Friday, 4 November 2011

Slide Culture Technique

Preparation of a slide culture can be useful in the study and identification of an unknown fungal isolate. While the 'sticky tape' preparation is quick and easy to perform, it can be disruptive, with salient features altered or destroyed. The slide culture when performed properly is more gentle allowing for the examination of intact structures & features as they naturally developed. The technique is quite easy to perform and I have outlined the basic steps in the photos that follow.

(1) Start by getting a plate of fungal media, Saboraud-Dextrose as shown above, and some means of cutting the agar. Pictured here is a sterile scalpel however they can be relatively pricey for such a small job. I prefer to use a microscope cover slip as the edge is thin, sharp and straight. A 20 X 20 mm cover slip can be used however the 20 X 40 cover slip allows for batter grip. Simply plunge or drag the edge of a cover slip into the agar surface, cutting out small blocks of agar somewhat about 1/2 to 3/4 of an inch square. as shown below.
(some people have used the open end of a sterile test tube as a punch to cut out a round plug of agar for the same purpose)

(2) Above you can see how I sliced the agar into squares that would be slightly smaller than a 20 X 20 mm glass microscope cover slip, using a glass cover slip as a knife. Cut as many squares as you require for the current and near future projects.

(3) A row of squares cut into the agar is seen above. Cut as many as you need for your project and the remaining plate can be placed into a plastic bag and refrigerated for future projects. (note the expiry date of the agar)

(4) Remove an agar square that you have cut into the plate using the same cutting tool (scalpel or cover slip) that you used to previously.

(5) Place the agar block onto a clean glass microscope slide. You can place one per slide or two per slide as I sometimes do. You can also place two different agars on the same slide. (eg. SAB and Corn Meal)

(6) The slide can then be placed in a clean petrie dish which will prevent contamination and preserve moisture during incubation. I like to raise the slide off the bottom of the petrie dish. Moisture in the between the plastic surface of the dish and the glass slide itself might create enough surface tension between the two making removal of the glass slide difficult without disrupting the delicate growth. You can use plastic or wooden stir sticks for this purpose. Some people sacrifice an uninoculated media plate such as blood agar and place the slide directly onto the agar surface. The agar acts as a source moisture during incubation however the surface tension may make remove of the slide somewhat more difficult.

(7) Using a sterile instrument (loop, needle, etc), transfer some of the fungus (spores, conidia etc) from the specimen being cultured to each of the four sides of the agar block. Just a quick touch of the isolate to the four edges as shown below.

(8) Transfer the fungus to the agar block's sides. It isn't necessary to smear the entire side but rather just touch the center of the agar block.

(9) After inoculation, place a clean cover slip on the surface of the agar block. (Remember to do this as it is easy to forget!!).

(10) A few drops of sterile water can be added to the petrie dish as an additional source of moisture which may be beneficial to slow growing fungi which may dry out with prolonged incubation. In general this is not necessary as the dish will be partially sealed up.

(11) The plate is now partially sealed with Parafilm™ or a bit of cellulose tape. If fully sealed the plate may fog up and moisture condense on specimen. The tape partially seals the plate, still allowing it to breath. The sealing of the plate also minimizes contamination of both the culture and the environment.

The slide culture is now ready for incubation. Incubate the slide at an appropriate temperature (room temperature to 30C for most fungi) and for an appropriate length of time. Fast growing fungi can overgrow the agar block very quickly so monitor the growth visually daily. One may wish to make several side cultures of the same fungus and examine one per day to observe how the structures develop.

To examine the slide culture, remove the slide from the petrie dish and then gently remove the cover slip from the agar block using plastic forceps or gloved fingers. The fungus should have adhered to some extent to the glass cover slip. Place a drop of LPCB* onto a clean microscope slide and then place the cover slip from the slide culture (growth down) onto the LPCB. The slide is now ready for examination under the light microscope. Don't throw the agar block away as if you remove it you will find that the fungus also adhered to the microscope slide. Just add LPCB and cover slip the growth. You get two cultures from one block - top and bottom.

*LPCB: Lacto Phenol Cotton Blue stain.