Microbiology of Water Determination and Water Quality Analysis Notes

LEARNING OBJECTIVES

• Students should be able to perform the three steps used to determine water quality.

• Students should be able to define coliform and explain why they are good indicator organisms of water quality.

• Students should be able to explain how the Most Probable Number (MPN) test is used in measuring water quality.

• Students should be able to discuss the selective and differential nature of Eosin Methylene Blue (EMB) agar.

INTRODUCTION TO WATER MICROBIOLOGY

• Water serves as a potential vehicle for the transmission of various diseases, including:     * Typhoid fever, caused by $Salmonella\,typhi$.     * Cholera, caused by $Vibrio\,cholerae$.     * Amoebic dysentery, caused by $Entamoeba\,histolytica$.

• Testing Challenges: Because these diseases can be initiated by a very low number of organisms, direct analysis for specific pathogens is extremely difficult.

• Indicator Organisms: Instead of testing for specific pathogens, scientists screen for indicator organisms that suggest the potential presence of pathogens.

• Regulatory Context: Both sewage water intended for release into the environment and drinkable (potable) water are subjected to these tests to ensure water supply safety and mitigate disease risk.

• Correlation: The primary goal of microbiological water analysis is to detect these indicator organisms and estimate their concentration. A higher concentration of indicator organisms indicates a higher likelihood that pathogens are present.

INDICATOR ORGANISMS AND COLIFORMS

• Indicator organisms are specifically those that indicate the presence of human wastes.

• This group of organisms is known as coliforms.

• Verbatim Definition of Coliforms: "Gram negative non-spore-forming, facultative rods that ferment lactose."

• $E.\,coli$: This specific coliform is considered especially indicative of human or animal waste contamination.

THE THREE-PART WATER QUALITY TEST SEQUENCE

• The standard water quality analysis consists of three parts, typically conducted over three consecutive lab sessions. The results of each part are used to inoculate the next.

• 1. Presumptive Test: Uses a statistical method to estimate coliform numbers.

• 2. Confirmed Test: Verifies the presence of lactose-fermenting Gram-negative rods using selective/differential media.

• 3. Completed Test: Verifies the presence of coliforms via Gram stain and biochemical testing (IMViC) to determine pathogenicity.

PART 1: THE PRESUMPTIVE TEST

• Objective: To estimate the number of coliforms in a water sample using the MPN (Most Probable Number) statistical method.

• Function: This test identifies whether lactose-fermenting organisms are potentially present in the water source.

• Materials and Sources:     * Water Sources: Tap water, drinking fountain water, toilet water, or pond water.     * Medium: Lactose broth (SSLB and DSLB).     * Tools: Durham vials (placed inside the broth), sterile serological pipettes, and pipettors.

• Medium Composition (Ingredients per liter of water):     * Peptone 190 (Pancreatic Digest of Gelatin): $5.0\,g$     * Beef Extract: $3.0\,g$     * Lactose: $5.0\,g$     * pH: $6.7 \pm 0.2$ at $25^{\circ}C$

• Methodology (Step-by-Step):     1. Inoculate three tubes of Single Strength Lactose Broth (SSLB) with $0.1\,ml$ of water each.     2. Inoculate three tubes of Single Strength Lactose Broth (SSLB) with $1\,ml$ of water each.     3. Inoculate three tubes of Double Strength Lactose Broth (DSLB) with $10\,ml$ of water each. DSLB is prepared using half the volume of liquid ($500\,ml$ instead of $1\,liter$) during media preparation.     4. Incubate all tubes at $37^{\circ}C$.

• Quantitative Interpretation:     * Positive growth is defined by signs of lactose fermentation, specifically the presence of gas in the Durham vial.     * MPN Index: Record the number of positive tubes for each volume group ($3 \times 10\,ml$, $3 \times 1\,ml$, $3 \times 0.1\,ml$) and match the combination to the statistical MPN table.     * Statistical Note: If all tubes are negative, the MPN is expressed as <3 per $100\,ml$, acknowledging the test evaluates the probability of organism concentration in a $100\,ml$ sample.

MOST PROBABLE NUMBER (MPN) STATISTICAL TABLE

• Data Source: Standard Methods for the Examination of Water and Wastewater, 12th edition (American Public Health Association).

• (Positive 10 ml, Positive 1 ml, Positive 0.1 ml) → MPN Index per 100 ml (95% Confidence Limits):     * (0, 0, 1) → $3$ (<0.5 - $9$)     * (0, 1, 0) → $3$ (<0.5 - $13$)     * (1, 0, 0) → $4$ (<0.5 - $20$)     * (1, 0, 1) → $7$ ($1$ - $21$)     * (1, 1, 0) → $7$ ($1$ - $23$)     * (1, 1, 1) → $11$ ($3$ - $36$)     * (1, 2, 1) → $11$ ($3$ - $36$)     * (2, 0, 0) → $9$ ($1$ - $36$)     * (2, 0, 1) → $14$ ($3$ - $37$)     * (2, 1, 0) → $15$ ($3$ - $37$)     * (2, 1, 1) → $20$ ($7$ - $44$)     * (2, 2, 0) → $21$ ($4.7$ - $47$)     * (2, 2, 1) → $28$ ($10$ - $150$)     * (3, 0, 0) → $23$ ($4$ - $120$)     * (3, 0, 1) → $39$ ($7$ - $130$)     * (3, 0, 2) → $64$ ($15$ - $380$)     * (3, 1, 0) → $43$ ($7$ - $210$)     * (3, 1, 1) → $75$ ($14$ - $230$)     * (3, 1, 2) → $120$ ($30$ - $380$)     * (3, 2, 0) → $93$ ($15$ - $380$)     * (3, 2, 1) → $150$ ($30$ - $440$)     * (3, 2, 2) → $210$ ($35$ - $470$)     * (3, 3, 0) → $240$ ($36$ - $1,300$)     * (3, 3, 1) → $460$ ($71$ - $2,400$)     * (3, 3, 2) → $1,100$ ($150$ - $4,800$)     * (3, 3, 3) → >1,400

