Isolation of Fecal Bacteria from Water Samples by Filtration

1. Water Sample Collection and Processing

1. Collect several 1-L water samples from the test water source (e.g. water fountains, swimming pools, reservoirs, water distribution systems, raw or treated sewage), and transport on ice to the laboratory for microbial analysis.
2. Sterilize or sanitize the membrane filtration manifold assembly prior to use by autoclaving, exposure to UV radiation (2 min), or ethanol-flame ignition.
3. Upon cooling of all parts, properly connect the manifold to a vacuum pump and vacuum filtration waste flask containing bleach.
4. Ethanol-flame sterilize a pair of forceps and with them, remove a sterile, gridded membrane filter from its packaging. Membranes 47 mm in diameter with a 0.45-µm pore size are typically used. However, alternate diameters and pore sizes can be employed provided that the pore size can sufficiently entrap the target microorganism(s), and at least 70% of the filter area is comprised of pore space.
5. Place the filter onto the center of the membrane filtration area of the manifold, and apply a sterile filter funnel to the unit and secure it into place.
6. Measure a desired volume of test water (e.g. 100 mL) and add it to the funnel (a 100-mL line mark is visible on the funnel).
7. Apply a partial vacuum [pressure differential of 34 to 51 kPa (kiloPascals)] in order to draw the test sample through the filter. Total suspended solid material, including bacteria and decaying organic matter, greater than the filter mean pore size of 0.45 µm are trapped on the filter. Smaller particles including viruses and dissolved solids such as small amounts of organic matter and salts, will pass through the membrane and into the waste container vacuum flask containing bleach.
8. Following complete passage of the sample through the filter, rinse the interior of the funnel with two to three 20-30 mL volumes of sterile water.
9. Power-off the vacuum and remove the funnel from the manifold upon closure of the final rinse.
10. With ethanol-flame sterilized forceps, immediately remove the membrane filter from the unit and promptly place it onto the appropriate growth agarose medium for the target microorganism (Table 1 lists the recommended growth media and incubation conditions for each).
11. Apply the membrane filter to the agarose surface with a roll-type motion to ensure complete contact of the membrane with the growth medium, and to avoid the entrapment of air bubbles.
12. Replace the used filtration funnel with a sterile unit between the processing of each sample, and ethanol-sanitize the stainless steel manifold in order to prevent cross-contamination.

2. Colony Enumeration

1. Following the incubation period, remove the growth plates from the incubator for enumeration.
2. Ideally, perform the colony counts under low power magnification using a cool, white light source.
3. Total coliforms
   1. Total coliform colonies are typically pink to dark-red in color with a metallic surface sheen. The sheen itself may either partially or completely cover the colony. Atypical total coliform colony morphologies may be dark red, mucoid, or nucleated without sheen.
   2. Colonies that are pink, blue, white, or colorless while lacking sheen are considered non-coliforms.
4. Fecal coliforms
   1. Fecal coliform colonies appear as various shades of blue.
   2. Nonfecal coliform colonies are grey to cream in color.
5. Fecal enterococci
   1. Fecal enterococci colonies range from pink to dark red in color.

3. Colony Verification

1. Total coliforms
   1. For a presence-absence verification, swab the entire membrane using a sterile inoculating loop. For colonies, it is preferable to verify at least five each of typical and atypical morphologies.
   2. Transfer the selected colony into a glass vessel containing lauryl tryptose broth with a Durham tube. Incubate the inoculated tubes at 35±0.5 °C for 48 h. The presence of turbidity indicating growth in conjunction with gas production verifies the colony as a coliform.
2. Fecal coliforms
   1. Transfer colonies blue in color aseptically into glass vessels containing sterile EC medium with a Durham tube. Incubate the inoculated tubes at 44.5 ± 0.2 °C for 24 h. The presence of turbidity indicating growth in conjunction with gas production verifies the colony as a fecal coliform.
3. Fecal enterococci
   1. Strike colonies typifying fecal enterococci morphology for isolation aseptically onto Brain-Heart Infusion Agar (BHIA), and incubate at 35 ± 0.5 °C for 24 to 48 h.
   2. Transfer growth from an isolated colony on BHIA onto two pre-cleaned glass slides.
   3. Add two to three drops of 3% hydrogen peroxide to prepared smears on the slides. The rapid appearance of bubbles indicates a “catalase positive” result, and the isolate is not a fecal streptococcus bacterium.
   4. Perform a Gram stain for isolates testing as “catalase negative” (no bubbles observed). Fecal enterococci are Gram-positive, ovoid, and appear mostly in pairs or short chains.

Fecal Bacterial Indicator	Recommended Media (Incubation Temperature, Time)1
Total coliforms	LES Endo Agar (35 ± 5 °C, 24 h) M-Endo Medium (35 ± 5 °C, 24 h)
Fecal coliforms	m-FC Medium (44.5 ± 0.2 °C, 24 h)
Fecal enterococci	m Enterococcus Agar (35 ± 0.5 °C, 48 h)

Table 1. Commonly-used culture growth media for the detection of fecal bacterial indicators in environmental samples
¹ As recommended by the Standard Methods for the Examination of Water and Wastewater (American Public Health Asssociation and the American Water Works Association, 22^nd Edition, 2012)