Biological Monitoring and Fecal Coliform Testing of Brush Creek
Objectives:
Activities:
Outline:
Fecal Coliform Bacteria -- Membrane Filter Method
Biological Monitoring -- Why Do It?
The quality of a stream's health can be determined in several ways. Physical monitoring gives information about the watershed of the stream and can help to identify possible sources of water quality problems. Chemical analysis provides information about selected parameters at one moment in time. In order to get an indication of stream conditions over a longer period of time, we need to look at the biological community the stream supports. A more complete assessment of water quality can be accomplished by evaluating the physical, chemical and biological aspects of a stream.
Biomonitoring involves sampling the biological community to determine the stream's health. After being collected, biological organisms are identified, the results are scored and the stream is given a quality rating. Benthic (or bottom-dwelling) macroinvertebrates are the biological indicators that you will be using as measures of the health of Brush Creek. Why should people who are concerned with the water quality of a stream study macroinvertebrates? There are several reasons:
1. Macroinvertebrates are non mobile, which means that they cannot escape the effects of water pollution.
2. There are many species of macroinvertebrates, and different species have different abilities to tolerate pollution. In clean streams, the macroinvertebrate community will be dominated by various pollution-intolerant (or pollution-sensitive) macroinvertebrates. In a polluted stream, the macroinvertebrate community will consist mostly of species that are pollution-tolerant (or pollution-insensitive).
3. Macroinvertebrates provide a way of monitoring the long-term conditions of water quality in a stream
4. Macroinvertebrates are easy to collect and relatively easy to identify.
7. The equipment needed to collect and identify macroinvertebrates is inexpensive, and no chemicals are needed to do it.
Macroinvertebrate Morphology (or Shape) (page 122, SFG)
The use of macroinvertebrates in water quality monitoring involves identifying
the various types and relative abundance of macroinvertebrates that live
on the bottom of a stream. Iidentification and classification of macroinvertebrates
is based upon their morphological (or shape) characteristics. Clams and
smails are perhaps the easiest "bugs" to identify to the class
level (Class Gastropoda for snails and Class Pelecypoda for clams) because
of their well-known characteristic shape. To identify something as an insect
(Class Insecta), crustacean (Class Crustacea), or a worm (Class Oligochaeta)
can be more difficult. The best clues to look for are
1) the presence of a head and legs
2) the number of legs, and
3) the presence of wings and exoskeleton (an outer covering that
provides support in lieu of an internal skeleton).
All adult insects have exactly six legs, while most crustaceans have more than six legs. Most adult insects have wings, while crustaceans do not. Both insects and crustaceans have heads and exoskeletons. Worms do not have any legs, heads wings, or exoskeltons.
The insect body is segmented and divided into three major regions; head, thorax, and abdomen. The head appears to be a single segment but is actually composed of several fused segments. Just below the head, the thorax is composed of three distinct segments, with one pair of legs attached to each (hence a total of six legs). If an insect has wings, they are also attached to the thorax. Just below the thorax, the abdomen is usually the longest region of an insect's body and is composed of several segments.
Unfortunately, aquatic insects can be difficult to identify as belonging to the Class Insecta because many are immature forms and thus are not fully developed. While many immature forms do have six legs, a distinct thorax and abdomen, and even undeveloped wings, many lack these features, and even appear headless.
In order to distinguish these immature insects from worms, look for other appendage-like attachments, such as gills, tails, filaments, and prolegs. Prolegs are fleshy, unsegmented, nubby leg-like structures attached to the thorax and/or abdomen of some immature insects. They are not to be confused with the regular legs of insects, which are segmented and usually longer and more slender. Some immature aquatic insects may have prolegs in addition to their six regular segmented legs.
