Alternative approaches for the qualitative detection of extracellular enzymatic properties
of bacteria are rare in the literature. Sizemore and Stevenson (1970) developed a marine
agar-milk double-layer plate technique for the detection of proteolytic marine bacteria
colonies. Paoni and Arroyo (1984) described a method which makes use of chromatogenic substrates (p-nitrophenyl-2-acetamido-2-deoxy-a-D-galactopyranoside) in detecting glycosidase activity of bacteria colonies on agar plates. Although these methods offer
some advantages in comparison with the selective media approach, it is somewhat
inconvenient to prepare double-layer agar plates. Furthermore, the quantitative assessment of chromatogenic reaction products (p-nitrophenol) is less precise than that of their
fluorogenic counterparts (e.g. 4-methylumbelliferone) (Taylor et al., 1977, Pettersson and
Jansson, 1978).
Quantitative estimates of enzymatic activities of bacteria in aquatic environments have
been made by several authors (e.g. Kim and ZoBell, 1974; Little et al., 1979). Recently
some investigators have applied fluorogenic substrates in highly sensitive methods suitable for direct in situ measurements of bacterial extracellular enzymatic activities (Hoppe
1983, Somville and Billen, 1983).
Among these fluorogenic substrates the butyryl ester of 7-hydroxy-4-methylcoumarin
was employed to identify esterase-positive fungal colonies on mixed culture plates (Pancholy and Lynd, 1971). Littel and Hartman (1983) reported using a large number of
fluorogenic substrates in order to differentiate species of fecal Streptococci. Now that
quantitative estimates of biochemical activities of bacteria (e.g. polymeric hydrolysis by
extracellular enzymes) can be made with high precision, the lack of adequate methods for
the qualitative determination of “physiological groups” and single colony enzymatic
properties is more apparent than ever.
In this report we describe a method for the detection of microbial extracellular enzymatic
activity on agar plates with fluorogenic MUF-substrates. In addition, comparative studies of the hydrolytic activity of bacterial pure cultures on selective agar plates and
enzymatic activity in a liquid medium supplemented with MUF-substrates are described.
MATERIAL AND METHODS
Bacteria inoculation
Natural water samples were diluted with sterile isotonic sea water and aliquot parts of
dilutions were transferred to ZoBell 2216 E agar plates (spread plate method). Plates were
incubated for 2 weeks at 20°C in the dark after which standard colony counts were
obtained. The plates were subsequently used for enzyme detection procedures as described below. In addition, 10 colonies were randomly selected from these agar plates and
each served to inoculate 7 agar plate parallels, which supplied material for 7 subsequent
enzyme detection treatments. After colonies had developed on these plates, they were
treated for extracellular enzyme production in the same manner as the standard agar
plates.
Substrate preparation
7 fluorogenic substrates were used for the qualitative detection of extracellular enzymatic
activities of bacteria. For example Methylumbelliferyl-α-D-glucopyranoside was used
for the α-glucosidase of bacteria and so on as listed in Tab. 1. To detect the lipase two
substrates were selected in order to check the difference between these two substrates
(MUF-butyrate and MUF-heptanoate). A fluorogenic substrate consists of a fluorescent
fluorophore (MUF) linked to an organic or inorganic substrate molecule (Fig. 1). The
176
of bacteria are rare in the literature. Sizemore and Stevenson (1970) developed a marine
agar-milk double-layer plate technique for the detection of proteolytic marine bacteria
colonies. Paoni and Arroyo (1984) described a method which makes use of chromatogenic substrates (p-nitrophenyl-2-acetamido-2-deoxy-a-D-galactopyranoside) in detecting glycosidase activity of bacteria colonies on agar plates. Although these methods offer
some advantages in comparison with the selective media approach, it is somewhat
inconvenient to prepare double-layer agar plates. Furthermore, the quantitative assessment of chromatogenic reaction products (p-nitrophenol) is less precise than that of their
fluorogenic counterparts (e.g. 4-methylumbelliferone) (Taylor et al., 1977, Pettersson and
Jansson, 1978).
Quantitative estimates of enzymatic activities of bacteria in aquatic environments have
been made by several authors (e.g. Kim and ZoBell, 1974; Little et al., 1979). Recently
some investigators have applied fluorogenic substrates in highly sensitive methods suitable for direct in situ measurements of bacterial extracellular enzymatic activities (Hoppe
1983, Somville and Billen, 1983).
Among these fluorogenic substrates the butyryl ester of 7-hydroxy-4-methylcoumarin
was employed to identify esterase-positive fungal colonies on mixed culture plates (Pancholy and Lynd, 1971). Littel and Hartman (1983) reported using a large number of
fluorogenic substrates in order to differentiate species of fecal Streptococci. Now that
quantitative estimates of biochemical activities of bacteria (e.g. polymeric hydrolysis by
extracellular enzymes) can be made with high precision, the lack of adequate methods for
the qualitative determination of “physiological groups” and single colony enzymatic
properties is more apparent than ever.
In this report we describe a method for the detection of microbial extracellular enzymatic
activity on agar plates with fluorogenic MUF-substrates. In addition, comparative studies of the hydrolytic activity of bacterial pure cultures on selective agar plates and
enzymatic activity in a liquid medium supplemented with MUF-substrates are described.
MATERIAL AND METHODS
Bacteria inoculation
Natural water samples were diluted with sterile isotonic sea water and aliquot parts of
dilutions were transferred to ZoBell 2216 E agar plates (spread plate method). Plates were
incubated for 2 weeks at 20°C in the dark after which standard colony counts were
obtained. The plates were subsequently used for enzyme detection procedures as described below. In addition, 10 colonies were randomly selected from these agar plates and
each served to inoculate 7 agar plate parallels, which supplied material for 7 subsequent
enzyme detection treatments. After colonies had developed on these plates, they were
treated for extracellular enzyme production in the same manner as the standard agar
plates.
Substrate preparation
7 fluorogenic substrates were used for the qualitative detection of extracellular enzymatic
activities of bacteria. For example Methylumbelliferyl-α-D-glucopyranoside was used
for the α-glucosidase of bacteria and so on as listed in Tab. 1. To detect the lipase two
substrates were selected in order to check the difference between these two substrates
(MUF-butyrate and MUF-heptanoate). A fluorogenic substrate consists of a fluorescent
fluorophore (MUF) linked to an organic or inorganic substrate molecule (Fig. 1). The
176