2 Concepts of Pure Culture and Selective Media

Transcript

Hello!! welcome to the second talk of the week that is pure culture and selective media. Pure culture is very very essential because it gives us an opportunity to identify the disease causing agents and help us to establish the relationship between symptom development and disease causing agent.

So it is very important to understand how pure culture is done for Fungi and Bacteria and let us see what are the major steps that needs to be follow for pure culturing of fungi that causes plant diseases. There are two basic methods that are used for pure culturing of fungal pathogens of plants. First one is: Single spore isolation method and the second one is Single hyphal tip method. In the Single spore isolation method, this method is applied to those pathogens of fungal origin which produce spores and which are coloured and bold. So these are some important criteria which are basically required for following this particular Single spore isolation method. The procedure involves that we normally take 3 tubes of plain agar which is melt and cool them to 50o C then transfer a loopful of spore suspension of the mixed to the first plain agar tube with an inoculation needle, then we should shake the suspension agar medium for uniform suspension of the spores, then transfer a loopful of the first dilution to the second tube and shake thoroughly. So this the step it is done here, likewise prepare third dilution as well so, we should go for second and third dilution and then pour the media with diluted spore into three separate petriplate and allow to solidify. Then after dilution we should pour the medium into petriplate and then we should allow the other medium to solidify. We should observe petriplates under low power objective of the microscope and locate isolated single spore. The single spores that are isolated on the other medium should be located and the single spore should then be transferred to PDA slant for obtaining a pure culture. This is the basic method for isolation of pure culture of fungal pathogens through single spore isolation method. Then second method is Single hyphal tip method – this method is employed for purifying fungi which either do not produce spores or produces small and hyaline spores which are difficult to isolate in a single state isolation method. The procedure is by using a cork borer we should take a disc of fungal colony and place it in the middle of a plain agar plate and incubate for 1 to 2 days. So once it is placed and incubated for 2 days then place the petriplate on compound microscope and locate a hyphal tip using the low power objective. Then this plate can be now placed in a microscope and we can see the hyphal tips under low power objectives. Then with help of a cork borer we can cut this single hyphal tip and remove it and place it into a PDA slant and maintain it as a pure culture. So, this can be transferred to a PDA slant and then it can be a pure culture from the pathogen. So, this is another simple method which we call it as single hyphal tip method for isolation of the plant pathogenic fungi.

Yeast purification can also be done but in a different way since yeast are single celled eukaryotic microorganism and produces slimy white soft colonies resembling the bacteria. The will not produce mycelial filaments. And that is why Purification can be done like that of bacterial cultures using Streak plate method pour plate method and spread plate methods. So yeast cells a separated in the similar way as in the case of bacterial pathogen. So, the methods used to isolate bacterial pure culture are also mostly the Streaking or Plating and Dilution or Plating method. In a Streak plate method what we do is we take a inoculation loop and then take a loopfull of bacterial suspension and then streak on agar plate in this direction followed by another direction, opposite to it, then another direction and then another direction. So, by doing this what we do is that we dilute the original pore suspension to an extent that finally it gives rise to scattered single spores and this spores when incubated on suitable agar medium they develop into a single spore colony. So this is the Streak plate method for isolation of bacterial culture.

In the Spread plate technique – we take the bacterial suspension and then take 0.1ml of the bacterial suspension on agar plate and then with the help of a sterilized glass rod we spread the spore suspension to rectangular suspension and then allow the plate to incubate for 24 to 48 hours. After 24 to 48 hours the spores that are separated by this method give rise to single spore colonies and these colonies can now be again transferred to slants for maintaining as a single spore isolation of the bacterial pathogen.

Then the concept of Selective media comes because all fungi and bacteria is difficult to separate from a common medium. So Selective media are used to isolate selective microbes whether this fungi or bacteria that causes certain plant diseases in a different way. So Selective media are used for the growth of only selected microorganisms. For example, if an microorganisms is resistant to a certain antibiotics, such as ampicillin or tetracycline, then that antibiotic can be added to the medium to prevent other cells, which do not possess the resistance, from growing. So use of antibiotics can be part of selective a microbial growth because it suppresses the sensitive microbes and it only allows the microbes that are tolerant or resistant to these antibiotics. So, examples of selective media include:

-Eosin methylene blue that contains dyes that are toxic to Gram-positive bacteria so, it is used for selective and differential medium for coliforms bacteria.

-YM(yeast extract, malt extract agar) has a low pH, deterring bacterial growth.

-Similarly, MacConkey agar is Gram-negative bacteria.

-Whereas, mannitol salt agar is selective for Gram-positive bacteria and differential for mannitol.

-Then Sabouraud’s agar is selective to certain fungi due to its low pH (5.6) and high glucose concentration. So by varying the composition of the medium we can develop certain selective media that is suitable for isolation of certain specific microorganisms whereas other microorganisms they don’t grow profusely on this medium and that is why they help us in selecting or isolating a specific micro organism based on selective media.

Actinomycetes have different types of selective media for their isolation like:

Yeast extract malt extract agar (ISP2)

Actinomycetes isolation agar (AIA)

Arginine Glycerol (AG agar)

Glycerol asparagines agar (ISP5)

and so on. So these media are very specific for growth and development for Actinomycetes very specifically when it was isolated from a mix cultures of other fungi bacteria. Then for a plant pathogenic fungi like Phytophthora we have selective medium like : V8 juice agar medium or Rye A agar medium that suitably used for isolation of phytophthoras species and suppressing other pathogens of fungal or bacterial origin.

So, here we have seen that how pure culture is obtained for fungal and bacterial cultures and what is the concept of selective medium, why they are used? And we have also used examples of selective medium, why they are used and how they are used for isolation of certain specific microbes that may be a causal agent for certain plant pathogens. So, with this we come to an end of lecture number 2 of this week and in the next lecture we will talk about microscopic techniques and straining methods for diagnosis of plant pathogenic fungi and bacteria.

Thank you very much.