Assay for Coliform Contamination in Ambient Water

Assay for Coliform Contamination in Ambient Water

Man has long recognized the dangers of drinking water which is contaminated with fecal waste. Major diseases which are spread in this manner include cholera, salmonellosis, dysentery, shigellosis, polio and many others. Mammalian fecal wastes carry large numbers of gram negative rod-shaped bacteria which are capable of using lactose (milk sugar) as a carbon source. Bacteria with these properties are collectively termed coliform bacteria. The presence of coliform bacteria is widely used as an indicator of potential fecal contamination. Escherichia coli is the most famous member of the coliform group.

Ecoli_green_sheen_P8011313
E. coli typically has a green sheen

A selective and differential medium which can be used to enumerate the number of coliform bacteria is Levine EMB Agar. It contains nutrients, 1% lactose, agar and two dyes, E osin and Methylene Blue. These dyes serve two purposes: first, as a selective medium, they inhibit the growth of gram positive bacteria, and second, as a differential medium, they cause colonies which ferment the lactose (“lac+”) to turn purple (E. coli typically has a green sheen ) while the “lac” colonies will be pink or uncolored.

Ohio_River_0.2mL_MaC_P8011311
lac + appear red, while lac – appear white

A second medium which may be used to enumerate coliform is MacConkey’s Agar which contains 1% Lactose, 0.15% Bile Salts, and the dyes Neutral Red and Crystal Violet. These dyes act much the same as the dyes in EMB agar, but lac + appear red, while lac – appear white .

By spreading a known quantity of aqueous sample (often 0.1 mL) on one of these media and incubating until colonies have formed, the number coliform will be equal to the number of lac + colonies, and indicates the degree of potential fecal contamination. A sub set of coliform, termed fecal coliform, are defined as those coliform which grow at the elevated temperature of 44.5 C instead of the usual 35 C.

RELATED PROTOCOLS:
Commonly Used Media
Sterile Delivery of Liquids by Pipet
Plate Spreading Technique

EQUIPMENT LIST:
sterile screw-capped culture tubes
Levine-EMB and/or MacConkey Agar plates
sterile pipets, 0.1, 0.2 and/or 1.0 mL
spreader, turntable
flame
35C incubator

1. COLLECT SAMPLE: Collect sample as close to plating time as possible, or keep refrigerated until plating. Use sterile screw-capped culture tubes (10 or 20 mL), fill 2/3 full from midstream of a flowing stream . Do not contaminate with sediment from stream bottom. Cap immediately . Write in pencil on the tube’s frosted area: location of collection point, date and your initials. Samples from above and below sewage treatment plant outfalls can be interesting, demonstrating whether raw or improperly treated sewage is contaminating the river.
2. PLATE OUT ON THE DIFFERENTIAL AGARS: Label plate with seat number, initials, source and aliquot volume. Using sterile technique as outlined in Plate Spreading Technique , spread 0.1 mL of sample on EMB and/or MacConkey agar. Plate 0.2 mL if the sample is thought to be unpolluted. If you believe the sample to be highly polluted, pipet 0.1 mL of sterile dH2O on the plate, then deliver 0.05, 0.01 or even 0.005 mL to the plate and spread. Incubate at 35o C for 48 hrs.
3. SCORE THE PLATES: Count and record the total colonies and also the number of coliform colonies/plate. Those which are lactose fermenting are purple. Calculate the number of these categories/100 ml of sample, enter into your notebook.
4. ENTER INTO THE CLASS DATA SHEET. Enter your data in the spreadsheet on the computer. After class data have been collected, mount the table in your note book.
RESULTS:
We have found some interesting (and disturbing) concentrations of bacteria, and coliform in particular in samples over the past few years.

ICE TEA:

We demonstrated in 1994 that restaurant ice tea often contained extremely high numbers of coliform. Here are some plates of restaurant ice tea (0.1 mL each) one from July 2001 , and another from July 2000 . These contain so many bacteria that they could not be accurately counted, but we estimate that they contain between 500,000 and 1 million bacteria per 100 mL, most of which are coliform. The standard for drinking water is 5 coliform per 100 mL, and for recreational waters, 5,000/100 mL. It would be illegal to swim in these samples of ice tea.

STREAMS AND RIVERS:

Here is a picture of an EMB lactose plate on which was plated out only 0.05 mL of “treated” sewage from the Nine Mile Sewage Treatment plant outfall . Here is a MaConkey Agar plate containing 0.2 mL of Ohio River water taken on the same day. Notice that the bacterial count is MUCH lower than that of ice tea…

Coliform in Drinking Water

Coliform in Drinking Water

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:
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

millipore filtering apparatus:
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:
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

Plate Ready for Incubation:
(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):

00_water_sample_P8141447

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):

02_Apply_mEndo_broth_P8141444

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:

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.

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

Once the platform is dry:
SET UP MEMBRANE FILTER: Flame off EtOH from blade forceps

pick up sterile membrane filter , (discard the blue spacer discs)

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

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

 

14_Flame_cylinder_P8131463md

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.

Grasp the outside of the sterilized cylinder with your fingers. (It should not be too hot if you touched off the EtOH before flaming.)

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:

FILTER WATER THROUGH:
Pour your 250 mL water sample into the cylinder , monitoring that it is not leaking at the clamped joint .

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.

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.)

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 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 . . .

. . . it drops down onto the pad.
If done properly, it will be
centered on the pad .

Ensure that the membrane is completely flat on the pad.

VII. INCUBATION AND CALCULATION OF RESULTS

28_colonies_P8161470

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.
Most chlorinated tap water will have no bacteria in 250 mL, as seen on the left membrane in the first of the nest set of pictures.
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

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…

12_Immerse_cylinder_EtOH_P8141447
grasp with fingers, then tongs: