Multiple Tube Method (MPN)

📌 Definition

The Multiple Tube Method (also called the Most Probable Number – MPN method) is a statistical method used to estimate the number of coliform bacteria in water by observing growth patterns in serial dilutions.

🧪 Principle (Core Concept)

• Coliforms (e.g., Escherichia coli) ferment lactose → acid + gas

• Gas is detected in Durham tubes

• Number of positive tubes → statistical estimation (MPN)

👉 Important:

MPN is not a direct count, but a probability-based estimate

🧪 Stepwise Procedure

1️⃣ Presumptive Test

• This is the first and most critical screening step in water bacteriology. It is designed to detect the possible presence of coliforms based on their ability to ferment lactose with gas formation.

  🔬 1. Principle

  
  

• Coliforms such as Escherichia coli ferment lactose → acid + gas

• Gas gets trapped in Durham tubes

• Acid production causes indicator color change

  👉 This step does NOT confirm coliforms, only suggests their presence.

  
  

  ⚙️ 2. Media Composition

  Lactose Broth Contains:

  • Lactose → fermentable substrate

  • Peptone → nutrients

  • Indicator dye → usually bromocresol purple

  Why Bromocresol Purple?

  • Turns purple → yellow in acidic pH

  • More stable and clear than some alternatives

    
    

  🧫 3. Tube Setup in 5-Tube Method

Group	Inoculum	No. of tubes	Medium
1	10 mL	5	Double-strength lactose broth
2	1 mL	5	Single-strength lactose broth
3	0.1 mL	5	Single-strength lactose broth

👉 Total = 15 tubes

Why Double Strength for 10 mL?

• Large inoculum dilutes the medium

• Double strength maintains proper nutrient concentration

  
  

🌡️ 4. Incubation Conditions

• Temperature: 37°C

• Duration:

  • 24 hours (first reading)

  • Extend to 48 hours if negative

👉 Mackie emphasizes:

Only gas formed within 48 hours is considered valid

✅ 5. Interpretation of Results

Positive Presumptive Test:

• Gas in Durham tube (essential)

• ± Acid production (color change)

  
  

Negative Presumptive Test:

• No gas

• No color change

  
  

🔍 How to Read Gas Formation

• Even a small bubble = positive

• Tube must be initially completely filled

• Air bubble at start = invalid setup

  
  

📊 6. Recording Results (Key for MPN)

Example:

Dilution	Positive tubes
10 mL	5/5
1 mL	3/5
0.1 mL	1/5

👉 Reported as: 5–3–1 combination

➡️ This goes for MPN table calculation

⚠️ 7. Limitations (Very Important Theory)

Presumptive test is not specific because:

False Positives:

• Non-coliform lactose fermenters(e.g., some environmental bacteria)

  
  

False Negatives:

• Injured coliforms

• Low bacterial count

👉 Hence: Confirmed test is mandatory

🧠 8. Subtle Technical Points (Mackie Pearls)

• Gas without acid → still considered positive

• Acid without gas → NOT coliform (usually ignored)

• Delayed gas (>48 hrs) → unreliable

• Turbidity alone → not sufficient

  
  

🧴 9. Variations / Special Tests

Elevated Temperature Test (E. coli detection)

• Incubation at 44°C

• Detects thermotolerant coliforms

👉 Indicates:✔️ Recent fecal contamination

🚨 10. Common Errors in Presumptive Test

• Improper Durham tube placement

• Air bubbles mistaken as gas

• Overfilling tubes

• Contamination during inoculation

  
  

📌 11. Clinical / Public Health Relevance

👉 Example:

• Water sample shows:

  • 5/5 tubes positive at 10 mL✔️ Suggests heavy contamination

But:❗ Could still be environmental coliforms → must confirm

🎯 12. Exam/Viva High-Yield Points

• First step of MPN method

• Detects lactose fermenters with gas

• Uses Durham tubes

• Not confirmatory

• Basis for MPN calculation

  
  

2️⃣ Confirmed Test

After a positive presumptive test, the confirmed test is performed to prove that the gas-producing organisms are true coliforms and not false positives.

🔬 1. Purpose (Why Confirmed Test?)

👉 Presumptive test limitations:

• Many non-coliform bacteria can ferment lactose and produce gas

👉 Confirmed test ensures:✔️ Growth of true coliforms✔️ Suppression of non-coliform organisms

⚙️ 2. Principle

• Coliforms are:

  • Gram-negative bacilli

  • Lactose fermenters

  • Bile salt tolerant

👉 Confirmed test uses selective + differential media to:

• Inhibit unwanted bacteria

• Demonstrate lactose fermentation clearly

  
  

🧪 3. Media Used (Very Important)

A. Brilliant Green Lactose Bile Broth (BGLB)

📌 Composition & Mechanism

• Lactose → substrate for fermentation

• Bile salts → inhibit Gram-positive organisms

• Brilliant green dye → suppress unwanted Gram-negative bacteria

👉 Only true coliforms survive and ferment lactose

✔️ Result Interpretation

• Gas in Durham tube = Positive

• No gas = Negative

👉 This is the most important confirmatory step

B. MacConkey Agar

📌 Mechanism

• Bile salts + crystal violet → inhibit Gram-positive bacteria

• Lactose → fermentable sugar

• Neutral red indicator → detects acid

  
  

