Tyndallization is a method of sterilization that uses repeated cycles of heating and cooling to destroy all microorganisms, including heat-resistant spores. It was developed by Irish physicist John Tyndall in 1877. Because sterilization is achieved across multiple sessions — not in a single step — it is also called fractional sterilization. This method is used for materials that cannot withstand high-temperature autoclaving.
What is Tyndallization? — Definition
- Tyndallization is a method of sterilization that uses repeated cycles of moist heat at 80–100°C.
- Each heating cycle lasts 20–30 minutes and is followed by incubation at 37°C for 24 hours.
- The entire process is repeated on 3 consecutive days.
- It destroys vegetative cells on Day 1 and kills germinated spores on Days 2 and 3.
- Also called Fractional Sterilization — sterilization is achieved in fractions across multiple sessions.
- Also called Intermittent Sterilization.
- Used for heat-sensitive, nutrient-rich media such as egg-based media, serum media, and gelatin.
Principle
Based on the inability of spores to resist germination and the sensitivity of germinated vegetative cells to heat.
- Vegetative bacteria are easily killed at 80–100°C during the first heating cycle.
- Bacterial spores are heat-resistant — they survive the first heating.
- During the 24-hour incubation at 37°C, surviving spores germinate into vegetative cells.
- Germinated vegetative cells are sensitive to heat and are killed in the next heating cycle.
- This cycle is repeated 3 times to ensure all spores have germinated and all vegetative cells have been destroyed.
- Result: complete sterilization without reaching autoclave temperature of 121°C.
- Sterilization is not achieved in a single step.
- It is achieved in fractions — each heating session destroys only a fraction of the microbial population.
- Day 1 → kills vegetative cells (Fraction 1).
- Day 2 → kills germinated spores from Day 1 (Fraction 2).
- Day 3 → kills any remaining germinated spores (Fraction 3).
- Only by combining all three fractions is complete sterilization achieved.
- Hence the name: Fractional Sterilization.
Mechanism
| Day | Step | Temperature | Duration | What Happens |
|---|---|---|---|---|
| Day 1 | Heating — Cycle 1 | 80–100°C | 20–30 min | All vegetative cells killed. Spores survive. |
| Day 1 | ⚠️ Incubation (Most Critical) | 37°C | 24 hours | Surviving spores germinate into new vegetative cells. |
| Day 2 | Heating — Cycle 2 | 80–100°C | 20–30 min | Germinated vegetative cells killed. Remaining spores may survive. |
| Day 2 | ⚠️ Incubation (Critical) | 37°C | 24 hours | Remaining spores germinate again into vegetative cells. |
| Day 3 | Heating — Cycle 3 | 80–100°C | 20–30 min | All remaining vegetative cells killed. Complete sterilization achieved. |
⚠️ The 24-hour incubation at 37°C is the most critical step. Without it, spores will not germinate and cannot be killed in the next heating cycle.
Procedure
Materials Required
- Water bath or steamer capable of reaching 80–100°C
- Incubator set to 37°C
- Containers with heat-sensitive media (e.g., Löwenstein-Jensen medium, Loeffler’s serum slope)
- Thermometer
Step-by-Step Method
- Prepare the heat-sensitive medium or material to be sterilized.
- Place containers in a water bath or steamer.
- Day 1 — Heat at 80–100°C for 20–30 minutes.
- Remove from water bath. Allow to cool to room temperature.
- Day 1 — Incubate at 37°C for 24 hours. This allows surviving spores to germinate into vegetative cells.
- Day 2 — Heat again at 80–100°C for 20–30 minutes.
- Day 2 — Incubate again at 37°C for 24 hours.
- Day 3 — Heat again at 80–100°C for 20–30 minutes.
- Allow to cool completely to room temperature.
- Store properly. Perform a sterility check by inoculating into broth and incubating at 37°C for 48 hours — no growth = sterile.
- Do not skip the incubation step — it is essential for spore germination.
- Maintain incubation at exactly 37°C — lower temperatures may prevent germination.
- All 3 heating cycles must be completed — stopping early leaves spores alive.
- Not suitable for non-nutrient fluids such as distilled water or saline — spores need nutrients to germinate. Use autoclaving or filtration instead.
- Temperature must not exceed 100°C — higher temperatures damage heat-sensitive media.