Identifying Macroinvertebrates -- the Dichotomous Key
Identifying macroinvertebrates is a relatively easy procedure that involves the use a certain type of key called a "dichotomous key." A dichotomous key consists of pairs of opposite choices or descriptive statements. The dichotomous key that you will be using to identify macroinvertebrates from Brush Creek is one that was developed by the Izaak Walton League of America (one of the country's first conservation organizations). To use the IWLA key, go to the key instructions web page by clicking on the following picture, read the directions, and proceed as directed. Be sure to tally the different types and numbers of macroinvertebrates that you find on the MACROINVERTEBRATE COUNT sheet provided by your T.A., and calculate a water quality rating for Brush Creek based on the macroinvertebrates that were collected at your monitoring site.
Introduction
Most streams contain a variety of microorganisms including bacteria, fungi, protozoa and algae. Most of these occur naturally and have little or no impact on human health. However, human health can be affected when disease-causing organisms (pathogens) enter streams.
Coliform bacteria can enter a stream as a result of atmospheric deposition, urban and rural runoff, wastewater discharges, septic tanks, and direct contamination by animals. The survival and growth of these and other bacteria in water depend on the physiological requirements of the bacteria and the physical, chemical, and biological conditions of the stream. Flow conditions, turbidity, water temperature, nutrients, oxidation-reduction conditions, presence of toxic substances, competition, and predation are some of the factors controlling the survival, growth, and reproduction of bacteria in water. Total bacteria counts in streams can range from several hundred to thousands of bacteria per milliliter (ml). Because bacteria tend to be attached to solids, the suspended concentration of bacteria in streams can be at least as variable as suspended sediment concentrations.
Bacteriological tests are used to assess the sanitary quality of water and the potential human health risk from waterborne disease. These tests are used to assure the safety of potable water and water used for recreation from waterborne diseases. Bacteriological tests also are used to determine the effectiveness of wastewater treatment.
Indicator Bacteria -- Fecal Coliform
Pathogenic organisms are relatively scarce in water, making them difficult and time-consuming to monitor directly. Because of the many kinds of pathogenic bacteria potentially present, monitoring for each on a routine basis is not practical. Instead, indicator bacteria, such as fecal coliform, are monitored. The presence of fecal coliform bacteria is an indication that pathogenic bacteria may be present. Fecal coliform bacteria by themselves are not pathogenic. Fecal coliform bacteria naturally occur in the digestive tract and feces of warm-blooded animals and aid in the digestion of food. Pathogenic organisms are found along with fecal coliform bacteria in infected organisms. Missouri Water Quality Standards list Fecal Coliform as the bacteria test to be used in Missouri waters. The following is an excerpt from the Rules of Department of Natural Resources, Chapter 7, Water Quality. "Fecal Coliform Bacteria: Protection of whole-body-contact recreation is limited to classified waters designated for that use. For periods when the stream or lake is not affected by storm water runoff, the fecal coliform count shall not exceed two hundred colonies per one hundred milliliters (200/100 ml) during the recreational season in waters designated for whole-body-contact recreation or at any time in losing streams. The recreational season is from April 1 to October 31."
Sources of Variation and Interpretation of Fecal Coliform Data
Fecal coliform levels in water are highly variable. The two greatest sources of variation are changes in stream flow and wastewater discharges. During rainfall which produces runoff, water running over the surface of the land picks up large numbers of fecal coliform, even in pristine watersheds such as the Eleven Point, Jack's Fork and Current rivers, resulting in high fecal coliform counts in the receiving stream. On April 13, 1994, the fecal coliform count on the Eleven Point River at Bardley was 2300 colonies/ 100 ml at a flow of 6,070 cfs. On August 10, 1994, at a flow of 656 cfs, the fecal coliform count was 2 colonies/100 ml. Even Greer Spring exhibited higher fecal coliform counts on these dates having a flow of 726 cfs and a fc count of 110 in April, and a flow of 494 cfs and a fc count of 12 in August.
Discharges of wastewaters are the other great source of fecal coliform variation in streams. Records on the Missouri River near St. Louis show that fc counts increase from about 4000 to about 10,000 over a 12 mile segment of the river where three large and seven smaller wastewater discharges occur. Since sewage treatment plant effluent, if not disinfected, may have fc counts of several hundred thousand per 100 ml, discharges to small streams with little dilution capacity can cause very high levels of fecal coliforms in these streams.