✔️ Colony Appearance

Organism	Appearance
Lactose fermenters (e.g., Escherichia coli)	Pink/red colonies
Non-lactose fermenters	Pale/colorless

👉 Confirms lactose fermentation ability

C. Endo Agar / EMB Agar

📌 Mechanism

• Contains dyes that:

  • Inhibit Gram-positive bacteria

  • React with lactose fermentation products

    
    

✔️ Key Feature

• Escherichia coli → Metallic green sheen (EMB) ✨

• Strong lactose fermenters → dark colonies

  
  

🧬 4. Stepwise Procedure

1️⃣ Select positive tubes from presumptive test

2️⃣ Inoculate into:

• BGLB broth (with Durham tube)

• MacConkey agar plate

3️⃣ Incubate:

• 37°C for 24–48 hours

  
  

✅ 5. Interpretation

Confirmed Positive:

• Gas production in BGLB

• Lactose fermenting colonies on agar

👉 Indicates true coliform presence

Confirmed Negative:

• No gas in BGLB

• Non-lactose fermenters on agar

👉 Suggests false presumptive result

⚠️ 6. Why These Media Work

Feature	Role
Bile salts	Mimic intestinal environment → select enteric bacteria
Brilliant green	Suppresses non-coliform Gram-negative bacteria
Lactose	Differentiates fermenters

🧠 7. Differentiation Insight

Organism	BGLB	MacConkey	EMB
Escherichia coli	Gas +	Pink	Metallic sheen
Klebsiella	Gas +	Mucoid pink	Dark colonies
Non-coliforms	No gas	Colorless	No sheen

🚨 8. Limitations

• Some environmental coliform-like organisms may still grow

• Requires completed test for final confirmation

• Cannot distinguish all species precisely

  
  

🔗 9. Link to Completed Test

👉 Confirmed test proves:✔️ Likely coliform

👉 Completed test proves:✔️ Definite coliform identity (microscopy + re-fermentation)

📌 10. Viva Pearls

• BGLB = key confirmatory medium

• Gas formation = essential criterion

• MacConkey = supportive evidence

• EMB metallic sheen = strong indicator of E. coli

• Confirmed test removes false positives from presumptive test

  
  

🧴 11. Clinical/Public Health Correlation

👉 Scenario:

• Presumptive test positive

• Confirmed test negative

✔️ Interpretation:→ Likely non-coliform lactose fermenters→ Water may not be fecally contaminated

3️⃣ Completed Test

🎯 1. Core Aim

To confirm:

1. True coliform organism

2. And if needed → specifically Escherichia coli (fecal origin)

   
   

🧪 2. Standard Completed Test Steps

Step 1: Colony Selection

• From MacConkey / EMB

• Lactose fermenting colony

  
  

Step 2: Gram Stain

• Gram-negative bacilli → ✔️ coliform group

  
  

Step 3: Lactose Broth Re-inoculation

• Gas in Durham tube → ✔️ lactose fermenter

  
  

➕ Step 4: Indole Test (Added for Speciation)

📌 Why Add Indole Here?

• Completed test confirms coliform

• Indole helps confirm fecal coliform (E. coli)

👉 Important distinction:

• Coliform ≠ always fecal contamination

• E. coli = definite fecal contamination

  
  

⚙️ 3. Indole Test — Mechanism

Principle:

Some bacteria degrade tryptophan → indole

Reaction:

Tryptophan → (tryptophanase enzyme) → Indole + pyruvate + NH₃

Procedure:

• Inoculate organism into tryptone broth

• Incubate at 37°C for 24 hrs

• Add Kovac’s reagent

Chemical Mechanism:

• Indole reacts with p-dimethylaminobenzaldehyde (in Kovac’s reagent)

• Forms red-colored compound (rosindole dye)

  
  

Why Tryptone broth is preferred over peptone water?

“Tryptone broth is preferred over peptone water for indole test because it contains a high and reliable concentration of tryptophan, ensuring accurate detection of indole production.”

