Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
The quality of water destined for use in agricultural, recreational, and domestic settings is of great importance due to the potential for outbreaks of waterborne disease. Microbial agents implicated in such events include parasites, bacteria, and viruses that are shed in high numbers in the feces of infected people and animals. Transmission to new and susceptible hosts may then occur via the fecal-oral route upon ingestion of contaminated water. Therefore, the ability to monitor water sources for the presence of pathogenic microorganisms is significant in order to ensure public health.
Due to the sheer number and variety of potential fecal-oral pathogens that may be present in water and their variable concentrations, it is impractical and expensive to assay directly for each one of them on a regular basis. Therefore, the microbiological assays for water quality monitoring employ coliform indicator bacteria. Coliforms comprise, in part, the normal intestinal microflora of warm-blooded mammals, are non-pathogenic, and are consistently excreted in the feces. Therefore, the detection of coliform bacteria in water means that a fecal release occurred, and that harmful pathogenic microorganisms may also be present.
The membrane filtration technique is used to assess the microbiological quality of water by assaying for fecal indicator bacteria. A quantity of water (e.g. 100 mL) is passed through a specialized membrane filter with a minimal mean pore size of 0.45 µm, facilitating the capture of bacteria, as they are approximately 1-µm in size. Following filtration, the membrane is carefully applied to a specialized agarose culture medium, and incubated under the conditions appropriate to culture the target microorganisms.
When applied for use in water quality monitoring, membrane filtration is most ideal for low turbidity sources such as drinking water, swimming pools, and natural recreational waters such as lakes and reservoirs. Waters high in particulate matter (e.g. raw sewage) will result in fouling of the filter; therefore, only smaller volumes (e.g. 100 mL) can be analyzed. Membrane filtration is also not practical for water sources with large numbers of background (or non-coliform) bacteria, which can increase the difficulty of enumerating the target coliform bacteria on the agarose medium following incubation.
This video demonstrates the collection of drinking water and environmental water samples, the membrane filtration of the samples, and the enumeration of several types of fecal indicator bacterial colonies using specialized agarose growth media including total coliforms, fecal coliforms, and fecal enterococci. Tests conducted further in order to verify presumptive colonies are also shown.
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1. Water Sample Collection and Processing
- 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.
- Sterilize or sanitize the membrane filtration manifold assembly prior to use by autoclaving, exposure to UV radiation (2 min), or ethanol-flame ignition.
- Upon cooling of all parts, properly connect the manifold to a vacuum pump and vacuum filtration waste flask containing bleach.
- 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.
- 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.
- 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).
- 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.
- 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.
- Power-off the vacuum and remove the funnel from the manifold upon closure of the final rinse.
- 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).
- 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.
- 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
- Following the incubation period, remove the growth plates from the incubator for enumeration.
- Ideally, perform the colony counts under low power magnification using a cool, white light source.
- Total coliforms
- 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.
- Colonies that are pink, blue, white, or colorless while lacking sheen are considered non-coliforms.
- Fecal coliforms
- Fecal coliform colonies appear as various shades of blue.
- Nonfecal coliform colonies are grey to cream in color.
- Fecal enterococci
- Fecal enterococci colonies range from pink to dark red in color.
3. Colony Verification
- Total coliforms
- 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.
- 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.
- Fecal coliforms
- 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.
- Fecal enterococci
- 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.
- Transfer growth from an isolated colony on BHIA onto two pre-cleaned glass slides.
- 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.
- 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, 22nd Edition, 2012)
Membrane filtration and the subsequent culturing of bacteria collected is a useful technique to assess the quality and cleanliness of a water source.
The quality of water destined for use in agricultural, recreational, or domestic settings is of great importance, due to the potential for outbreaks of waterborne disease. If water is contaminated with fecal matter from animals or humans, then pathogenic parasites, bacteria, or viruses may be spread to new hosts upon their ingestion. Monitoring water sources for such disease-causing organisms is therefore critical to ensure public health.
The sheer number and variety of fecal-oral pathogens that may be present in a water source makes it impractical to assay for each independently and on a regular basis. Instead, common microbiological assays for water quality utilize coliform indicator bacteria. For more information on this process, see this collection's video on indicator organisms.
This video will illustrate the process of membrane filtration on an environmental water sample, demonstrate how to culture several types of fecal indicator bacteria including total coliforms, fecal coliforms, and fecal entercocci, and describe how to verify the presence of fecal contamination.
Membrane filtration technique utilizes negative pressure to draw water samples across a filter and trap bacteria. The filter is a specialized membrane with a minimal mean pore size of 0.45 μm that allows the capture of bacteria, which are typically around 1 μm in size. After filtration, the membrane is applied to agarose growth media, and incubated at conditions appropriate to culture the target microorganisms.
This technique is most ideal for low turbidity sources such as drinking water, swimming pools, or lakes and reservoirs. Water high in particulate matter content can result in fouling or clogging of the filter, limiting the volume that can be processed. Additionally, membrane filtration is not practical for water sources containing large numbers of background, or non-coliform bacteria, like raw sewage, as this can increase the difficulty of enumerating target coliforms upon culture and incubation.
Once bacterial samples have been trapped in the filter, they can be transferred to growth plates to determine the types of indicator bacteria present in the water samples. Plating on different media types selects for different bacterial types, and can allow for rapid identification.
After growth on culture specific plates, further confirmation of indicator bacteria identities can be carried out using techniques such as picking colonies into liquid media and using Durham tubes to capture gases, which should only be produced in the presence of fecal coliforms or total coliforms. Additionally, suspected fecal enterococci can be confirmed by a combination of a positive Gram staining, along with a negative hydrogen peroxide-catalase test.