A third type of variation in fecal coliform counts, which should be apparent from disparity between fc counts on the Eleven Point and on the Missouri, is that some streams have intrinsically higher fecal coliform counts than others, and this is usually a function of differences in land use and volume of wastewater discharges. In Missouri, the Mississippi and the Missouri rivers seem to have the highest fc counts, followed by northern Missouri streams. Streams in the Ozark plateau have the lowest counts. The attached sheet lists average levels of fecal coliforms and several other water quality constituents for selected Missouri streams.
For the Membrane Filter Method, which is the method used by the Missouri Volunteer Monitoring Program, the fecal coliform group of bacteria is defined as all organisms that produce blue coliform colonies within 24 ± 2 hours when incubated at 44.5 ± 0.2°C on M-FC medium. If fecal coliform counts are high (more than 200 colonies/100 ml of water sample) there is a greater chance that pathogenic organisms are also present. A person swimming in such waters has a greater chance of getting sick from swallowing disease causing organisms, or from pathogens entering the body through cuts in the skin, the nose, mouth, or the ears. Diseases and illness such as typhoid fever, hepatitis, gastroenteritis, dysentery, cholera, and ear infections can be contracted in waters with high fecal coliform counts.
The purpose of your monitoring will determine when and where you collect water samples. If the purpose of sampling is to determine fecal coliform levels in a river reach, then samples should be taken beneath the water surface and in the current. If the purpose of sampling is to confirm suspected sources of fecal coliform contamination, then samples should be taken just downstream from the suspected source and upstream from the source as a control.
If you are monitoring non-point storm water runoff, then you should sample during and/or just after a rainstorm. Non-point runoff samples can then be compared to samples taken during a period of dry weather. Typically, the runoff samples will contain higher levels of fecal coliform than samples collected during dry weather.
Sample Collection
1. All samples should be collected in sterile 3" x 71/4" 180 ml Whirl-Pak Sampling Bags.
2. Using a permanent marker, record the Time, Date and Location on the Whirl-Pak.
3. To collect a sample, tear the perforated seal at the top of the Whirl-Pak just before sampling and do not touch the inside of the bag. If sampling by hand, wear rubber gloves. Face upstream, grasp the pull tabs and submerge the Whirl-Pak, with the opening pointed downward, below the water surface. Open the Whirl-Pak under the water by the pulling the tabs to allow the bag to fill while pointed into the current. Do not let the water run over your hands before it enters the Whirl-Pak.
4. Prior to removing the Whirl-Pak from the water close the bag with the pull tabs. Remove the Whirl-Pak with the opening of the bag pointed upward and seal by tightly folding the tab over three times. Avoid sampling the water surface because the surface film often contains greater numbers of fecal coliform bacteria than is representative of the river. Avoid sampling the sediments for the same reason.
5. Samples must be analyzed as quickly as possible after collection (preferably within 1 hour and not more than 6 hours) to minimize changes in the bacterial population. Samples should be iced or refrigerated, but never frozen, and kept in the dark until analyzed. Samples should not be exposed to direct sunlight.
Sterilization of the Membrane Filter System
Bacteria monitoring requires aseptic (sterile) technique. This means you must wash your hands before analyzing samples or handling filtration equipment. Always wear gloves when sampling from water suspected to be contaminated with sewage or any other contaminant. Use alcohol or a 10% bleach solution to clean your work space or counter area. Never touch the inside of funnels or the inside of sample containers. It is essential to use sterilized equipment for the entire fecal coliform monitoring procedure. All of the items will be provided to you sterilized and packaged.
Preparing the Filtration System
1. Attach the hand pump to the filter flask.
2. Attach the filter apparatus to the filter flask with the adapter provided.
3. Do not touch the inside of the filter apparatus or the inside of the lid. Make sure the lid is replaced to prevent contamination.