✔️ Results

Result	Observation
Positive	Red ring on top 🔴
Negative	No color change

🧠 4. Interpretation in Water Bacteriology

Test Component	Meaning
Gram-negative rod	Coliform group
Lactose + gas	True coliform
Indole positive	E. coli → fecal contamination 🚨

📊 5. Differentiation

Organism	Lactose	Gas	Indole
Escherichia coli	+	+	✅ Positive
Klebsiella	+	+	❌ Negative
Enterobacter	+	+	❌ Negative

👉 This is why indole is powerful:

It differentiates true fecal coliform (E. coli) from environmental coliforms

🔗 6. Final Integrated Completed Test Criteria

A fully confirmed fecal coliform (E. coli) will show:

✔️ Gram-negative bacilli✔️ Lactose fermentation with gas✔️ Growth on selective media✔️ Indole positive

⚠️ 7. Important Exam Clarification

• Classical Mackie completed test:

  • ❗ Does NOT mandate indole

• But:

  • ✔️ Frequently added in practice

  • ✔️ Important in modern interpretation

👉 So in exams, write:

“Indole test may be performed additionally to identify E. coli”

🚨 8. Clinical / Public Health Significance

👉 Scenario:

• Completed test positive

• Indole positive

✔️ Interpretation:

Recent fecal contamination (human/animal origin)

📌 9. Viva Pearls

• Indole test = not compulsory but highly useful

• Detects tryptophanase activity

• Red ring = positive

• Helps identify E. coli (gold standard fecal indicator)

• Differentiates from Klebsiella & Enterobacter

📊Reading Results (Very Important)

After incubation, record number of positive tubes:

Example:

Dilution	Positive tubes (out of 5)
10 mL	5/5
1 mL	3/5
0.1 mL	1/5

👉 Express as: 5–3–1 combination

📈MPN Calculation (McCrady Table)

• In the Multiple Tube Fermentation (MPN) method, results from the presumptive test (e.g., 5–3–1) are converted into a numerical estimate of coliforms using statistical tables.

  🎯 1. Why Tables Are Needed

  
  

  • Bacteria are randomly distributed in water

  • Each tube = probability event (growth / no growth)

  • Exact count is impossible

  👉 Hence:

MPN = statistical estimation based on probability theory

📘 2. McCrady’s Probability Table (Most Important)

🔬 Basis:

• Derived from Poisson distribution

• Uses:

  • Number of tubes

  • Number of positives at each dilution

📊 How to Use (Step-by-Step)

Example (5-Tube Method):

Dilution	Positive tubes
10 mL	5/5
1 mL	3/5
0.1 mL	1/5

👉 Combination = 5–3–1

Steps:

1. Locate 5–3–1 in McCrady table

2. Read corresponding value

3. Express as:

👉 MPN per 100 mL

📌 Example Value:

• 5–3–1 → approx 110 MPN/100 mL

  
  

  (varies slightly with table version)

📈 3. Confidence Limits (Very Important)

McCrady table also provides:

• Lower confidence limit

• Upper confidence limit

👉 Because:

MPN is an estimate, not an exact count

Example:

110 MPN/100 mLRange: ~40 to 250

📚 4. Other Tables Used in MPN

A. 3-Tube Table (Standard Routine)

• Uses combinations like:

  • 3–2–1

  • 2–1–0

👉 Less accurate than 5-tube👉 Used in routine labs

B. 5-Tube Table (More Accurate)

• Used in:

  • Research

  • Reference labs

• Provides:

  
  

  ✔️ Narrower confidence limits

  
  

  ✔️ Better sensitivity

  
  

C. Tillett’s Table

📌 What is it?

• A simplified probability table

• Sometimes used in teaching

👉 Less commonly used than McCrady

D. Thomas’ Formula (Rare but Important)

• Mathematical alternative to tables

Formula (conceptual):

MPN ≈ function of:

• Number of positive tubes

• Volume of sample

👉 Not used routinely due to complexity

🧠 5. Conceptual Understanding

Relationship:

• More positive tubes → higher MPN

• Positives at higher dilution → heavy contamination

Example Patterns:

Pattern	Interpretation
5–5–5	Very high contamination
5–0–0	Moderate contamination
0–0–0	Safe water

⚠️ 6. Limitations of MPN Tables

• Statistical estimate only

• Requires correct technique

• Wide confidence intervals at extremes

  
  

🔬 7. Why McCrady Table is Preferred

• Based on sound statistical model

• Widely validated

• Standard in Mackie & McCartney

  
  

📌 8. Viva Pearls

• MPN = Most Probable Number

• Based on Poisson distribution

• Expressed as per 100 mL

• 5-tube method = more accurate

• McCrady table = standard reference

  
  

⚠️Interpretation

MPN / 100 mL	Interpretation
0	Excellent (safe)
1–10	Acceptable (doubtful safety)
>10	Contaminated
Presence of E. coli	Recent fecal contamination 🚨

🧠Important Technical Points

• Double-strength broth used for 10 mL inoculum

• Durham tube must be completely filled (no air bubbles initially)

• Gas within 48 hrs only counts

• Late gas → false positive risk

  
  

❌Sources of Error

• Air bubbles mistaken for gas

• Non-coliform lactose fermenters

• Improper sterilization

• Overgrowth masking results

  
  

🔬Why MPN Still Matters (Despite Modern Methods)

• Works even with turbid water

• Detects low bacterial counts

• No need for advanced equipment

👉 But:

• Time-consuming

• Less precise than membrane filtration

  
  

🧾Clinical / Field Correlation

👉 Example:

• Village outbreak of diarrhea

• MPN result: >180/100 mL + E. coli detected

  
  

  ✔️ Confirms fecal contamination of water source