Now that we are familiar with the principles behind the membrane filtration of water samples, let's take a look at how this procedure is carried out.
To begin the procedure, first collect water samples from test water sources of interest. Ensure the samples are collected in sterile 1-L bottles. Once collection is complete, put the samples on ice, and transport them to the laboratory for microbial analysis.
To begin the analysis, first sterilize a membrane filtration manifold. Next, connect the manifold to a vacuum pump and filtration waste flask containing bleach.
Ethanol flame-sterilize forceps and remove a sterile gridded membrane from the packaging. Place the filter onto the center of the membrane filtration area of the manifold, and apply a sterile filter funnel to the unit, then secure in place.
Measure out a desired volume of test water into the funnel. Apply a partial vacuum to draw the test sample through the filter. Suspended solid material, including bacteria and other organic matter, greater than 0.45 μm will be trapped on or within the filter, while smaller particles, viruses, and dissolved solids will pass though into the waste flask containing bleach.
After the sample has passed through the filter, rinse the interior of the funnel with 25 mL of sterile water 3 times, allowing this to pass through the filter. When the final rinse is complete, disconnect the vacuum and remove the funnel from the manifold.
Next, ethanol flame-sterilize forceps and immediately remove the membrane filter from the unit. Place it onto the appropriate growth plate for the target microorganism using a rolling motion to ensure complete contact with the surface and avoid trapping air bubbles.
For the processing of each further sample, sanitize the stainless steel manifold and use a sterile funnel to prevent cross contamination. Finally, place the plates into an incubator for the appropriate incubation period.
Following the incubation period, remove the plates from the incubator for enumeration. If possible, perform the colony counts under low power magnification using a cool white light source. To determine total coliforms, identify and count colonies that appear pink to dark red in color, and have a metallic surface sheen fully or partially covering the colony. Atypical total coliform colonies may appear dark red, mucoid, or nucleated without sheen.
Colonies that appear blue, white, colorless, or pink without sheen are considered non-coliforms, and should not be included in the total coliforms count.
Fecal coliform colonies will appear as various shades of blue, and these should be counted as a separate category. Non-fecal coliform colonies are typically grey to cream in color, and should also be recorded in an individual category. Finally, fecal enterococci colonies will range from pink to dark red in color and should be counted separately.
To verify total coliform colonies, apply a sterilized and cooled inoculating loop to a single colony of interest. Transfer the selected colony into a glass vessel containing lauryl tryptose broth and a Durham tube. Next, place the cultures into an incubator. The presence of turbidity along with gas production captured by the Durham tube verifies the colony as a total coliform.
For fecal coliform verification, aseptically transfer colonies blue in color into glass vessels containing sterile EC medium and a Durham tube. Place the inoculated tubes into an incubator. After incubation, turbid inoculates in conjunction with gas production confirm the colony to be a fecal coliform.
To confirm fecal enterococci, aseptically transfer suspected colonies with the correct morphology onto Brain-Heart Infusion Agar plates, and incubate. Next, transfer growth from an isolated colony on BHIA onto two sterile glass slides.
Add 2-3 drops of 3% hydrogen peroxide to one of the glass slides. Rapid gas production indicates a catalase-positive bacterium such as Citrobacter. Fecal enterococci bacteria are catalase negative; therefore, no bubbling is observed. For catalase-negative colonies that don't display bubbling, perform a Gram stain. As fecal enterococci, these should appear Gram positive, ovoid in shape, and be grouped mostly in pairs or short chains.
Finding any of these indicator bacteria in a water source indicates the presence of a contamination. If more than 5% of samples are found to be contaminated over a one-month period, the source may be considered unfit for human consumption.
Membrane filtration is commonly used in a number of biological applications, and fecal indicator organisms can also be detected by other experimental procedures. Some of these applications are explored here.
Membrane filtration can also be used in virus capture from water samples. As viruses will typically be present at very low levels, water samples must be concentrated in order to capture them for analysis. Captured viruses can then be released from the filters, and identified using techniques such as cell culture infectivity assays or PCR.
Membrane filtration is also utilized in the production of high purity water for industrial or laboratory use. Many industries require highly purified water for their operational processes, and membrane filtration can serve to remove contaminants, including unwanted dissolved metals and salts from water. It can also be used in the desalination of salt water to produce potable water.
You've just watched JoVE's introduction to identifying indicator organisms in water by membrane filtration. You should now understand how to membrane filter water samples, how to culture several types of fecal indicator bacteria from the membrane, and how to confirm these as indicator organisms. Thanks for watching!
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Applications and Summary
Membrane filtration is used in virus capture and concentration from water. Human pathogenic viruses carry a net negative charge in aquatic solutions, and are often present at low levels in water sources. Therefore they must be concentrated prior to analysis. Membrane filtration is but one capture method for this purpose, and employs a negatively-charged filter. Water samples (e.g. 1-L) of interest are amended with a salt solution (e.g. magnesium chloride) to impart a positive charge to the viruses, thereby facilitating their adsorption to the negatively-charged HA membrane filter as the water is filtered. A low concentration acid solution is used to rinse the membrane and rid it of excess salts. A low concentration and volume of sodium hydroxide is then used to release the viruses from the filter prior to further concentrations and analyses (e.g. cell culture infectivity assays or quantitative PCR).
Membrane filtration is also utilized in the production of high-purity process water for industrial use. Many industries require highly purified water for their operational processes. Membrane filtration (e.g. nano-filtration) serves to remove contaminants including dissolved metals and salts from water. Membrane filtration is also used in the desalination of salt water to produce potable water.
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