Smear (microbiology)

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Smear (also dilution plating ) referred to in the microbiology a method for separating microorganisms on the surface of solid nutrient media (Gelnährmedien).

purpose

Under smear is understood in the microbiology , the distribution of microorganisms on the surface of a gel-like culture medium (nutrient medium). This is intended to distribute the microorganisms as individually as possible on the surface. This enables colonies with a large number of individuals to be formed during their subsequent propagation , which in the ideal case each have developed from a single individual, i.e. represent a clone (several genetically identical individuals), a so-called pure culture. The method is mainly used for three purposes: (a) Obtaining pure cultures that are available for further investigations. (b) Determination of properties of the microorganisms grown in separate colonies. (c) Overview of the various members of a microorganism population , insofar as they multiply under the culture conditions used. This is possible because the colonies of different microorganisms can almost always be distinguished by their characteristics.

principle

A smear to isolate microorganisms is usually made on the smooth surface of a nutrient gel, so-called plating. For the distribution of microorganisms on the surface of a gel-like nutrient medium by spreading it out, it is necessary that the distributing tool ( inoculation loop , glass spatula) glides smoothly on this surface. This is due to the fact that a very thin layer of liquid forms on the surface of an agar gel through syneresis , which enables solid objects to slide on the gel surface. By isolating, pure cultures can be produced by allowing the isolated microorganisms to multiply under suitable culture conditions and thereby allow colonies to form on the surface of the agar gel. The colonies have a characteristic appearance depending on the type of microorganism, type of culture medium and other culture conditions: color, shape (for example smooth or rough edge), surface (for example glossy or matt). These features can - among others - be used to differentiate between different microorganisms and to identify them, i.e. to assign them to a species. In this process, the nutrient gel is usually in round, flat plastic or glass dishes with overlapping lids, so-called Petri dishes . The agar gel covers the soil in it and forms a so-called agar plate , often referred to as a plate for short . Smear tools, Petri dishes and culture medium must be sterile . There are various methods of isolating the microorganisms on the gel surface.

Smear with an inoculation loop

Different types of smear with an inoculating loop
Principle of smear with an inoculation loop

A small amount of the microorganisms or the microorganism-containing material is picked up with a sterile inoculation loop and distributed on the smooth surface of the nutrient medium gel by serpentine sideways movements of the inoculation loop. The continued stripping of the mass of microorganisms from the inoculation loop results in a dilution effect and the number of microorganisms released per area decreases until individuals come to lie so far apart that, when incubated under suitable conditions, isolated colonies develop from each other ideally have developed from a single individual through its reproduction, i.e. represent clones. Instead of an inoculation loop, sterile cotton swabs are sometimes used. The advantage of the inoculation loop smear is that you only need one agar gel plate for each separation and that you can work with only one inoculation loop, which can be sterilized by annealing in a flame between each work step. The disadvantage is that the microorganism individuals often cannot be separated, i.e. isolated, if they adhere firmly to one another, for example through very consistent mucus coats. Furthermore, compared to the method with a Drigalski spatula, fewer isolated colonies are obtained and thus a not so comprehensive overview of the various microorganisms contained in the material.

Smear with a Drigalski spatula

Smear with a Drigalski spatula, last plate in the series

A small amount of the microorganisms or the material containing microorganisms is placed on the surface of an agar plate and distributed as evenly as possible with a correspondingly shaped glass rod (see picture), a so-called Drigalski spatula . With the same spatula and its same side, a second, sterile agar plate is smeared without microorganism material having been applied to this second plate beforehand. Microorganisms still adhering to the spatula are distributed on the agar plate. This is now repeated on a third sterile agar plate. With this method (“streaking out in descending order”) only a very small part of the microorganism material is transferred to the next agar plate, so that the microorganisms are usually already isolated on the second, but in any case on the third agar plate, so that isolated colonies develop. The advantage of the Drigalski smear is that the microorganism individuals are better separated from each other by being squeezed between the glass spatula and the gel surface than with the inoculating loop smear and that a larger number of isolated colonies is obtained compared to the vaccination loop smear and thus a more comprehensive overview of the various microorganisms contained in the material and their approximate proportions. The disadvantage is that three agar plates are required for each smear instead of just one, and that the Drigalski spatulas have to be sterilized in large numbers beforehand by heating them in a heating cabinet or in a sterilization autoclave.

Distribution with glass beads

In the glass bead process, sterile glass beads are used to distribute and separate the microorganisms .

Applications

Most often, a smear is used to isolate a clonal colony. The colonies also have a characteristic appearance depending on the type of microorganism, type of culture medium and culture conditions: color, shape (smooth or rough edge), surface (glossy or matt). These features can be used to identify the microorganism, i.e. to assign it to a species . Smears with Drigalski spatulas or glass beads are used, among other things, to determine the number of living cells by counting the plates, since, in contrast to the use of an inoculation loop, they lead to an even distribution of the microorganisms.

Using a special process, the so-called stamping technique , the microorganism colonies obtained by smearing and incubation can be transferred to the surface of other gel-based culture media while maintaining the positions of the colonies in relation to one another. Separate colonies are required for this, as they arise after a smear with a Drigalski spatula.

Delimitation of the term

In the hematology smears for direct microscopic evaluation of cells used partly in connection with a previous histological or immunological staining, z. B. a blood smear .

history

The smear of microorganisms on nutrient media in Petri dishes was first carried out by Friedrich Loeffler and Georg Gaffky in Robert Koch's laboratory.

Web links

Commons : Smear  - collection of images, videos and audio files

Individual evidence

  1. ^ Georg Fuchs, Thomas Eitinger, Hans Günter Schlegel: General microbiology. Georg Thieme Verlag, 2007. ISBN 9783134446081 . P. 168.
  2. ↑ Katharina Munk: Microbiology. Georg Thieme Verlag, 2008. ISBN 9783131520111 . P. 297.
  3. ↑ Barbara J. Bain, Dieter Huhn: Roche Basic Course in Hematological Morphology. Volume 7 of Ex libris Roche, Georg Thieme Verlag, 1997. ISBN 9783894122997 . Pp. 43-45.
  4. ↑ Axel M. Gressner, Torsten Arndt: Lexicon of Medical Laboratory Diagnostics . 2nd edition, Springer-Verlag, 2013. ISBN 9783642129216 . P. 480.