SDCK LABS
BACTERIA ANALYSIS: WATER SAMPLES
INTRODUCTION
The overall procedure of bacterial analysis includes diluting the sample,
transferring either the Colisure or Enterolert reagent into the sample, pouring
the sample into an IDEXX tray, sealing the IDEXX tray, and incubating the
sealed IDEXX tray. There are two incubators in the lab. One is set at 35°C
and another at 41°C. Samples mixed with the Colisure reagent should be
placed in the 35°C incubator. Samples that are mixed with the Enterolert
reagent should be placed in the 41°C incubator. All samples should be
prepared ahead of time placed in the incubators all at once because opening
the incubators causes temperature fluctuations. Both incubators should be
within one(1) degree of the desired temperature.
MATERIALS
1 - gallon distilled water
1 - bottle of sanitizing solution (alcohol hand sanitizer or anti-bacterial soap)
1 - roll of paper towels
1 - 100ml whirlpack water sample
1 - 100ml whirlpack BLANK (reagent and distilled water)
2 - reagent capsules
- Colisure
- Enterolert
2 - 120 ml sterile plastic specimen containers
2 - sterile 10ml plastic pipettes
2 - pipette bulbs
2 - IDEXX Quanti-Tray®/2000 sample trays
1 - Sealer
1 - silicon feeder for sealer
2 - incubators
2 - thermometers
1 - long wave UV lamp @ 365nm
1 - autoclave
1 - disposable 2ml plastic pipette
***IDEXX anti-foam if possible.
PROCEDURE
I. PREPARING WORK AREA
1) The two incubators need to be set to 41°C(Entero) and the other to
35°C(E.Coli).
2) Wash hands with anti-bacterial soap or hand sanitizer.
3) Clean the work area with sanitizing solution.
4) Place sterile paper towels down covering the work area.
5) Have sterile pipette (still sealed), pipette bulb, specimen container,
100ml whirlpack water sample, and reagents out and ready to process.
6) Turn on the Sealer and let it warm up for at least 15 minutes.
II. PREPARING SAMPLE
5) Label trays with...
a. Date
b. Site ID
c. Temperature of Incubation
d. Name of Lab Technician
e. Lab Location
f. Dilution
g. Colisure Grid or Enterolert Grid
Colisure Grid for Total Coliform and E-Coli(Figure 1)
MPN
Large Small (most probable
Cells Cells number)
Total
Coliform
E. Coli
(24
hours)
Figure 1: Example grid label written directly onto the trays using
the Colisure reagent. Grid is used to count the number of positive
and negative cells at the end of the incubation period. Positive cells
are then then matched to the IDEXX Quanti-Tray®/2000 MPN
Table(step 23).
Enterolert Grid for Enterococci(Figure 2)
MPN
Large Small (most probable
Cells Cells number)
Enteroco
cci
(24
hours)
Figure 2: Example grid label written directly onto the trays using
the Enterolert reagent. Grid is used to count the number of positive
and negative cells at the end of the incubation period. Positive cells
are then then matched to the IDEXX Quanti-Tray®/2000 MPN
Table(step 24).
6) Open sterile specimen container and add approximately 20 ml of
distilled water.
7) Empty one reagent capsule into the specimen container by cracking it
directly above the container. See example video 1 below.
Example Video 1 @ 2 minutes:
http://www.idexx.com/water/colilert/colilert300_en.jsp
8) Gently swirl the specimen container to dissolve the reagent completely.
9) Next take the 100ml whirlpack water sample and gently twirl the
whirlpack to homogenize the sample. You can also squeeze the bottom of
the whirlpack gently with your hand.
10) Open the sterile plastic pipette and attach bulb.
12) Using the pipette extract 10ml from the homogenized 100ml whirlpack
water sample. Release the 10ml into the specimen container.
13) Fill the specimen container up to the 100ml mark as accurately as
possible with distilled water. You can do this by using the disposable 2ml
plastic pipette. Minimize bubbles and froth, otherwise the tray will not
seal correctly.
14) Open IDEXX tray. Hold tray with left hand, while cells are facing up, and
with your right hand pull on the white tab.
15) With the tray open, have partner pour the 100ml solution from the
specimen container into the tray.
16) Place tray into silicon feeder by matching up the cell sizes. Make sure
the notch in the feeder is situated towards the bottom right of the small
cells on the tray.
17) With the white plastic facing up, the feeder should be inserted into the
sealer with the small cells going in first. This will ensure that the sample
solution in the tray does not spill out.
18) Repeat steps 5-17 to prepare all trays for all sampling sites. All trays
should be placed into the incubators at the same time!
19) Finally, once all trays have been prepared, record onto each tray
(where previous information has been recorded) the ‘time of the
start of incubation’!!!
20) Place trays into their corresponding incubators.
- Colisure reagent trays into the incubator set to 35°C. These will
incubate for a period no less the 24 hours.
- Enterolert reagent trays into the incubator set to 41°C. These will
incubate for a period no less the 24 hours.
*Do not stack trays more then ten high!!! Be careful not to block
air circulation in the incubators!!!
III. POST-INCUBATION: Recording the Results (24 hours later)
21) Turn on UV lamp to allow for a 10min warm-up.
22) Remove the trays form the incubators and add to the record the time
and date of removal on each tray.
23) Recording and interpretation of the results for the Colisure trays
are as follows. These results must be interpreted within 24 hours after
the incubation period.
a. A positive signal for the total coliform will have a magenta color.
Count the number of
large cells and small cells that have turned magenta. Record onto
the grid along the Total Coliform row(see figure 1).
b. A positive signal for the E-Coli will have, in addition to the
magenta color, a fluorescent
blue color. Any cells that do not have the magenta color and
fluoresces does not count as a positive! Using the UV-lamp @
365nm carefully shine the light onto the tray by holding it a few
inches above the cells. Make sure that the lamp is not directed
at anyone’s eyes! Count the number of large cells and small cells
that fluoresce blue. Record onto the grid along the E-Coli row(see
figure 1).
c. Refer to the IDEXX Quanti-Tray®/2000 MPN Table and record the
MPN number in
the last column of the grid. Link below.
http://www.idexx.com/water/refs/096323501.pdf
24) Recording and interpretation of the results for the Enterolert trays
are as follows. These
results must be interpreted within 4 hours after the incubation
period.
a. A positive signal for the Enterococci will have a fluorescent blue
color. Using the UV-
lamp @ 365nm carefully shine the light onto the tray by holding it a
few inches above the cells. Make sure that the lamp is not
directed at anyone’s eyes! Count the number of large cells and
small cells that fluoresce blue. Record onto the grid along the
Enterococci row(see figure 2).
b. Refer to the IDEXX Quanti-Tray®/2000 MPN Table and record the
MPN numbers in
the last column of the grid. Link below.
http://www.idexx.com/water/refs/096323501.pdf
Clean-up:
To be determined. For now simply store the trays in a cool container with a
lid.
