permaculture

[Lawrence London <lfljvenaura@gmail.com>]

---------- Forwarded message ----------
From: mpicassine@yahoo.com.mx [compost_tea] <compost_tea@yahoogroups.com>
Date: Mon, Dec 21, 2015 at 9:48 PM
Subject: [compost_tea] Microscope to assess soil, compost, tea foodweb
To: compost_tea@yahoogroups.com


Hi, I have followed closely this group, and now it’s time to ask for your
help:

I want to perform the food web analysis, so first some general questions on
microscopy:



 I want to use both brigthfield and darkfield illumination (I find that in
darkfield it's much easier to differentiate stuff)



 Also a 60x objective instead of the unused 100x



The first thing I have doubt of, is the quality of the objectives needed,
there are 3 options:



 just achromatic (60% aberration free field of view)

semi plan (80% aberration free field of view)

plan (100% aberration free field of view)



Is there any significancant difference when measuring hyphal lenght and
width?



 Second, as the active bacteria&fungi requires fluorescence capability, so,
How important is that piece of information to be known?*



So I if fluorescence capability for the fluorescin-bearing-active
bacteria&fungi assay is needed, the microscope price escalates a lot, so...
starting with the cheapest,



 I plan to adapt this(1)  reflected(epi)&transmitted illuminated OMAX
microscope to fluorescence by replacing the halogen epi lamp by the
adequate wavelenght & power LED lamp. (2)



Drawbacks: first: blue light will be reflected along with fluorescent green
light, possible lack of contrast to discern green, fluorescent bacteria
&fungi, Can't find a way to put a removable barrier filter. Anybody knows
about this?

Second: As  the objectives are not labeled fluorescence, they may not
perform as intended, Anybody knows if they will perform acceptably?



Next option is to buy a cheap EPI Fluorescence

scope (3), pros no blue light reflected alog with green

 Drawbacks: no PLAN objectives, $60-100hourlifetime-lamp is made of mercury
(had to deal with hazardous waste)



Another option: buy a not so cheap EPI Fluorescence

 scope (4), pros: plan objectives

cons: $60-100hourlifetime-lamp is made of mercury (had to deal with
hazardous waste)



Further, If i can get a  EPI Fluorescence

 intended scope with the lamphouse not working by a reduced price, I could
refurbish it for the LED source



So, if you have some ideas, experiences, etc I will appreciate them a lot,
thanks.



 1.
http://www.microscopenet.com/omax-40x2000x-infinity-trinocular-metallurgical-microscope-transmittedreflected-light-p-9059.html?products_id=9059&currency=USD

2.
http://www.leds-global.com/50w-460nm-blue-high-power-led-for-plant-growing-and-aquarium-p-374.html

3.
http://www.microscopenet.com/omax-40x1600x-epifluorescence-trinocular-biological-microscope-with-cmos-digital-camera-p-10657.html

or

http://www.amscope.com/40x-2500x-infinity-extreme-widefield-epi-fluorescent-microscope.html

4.
http://www.amscope.com/40x-2500x-plan-infinity-extreme-widefield-epi-fluorescent-microscope.html

 * If the active portion is too high, it can "compete" with plants for
nutrients? can we just dilute it more and assure an a
activebacteria:protozoa and an a fungi:fungal-feeding-nematode safe ratio?
Or in the case of tea, based on motility?


Reply from Elaine Ingham:



You really want to use the shadowing method that you can much less
expensively obtain with an ABBE condensor, rather than using dark field.
 It is very hard to assess diameter's on organisms using dark field.
 Whereas with the shadowing method, it is quite easy.   Way less expensive
too.

Using a 60X objective rather than a 40X objective means you get to re-do
all the calculations on the spreadsheet.

The objectives you need are 4X scanning lens, 10X or 20X lens, and a 40X
lens.  There is no need at all for a 100X objective.  You will be able to
see all the organisms using the 4X objective, but to differentiate fungal
hyphae from actinobacteria strands, or testate amoebae from cysts or
spores, you need to do most of the work using a 40X objective.

Just remember that 10X eyepieces are also used, at all times.  So the total
magnification we use is 40X, 100X, and 400X.

Plan lens are best, semi-plan second, achromatic - well, ah, you are going
to have some curvature happening, and that can make some people get "car
sickness" when they use those lenses

Think of fungal hyphae as being long flexible tubes.  The length of the
tube is quite a different thing from the diameter, or width of the tube.
The tube may be 1 mile long, but the diameter or width maybe by 2
micrometers, or 10 micrometers.

If you are doing certain types of experiments, it can be very important to
differentiate active bacteria, or active fungi, from not-active, or
slow-growing or dormant bacteria or fungi.  Most growers do not need that
degree of fine-tuning, however.  And we can get around that limitation when
using shadowing methods by looking at how many more bacteria we have after
a day, or three, or ten, or 100.  Fluorescence work is for when you need to
precisely understand how much CO2 is being evolved by fungi versus bacteria
at a single instant in time.

Are you doing work that needs assessment of activity at single instants in
time?  If not, then don't worry about the epi-fluorescent microscopes.

Have you had experience with dark field microscopy?  I encourage you to try
the shadowing method in comparison to dark field.  My experience shows
shadowing to be much preferable.  If you want the same expensive version of
shadowing, check out Differential Interference microscopy.  Not much
difference.

Elaine R. Ingham
President, Soil Foodweb Inc.       Soil Life Consultant
Since I travel a great deal, please consider that I may miss e-mails so if
I don't answer within 3 days, please try again.
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-- 
Lawrence F. London
lfljvenaura@gmail.com
https://sites.google.com/site/avantgeared/