LAB 2

Topic: Methods of visualization of conidiophores for microscopy

Objective:

Learn the Riddell mount method to prepare the specimen which is allowed to observe the intact conidiophores.

Materials:

 1.        Cultures of fungi with spores. Several species of Aspergillus were support in this practice, include Aspergillus oryzae, Aspergillus niger, Aspergillus flavus, and Aspergillus sojae.

 2.        Olympus compound microscope.  One is available with an attached camera.

 3.        A kit containing microscope slides, cover slips, needles, and transfer loops.

 4.        Immersion oil and dropper bottles for water to suspend specimens. 

             5.       Lens paper for cleaning objectives, and Kim wipes for working with microscopic slides and specimens.

             6:        Bunsen burner, Petri dish, bend plastic rod, filter paper.

Procedures:

1: There are two different ways to process Riddell mount.

A: Subculture the fungi on agar bed. As the Fig.1 shown, I directly cut a agar block from the agar plate, placed it on the agar bed, and used the transfer loop to inoculate Aspergillus on the edge of agar block from culture plate.  Gently covered a cover slip on the top of agar block, leave some space to avoid the contact of agar block which allow the hyphae to grow around the cover slip.

B: Subculture the fungi on slide bed.  As the Fig.2  shown, firstly I placed a piece of filter paper underneath the Petri dish, and pre-wet with distill water to keep the Petri dish humid, then a triangle plastic rod was placed on the paper to support a slide and avoid to touch the pre-wet filter paper.  A properly size of agar block was cut from agar plate and placed on the slide, and subculture the fungi on this block as described previously.  A cover slip was used to cover the agar block and allow the hyphae to grow around.

2:  One week later, the cover slip was gently took out and place on the slide for microscope observation.  To avoid accidently squash the specimen and distort the intact conidiophore, some broken cover slips were used to pad up the cover slip which with fungi growth on it.

3: The procedures of observation the specimen under the microscope were described last report.

Observation:

  Fig.1 Agar bed based Riddell mount

  

Fig.2 Slide bed based Riddell mount

    

Fig.3 Aspergillus flavus conidiaphore

 

Fig. 4 Aspergillus paraititis conidiaspore
 

Fig. 5 Aspergillus sojae

 

Fig.6 Aspergillus nidulans conidiaphore

Fig.7 Aspergillus tamari conidiaphore

Discussion:

Riddell mount is a powerful method to observe the intact conidiophores for a lot of fungi. The principle of this method is to mimic the hard and hydrophobic surface of plant surface and promote fungi to form the conidiophores as in nature.  The advantage of Riddell mount, when compare with tape mount or slide mounting, is that the hyphae grow on the surface of the cover slip and form the intact conidiophores

In this class we also have the chance to learn how to choose medium to culture the fungi as different medium have different features. For particular specie and purpose usually need to use the specific medium.  A variety of medium were used to culture fungi in the lab and PDA (potato dextrose agar) was most widely used medium for fungi culture, for different purpose, people used different concentration of PDA. ½ PDA, ¼ PDA were used to culture some particular fungi as they have much limit nutrients.  Because PDA medium is too rich nutrients, so that excessive mycelial growth is obtained at the expense of sporulation. Water agar plate also widely used in lab as fungi and bacteria all grow on this plate but at such slow rate, and target fungi will be much easy to isolate from this kind of medium.