Determining the Volumes of Sample to Filter
Ideally the volumes of a sample to be filtered for monitoring fecal coliform bacteria should be such that, after incubation, at least one of the membrane filters will contain from 20 to 60 fecal coliform colonies. The following sample volumes are suggested for filtration:
Assuming water is polluted -- Run tests on volumes of 1.0 and 10.0 ml of diluted sample:
[Dilution Procedure: Put 1.0 ml of raw sample into 99 ml of the dilution water that is provided. When preparing dilutions, use a sterile pipet for each dilution. After each transfer between bottles, close and shake the sample bottle vigorously at least 25 times. Diluted samples should be filtered within 20 minutes after preparation. ]
1) Filter 1.0 ml of diluted sample (this is equivalent to filtering
0.01 ml of raw sample) -- then multiply your final colony count by 10,000
to determine number of fecal coliform colonies per 100 ml
2) Filter 10.0 ml of diluted sample (this is equivalent to filtering
0.1 ml of raw sample) -- then multiply your final colony count by 1,000
to determine number of fecal coliform colonies per 100 ml
Assuming water is unpolluted -- Run tests on volumes of 1.0, 10.0, and 100.0 ml of raw (undiluted) sample:
1) Filter 1.0 ml of raw sample -- then multiply your final colony
count by 100 to determine number of fecal coliform colonies per 100 ml
2) Filter 10.0 ml of raw sample -- then multiply your final colony
count by 10 to determine number of fecal coliform colonies per 100 ml
3) Filter 100.0 ml of raw sample -- then multiply your final colony
count by 1 to determine number of fecal coliform colonies per 100 ml
The Step-By-Step Proceedure:
1. Label the BOTTOM of the petri dish prior to filtration (this will ensure that the colonies are not obscured from sight while counting) with the time, location and, the dilution of sample tested. Also note which site the sample came from.
2. Shake the sample vigorously, at least 25 times, before each volume is withdrawn and immediately withdraw the sample volume to be filtered with sterile plastic 10 ml capacity pipets. FILTER THE SMALLEST SAMPLE VOLUME (GREATEST DILUTION) FIRST.
DO NOT PIPET BY MOUTH!
3. If the volume of a sample to be filtered is between 1.0 and 10.0 ml, pour about 20 ml of sterile dilution water into the filter funnel before adding the sample. This procedure facilitates distribution of organisms on the membrane filter.
4. After the sample has been transferred to the filter funnel, apply vacuum with the hand pump. Do not exceed a pressure of 25 cm to avoid damage to bacteria.
5. After filtration, remove the filter riser from the dish without touching the inside of the dish. Release the pressure from the hand pump. Pour one ampule of m-FC Broth on the filter pad. Pump the hand pump once to remove excess media. Replace the lid on the petri dish. Do not touch any of the inner parts of the filter apparatus, the filter pad or dish.
6. Remove the petri dish from the filter flask and invert. Plug the dish with the blue plug provided.
8.After the sample volumes have been filtered, incubate the cultures in the petri dishes for 24 ± 2 hours in an incubator or water bath preheated to and maintained at 44.5 ± 0.2°C. Filters need to be incubated within 20 minutes after placement in the petri dishes. Before incubation in a water bath, double seal the petri dish by placing it in a Whirl-Pak bag and then enclosing the Whirl-Pak in a zip lock freezer bag. Make sure the petri dishes are fully submerged and upside down.
9. Continue with filtration of the other volumes of samples, in order of increasing volume. Record on the Fecal Coliform Field Sheet the volumes filtered.
10. Run a sample with a positive control from a known contaminated source. A positive control is a quality assurance check for the media. This will ensure that the media will grow fecal coliform bacteria. Be sure to use sterile technique and all safety procedures when testing a positive control.
11. Run a negative control sample after completing filtration of all samples to assure that your sterile technique is satisfactory. Run a negative control by filtering 20 - 30 ml of dilution water.
