GERBAM — Deuxième Colloque International de Bactériologie marine
IFREMER, Actes de Colloques, 3, 1986, pp. 175-183
CNRS, Brest, 1-5 octobre 1984
19
MICROBIAL EXTRACELLULAR ENZYME DETECTION ON AGAR PLATES
BY MEANS OF FLUOROGENIC METHYLUMBELLIFERYL-SUBSTRATES
S.-J. KIM and H.-G. HOPPE
Institut Für Meereskunde, Düsternbrooker Weg 20, 2300 KIEL (FRG)
ABSTRACT - A rapid and sensitive method to detect the extracellular enzymatic activity of bacteria colonies
grown on agar plates is described. Selective agar media supplemented with protein, starch, chitin, Tween-80, etc.
are conventionally used to detect biochemical properties of bacteria. It has been experimentally demonstrated
with bacteria pure cultures that fluorogenic Methylumbelliferyl (MUF) -substrates are excellent substrate
analogues for normally occurring polymers. Based on MUF-substrate hydrolysis the new method provides
reliable qualitative estimates of extracellular enzymatic properties of bacteria within minutes using pure
cultures as well as agar plates prepared for colony counts.
Key words: microbial extracellular enzyme detection, methylumbelliferyl-substrates, agar plate.
RÉSUMÉ - Une méthode rapide et sensible de détection de l’activité enzymatique extracellulaire de colonies
bactériennes se développant sur milieu gelosé est décrite. Des milieux sélectifs enrichis en protéines, amidon,
chitine, Tween 80, etc. sont généralement utilisés pour détecter les propriétés biochimiques des bactéries. Il a été
démontré expérimentalement sur cultures pures de bactéries, que les substrats au Methylumbelliferyl fluorogénique sont d’excellents analogues des substrats polymériques courants. Basée sur l’hydrolyse des substrats MUF
cette nouvelle méthode fournit des estimations qualitatives fiables sur les propriétés des enzymes extracellulaires
des bactéries. Le résultat est obtenu en quelques minutes en utilisant des cultures pures aussi bien que des
colonies ensemencées sur agar.
Mots clés : détection d’enzymes microbiennes extracellulaires, substrats Methylumbelliferyl, milieu gélosé.
INTRODUCTION
Microbial hydrolysis of high molecular weight organic matter plays an important role in
the substrate turnover of aquatic environments. It is assumed that high molecular weight
organic matter is not directly utilized by bacteria but undergoes enzymatic hydrolyzation
to low molecular weight substances before incorporation.
Many microbiologists have tried to identify bacteria on the basis of “physiological
groups” for a more biochemically oriented analysis of the composition of a bacterial
population. Sieburth (1971) and Kjelleberg and Hakansson (1977) have investigated the
distribution of bacterial physiological groups in the sea on the basis of selective agar
media supplemented with starch, protein, other polymers or Tween-80. In freshwater
lakes Jones (1971) showed a seasonal fluctuation in separate populations of protease-,
amylase- and lipase-producing bacteria. This methodological approach was developed,
modified and also extensively used by many other microbiologists (Fred and Waksman,
1928 ; ZoBell, 1946 ; Sierra, 1957 ; Skerman, 1967 ; Holding and Collee, 1971). However,
the selective media technique is rather time consuming and zones of enzymatic activity
around colonies may very possibly overlap (e.g for amylase). Consequently, incubation
times for plates must be shortened, and results cannot be directly compared to standard
colony counts in every case.
175
IFREMER, Actes de Colloques, 3, 1986, pp. 175-183
CNRS, Brest, 1-5 octobre 1984
19
MICROBIAL EXTRACELLULAR ENZYME DETECTION ON AGAR PLATES
BY MEANS OF FLUOROGENIC METHYLUMBELLIFERYL-SUBSTRATES
S.-J. KIM and H.-G. HOPPE
Institut Für Meereskunde, Düsternbrooker Weg 20, 2300 KIEL (FRG)
ABSTRACT - A rapid and sensitive method to detect the extracellular enzymatic activity of bacteria colonies
grown on agar plates is described. Selective agar media supplemented with protein, starch, chitin, Tween-80, etc.
are conventionally used to detect biochemical properties of bacteria. It has been experimentally demonstrated
with bacteria pure cultures that fluorogenic Methylumbelliferyl (MUF) -substrates are excellent substrate
analogues for normally occurring polymers. Based on MUF-substrate hydrolysis the new method provides
reliable qualitative estimates of extracellular enzymatic properties of bacteria within minutes using pure
cultures as well as agar plates prepared for colony counts.
Key words: microbial extracellular enzyme detection, methylumbelliferyl-substrates, agar plate.
RÉSUMÉ - Une méthode rapide et sensible de détection de l’activité enzymatique extracellulaire de colonies
bactériennes se développant sur milieu gelosé est décrite. Des milieux sélectifs enrichis en protéines, amidon,
chitine, Tween 80, etc. sont généralement utilisés pour détecter les propriétés biochimiques des bactéries. Il a été
démontré expérimentalement sur cultures pures de bactéries, que les substrats au Methylumbelliferyl fluorogénique sont d’excellents analogues des substrats polymériques courants. Basée sur l’hydrolyse des substrats MUF
cette nouvelle méthode fournit des estimations qualitatives fiables sur les propriétés des enzymes extracellulaires
des bactéries. Le résultat est obtenu en quelques minutes en utilisant des cultures pures aussi bien que des
colonies ensemencées sur agar.
Mots clés : détection d’enzymes microbiennes extracellulaires, substrats Methylumbelliferyl, milieu gélosé.
INTRODUCTION
Microbial hydrolysis of high molecular weight organic matter plays an important role in
the substrate turnover of aquatic environments. It is assumed that high molecular weight
organic matter is not directly utilized by bacteria but undergoes enzymatic hydrolyzation
to low molecular weight substances before incorporation.
Many microbiologists have tried to identify bacteria on the basis of “physiological
groups” for a more biochemically oriented analysis of the composition of a bacterial
population. Sieburth (1971) and Kjelleberg and Hakansson (1977) have investigated the
distribution of bacterial physiological groups in the sea on the basis of selective agar
media supplemented with starch, protein, other polymers or Tween-80. In freshwater
lakes Jones (1971) showed a seasonal fluctuation in separate populations of protease-,
amylase- and lipase-producing bacteria. This methodological approach was developed,
modified and also extensively used by many other microbiologists (Fred and Waksman,
1928 ; ZoBell, 1946 ; Sierra, 1957 ; Skerman, 1967 ; Holding and Collee, 1971). However,
the selective media technique is rather time consuming and zones of enzymatic activity
around colonies may very possibly overlap (e.g for amylase). Consequently, incubation
times for plates must be shortened, and results cannot be directly compared to standard
colony counts in every case.
175