Hi, Yuke Cen.

I have no experience fungi culture although they grow in my contaminated culture at early days. However, I have a suggestion for you.

You could stain cells with DNA-specific fluorescent dye, Hoechst 33322 (blue) for 20 min.

Carefully remove 2/3 of the fluid and add new buffer with a pipette tip, then remove the same. Repeat this procedure for 3 times.

Record the image under low power fluorescent microscope (20X objectives). This will help focus every cells in the field (or even 10X objective). You could expose separate fields, then combine them in PPT. This is the cell number.

Remove the remaining sol carefully not to lose the cells. Do MTT assay.

You could do this MTT first for cell viability. Then similarly use propidium iodide (PI for nuclear staining, red) during incubation time or immediately after starting MTT by add a few ul of PI stock solution (2,000~5,000x). It's quick. You have to check wheteher reacting cells are not damaged. Do a preliminary expt.

Best of luck.

The standard method for measuring filamentous fungi growth in liquid culture is by dry weight.

Cell-counting in filamentous fungi, while feasible, it isn't representative of the whole culture. By it's nature you cannot homogenise the culture, and therefore not a proxy for cell concentration.

Cell counting can be used when you are interested in relative abundances such the proportion of live/dead cells. If you have access to a fluorescence microscope, a dual staining FDA/PI works well (see paper)

Article Uranium Binding Mechanisms of the Acid-Tolerant Fungus Conio...

There are typical direct methods which are based on fungal biomass content. Normally, as other researchers pointed out, the standard or election method is dry weight. This may take, however, some time for drying, which implies that results are not rapidly available (at least overnight drying, or until constant weight) at 105°C. The method of CFU counting is also widely spreaded, but I would preferably recommend it for bacteria or yeasts. Nevertheless, it also has some drawbacks as for example, several aggregated cells may give rise to a unique colony.

I have read once about a proposal to accelerate drying time by using microwave drying, but it did not work nicely (reproducibly) for me, at least. So, I would recommend a method for separating the biomass (centrifugation or filtration, usually), and then, pour it into pre-weighed trays and try drying until constant weight at 105°C. Other technique that normally works for bacteria or yeasts is OD, but it does not work in a reliable way for fungi (at least not for me, according to my several trials on this).

Otherwise, you may try indirect methods, like measuring a component of biomass. For instance, glucosamine or ergosterol. The main point on this is that the chosen component for monitoring biomass should be more or less constant during growth. Sometimes, this is not applicable for proteins or DNA. There are 3 critical points on this: 1) you have to measure the biomass component in advance, preferably along cultivation time. 2) the indirect marker should be ~ constant with time (also take into account culture medium - this may change cell composition sometimes). 3) you should check that have no interference for components of culture medium on your determination. Obviously, you normally wash your cells before quatifying!

If you try direct methods like CFU or OD, you can construct a calibration curve vs. dry weight, and if linear with good correlation, you can become independent of dry weight, but you may convert your measurements to dry weight, when necessary.

Lots of luck on your work!