Nitrogen: they are fundamentally needed for protein synthesis, that is, to form the amino group of amino acids, it is also used to create DNA and RNA. to synthesize amino acids and vitamins and to synthesize nucleic acids and phospholipids from nuclear membranes.

Potassium, Magnesium, Calcium: Their main function is that they are necessary as cofactors of the enzymes. An enzyme has a protein part (apoenzymes) and a non-protein part (cofactor). The latter can be a metal ion or a complex organic molecule called a coenzyme. Cofactors can give or accept atoms required by the substrate.

Trace elements: Iron, Copper, Zinc ,. They participate as cofactors of enzymes.

Organic growth factors: Essential organic compounds that the body is unable to synthesize.

Culture of strict aerobic microorganisms

In petri dishes on solid media. The surface of the culture medium is in contact with atmospheric oxygen, therefore there is no problem in cultivating this type of microorganisms. In a liquid medium, the microorganism grows in the solution, where the oxygen concentration is lower, and decreases depending on the growth of the microorganism. To avoid this decrease in oxygen, we use two resources:

Agitation
Adding sterile air or O2 by bubbling.

Culture of strict anaerobic microorganisms

In liquid medium, we add reducing agents to the culture medium that take the O2 dissolved in the water and form H2o. These agents are cysteine or thioglycolate, for example. Another way is by pumping 02 free CO2 or N2 into the medium, by bubbling. In solid medium, anaerobiosis jugs or Gas-Pak jugs are used. Its size is similar to that of a thermos. The inverted petri dishes are placed inside. The container is closed with an airtight lid. At the bottom is a palladium catalyst. To achieve anaerobiosis, we introduce anaerobiosis sachets, which contain NaCO3 and sodium borohydride. By adding water to the envelope, H2 and CO2 are released. The oxygen present is reduced to water. To verify anaerobiosis there is an indicator, which consists of a strip of paper impregnated with methylene blue. In aerobic conditions, the strip is blue, but if not, it is transparent. On the lid there is a manometer with which we measure the internal pressure, since CO2 and H2 are being released.

Conservation of crops

The preservation of microbial cultures is not an easy task and must guarantee the viability, purity and genetic stability of the cultures, characteristics that coincide with the objectives of a good conservation method. The knowledge of the peculiarities of the different existing preservation techniques for their correct application, as well as the continuous monitoring of the properties of the strains, favor their use as a reliable inoculum in industry, teaching and research.

Choosing the most suitable technique for preserving microbial cultures is often difficult, since the criteria of viability and purity of the strains, population and genetic changes, number and value of the crops, cost, supply and transport of strains must be taken into account. , as well as the frequency of the use of the crops.

The conservation method chosen must guarantee the survival of at least 70% of the cells for a considerable period of time, so that the surviving population is as similar to the original as possible, preserving the important properties of the cells. crops and minimize the occurrence of genetic events. Likewise, it must minimize the risk of contamination and allow the purity of the culture to remain unchanged.

Various techniques are available for the conservation of microorganisms and for that the recommendations of the World Federation of Culture Collections (WFCC), in its general guidelines, must be considered, which states that for safety and to minimize the probability of Strain loss, each must be maintained by at least 2 different procedures. In general, there are 3 categories in which the preservation methods are grouped according to the time in which the conserved cells remain viable, which are the long, medium and short-term preservation methods.

Freezing (at –70 ° C and –196 ° C) and lyophilization 1-4 are classified in the first of these categories as techniques that minimize the risk of genetic change in cells and keep them viable for 10 years or more. , advantages that have conditioned its extensive use to preserve dissimilar biological materials (cultures of fungi, bacteria and yeasts, algae, serum, blood cells, among others) and that are recognized as techniques of choice. The high cost of the equipment used by these methods makes it difficult to implement them in many institutions, which sometimes make use of the maintenance and conservation services through these, which provide recognized crop collections.

In the medium term, it is the term that groups the techniques with which it is possible to maintain the viability of the crops between 2 and 5 years. Desiccation in this group stands out in different supports (sand, silica gel, glass beads) where growth is paralyzed by elimination of available water , 1-3,9) as well as storage in soil, liquid paraffin and suspension in sterile water (distilled or sea), well-documented methods especially for fungi.

Periodic reseeding is a technique that allows the survival of crops in short periods of time, which is why it is recognized as a short-term conservation method.2,9 It is based on transferring the crop from the dry medium to a fresh one, providing the conditions optimal growth, which determines the high risk of contamination and variability of the characteristics of the strains, which are its main disadvantages.

Finally, it is frequent to find that a preservation technique is more effective for one microbial group than for another, which indicates that in addition to considering the above aspects, it is necessary to review the available literature on the microorganisms in question and the maintenance alternatives used, and adapt them to the real situation of the organization. We can therefore highlight that there is no universal technique to properly conserve all microorganisms.