Subject: [compost_tea] Re: Observing Microbial Biofilms on Leaf Surfaces + Isolation of Culturable Micr

From: "Steve Diver" <[email-masked]>
Date: Sat, 27 Mar 2004 06:24:30 -0000

Hi Bob -

SFI uses fluorescein diacetate (FDA) in combination with 
epifluorescent microscopy to measure active microbial
biomass, and a range of microscopy methods for all the
different parameters they measure.  Elaine can provide exact 
details.   

Elaine's methodologies were developd through graduate and 
post-graduate research going back to early 1980s.

See a bibliography of her papers at:

Sunburst Nominees with SFI | Australia
http://www.sunnom.com.au/sfi/members.htm

For example: 

Ingham, E. R., and D. A. Klein. 1984. Soil fungi: relationships
between hyphal activity and staining with fluorescein
diacetate. Soil Biology and Biochemistry 16:273-278. 

Ingham, E.R. and D.A. Klein. (1984). Soil fungi: Measurement 
of hyphal length. Soil Biol. Biochem. 16:279-280.

Stamatiadis, S., J.W. Doran and E.R. Ingham. (1990). Use of 
staining and inhibitors to separate fungal and bacterial 
activity in soil. Soil Biol. Biochem. 22:81-88.

Mary Appelhof published an article in WormEzine (August 2003) 
that summarized the SFI lab techniques:

"Another capability that Dr. Ingham has is to determine which 
cells are active at the time the lab receives the samples. 
Many bacteria, protozoa, and fungi would be in a dormant 
or inactive state. Others are actively metabolizing at the 
time they make the preparation. By adding fluorescein diacetate 
(FDA) to the preparations, any cell whose mitochondria are 
active take up the FDA. When the technicians look at the sample 
using ultraviolet light with the microscope, the active cells 
show up a bright fluorescent yellow/green. By using a dark 
field on the microscope the bright yellow-green cells show up 
very readily, enabling the viewer to ascertain the number 
of active bacteria, fungi, or protozoa as compared with the 
total numbers or hyphal length." 

Etc... keep reading, this article provides an excellent 
summary of SFI procedures. 

WormEzine Vol. 2, No. 7
August 2003
http://www.wormwoman.com/acatalog/WormEzine_2-7.pdf

FDA and acridine orange (used in the French study) are 
fluorochromes. There's a whole bunch of the fluorochromes.

See:

Nikon Microscopy: Yellow Fluorescent Protein (YFP) Filter Set
http://www.microscopyu.com/articles/fluorescence/f
iltercubes/yfp/yfphome.html

Fluorochromes and recommended filter pairs
http://www.mtxlsi.com/fluorofilters.htm

Regards,

Steve Diver 

--- In [email-masked], "bobkacalek" <bob@s...> wrote:
> Hi Steve,
> Thanks for sharing, very interesting.
> The advantage of quality ACT is the abundance of diversity in Micro-
> organisms delivered vs one or two specific cultures. 
> Are the methods described (Epifluorescence microscopy, scanning 
> electron microscopy, and confocal laser scanning microscopy were
used 
> to observe microbial biofilms directly on leaf surfaces)similar to 
> methods used by SFI?  Maybe Dr. E will respond?
> Thanks Again,
> bob 
> 
> 

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---

From: "Steve Diver" <[email-masked]>
Date: Sat, 27 Mar 2004 06:24:30 -0000

Hi Bob -

For example: 

WormEzine Vol. 2, No. 7
August 2003

See:

Regards,

Steve Diver 

used 

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