BACTERIA In the Environment

DIY Bacterial Screening Test

General Information:  

Bacteria are among the simplest, smallest, and most abundant organisms on earth.

Most bacteria are only 1 micrometer (µm) in diameter, but they can range in size from 0.1 µm to greater than 10 µm. Bacteria are procaryotic organisms; that is, they do not have an organized nucleus surrounded by a nuclear membrane. Procaryotic microorganisms include bacteria and blue-green algae (cyanobacteria). Bacteria contain a single strand of DNA, and they typically reproduce by binary fission. During binary fission, a single cell divides transversely to form two new cells called daughter cells. Each daughter cell contains an exact copy of the genetic information contained in the parent cell. The process continues with each daughter cell giving rise to a generation of two new cells. The way that bacteria populations increase is an example of geometric progression: 1 cell 2 cells 4 cells 8 cells 16 cells, etc. The generation time is the time required for a given population to double in size. This time can be as short as 20 minutes for some bacteria species (e.g., Escherichia coli).

All bacteria are unicellular (single-celled) organisms. Two methods by which bacteria can be grouped are by cell shape and by differential stains. There are three key shapes: bacilli (rods), cocci (spherical or spheroid), and spirilla (spiral or corkscrew). Cells can occur either individually or as groups of cells. Grouped cells neither communicate nor cooperate with each other; however, the configurations that are observed for a particular species are fairly constant. Arrangements of spherical cells can be used in taxonomy: diplococci (paired cells), streptococci (cell chains), tetracocci (four cells arranged in a square), and staphylococci (grape-like clusters).

There are two key types of stains used on bacteria: simple stains and differential stains. Simple stains serve to increase color contrast in cells. They are generally able to react with all types of bacteria. Aniline (coaltar) dyes, such as methylene blue, crystal violet, basic fuchsin, eosin Y, and safranin O, are examples of simple stains. These are basic dyes, as opposed to acidic dyes, and bind with the acidic portions of cells.

Other stains do not react equally with all types of bacteria. Such stains can be used to differentiate among bacterial types; hence, the name differential stain. Differential stains are also used to detect differences among structures within cells. The Gram stain is probably the most commonly used differential stain. The procedure was developed in 1884 by Christian Gram, a Danish physician. The staining method successfully separates many types of bacteria into two groups: gram positive or gram negative. It works best when used on young, actively growing bacteria. A four-step process is followed: (1) apply a primary stain (crystal violet), (2) apply an iodine solution (acts as a mordant: it causes the primary stain to adhere to the cells better), (3) rinse with 95% ethyl alcohol (decolorizer), then (4) apply a counterstain (can use eosin Y, safranin O, brilliant green, or Bismarck brown). Gram positive cells retain the crystal violet stain. Gram negative cells lose the crystal violet during the decolorizing step and are then colored by the counterstain.

Bacteria are able to survive in a wide range of environmental conditions. The preference for certain conditions leads to further classification:

"Obligate (or strict) aerobes: the presence of oxygen is required (e.g., Pseudomonas fluorescens). Obligate (or strict) anaerobes: the absence of oxygen is required, oxygen is toxic to the cells (e.g., Clostridium botulinum, C. tetani). Facultative anaerobes: can survive with or without oxygen (e.g., Escherichia coli). Microaerophiles: require low concentrations of oxygen and don't do well either at atmospheric oxygen concentrations or without oxygen (e.g., Sphaerotilus natans, Enterobacter aerogenes)"

Bacteria cannot control their own temperature. The range of temperatures that they can withstand categorizes bacteria as either stenothermal or eurythermal:

"Stenothermal: can only survive over a very narrow temperature range (< 10 C range). Eurythermal: can survive over a wide temperature range"

The optimum temperature for growth is the temperature at which the fastest growth rate occurs. Bacteria are classified as psychrophiles, mesophiles, or thermophiles based on their optimum growth temperature:

