Biotechnology Letters Vol 8 N o 8 579-580 (1986)

Received as revised July

LIMITATIONS OF THE CONGO-RED STAINING TECHNIQUES FOR THE DETECTION OF

CELLULOLYTIC ACTIVITIES.

P. Sharma*, S. Pajni, N. Dhillon, D.V. Vadehra and D.K. Dube.

Department of Microbiology, Panjab University, Chandigarh, India - 160 O14.

The interaction of Congo-Red with polysaccharide containing /B(I.4) or

/B(I.3) linkages gives an intensely red complex (Teather and Wood, 1982)
whose formation is prevented by the action of /B-glucanases. This
phenomenon has been extensively applied in an agar plate diffusion
technique for isolation of mutants with cellulolytic activity (Gilkes et
al., 1984); ihas facilitated work in cloning the cellulase genes (Collme-~-
and Wilson, 1983; Cornet et al., 1983) and has been used for localising
endoglucanases in polyacrylamide gels (Bartley et al., 1984) and for
differentiating endo- and exo-glucanase activities (G---abr--{el,1971).

We observed that certain cell lysates prepared with Triton-X-lO0 and/or

lysozyme mimicked the cellulase activity on Congo Red plates, thus giving
false positive results. This prompted us to study interferences with the
Congo Red staining caused by a number of chemicals.

Preparation of agar plates containing CMC or xylan: CMC agar plates were
prepared with 0.5% carboxymethyl cellulose (7 MT, Hercules Inc. Wilmington,
Delaware, USA) (0.5 degree of substitution) and 0.8% agar powder. Xylan
suspension was prepared by grinding l.Og larch-wood xylan (Sigma Chem. Co)
with O.I N NaOH and neutralising with 0.I N HCI. The mixture was
centrifuged (5,000 g x 30 min) and the clear supernatant added to molten
agar at 45~ to give a final concentration of 0.8% agar and 0.5% xylan.

P[eparation of interferin 9 reagents: Molar solutions of inorganic salts or

oxidising and reducing agents were used. Tween-60, Tween-80 and
Triton-X-lO0 were used undiluted and sodium lauryl sacodinate as a 50%
(w/v) solution. Compounds exhibiting a positive interference effect were
tested at i0, 0.5, 0. i and 0.01% concentrations.
Commercially available preparations of cellulase and xylanase (1.0%)
(Enzyme Development Corp. Pen, Plaza, N.Y) served as positive controls and
distilled water as negative control.

Detection of activity/interference on plates: Wells of 0.8 cm diameter

were cut in the plates and 150 /ul of various test solutions was charged in
each well. The plates were incubated at 55~ for 4 hr, then flooded with a
1 mg/ml solution of Congo Red for 15 min. and then washed with 1 M NaCl.
Yelow zones of clearance against a red background were read as positive
results.

RESULTS

Of all the inorganic salts tested, only Ba(OH)2 and BaCl 2 gave false
positive results (Table i). ZnCI 2, HgCI 2, Pb(N03)2, PbO, CdCI2, CuCl and
FeCl 3 precipitated the dye which subsequently failed to bind the
polysaccharide. Cysteine hydrochloride, ascorbic acid, 2-mercaptoethanol
and an~nonium persulphate gave similar results. Neutralised solutions of
NH4CI, KCl, NaCI, LiCI, (NH4)2S04, CaCI 2 and acetamide had no effect. A
variety of surfactants (see Table) showed varying degrees of false positive
results.
The interference was concentration dependent (Fig 1 and 2).

579
Fig.l: CMC-Congo Red Plate showing
zones of clearance. Well # i, un-
diluted Triton-X-lO0; 2, undiluted
Tween-80; 3, IM acetamide; 4, 1%
CMCase (positive control); 5, dis-
stilled water (negative control).
Fig.2: CMC-Congo Red plate showing
the effect of Triton-X-lO0 conc. on
clearance zone diameter. Well #I,
O.O1%; 2, O.1%; 3, 0.5%; 4, 1%;
5, distilled water (-re control);
1 % CMCase (positive control).

TABLE l:Effect of concentration of compounds on the de~ree of interference*

Interfering CMC-Agar plas Xylan-Agar plates
agents (concentrations %)
1.0 0,5 0.i 0.O1 1.0%
Triton-X-lOO III ++ + + +
Tween-20 ::: ++ - - ++
Tween-60 III + - - ++
Tween-80 ++ + + - +Jr
Sodium taurocholate III ++ ~ - ++
Taurodeoxycholic acid Itl +++ ~ - ++
Sodium lauryl sarcodinate ++ + - - +++
Sodium dodecyl sulphate ++ + - - ++
Barium hydroxide ~ ++ + - ND
Barium chloride ++ + - - ND

* +, ++, +++ indicate zone diameters of > 15, 20 and 30 mm respectively.

Although the Congo Red technique has been widely used forscreening
cellulolytic bacteria, its underlying principle is not clear, so that the
interference caused by some commonly used chemicals and enzymes cannot be
explained at the moment. However, care must be taken in the use of certain
possibly interfering agents in the use of this technique for screening, to
avoid false positve results.

References:

Bartley, T.D., Holland,K. and Eveleigh, D.E. (1984). Anal Biochem. 140,
157.
Collmer, A. and Wilson, D.B. (1983). Biotechnol. 1 (7), 594.
Cornet, P., Millet,J., Beguin, P. and Aubert, J.P. (1983). Biotechnol. 1
(7) , 589.
Gabriel, O. (1971). in Methods in Enzymology. (Jakoby, W.B. ed.) Vol. 22,p
578-604, Academic Press, New York.
Gilkes, N.Ro, Kilburn, D.G. Miller, R.C. and Warren, R.A.J. (1984).
Biotechnol. 2 (3), 259.
Teather, R.M. and Wood, P.J. (1982). Appl. Environ. Microbiol. 43, 777.

580