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COLIFORM IN DRINKING WATERProfessor of Biology and Chemistry University of Cincinnati Clermont College Batavia OH 45103 |
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(apparatus with labels) |
This page has been accessed
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"filtered water" is on the right... |
Modified from: Standard Methods for the Examination of Water and Wastewater, 14th Ed, (1975). pages 928-935.
Students can test the drinking water
from their homes, school, etc using this protocol. The health
standard for
drinking water allows no more than 5 coliform per hundred mL. For
this
reason, bacteria from 250 mL will be collected on a membrane filter,
and
grown on top of m Endo medium. Coliform bacteria appear as red
colonies
on this medium. More than 13 colonies with this assay does not
meet
health standards.
One student in 2001 tested her tap water
before
and after filtration through a water "purification" device.
The image shows two plates
, the one on the left is tap water, the plate on the right is water
filtered
through the filter. Clearly the filter is contributing bacterial
load
to the water. These devices must be regularly changed and
maintained
in order to prevent such contamination.
| EQUIPMENT AND SUPPLIES: | millipore filtering apparatus: |
| Sterile 250 mL capped bottles
(1/student) sterile 47 mm petri dishes (1 per student) sterile 47 mm memb. filters, 0.45 µm pores sterile 47 mm millipore pads vacuum pump 3 vacuum hoses joined with "T" joint 2 strong hose clamps 1000 mL side arm filter flask m-Endo Broth MF powder sterilized repipet in 250 mL bottle |
sintered glass platform in #8
stopper glass cylinder, 300 mL capacity 800 mL beaker with 400 mL EtOH 150 mL beaker with 100 mL EtOH triangle-tipped Tongs forceps with bent tipped blade Bunsen burner protective eye wear |
MAKE THESE TWO ILLUSTRATIONS WITH
LISTED FEATURES LABELED:
(pay close attention to the location of
the
valves)
| Vacuum Filtering Apparatus: | Plate Ready for Incubation: | |
apparatus:  note the labeled valves:  |
vacuum pump on/off power switch main vacuum line main vacuum line clamp T joint relief valve clamp filter flask #8 stopper screen platform membrane filter glass cylinder, 300 mL cylinder clamp |
(exploded view)
50 mm petri dish top 47 mm membrane filter 47 mm pad with 2 mL m-Endo MF 50 mm petri dish bottom |
I. COLLECTION OF WATER
(Collect the same day as performance of assay):
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Determine the precise name of
your water district, record it in your notebook.
Run tap water until it is cold (to clear out pipes, at least a minute or so). Fill sterile 250 mL capped bottle with water, rinse several times, finally fill to neck, cap securely, maintaining sterility . |
II. PREPARATION OF 50 mL OF MEDIUM
(for 20 assays):
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a: For up to 20
determinations, weight
out 2.4 g m-Endo Broth MF powder into 150 mL beaker. b: Add 49 mL dH20 and 1 mL EtOH. c: Bring to boil, remove from heat immediately. Using sterile technique, pour into sterilized repipet vessel. Securely screw down repipet to container. Clamp to ring stand for stability. |
III. PREPARATION OF PAD:
sterile pad in petri dish:  |
add 2.0 mL m-Endo Broth |
Label 47 mm top of sterile petri
dish with
initials, seat number, date & source.
Flame off EtOH from bent blade-tipped forceps, pick up sterile pad, place in bottom of sterile petri dish , replace cover. Repipet 2.0 mL m-Endo Broth sterilely onto pad , replace lid, keep bottom down. |
IV. SET UP PLATE: Note: Wear safety
goggles,
tie back hair, keep flammable materials away from flame.
inadequate head space:  |
close relief valve:  |
PRELIMINARY: Check for
adequate head space in the filter flask to contain the water you
are about to filter. If inadequate, empty out the
flask into the sink, and replace the support platform. If necessary,
re-sterilize
surface of support platform with EtOH, turn on vacuum briefly to dry,
then
tightly clamp the main vacuum line.