"Psychrophiles: optimum temperature occurs between 0 and 20 C. Mesophiles: optimum temperature occurs between 20 and 45 C. Thermophiles: optimum temperature occurs between 45 and 60 C"

Many bacteria are able to survive, if not proliferate, at low temperatures; however, only some bacteria are able to survive at elevated temperatures. Thermus aquaticus and Bacillus stearothermosphilus are non-sporeforming bacteria that can grow in hot springs at temperatures above 70 and 55 C, respectively. Some bacteria are able to survive high temperatures because they form spores. Spores are special cells that are resistant to harsh environmental conditions. Once conditions become favorable, the cells return to the vegetative (or actively growing) state.

Bacteria can be useful to humans in many ways. Bacteria decompose many types of organic substances and are currently being investigated as a means of decomposing unwanted synthetic chemicals (e.g., pesticides, dyes, and petroleum) that are released into the environment.
 

Table A: Human pathogenic bacteria

Some of the better known waterborne diseases, caused by bacteria, are: cholera, bacillary dysentery, shigellosis, and typhoid fever.
 

Bacteria and water:

Coliform bacteria have been used to evaluate the general quality of water.  Testing for coliform bacteria is faster and cheaper than testing for specific organisms and pathogens.  U.S. Public Health Service established a standard in 1914.  Coliforms include all aerobic and facultatively anaerobic, gram-negative, non-sporeforming bacilli that, when incubated at 35 C, can ferment lactose and produce gas (CO2) within 48 hrs.

Fecal coliforms are the coliform bacteria that originate specifically from the intestinal tract of warm-blooded animals (e.g., humans, beavers, racoons, etc.). They are cultured by increasing the incubation temperature to 44.5 C and using somewhat different growth media.

Two other groups of bacteria that are present in feces are: fecal streptococci and Clostridium. Clostridia spores can survive a long time during adverse conditions. This genera occurs naturally in soils and polluted waters; it is not used for monitoring purposes. Fecal streptococci and enterococci are terms that have been used interchangeably; however, there are some differences between the two groups (Table B). Fecal streptococci indicate the presence of fecal contamination by warm-blooded animals. Unlike coliforms, fecal streptococcal bacteria are not known to multiply in the environment. Also, they tend to die-off more quickly than coliforms.

The ratio of fecal coliforms to fecal streptococci (FC/FS) can provide information on the source of contamination (Table C); however, several precautions are in order when using these ratios: 

(1) Bacterial concentrations can be greatly variable if the pH is outside of the 4.0 to 9.0 range, 

(2) The faster die-off rate of fecal streptococci will alter the ratio as time from contamination increases, 

(3) Pollution from several sources can alter the ratio and confuse the issue, 

(4) FC/FS ratios have been of limited value in identifying pollution sources in irrigation returns, bays, estuaries, and marine waters, and 

(5) Ratios should not be used when fecal streptococcal counts are less than 100/100 mL.
 

Table B: Fecal streptococci 

Table C: FC/FS ratios 

Water quality criteria, guidelines, and standards: A health effects recreational water quality criterion is defined as a measurable relationship between the quantity of the indicator in the water and the potential risk to human health associated with using the water for recreational purposes. A water quality guideline, obtained from the criterion, is a suggested upper limit on the quantity of the indicator in the water that is associated with an unacceptable level of health risk. A water quality standard, obtained from the criterion, is a guideline set by law.

