Detection of specific extracellular enzymatic properties of bacteria colonies
Chromatography filter papers of the same size as the Petri dishes used were soaked with
MUF-substrate working solutions. The soaked filter papers were then carefully layed on
bacteria colonies grown on the standard agar plate (c.f. bacteria inoculation). One of the
standard agar plates parallels was used for one MUF-substrate only. Care must be taken
to avoid air bubble formation between the agar layer and the soaked chromatography
filter paper because entrapped air bubbles result in decreased sensitivity. After ca. 3 minutes at room temperature the filters were removed from the agar surface and placed on a
glass plate or a clean and empty Petri dish. The filter papers were then observed under an
HBO fluorescence microscope lamp (365 nm) and the resultant fluorescent spots were
counted. A conventional UV lamp could not be used for this purpose.
Because MUF has its maximal fluorescence at alkaline pH values (optimal pH 10.3) the
filter papers were exposed to concentrated NH3 vapors before observation to enhance
fluorescence intensity (Fink and Koehler, 1970, Guilbault 1973). Exposure to concentrated NH 3 in a desiccator lasted about 1 minute. NH3 treatment was especially important
when MUF-glucosaminide was applied because the working solution for this substrate
has a pH of 4.95 which permits only a very low fluorescence emission. With other
MUF-substrates the working solution pH is already near the pH of maximal fluorescence
and only minor improvement of fluorescence intensity was observed.
Substrate analogue tests
To test whether or not MUF-substrates are analogues for polymeric substrates commonly
used for the detection of biochemical properties of bacteria (e.g. gelatin, starch, chitin and
Tween-80 (sorbitan monooleate)) we examined the response of pure cultures towards the
two types of substrates. The pure cultures were isolated from the Baltic Sea and from the
Atlantic Ocean (The Azores, Portugal) and cultivated in ZoBell 2216 E liquid medium
with a salinity of 0.8 % and 2.4 %, respectively. For this purpose the cells were harvested
and washed three times with autoclaved brackish water (sea water/ distilled water mixture
1: 3) via moderate centrifugation. The washed bacteria pure culture were suspended in
autoclaved brackish water and separated into four 20 ml portions. One of the substrates
was then added aseptically to one of the subsamples. The final concentration of MUFsubstrates was 2//Ml' 1 . Incubation was performed at 20°C in the dark. Relative fluorescence intensity of samples was measured three times, i.e. immediatly after substrate
addition, after 4 hours and after 24 hours, with a spectrofluorometer (Jasco model
FP-1050, excitation wavelenth 364 nm, emission wavelength 445 nm). Growth patterns of
the same bacteria cultures were comparatively studied by the use of ZoBell agar medium
supplemented with one of the following substrates: 0.4 % gelatin, l % soluble starch,
precipitated chitin (precipitated chitin was added until the agar medium became milky in
appearance) or 1 % Tween-80. The supplemented media were autoclaved at 125°C for 20
minutes and poured into sterile Petri dishes. The liquid pure cultures were streaked on the
prepared agar plates with a loop. Inoculated agar plates were incubated at 20°C for 2
weeks. Chitinase and lipase activity of each colony on the chitin-and Tween-80containing media was observed directly, as indicated by a distinct clear zone and halo
zone around active colonies, respectively. Protease was detected by applying a layer of
mercuric chloride-hydrochloric acid coagulant solution to the plate resulting in a halo
around active colonies. Amylase was similarly determined using dilute Lugol’s solution
leading to a starch-iodine reaction indicating zones of hydrolysis.
178
Chromatography filter papers of the same size as the Petri dishes used were soaked with
MUF-substrate working solutions. The soaked filter papers were then carefully layed on
bacteria colonies grown on the standard agar plate (c.f. bacteria inoculation). One of the
standard agar plates parallels was used for one MUF-substrate only. Care must be taken
to avoid air bubble formation between the agar layer and the soaked chromatography
filter paper because entrapped air bubbles result in decreased sensitivity. After ca. 3 minutes at room temperature the filters were removed from the agar surface and placed on a
glass plate or a clean and empty Petri dish. The filter papers were then observed under an
HBO fluorescence microscope lamp (365 nm) and the resultant fluorescent spots were
counted. A conventional UV lamp could not be used for this purpose.
Because MUF has its maximal fluorescence at alkaline pH values (optimal pH 10.3) the
filter papers were exposed to concentrated NH3 vapors before observation to enhance
fluorescence intensity (Fink and Koehler, 1970, Guilbault 1973). Exposure to concentrated NH 3 in a desiccator lasted about 1 minute. NH3 treatment was especially important
when MUF-glucosaminide was applied because the working solution for this substrate
has a pH of 4.95 which permits only a very low fluorescence emission. With other
MUF-substrates the working solution pH is already near the pH of maximal fluorescence
and only minor improvement of fluorescence intensity was observed.
Substrate analogue tests
To test whether or not MUF-substrates are analogues for polymeric substrates commonly
used for the detection of biochemical properties of bacteria (e.g. gelatin, starch, chitin and
Tween-80 (sorbitan monooleate)) we examined the response of pure cultures towards the
two types of substrates. The pure cultures were isolated from the Baltic Sea and from the
Atlantic Ocean (The Azores, Portugal) and cultivated in ZoBell 2216 E liquid medium
with a salinity of 0.8 % and 2.4 %, respectively. For this purpose the cells were harvested
and washed three times with autoclaved brackish water (sea water/ distilled water mixture
1: 3) via moderate centrifugation. The washed bacteria pure culture were suspended in
autoclaved brackish water and separated into four 20 ml portions. One of the substrates
was then added aseptically to one of the subsamples. The final concentration of MUFsubstrates was 2//Ml' 1 . Incubation was performed at 20°C in the dark. Relative fluorescence intensity of samples was measured three times, i.e. immediatly after substrate
addition, after 4 hours and after 24 hours, with a spectrofluorometer (Jasco model
FP-1050, excitation wavelenth 364 nm, emission wavelength 445 nm). Growth patterns of
the same bacteria cultures were comparatively studied by the use of ZoBell agar medium
supplemented with one of the following substrates: 0.4 % gelatin, l % soluble starch,
precipitated chitin (precipitated chitin was added until the agar medium became milky in
appearance) or 1 % Tween-80. The supplemented media were autoclaved at 125°C for 20
minutes and poured into sterile Petri dishes. The liquid pure cultures were streaked on the
prepared agar plates with a loop. Inoculated agar plates were incubated at 20°C for 2
weeks. Chitinase and lipase activity of each colony on the chitin-and Tween-80containing media was observed directly, as indicated by a distinct clear zone and halo
zone around active colonies, respectively. Protease was detected by applying a layer of
mercuric chloride-hydrochloric acid coagulant solution to the plate resulting in a halo
around active colonies. Amylase was similarly determined using dilute Lugol’s solution
leading to a starch-iodine reaction indicating zones of hydrolysis.
178