Result Interpretation
| After | Observation | Interpretation | Status |
|---|---|---|---|
| Day 1 — After incubation | Turbidity may appear in medium | Spores have germinated — vegetative cells growing | ❌ Not sterile |
| Day 2 — After incubation | Reduced turbidity | Most vegetative cells killed. Some spores remain and germinate. | ❌ Not sterile |
| Day 3 — After heating | No turbidity | All vegetative cells and germinated spores destroyed | ✅ Sterile |
| Sterility check | Inoculate into broth — no growth after 48 hours at 37°C | Confirms complete sterilization | ✅ Confirmed sterile |
Tyndallization is not effective for non-nutritive fluids such as distilled water or simple saline. Spores require nutrients to germinate. Without germination, spores cannot be killed in subsequent heating cycles. Use autoclaving or membrane filtration for such materials.
Diagram — Tyndallization Workflow
Comparison Table
Tyndallization vs Autoclaving vs Pasteurization
| Feature | Tyndallization | Autoclaving | Pasteurization |
|---|---|---|---|
| Developed by | John Tyndall (1877) | Charles Chamberland (1879) | Louis Pasteur (1864) |
| Temperature | 80–100°C | 121°C | 63°C (LTLT) / 72°C (HTST) |
| Pressure required | No | Yes — 15 psi | No |
| Number of cycles | 3 cycles over 3 days | Single cycle | Single cycle |
| Kills vegetative cells? | Yes ✅ | Yes ✅ | Yes ✅ |
| Kills spores? | Yes ✅ (via germination) | Yes ✅ (direct heat) | No ❌ |
| Result | Sterile | Sterile | Not sterile |
| Time required | 3 full days | 15–20 minutes | Seconds to 30 minutes |
| Suitable for | Heat-sensitive nutrient media | Most lab materials | Milk, beverages, food |
| Limitation | Needs nutrients — fails for non-nutrient fluids | Damages heat-sensitive materials | Does not achieve sterility |
Tyndallization vs Inspissation
| Feature | Tyndallization | Inspissation |
|---|---|---|
| Temperature | 80–100°C | 75–80°C |
| Purpose | Sterilization of heat-sensitive liquid media | Sterilization + solidification of egg/serum media |
| Number of cycles | 3 heating + incubation cycles | 3 heating cycles |
| Incubation between cycles? | Yes — 37°C for 24 hours | Not always required |
| Equipment | Water bath / steamer + incubator | Inspissator (slanted oven) |
| Media example | Löwenstein-Jensen broth, serum broth | Löwenstein-Jensen agar, Loeffler’s serum slope |
Why Tyndallization = Fractional Sterilization
| Fraction | Day | Action | What Is Killed | What Survives |
|---|---|---|---|---|
| Fraction 1 | Day 1 | Heat + Incubate | All vegetative cells | Spores (now germinating) |
| Fraction 2 | Day 2 | Heat + Incubate | Germinated vegetative cells from Day 1 | Remaining spores (germinating) |
| Fraction 3 | Day 3 | Heat only | All remaining vegetative cells | Nothing — complete sterility ✅ |
| All 3 fractions combined → Complete Sterilization → Hence called: Fractional Sterilization | ||||
Summary
- 📌 Tyndallization developed by John Tyndall, 1877.
- 📌 Temperature: 80–100°C — well below autoclave level (121°C).
- 📌 3 cycles of heating + incubation over 3 consecutive days.
- 📌 Incubation at 37°C for 24 hours between each cycle — essential for spore germination.
- 📌 Also called Fractional Sterilization — sterilization occurs in fractions across 3 days.
- 📌 Also called Intermittent Sterilization.
- 📌 Day 1 kills vegetative cells. Days 2–3 kill germinated spores.
- 📌 Spores cannot be killed directly — they must first germinate into vegetative cells.
- 📌 Used for heat-sensitive nutrient media — Löwenstein-Jensen medium, Loeffler’s serum slope.
- 📌 Not effective for non-nutrient fluids — spores need nutrients to germinate.
- 📌 No pressure required — unlike autoclaving.
- 📌 Main limitation: time-consuming — takes 3 full days.
- 📌 Mnemonic: H–I–H–I–H (Heat · Incubate · Heat · Incubate · Heat).
- 📌 Key phrase: “Sterilization is achieved in fractions — each cycle destroys one fraction of the microbial population.”
Also read:
Autoclaving ·
Pasteurization ·
Sterilization Methods in Microbiology ·
Inspissation ·
Culture Media Types