
j. Disposable wipes.**
k. Hydrochloric acid, HCl, and sodium hydroxide, NaOH, 1M
each, for pH adjustment.
l. Potassium iodide, KI. Dissolve 10 g KI in 100 mL reagent
water.
m.Starch indicator solution. Either use commercial product or
prepare as follows: to 5 g starch (potato, arrowroot, or soluble),
add a little cold water and grind to a thin paste in a mortar. Pour
into 1 L boiling water, stir, and let settle overnight. Use clear
supernate, preserving with 1.25 g salicylic acid and 4 g zinc
chloride, or a combination of 4 g sodium proprionate and 2 g
sodium azide per L.
4.
Procedure
a. Sample collection, handling, and treatment: Collect 250 to
500 mL sample in glass, polyethylene, or polypropylene jars or
bottles, preferably leaving no airspace. Test sample as soon as
possible, preferably within 24 h of collection. Before use, store
sample in the dark at 4 ⫾2°C. Prompt testing avoids possible
changes in sample toxicity.
Centrifuge sample if significant turbidity is present. Transfer
cleared sample to a new container. (NOTE: The material causing
turbidity may also have toxic effects.) Other tests are available
(i.e., solid-phase testing procedures
3
) if turbid samples must be
analyzed as received.
Luminescent bacteria are adversely affected by pHs below 6.0
and above 8.0, so adjust sample pH to between 6 and 8 with
either NaOH or HCl, as needed. If over-titration occurs, discard
sample and start over.
Osmotically adjust samples to a final concentration of 2.0%
(M/M) as NaCl. Pour 50 g homogenized sample into a 100-mL
beaker. Tare out the sample-filled beaker and add 1.00 g solid
analytical reagent (AR)-grade NaCl crystals to the beaker. Stir
with a disposable glass pipet until all crystals have completely
dissolved.
Luminescent bacteria are sensitive to chlorine, so if necessary,
dechlorinate samples with sodium sulfite. Determine amount of
sulfite solution required on a 100- to 1000-mL portion of neu-
tralized sample by adding 10 mL 1 ⫹1 acetic acid, 10 mL KI
solution (10 g/100 mL) per 1000 mL sample, and titrating with
Na
2
SO
3
solution to starch–iodide endpoint (blue color is dis-
charged). Add to neutralized sample the proportional volume of
Na
2
SO
3
solution determined by the above residual test, mix, and
check for residual chlorine after 10 to 20 min.
b. Prepare positive control: As part of the basic test protocol,
include positive controls of either phenol or ZnSO
4
and analyze
them at least once a day (frequency depends on the number of
samples to be tested). Phenol ordinarily has an IC
50
between 13
and 26 mg/L after 5 min exposure, while ZnSO
4
has an IC
50
between 5 and 12 mg/L after 5 min exposure.†† Prepare a
100-mg/L stock solution of either phenol or ZnSO
4
by accurately
weighing and transferring 100 mg of chemical into a clean,
rinsed (with dilution water), 1000-mL volumetric flask. Protect
the phenol standard from light and prepare in an amber-colored
or aluminum-foil-wrapped flask. Although both standards will
last for 3 to 4 months when stored at 2 to 8°C, preferably prepare
standards on each day of testing. Alternatively, analyze stan-
dards on testing day to determine actual concentration.
c. Prepare analyzer and bacteria (Vibrio fischeri): Turn on the
analyzer at least 30 min before testing begins to allow the unit to
bring reagent, incubator, and read wells to their preset temper-
atures. Follow manufacturer’s instructions for calibrating and
operating instrument.
Place clean, unused cuvettes in the reagent well (maintained at
5.5 ⫾1°C) and sample wells of the incubator block (maintained
at 15 ⫾0.5°C)—as many as required for the number of samples
to be analyzed. When handling cuvettes, hold them near the open
end so light measurements are not affected by finger or hand-
prints. To set up analyzer for acute toxicity testing of one sample
at multiple dilutions, follow the incubator diagram (Figure 8050:
1).
Place 1000
L reconstitution solution (ultrapure water) into
the cuvette in the reagent well. Allow 10 min for solution to
reach the temperature in the well. Take a reagent vial from the
freezer, tap the pellet to bottom of vial, and remove seal.
Take water-filled cuvette from reagent well, and place cuvette
lip on top of reagent vial. Quickly pour distilled water into
reagent vial (slow reconstitution causes bacteria lysing and low
reagent light levels). Swirl reagent vial only three or four times
(overmixing will warm reagent), pour mixture back into cuvette,
and place cuvette back in reagent well.
Use the 500-
L pipettor to mix reconstituted reagent by filling
and dispensing the pipettor 20 times for uniform dispersal of
reagent. Allow 20 min for reconstituted bacterial reagent to
stabilize.
d. Prepare samples: Pipet 500
L diluent (2% NaCl solution)
into each cuvette in Wells B1, B2, B3, B4, B5, D1, and D2. Pipet
1000
L diluent into each cuvette in Wells A1, A2, A3, A4, A5,
and C1. The cuvette in Well C2 receives no diluent.
Then, pipet 1000
L osmotically adjusted sample into cuvette
in Well C2 (which is empty) and into cuvette in Well C1 (which
contains diluent). Mix cuvette in Well C1 using pipettor.
To prepare the series of 1:2 dilutions, transfer 1000
L from
C1 to A5 and mix using pipettor. Then, transfer 1000
L from
A5 to A4 and mix using pipettor. Transfer 1000
L from A4 to
A3 and mix using pipettor. Then, transfer 1000
L from A3 to
** Kimwipes
®
, or equivalent.
†† Reagent Certificates of Performance, which report IC
50
values for phenol and
ZnSO
4
, are available from Strategic Diagnostics, Inc. (formerly Azur Environ-
mental) for each lot number of bacteria.
Figure 8050:1. Incubator diagram for acute toxicity testing of one sam-
ple at multiple dilutions.
BACTERIAL BIOLUMINESCENCE (8050)/Bacterial Bioluminescence Test
2
BACTERIAL BIOLUMINESCENCE (8050)/Bacterial Bioluminescence Test