Exhaustive Guide to Applied Water Bacteriology and Quality Testing

General Principles of Applied Water Bacteriology

• Characteristics of Water: Water is defined as a liquid that is transparent, odorless, and tasteless.
• Sources of Water Supply: Water is derived from five primary sources:     * Rain     * Rivers     * Surface-water or shallow wells     * Deep wells     * Springs
• Natural Purity and Contamination:     * Water is never found in a state of absolute purity in nature.     * It is considered nearly pure under specific conditions: when gathered in an open field, immediately following a heavy rainfall, or when sourced directly from springs.     * For city and town supplies, water requires frequent and careful analysis to ensure it is not polluted with disease-causing microorganisms.
• Fecal Contamination and Pathogens:     * The primary risk for water supply contamination involves fecal discharges from sewage, farm animals, and wild animals.     * This contamination can transport various pathogens into drinking water, including bacteria, viruses, and protozoans.
• Global Health Impact: The World Health Organization (WHO) estimates that waterborne diseases are responsible for $10 \times 10^{6}$ deaths (10 million) annually on a worldwide scale.

The Use of Indicator Organisms in Water Analysis

• Rationale for Indicators:     * Direct detection of every potential waterborne pathogen is difficult, time-consuming, and expensive.     * Consequently, water quality surveys utilize various indicators of fecal contamination to signal the potential presence of pathogens.
• Definition of Indicator Organisms:     * These organisms are generally not considered pathogens themselves (though specific strains like $E. coli$ can cause illness if ingested).     * They represent a group of organisms that are easily detected and enumerated.     * They serve as proxies for the presence of sewage.
• The Coliform Group: "Coliform" is a broad term encompassing several related groups including total coliforms, thermotolerant (fecal) coliforms, and $Escherichia~coli$.

Categorization and Criteria for Coliform Bacteria

• Total Coliforms Criteria: This group of microorganisms is defined by the following specific characteristics:     * Aerobic and facultatively anaerobic.     * Gram-negative staining.     * Non-spore forming.     * Rod-shaped (bacilli).     * Ability to ferment lactose with the production of gas and acid within a duration of $24$ to $48$ hours at a temperature of $35^\circ C$.
• Modern Diagnostic Criteria: With the advent of commercial test kits and media, two additional criteria are now used:     * Cytochrome oxidase negative.     * $\beta$-galactosidase positive.
• Taxonomy and Origin:     * These bacteria belong to the family $Enterobacteriaceae$.     * They are typically a small part of the intestinal micro-flora of warm-blooded animals and are thus associated with fecal material.     * Limitations (False Positives): Some members can originate from nonenteric (environmental) sources, which may result in a false positive for pathogen presence.     * Limitations (False Negatives): Coliforms are generally more susceptible to disinfectants like chlorine or ozone than certain viral or protozoan pathogens, potentially leading to false negatives regarding those specific pathogens.

Thermotolerant (Fecal) Coliforms and Escherichia coli ($E. coli$)

• Thermotolerant (Fecal) Coliforms:     * This is a subset of the total coliform group.     * In addition to standard coliform criteria, they must be capable of growing and fermenting lactose with acid and gas production at a temperature of $44.5^\circ C$.     * There is a generally excellent correlation between these organisms and fecal contamination.     * Exceptions: Certain members, such as $Klebsiella$, can sometimes be isolated from environmental samples where no fecal pollution is present.
• Escherichia coli ($E. coli$):     * A Gram-negative rod usually found in the human intestine.     * It serves as the definitive indicator organism; its presence indicates the water has likely been contaminated with human feces.     * Biochemical Differentiation: $E. coli$ meets all criteria for total and thermotolerant coliforms. It is distinguished from other fecal coliforms by:         * Lack of the urease enzyme.         * Presence of the $\beta$-glucuronidase enzyme.

Methodologies for Water Quality Testing

• 1. Gene Probe Tests:     * Considered the most sophisticated tests for water bacteriology.     * Specifically designed to detect the presence of $Escherichia~coli$.     * Process: Water is treated to disrupt bacteria and release their nucleic acids. A specific $E. coli$ DNA probe (a fragment of DNA that seeks out complementary fragments) is added.     * Detection: If the probe unites with E. coli DNA, a radioactive signal is emitted. If no radioactivity is detected, the gene probe failed to find matching DNA, and $E. coli$ is presumed absent.
• 2. Membrane Filtration (MF) Method:     * Simpler than the MPN procedure; it is based on the entrapment and concentration of bacterial cells.     * Process: A $100\,ml$ sample of water is passed through a filtration apparatus containing a cellulose membrane filter with a pore size of $0.45\,\mu m$.     * Retention: Since typical bacterial cells are larger than $1\,\mu m$, they are retained on the filter surface, while viruses and dissolved substances pass through.     * Incubation: The filter pad is transferred to a bacteriological growth medium and incubated. Bacteria grow into discrete, visible colonies.     * Quantification: Counting the colonies provides an estimate of the number of bacteria in the original $100\,ml$ sample.
• 3. Standard Plate Count (SPC):     * Samples are diluted in jars containing $99\,ml$ of sterile water.     * Diluted samples are placed in Petri dishes with nutrient agar or other nutritious media.     * Following incubation, the colony count is multiplied by the dilution factor to determine the total bacteria per $ml$ of the original sample.     * Specific Media:         * Violet Red Bile Agar (VRBA): Used to encourage the proliferation of coliform bacteria.         * Eosin Methylene Blue (EMB) Agar: Used specifically for $E. coli$. On this medium, $E. coli$ colonies appear green with a characteristic metallic fluorescent sheen.
• 4. Most Probable Number (MPN) Test:     * The traditional technique for detecting coliforms in drinking water using multiple tube fermentation.     * Procedure: Tubes of lactose broth are inoculated with water samples in volumes of $10\,ml$, $1\,ml$, and $0.1\,ml$.     * Indications: During incubation, coliforms produce gas.     * Interpretation: Based on which tubes display gas, an MPN table is used to determine a statistical range of the number of coliform bacteria.     * Pros/Cons: It is easy to perform and interpret but does not provide an exact bacterial count like the standard plate count does.

Document Information

• Compiler: Lynnae D. Navarette, RMT