FDA hydrolysis activity test

Made by: Zch Attila

Introduction

The FDA is the short form of fluorescein-diacetate (!-"!

diacetyl-fluorescein#$

Many en%ymes (&roteases' li&ases' esterases# are able

to hydroly%e FDA$

The &roduct is fluorescein-diacetate$

FDA is used for determine total microbial activity$ This is

a (ood (eneral measure of or(anic matter turnover in
natural habitats (reason: more than )*+ of the ener(y
flo, &asses throu(h microbial decom&osers#$
A brief descri&tion of the method

Determination of total microbial activity$

Active fun(i' bacteria -uantitative determination' locate

acetyl-esterase in livin( &rotist cells and e.amine the
en%ymatic activity of animal cells$

/revious studies revealed that all fun(i' most bacteria'

&roto%oa and al(ae sho, some FDA hydrolytic activity$

0a&id' sensitive and sim&le method$

The result of fluorescein &roduced by microbial activity'

and this is measured by fluorescence microsco&y'
fluorimetric' or by s&ectro&hotometry$
1ome e.am&les for test materials
FDA-hydrolysis activity of various sam&les could be
e.amined$
1oil:
2
the sam&le is ta3en from a(ricultural land' different
various layers:
to&soil
sandy loam
clay layer
2 1oil sam&les are sterili%ed by autoclavin( at 45*67 for 5* min
on 5 consecutive days$

1tra,:
2 barley stra,
2 cut into 5 cm &ieces' store at room tem&erature
2 Autoclave at 44* 67 for * minutes

/ure microor(anism cultures' e.am&les:

2
Pseudomonas denitrificans
2
Fusarium culmorum
Determination of FDA hydrolysis

Dissolve FDA in acetone and store as a stoc3 solution (5

m(8ml# at -5*67$

Acetone is used for:

2 ensure the end of the en%ymatic reaction
2 &roduce a clear solution ,ith a lo, bac3(round absorbance
2 It hel&s to dissolve the cell-membranes
2
It is difficult to remove colloids from the su&ernatants of clay
sam&les ,ithout acetone
The hydroly%ed FDA is measured at absorbance of 9)* nm' by a
:ec3man s&ectro&hotometer
If the sam&les! absorbance is more than *$;' these sam&les must be
attenuated before the measurement

Add 4* <(8ml FDA and "*mM sodium &hos&hate buffer to the

sam&les' so the &= is set to >$"' and the resultin( mi.ture is
incubated at 59 67 on a rotary sha3er

In the e.&eriments ,ith stra,' A9)* values can be

determined directly in buffer sam&les ta3en from the
incubation flas3s

In the e.&eriments of &ure cultures:

2 P. denitrificans: cells are removed from the incubation solution
by centrifu(ation for ? min at "'*** r&m' and the su&ernatant is
filtered throu(h Mun3tell no$ filter &a&er' after this &rocedure
,e can measure the absorbance
2
F. culmorum: the mycelium is removed by centrifu(ation at "'***
r&m for ? min follo,ed by filtration of the su&ernatant throu(h a
<m membrane filter' after this ,e can measure the absorbance

1oil is removed from the incubation solution by

centrifu(ation for ? min at "'*** r&m follo,ed by filtration
throu(h Mun3tell no$ filter &a&er$
@valuation

1oil:
2
FDA hydrolysis activity increases linearly ,ith time
and the amount of soil
2
Autoclaved soils does not sho, any FDA activity
2
The sand and the clay sam&les sho, no FDA activity

1tra,:
2
Autoclaved sam&le sho,s less activity

/ure cultures:
2
P. denitrificans: the concentration-absorbance
relationshi& is linear
2
F. culmurum: the concentration-absorbance
relationshi& is linear
=ydrolysis of FDA by different amounts
of soil
1ource: Aohan 1chnBrer and Thomas 0oss,all' (4);5 Aune#' Fluorescein Diacetate
=ydrolysis as a Measure of Total Microbial Activity in 1oil and Citter' Applied and
Environmental Microbiology' Dol 9("#' 45?;
=ydrolysis of FDA by soil sam&les over
time
1ource: Aohan 1chnBrer and Thomas 0oss,all' (4);5 Aune#' Fluorescein
Diacetate =ydrolysis as a Measure of Total Microbial Activity in 1oil and
Citter' Applied and Environmental Microbiology' Dol 9("#' 45?;
7onditions of this method
/ro&er &=
&=E>'" by sodium-&hos&hate buffer
the reason of the &=-sensitivity is that on lo, and hi(h
&= there are no FDA hydrolytic activity
/ro&er tem&erature
sam&les are incubated at 59 67 on a rotary sha3er
0es&iration (F
5
-sensitivity#
There is a relationshi& bet,een F
5
and the FDA
hydrolytic activity' the best activity of soil sam&les is in
the to&soil' the activity decreases as the de&th
increases$
res&iratory rate' as the consum&tion of F
5
,as defined at
4? 67' by Gilson res&irometer
Disadvanta(es

More suitable for determinin( the heterotro&hic activity'

as determine biomass -uantification$
2 reason: the active biomass is not directly &ro&ortional to the
measured FDA hydrolysis activity
usin( the method described above
2 FDA microsco&e can Hust ma3e difference bet,een active and
inactive biomass

/ro&er and accurate &re&aration is re-uired:

2
to avoid AT/ hydrolysis
2
a&&ro&riate incubation' &=

=o,ever' this is the only method for determinin( the total

microbial activity' and it has the (reatest &romises$
1ources
1chnBrer' A$ and 0oss,all' T$ (4);5#' Fluorescein Diacetate
=ydrolysis as a Measure of Total Microbial Activity in 1oil and Citter'
Applied and Environmental Microbiology' Dol 9("#' &$45?"-45"4