Enumeration of Spores
There are various ways to count the number of spores in a sample; we use two methods, depending on whether spore density is high or low.
Ocular field (high density)
1. Using a fine ruler, determine the diameter of the ocular field of your stereomicroscope at a magnification where spores can be easily distinguished from mineral particles and organic debris (some of which can be easily mistaken for spores by the inexperienced person). Then calculate the area of the spherical field (circle) at that magnification.
In our Nikon stereomicroscope, we choose a magnification of 4x for a comfortable field of view (diameter of 5mm). The area of the field is 19.6 mm2 (radius = 2.5 mm).
2. We always use plastic petri dishes to count spores because the base of the plate is flat. Because the dish also is hydrophobic, enough water must be added to have complete coverage of the base. Dishes vary in diameter; we use those which are 85 mm across.
The area of the base is calculated to be 5672 mm2. From this datum, and the area of the ocular field, total number of fields in the dish is: 5672/19.6 = 289.
3. Extract spores and use sieves to separate out as much root and organic detritus as possible, add the spore suspension to a petri dish and then randomly rotate the dish to spread out spores as evenly as possible. Note: This method will not work for extracted spores that have been stored more than 24 hr because aggregates invariable will form.
If there are less than 30-40 spores per field, count them in 40 fields randomly chosen over the area of the dish. Calculate average number of spores per field and multiply this number by 289 (# fields/dish).
If there are more than 30-40 spores per field, then place spore extract in a test tube, dilute 1:1 with water. While vortexing, remove a specific volume (10 ml works well), transfer to petri dish, and recount. Make sure to keep track of each dilution to calculate spores in total sample.
Spores are extracted from a 50 cm3 soil sample and suspended in 15 mL water.
In petri dish, too many spores are observed in each field to be counted. This volume then is diluted 1:1 to 30 mL. Spores are counted from a 10 ml aliquot of this dilution.
An average of 28 spores were found in 40 fields of view.
28×289 = 8092 spores in the 10 mL sample.
8092×3 = 24,276 spores in the total sample (30 mL and by extension, the original 50 cm3 soil from which spores were extracted).
24276/50 = 486 spores/cm3
Direct counts (low density)
When there are less than 3-4 spores some fields of view and no spores in others (25-50% of fields examined), then the method above cannot be used because it will overestimate total number of spores in the sample. In this case, spores often are few enough to be counted directly.
- Transfer spore suspension to a test tube, vortex, and transfer 1 ml to a watch glass. Perform this step three more times to count spores in four replicates.
- Swirl water in watchglass (clockwise or counterclockwise) to concentrate spores in the center. Expand field of view to see all spores and make a direct count. Average the counts from four watchglasses and multiply result by dilution factor (1/x total mls in test tube).
- If number of spores in watchglass is too many to count comfortably, then increase the dilution and recount.
Rating of spore abundance
Within 30 days of harvest, a rating of sporulation abundance is assigned to each active culture in the collection. We use this is relative measure as an indicator of culture productivity (see notes section of the searcheable culture database) and it has served us well for over a decade.
- A 50 cm3 sample is removed from a pot culture and spores extracted.
- The spore extract is transferred to a petri dish and the contents shaken/swirled to distribute spores as evenly as possible.
- A rating is assigned to each of five fields randomly selected across the dish. This rating varies with size of spores: (i) small (<100 Ám), (ii) medium (>100 and <250 Ám), and (iii) large (>250 Ám).
small = >300 spores/field
medium = >200 spores/field
large = >100 spores/field
small = >100 and <300 spores/field
medium = >50 and <200 spores/field
large = >25 and <100 spores/field
small = <100 spores/field
medium = <50 spores/field
large = <25 spores/field
|click here for photos of fields exemplifying ratings of small-spored Acaulospora morrowiae||click here for photos of fields exemplifying ratings of medium-spored Archaeospora leptoticha.||click here for photos of fields exemplifying ratings of large-spored Gigaspora gigantea|