12. After incubating for 24 ± 2 hours, remove the petri dishes from the incubator. Count the number of blue dots on each filter and record the number on the Fecal Coliform Data Sheet. Each blue dot indicates one fecal coliform colony. It is a good idea to have 2 or more people verify the count. Count using a preset plan (a side-to-side pattern along grid lines is suggested). Count until replicate counts for each filter agrees within 5 percent. Counts should be completed within 20 minutes after the petri dishes have been removed from the incubator. Bacterial colonies that are not fecal coliform will be gray to cream colored. Do not count these bacterial colonies. It is best to count with the aid of 10x to 15x magnification.
13. Count and record the number of colonies for all dilutions. The most reliable results are obtained if the number of fecal coliform colonies counted is between 20 and 60. Counts of less than 20 (<20) and greater than 60 (>60) may also be used but are considered less reliable. Two hundred (200) is the maximum number of coliform colonies that is considered to be countable. Results above this are reported as Too Numerous To Count (TNTC). In order to produce a countable plate (1-200) a wide range of dilutions should be prepared.
14. When multiple dilutions are prepared the results will not be the same between dilutions, so you must choose and report the best number available. Use the qualifying statements in the Appendix and guidelines in your selections.
15. Properly dispose of the petri dish, Whirl-Paks, and filter apparatus. To do this, first place them in a 10% bleach solution for 10 minutes and them discard them with the regularly trash
Qualifying Statements
A. Acceptable Result -- 20-60 blue coliform colonies per plate.
B. Estimated Count -- <20 blue coliform colonies per plate.
C. "<"Value -- no Colonies -- value based on largest single volume filtered
D. Estimated Count --> 60 countable blue coliform colonies per plate.
E. ">" Value -- coliform colonies too numerous to count (TNTC).
If one or more dilutions are between 20-60:
THEN calculate colonies/100 ml for each of these plates and determine the arithmetic average of these numbers. Disregard counts not between 20 and 60.
REPORT the arithmetic mean with qualifier A.
If all counts are less than 20:
THEN use the highest count observed, a single plate count. Disregard the other counts; do not calculate an average.
REPORT the actual number calculated with qualifier B.
If there are no Colonies, zero counts:
THEN assume one (1) count was observed on the LARGEST volume filtered and calculate colonies/100 ml.
REPORT less than (<) the calculated number with qualifier C.
If all counts are greater than 60 but still countable (less than 200):
THEN use the counts from the SMALLEST sample volume filtered and disregard the others.
REPORT the number calculated with qualifier D.
If all plates are TNTC:
THEN assume 60 counts were observed on the SMALLEST sample volume filtered and calculate colonies/100 ml.
REPORT greater than > the number calculated with qualifier E.
If there are counts less than 20 and greater than 60:
THEN use the count from the LARGEST sample volume filtered to calculate colonies/100 ml.
REPORT the number with qualifier B or D.
REFERENCES
Code of State Regulations. 1994. Rules of Department of Natural Resources, Division 20-Clean Water Commission, Chapter 7-Water Quality, 10 CSR 20-7.
Crawford, R. Field Analysis of Fecal Coliform Bacteria. SOP # MDNR-FSS-206, Missouri Department of Natural Resources, Division of Environmental Quality, Standard Operating Procedures.
Adams, I. 1993. Technical Instructions for the Colorado River Watch Network. Lower Colorado River Authority, Austin, Texas.
Lehrer, A. 1992. Bacterial Monitoring of Surface Waters. Fact Sheet No. 92-1, Natural Resources Facts, University of Rhode Island, College of Resource Development, Department of Natural Resources, Cooperative Extension.
Buratti, J. And E. Brown. 1993. Building Your Own Water Bath Incubator. The Volunteer Monitor. The National Newsletter of Volunteer Water Quality Monitoring. Vol. 5, No 1,pg 15.
American Public Health Association, American Water Works Association, and Water Pollution Control Federation. 1995. Standard Methods for the Examination of Water and Wastewater, 19th Ed. American Public Health Association, Washington DC.