PART 2: THE CONFIRMED TEST

• Purpose: To verify the presence of lactose-fermenting Gram-negative rods found in positive presumptive samples.

• Materials: Eosin-Methylene Blue (EMB) agar.

• EMB Selective Nature: The dyes in the medium (Eosin and Methylene Blue) diffuse through the peptidoglycan layer of Gram-positive cells to reach the cell membrane, killing them. Gram-negative bacteria possess an outer membrane that blocks this access, allowing them to grow.

• EMB Differential Nature:     * The agar contains lactose.     * Fermentation of lactose produces acid, lowering the local pH.     * Low pH causes the precipitation of dyes onto the colony.

• Visual Results:     * Lactose fermenters: Blue-black colonies, often with a metallic green sheen (e.g., $E\,coli$).     * Non-lactose fermenters: Colorless or translucent colonies (e.g., $Proteus\,vulgaris$).

PART 3: THE COMPLETED TEST

• Purpose: To confirm coliform identity and differentiate between fecal contamination and common environmental organisms.

• Methodology: Select an isolated colony from an EMB plate showing lactose fermentation and inoculate an IMViC series.

• IMViC Series Components:     1. Indole test.     2. Methyl Red test.     3. Voges-Proskauer test.     4. Citrate test.

• Differential Outcomes: This series distinguishes between $E.\,coli$ (truly indicative of fecal contamination) and $Enterobacter\,aerogenes$ (an environmental organism).

LAB DISCUSSION QUESTIONS

• Presumptive Test Specifics:     * Medium Classification: Whether the medium is selective, differential, or both.     * Durham Vial Contents: The specific molecules (gases) present if lactose is fermented.     * Dilution Math: After adding $10\,ml$ of water to $10\,ml$ of DSLB, the final lactose broth concentration becomes 1x (single strength) in the $20\,ml$ total volume.     * Sampling Volume: Total volume of water tested per sample equals $33.3\,ml$ ($10 \times 3 + 1 \times 3 + 0.1 \times 3$).

• Confirmed/Completed Test Specifics:     * Alternative Media: Knowledge of other media selective for Gram-negative organisms and differential for lactose fermentation (e.g., MacConkey agar).     * Hypotheses: Evaluation of whether class data supports initial predictions regarding which water source (tap, fountain, toilet, pond) contains coliforms.

Presumptive Test Specifics:

1. Medium Classification:
      - The medium used in the presumptive test, such as Lactose Broth, is considered both selective and differential. It is selective because it promotes the growth of lactose-fermenting bacteria while inhibiting the growth of non-lactose fermenters and other bacteria. It is differential because it allows for the observation of gas production, indicating lactose fermentation.

2. Durham Vial Contents:    - In positive tests, the Durham vial captures gas produced by lactose-fermenting bacteria. The gas can be carbon dioxide (CO₂), resulting from the breakdown of lactose by coliform bacteria. The presence of gas bubbles in the Durham vial is a clear sign of fermentation and potential coliform presence.

3. Dilution Math:
      - The final concentration of the lactose broth after dilution is important for interpreting results. The total volume for testing was 33.3 ml across different tube dilutions. When water is added to lactose broth, the effective concentration of lactose changes, which plays a crucial role in estimating the Most Probable Number (MPN) of coliforms in the original water sample.

4. Sampling Volume:
      - The total volume of water tested per sample, which is calculated as $10 imes 3 + 1 imes 3 + 0.1 imes 3$, equals 33.3 ml. This total volume provides insight into the representativeness of the sample and how it relates to possible contamination levels in the source water.

Confirmed/Completed Test Specifics:

1. Alternative Media:
      - Besides Lactose Broth, other media such as MacConkey agar and XLD (Xylose Lysine Deoxycholate) agar are used for differentiating Gram-negative organisms. MacConkey agar is selective for Gram-negative bacteria and differentiates lactose fermenters (which appear pink) from non-fermenters (which remain colorless), providing critical insights into the microbial composition of a sample.

2. Hypotheses:
      - Evaluating class data against initial hypotheses about coliform presence helps students analyze their findings in light of expected results. For instance, if the hypothesis was that pond water would contain more coliforms than tap water, the observed results in each source can lead students to conclusions about contamination rates, microbial ecology, and public health implications.