Decomposition: Relative Bacterial Heterotrophic Activity
273
be needed. Swirl the samples gently several times during the incubation or place on
a very slowly turning rotary shaker.
7. Terminate the incubation by killing the bacteria. Carefully inject 0.5 ml per 10 ml
of sample of 2N sulfuric acid solution into the sample through the serum stopper
[or through the side-arm port when these modified flasks are used (see "Apparatus
and Supplies," p. 278)]. Avoid contact with the cup assembly. Note the time of
injection for each flask.
8. Allow the sealed flasks to stand for a few minutes with gentle shaking on a rotary
shaker. Add 0.2 ml of monoethanolamine directly to the filter paper in the cup by
injection through the serum cap. Incubate with shaking for 1 h.
9. Carefully remove the cup assembly and, with it poised over a labeled scintillation
vial, clip the support rod (Fig. 20.1) and allow the cup and filter paper to fall into
the vial. Add 15 ml of scintillation cocktail (PPO-bis-MSB-toluene-methanol, see
p. 278) and seal the vial. Mix well.
10. Filter the entire water sample onto 0.45-llm pore size membrane filters (e.g.,
Millipore HA). Keep an accurate account of the order of the samples since the
edges of the filters cannot be written on (interferes with radioassay). Rinse each
flask with 10 ml of prefiltered (0.45-llm pore size) water sample and filter with
the sample. The coating of the filtration funnels with a silicone compound (e.g.,
Beckman Desicote) prior to use prevents adhesion of cells to the walls.
11. Using forceps, place the wet filters directly into labeled scintillation vials, when
Instagel or other dioxane-based scintillation cocktails are used (see p. 278). The
filters must be dried under desiccation prior to radioassay when toluene-based
cocktails are used [e.g., Hobbie and Crawford (1969a)].
12. Radioassay the samples with a liquid scintillation counter, correcting for
quenching with the channels ratio method to yield the counting efficiency for each
sample. Convert the counts per minute values to disintegrations per minute.
Calculations
1. The uptake of organic compounds should exhibit saturation kinetics. With
saturation kinetics, the uptake velocity approaches a constant value in spite of
increasing concentrations (Wright and Hobbie, 1965, 1966), and a plot of uptake
velocity versus concentration demonstrates a hyperbolic relationship. Although the
measured activity likely resulted from bacterial enzyme-mediated transport systems,
the observed activity is a measure of a community response to the substrate.
2. The uptake can be analyzed by the well-known Michaelis-Menten kinetics for
enzyme-substrate systems:
(Vrnax)(S)
v=~-~Kt+S
(1)
where v = uptake velocity, Vrnax = theoretical maximum uptake velocity when all
uptake sites are saturated, S = substrate concentration (natural plus added), and
K t = transport constant~by definition, the substrate concentration at which
v = V rnax /2. This equation can be transformed by inversion and multiplication of
both sides of the equation by S to derive the Lineweaver-Burke equation:
(2)
273
be needed. Swirl the samples gently several times during the incubation or place on
a very slowly turning rotary shaker.
7. Terminate the incubation by killing the bacteria. Carefully inject 0.5 ml per 10 ml
of sample of 2N sulfuric acid solution into the sample through the serum stopper
[or through the side-arm port when these modified flasks are used (see "Apparatus
and Supplies," p. 278)]. Avoid contact with the cup assembly. Note the time of
injection for each flask.
8. Allow the sealed flasks to stand for a few minutes with gentle shaking on a rotary
shaker. Add 0.2 ml of monoethanolamine directly to the filter paper in the cup by
injection through the serum cap. Incubate with shaking for 1 h.
9. Carefully remove the cup assembly and, with it poised over a labeled scintillation
vial, clip the support rod (Fig. 20.1) and allow the cup and filter paper to fall into
the vial. Add 15 ml of scintillation cocktail (PPO-bis-MSB-toluene-methanol, see
p. 278) and seal the vial. Mix well.
10. Filter the entire water sample onto 0.45-llm pore size membrane filters (e.g.,
Millipore HA). Keep an accurate account of the order of the samples since the
edges of the filters cannot be written on (interferes with radioassay). Rinse each
flask with 10 ml of prefiltered (0.45-llm pore size) water sample and filter with
the sample. The coating of the filtration funnels with a silicone compound (e.g.,
Beckman Desicote) prior to use prevents adhesion of cells to the walls.
11. Using forceps, place the wet filters directly into labeled scintillation vials, when
Instagel or other dioxane-based scintillation cocktails are used (see p. 278). The
filters must be dried under desiccation prior to radioassay when toluene-based
cocktails are used [e.g., Hobbie and Crawford (1969a)].
12. Radioassay the samples with a liquid scintillation counter, correcting for
quenching with the channels ratio method to yield the counting efficiency for each
sample. Convert the counts per minute values to disintegrations per minute.
Calculations
1. The uptake of organic compounds should exhibit saturation kinetics. With
saturation kinetics, the uptake velocity approaches a constant value in spite of
increasing concentrations (Wright and Hobbie, 1965, 1966), and a plot of uptake
velocity versus concentration demonstrates a hyperbolic relationship. Although the
measured activity likely resulted from bacterial enzyme-mediated transport systems,
the observed activity is a measure of a community response to the substrate.
2. The uptake can be analyzed by the well-known Michaelis-Menten kinetics for
enzyme-substrate systems:
(Vrnax)(S)
v=~-~Kt+S
(1)
where v = uptake velocity, Vrnax = theoretical maximum uptake velocity when all
uptake sites are saturated, S = substrate concentration (natural plus added), and
K t = transport constant~by definition, the substrate concentration at which
v = V rnax /2. This equation can be transformed by inversion and multiplication of
both sides of the equation by S to derive the Lineweaver-Burke equation:
(2)