The first federal water quality criteria recommendations were proposed, in 1968, by the National Technical Advisory Committee (NTAC). The criterion for bathing waters was based on studies, conducted during the late 1940's and early 1950's by the Public Health Service, of total coliform concentrations. The criterion was converted to fecal coliform concentrations using Ohio River data collected, during the original study, in 1949. The NTAC recommended that:
 

"Fecal coliforms should be used as the indicator organism for evaluating the microbiological suitability of recreation waters. As determined by multiple-tube fermentation or membrane filter procedures and based on a minimum of not less than five samples taken over not more than a 30-day period, the fecal coliform content of primary contact recreation waters shall not exceed a log mean of 200/100 mL, nor shall more than 10% of total samples during any 30-day period exceed 400/100 mL." (NTAC, 1968)

The above criterion was again recommended, in 1976, by the USEPA, even though several aspects of it had been criticized by a number of researchers (USEPA, 1986). Since then, researchers have conducted several studies in an effort to determine acceptable bacteria concentrations in recreational waters. Evaluations of such studies are presented in: Health Effects Criteria for Fresh Recreational Waters (Dufour, 1984) and Health Effects Criteria for Marine Recreational Waters (Cabelli, 1983). The Ambient Water Quality Criteria for Bacteria - 1986 (USEPA, 1986) had the following recommendations for recreational bathing waters:
 

"Based on a statistically sufficient number of samples (generally not less than five samples equally spaced over a 30-day period), the geometric mean of the indicated bacterial concentrations should not exceed one or the other of the following:

Freshwater:

Marine Water:

In shellfish harvesting areas, the geometric mean fecal coliform concentration must not exceed 14 bacteria per 100 mL, with not more than 10% of the samples exceeding 43 bacteria per 100 mL (USEPA, 1987; Mueller et al., 1987). Total coliform bacteria should not exceed 70 per 100 mL, with not more than 10% of the samples taken during any 30-day period exceeding 230 colonies per 100 mL (Mueller et al., 1987). Diseases such as paratyphoid and infectious hepatitis may be spread through the consumption of bacteria-contaminated shellfish. For more information on shellfish, please refer to the Shellfish section.

The standards actually utilized by the various states differ (USEPA, 1988a; USEPA, 1988b). The original criterion, based on fecal coliform concentrations, is still in use by many states. In many cases, water quality classifications have been developed that change the concentration of fecal coliforms required to meet the intended use of the water (e.g., primary contact, secondary contact, shellfish, etc.). A more recent report (Francy, Myers, and Metzker, 1993), conducted using water samples collected from several areas in Ohio, concluded that:
 

"The difference between the use of E. coli and fecal coliform bacteria is that E. coli can be used to establish guidelines and standards on the basis of an acceptable level of risk as determined by a regulatory agency and the public. The relation between fecal coliform bacteria and E. coli concentrations can vary, whereas the epidemiological literature shows that the relation between E. coli and swimming- associated illness is strong and consistent over geographic boundaries."

Drinking water requirements: The EPA has set the following maximum contaminant levels (MCLs) on treated drinking water:

"For systems that analyze at least 40 samples per month, no more than 5% of the samples may be total coliform positive.For systems analyzing fewer than 40 samples per month, no more than 1% of the samples may be total coliform positive" (AWWA, 1990b).

The number of samples collected each month is based on population (Table 4).
 

Table 4: Drinking Water Sampling (abbreviated table adapted from Bordner and Winter, 1978)

Irrigation effects: Human diseases can occur from the consumption of crops that have been irrigated with polluted water. Crops that are eaten raw (e.g., celery, lettuce, tomatoes, peppers) are especially dangerous for the transmission of disease-causing organisms. Because some bacteria will dessicate (or dry-out) and die from prolonged exposure to air, the risk for illness can be decreased by delaying the harvest and consumption of crops (IHD-WHO, 1978).

Recreation effects: Immersion in bacteria-contaminated water can result in infections of the eyes, ears, nose, and throat (Mueller et al., 1987). Epidemiological studies that associated the occurrence of gastrointestinal illness with coliform concentrations were used to develop the criteria for fresh and marine recreational waters that were previously discussed. From bacteriological data, it was estimated that fecal coliform concentrations of 200 per 100 mL would cause 8 illnesses per 1,000 swimmers at fresh water beaches and 19 illnesses per 1,000 swimmers at marine beaches (USEPA, 1986).