Also, apply vacuum to the platform to remove any water or alcohol remaining from the previous filtrations by closing the relief valve, opening the main valve, then closing the main valve. |
open relief valve:  |
tongs in alcohol:  |
Once the platform is dry: SET UP MEMBRANE FILTER:
Flame off EtOH from blade forceps |
flame off alcohol 
|
pick up sterile membrane  |
pick up sterile membrane filter , (discard the blue spacer discs) |
apply to platform  |
center the membrane:  |
center it on screen platform . Open main vacuum line to hold membrane in place (turn on pump if not on yet). |
V. THE DANGEROUS STEP: STERILIZING AND FLAMING THE CYLINDER
| grasp with fingers, then tongs:  |
touch off excess alcohol  |
STERILIZE THE CYLINDER (Dangerous
step): Ensure that
all surfaces of the glass cylinder are immersed in 95% EtOH
, pick up with fingers (touch outside of the cylinder only),
invert.
Grasp cylinder upside down with triangle-tipped tongs (Ekco, for instance) and allow excess EtOH to drip into 800 mL beaker, touching off the last drops on a paper towel. |
Pass through flame to ingnite, hold away from people and
beakers of alcohol!  |
Maybe ignited too soon?  |
CAREFULLY flame off the EtOH away from EtOH beakers and other students). It will flame up fairly high, but should burn off in a few seconds. Pass quickly through the flame once more to ensure that all of the EtOH has been removed. |
flaming stopped, grasp with fingers  |
Grasp the outside of the sterilized cylinder with your fingers. (It should not be too hot if you touched off the EtOH before flaming.) | |
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SET UP FILTER APPARATUS:
Place sterile cylinder centered over the membrane filter and
support platform. Clamp in place with spring clamp , vacuum still on. |
VI. FILTERING THE WATER AND APPLYING MEMBRANE TO PREPARED
PAD:
Pour water into apparatus  |
FILTER WATER THROUGH:
Pour your 250 mL water sample into the cylinder
, monitoring that
it is not leaking at the clamped joint
. |
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the vacuum draws it through  |
empty all 250 mL into cylinder  |
The vacuum will draw the water through, and all bacteria which may be in the 250 mL sample will be trapped on the surface of the membrane filter. |
unclamp cylinder  |
remove cylinder  |
When all of the water has been drawn through, unclamp and remove the cylinder , place it carefully back in the EtOH, top down. (Do not let it drop into the beaker.) |
slip tweezers under membrane  |
gently lift membrane  |
RELEASE VACUUM ON MEMBRANE
FILTER
: Clamp the main vacuum line shut and open the relief valve to release the vacuum in the flask. With sterile, EtOH-free blade forceps, gently lift the edge of the membrane filter and remove from the screen platform . (Caution: the membrane filter is brittle.) |
transfer to prepared dish  |
slide over edge to drop onto pad  |
TRANSFER MEMBRANE FILTER TO PREPARED PAD , avoiding bubbles by lowering from one side first. Rest it on far edge of petri dish, slowly pull it across the edge down toward you until . . . |
do not let it rest on edge  |
centered membrane on pad  |
. . . it drops down onto the pad.
If done properly, it will be Ensure that the membrane is completely flat on the pad. |
VII. INCUBATION AND CALCULATION OF RESULTS
| INCUBATE the plate
without inverting (pad-side down) at 35C for 24 hours.
COUNT THE COLONIES:
Record the total number of colonies, and the number of coliform (red
colonies)
. Divide by 2.5 to yield the number of bacteria per 100 mL. According
to national
health standards for drinking water, the number of coliform/100 mL
should
not exceed 5. Enter your data in this sequence and format into your notebook, and then into the spreadsheet for the class table in the computer Desk No. Initials Source in detail (water district) Coliform/100 mL |
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| tap water vs. "filtered" water
Williamsburg OH water |
TOP: tap water and
"filtered" water from the same home. Note that filters must be
changed regularly, or else they become "nesting" places for bacteria.
MIDDLE: The two water samples shown are both heavily contaminated. BOTTOM: For years, Williamsburg had problems with their water, as can be seen in the 8/14/00 sample. We even had a village official tell a student it was against the law for her bring in a sample of her own water to test... |
