In this video, we describe a label free live imaging protocol to capture images of the model fungus aspergillus nidulans, using transmitted light microscopy techniques. We use this images to analyze and quantify growth kinetics of aspergillus nidulans in both liquid and solid media. A visual representation of this protocol allows users to become familiar with significant steps of image capturing and microscopy image processing.
A common method used to measure filamentous fungal growth is two inoculate fungal spores in a Petri dish containing nutrient agar and to measure the diameter of the developing colony a few days later. However, this approach is not sensitive enough to quantify fine growth differences. Therefore, single cell measurements using time lapse microscopy were developed.
This measurements are able not only to provide accurate and quantitative results, but also to reflect growth dynamics of different cells within a population. Moreover, this approach aims to better understanding of the molecular mechanisms involved in fungal growth responses to endogenous and environmental signals. Begin by pouring 15 milliliters of liquid nutrient agar into Petri dishes.
Place the lid onto the top and allow it to cool. Use UV radiation to sterilize plates. Before streaking out the fungal strain of interest from a stock, heat the nodulating loop in the Bunsen burner until it is red hot, cool the loop by stabbing it into the agar.
Vortex the tube and streak the loop across the surface of the agar minimal medium supplemented with the appropriate nutritional requirements. Incubate plates for two to three days at 37 degrees Celsius. Using a sterile toothpick, transfer a small number of conidia by gently touching a single colony to plates of complete media.
Incubate plates for three to four days at 37 degrees Celsius. Conidia of aspergillus nidulans, are harvested in a sterile 1.5 milliliter vortex tube, with 1.0 milliliter of autoclave distilled water containing 0.05%volume per volume, Tween 80, for reducing the number of conidia clumps. A modified version of the inverted agar method is used for imaging filamentous fungi on agar medium surface.
Initially spot 10 microliter aliquots of vigorously vortex conidial suspension, approximately two times 104 cells per milliliter onto Petri dishes of 15 milliliters minimal medium with one weight per volume agar. Incubate the experimental culture according to the developmental stage to be investigated. Here we incubate for three days at 30 degrees Celsius.
After, slice out a block of agar containing the colony, using a sterile scalpel. Here we cut out a wedge of agar at the colony margin, invert and place the agar slice into an eight, well slide. Transfer 10 microliter aliquots of vigorously vortex conidial suspension of approximately two times 10 in the power of four in the wells of a well slide containing 200 microliters of minimal medium with the appropriate supplements.
Incubate for the time and temperature to be examined each time. The choice of microscope depends upon the available equipment. In any case, the microscope set up should include an inverted stage, an environmental chamber, or at least a room with precise air temperature control.
Initially preheat the thermostat and microscope chamber to stabilize the desired temperature. Here we set the temperature at 30 degrees Celsius. Turn on the microscope, the scanner power, the laser power and computer, and load the imaging software.
Place the previously prepared well slide in the microscope stage and focus. Find fields of view that contain isolated, not overlapping cells, or at least not overcrowded, to facilitate growth measurements during image analysis. Select the transmitted light microscopy technique to be used and activate the transmitted light detector as well as detectors for fluorescence when necessary.
Set the microscope to acquire images at desired time intervals and start time series acquisition. Loading, visualization and processing of images is accomplished with the open source imageJ Fiji software. Begin by importing the images to Fiji using plugins by a format.
Select the default color mode and auto scale. In order to visualize a timestamp and a scale bar as overlay go to analyze, tools, scale bar. Set the desired parameters concerning width, height, color, and location of the scale bar.
Go to image, properties, to display image properties in imageJ Fiji software. Observe frame interval information. Here we use a time interval of approximately 15 minutes and press okay.
Then go to image, stacks, label, and set the correct information to interval. Set location of the label by modifying X and Y.Set the measurement unit in the field text and press preview. Use histogram matching for illumination correction between different frames by selecting image, adjust, bleach correction, histogram matching.
A new window with corrected illumination appears. Use MtrackJ plugin at plugins, MtrackJ, to track growing hyphal tip To add the track, select the add button in the toolbar and place the first point at the hyphal tip using the left click of the mouse. The time series will automatically move to the next frame.
To complete the tracking process, double click the mouse on the final point, or press the escape key. Scrolling the output table, the most important column for calculating hyphal tip growth, is the speed at any given frame. Safe track measurements, add file, save as, to the file format of your choice, for example, CSV, import the saved file into your spreadsheet program and compute the visualization and further tests.
Press the movie button to generate a movie, RGB color type, containing the frames with the tracks drawn into them, which can be visualized in any standard multimedia player. Following this protocol, we captured and analyzed various images corresponding to different growth and developmental stages of the filamentous fungus aspergillus nidulans. Figure shows the comparison of wild type and azhAD Delta ngnA Delta growth rate measurements.
The azhAD Delta ngnA Delta is a double deleted strain in two genes implicated in the detoxification and assimilation of the toxic fighter product L as added in two carboxylic acid. It shows statistically significant lower growth rate measurements compared to the wild type strain in submerged liquid cultures. This difference in growth is not detectable by measuring the colony area of these two strains in solid minimal medium.
Single cell, long-term live imaging is of significant value in the effort to obtain spacial and temporal information of cellular proteins dynamics. This protocol is suitable also for carrying out long-term live cell imaging. Here we see germination of conidia co-expressing the GFP labeled core eisosomal protein PilA and the mRFP histonen H1.Here in we present a protocol for analyzing fungal growth kinetics in a reproducible and reliable manner without the need of any prior image analysis experience from the user.
This protocol allows objective accurate quantification of fungal growth.