Bacteria sources: Bacteria can enter water via either point or nonpoint sources of contamination. Point sources are those that are readily identifiable and typically discharge water through a system of pipes. Sewered communities may not have enough capacity to treat the extremely large volume of water sometimes experienced after heavy rainfalls. At such times, treatment facilities may need to bypass some of the wastewater. During bypass or other overflow events, bacteria- laden water is discharged directly into the surface water as either sanitary sewer overflow (SSO) or as combined stormwater overflow (CSO). Power outages and flooding can also contribute to the discharge of untreated wastewater. Treated wastewater and treatment plant residuals can also have adverse impacts on sensitive areas. Estuaries may be particularly susceptible to contamination from offshore sewage sludge dumping and offshore sewage pipe outfalls (Kennish, 1992).

Nonpoint sources are those that originate over a more widespread area and can be more difficult to trace back to a definite starting point. Typical nonpoint sources include agricultural, residential and urban areas. Agricultural sources include livestock excrement from barnyards, pastures, rangelands, feedlots, and uncontrolled manure storage areas. Land application of manure and sewage sludge can also result in water contamination, which is why states require permits, waste utilization plans, or other forms of regulatory compliance.

Failed on-site wastewater disposal systems (septic systems) in residential or rural areas can contribute large numbers of coliforms and other bacteria to surface water and groundwater. Stormwater runoff from residential, rural, and urban areas can transport waste material from domestic pets and wildlife into surface waters.

Bacteria transport: Bacteria-laden water can either leach into groundwater and seep, via subsurface flow, into surface waters or rise to the surface and be transported by overland flow. Bacteria in overland flow can be transported freely or within organic particles. Overland flow is the most direct route for bacteria transport to surface waters. Underground transport is less direct, because the movement of water and bacteria is impeded by soil porosity and permeability constraints.

Analytical methods: There are a variety of methods and media available for the detection and enumeration of indicator organisms. Two manuals that go into great detail describing these techniques are Standard Methods for the Examination of Water and Wastewater (AWWA, APHA, and WEF, 1992) and Microbiological Methods for Monitoring the Environment: Water and Wastes (Bordner and Winter, 1978). A brief listing of the method types is presented below:

Total and fecal coliforms: Total coliforms are incubated at 35±0.5 C, whereas fecal coliforms are further distinguished by incubation at 44.5±0.2 C.
 

a. Multiple-tube most probable number (MPN) fermentation technique: This method is applicable for treated and untreated water; however, untreated water will likely require a greater dilution range. Statistical tables (MPN tables) are utilized to determine the number of bacteria present and the range in the 95% confidence interval based on the number of positive culture tubes. The procedure has three steps, presumptive, confirmed, and completed, that can take seven days to complete.

b. Membrane filter procedure: This method will result in discrete bacterial colonies that may be further identified. Highly turbid water and noncoliform bacteria can interfere with the test. This test can require the processing of several sample dilutions in order to obtain filter plates with the appropriate range of colonies for valid enumeration.

c. Presence-absence test: A qualitative test, rather than quantitative, that can be used on routine water distribution samples. Positive drinking water samples would be further analyzed.

d. Defined-substrate: Media to which special, selective components have been added. Such additives help identify the target organism through color changes or other responses. The responses are brought about through metabolic or enzymatic processes specific to the target organism.

Fecal streptococci: As with fecal coliforms, these organisms undergo incubation steps at both 35±0.5 C and 44.5±0.2 C.

a. Fecal streptococci most probable number procedure: This method is similar to that for coliforms; however, different media are utilized.

b. Fecal streptococci membrane filter procedure: Again, this test is similar to that used for coliforms, except for the type of media used.

Methods are continually being revised, streamlined, and updated in an effort to: reduce analysis time, reduce costs, and increase accuracy.
 

School Science Fair DIY Screening Tests

For coliform testing information,
please contact bfenviro@ptd.net .

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