🧫 Total Viable Count at 30°C (TVC)
Pour plate method for enumeration of aerobic mesophilic total count in food. A general hygiene indicator.
Overview
Total Viable Count (TVC / total plate count) at 30°C is one of the fundamental indicators of microbiological quality of food. It is not a test for specific pathogens but rather a measurement of overall microbiological contamination — the so-called "general hygiene" of a product.
A high viable count indicates: inadequate production hygiene, cold chain interruption, excessively long storage, improper storage conditions, or insufficient heat treatment.
The pour plate method involves mixing a diluted sample with molten PCA (Plate Count Agar), incubation at 30°C for 72 hours, and counting the grown colonies. The result is expressed in CFU/g (colony-forming units per gram).
This is the reference method required by Regulation (EC) 2073/2005 for many food categories (dairy products, ready-to-eat foods, frozen foods, juices).
Method principle
Pour plate technique: 1. The sample is homogenized and decimal dilutions (10⁻¹, 10⁻², 10⁻³...) are prepared in peptone saline diluent. 2. 1 mL of each dilution is transferred to a sterile Petri dish. 3. Approximately 15 mL of molten PCA (cooled to 44–47°C) is poured over. 4. Mixed with circular motions (8× clockwise, 8× counterclockwise) and left to solidify. 5. Inverted plates are incubated at 30±1°C for 72±3 hours. 6. Colonies are counted on plates containing 10–300 colonies.
Result [CFU/g] = mean colony count × reciprocal of dilution factor.
Applications
- Ready-to-eat food quality control
- Milk and dairy product testing (process hygiene criterion)
- Meat and meat product control
- Beverages, juices, and bottled water testing
- Production facility hygiene monitoring (swabs)
- Incoming material quality control — microbiological assessment of deliveries
Key parameters
| Parameter | Value |
|---|---|
| Incubation temperature | 30 ± 1°C |
| Incubation time | 72 ± 3 hours |
| Counting range | 10–300 colonies per plate |
| Result unit | CFU/g or CFU/mL |
| Limit of detection | <10 CFU/g (at 10⁻¹ dilution) |
| Culture medium | PCA — Plate Count Agar (tryptone-glucose) |
Standard
- Standard number
- PN-EN ISO 4833-1:2013
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania liczby drobnoustrojów — Część 1: Oznaczanie liczby metodą płytkową w temperaturze 30°C
- Title (EN)
- Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — Part 1: Colony count at 30°C by the pour plate technique
Step-by-step procedure
1. PCA medium preparation
Dissolve PCA in distilled water. Sterilize in autoclave at 121°C/15 min. Cool in a water bath to 44–47°C (agar must remain molten but not hot).
2. Sample preparation
Weigh 10 g of sample into a stomacher bag. Add 90 mL of diluent (1:10 = 10⁻¹ dilution). Homogenize for 1–2 min.
3. Serial dilutions
Transfer 1 mL of 10⁻¹ suspension to 9 mL of diluent → 10⁻². Repeat: 10⁻³, 10⁻⁴... Use a fresh pipette tip for each dilution. Vortex between transfers.
4. Inoculation — transfer to Petri dishes
Transfer 1 mL from selected dilutions to sterile Petri dishes (2 dishes per dilution = duplicate). Work near a Bunsen burner or in a laminar flow cabinet.
5. Pouring PCA
Pour approximately 15 mL of molten PCA (44–47°C) onto each dish. Check internal temperature with the back of the hand — warm, not hot. Immediately mix with circular motions: 8× CW, 8× CCW, 8× back-and-forth, 8× side-to-side.
6. Agar solidification
Leave plates on a flat surface until completely solidified (~15 min). Do not move the plates during this time!
7. Incubation
Invert the plates (lid down, to prevent condensation dripping onto colonies). Incubate at 30±1°C for 72±3 hours.
8. Colony counting
After 72 h, count colonies on plates containing 10–300 colonies. Use a colony counter with magnifying lens. Count ALL colonies — small and large, of various morphologies.
9. Calculations
N = ΣC / [V × (n₁ + 0.1×n₂) × d], where ΣC = sum of colonies, V = inoculum volume (1 mL), n₁ = number of plates at lower dilution, n₂ = at higher dilution, d = lower dilution. Result in CFU/g.
10. Quality control
Control plate (blank): PCA agar only — no growth expected. Check diluent and medium sterility with each batch.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 30±1°C | Memmert IN110, Binder BD 115, POL-EKO ST 2 | 5 000–15 000 PLN |
| Stomacher (homogenizer) | Seward Stomacher 400 Circulator | 12 000–25 000 PLN |
| Autoclave | Tuttnauer 3870ELV | 30 000–80 000 PLN |
| Water bath 44–47°C | GFL 1003, Memmert WNB 14 — for maintaining PCA in molten state | 3 000–8 000 PLN |
| Automatic pipettes 1 mL | Eppendorf Research Plus, Brand Transferpette S | 800–1 500 PLN |
| Petri dishes ø 90 mm | Sterile, disposable, polystyrene | 150–300 PLN / 500 pcs. |
| Colony counter | Interscience Scan 500, Stuart SC6+ | 3 000–15 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Plate Count Agar (PCA) | — | Tryptone-glucose agar: tryptone 5 g + yeast extract 2.5 g + glucose 1 g + agar 15 g per litre. Merck 1.05463, Oxoid CM0325. |
| Diluent — peptone saline | — | NaCl 8.5 g + peptone 1 g per litre of water. Merck 1.12535. For decimal dilutions. |
| Diluent tubes 9 mL | — | Sterile, with screw caps, for 10⁻¹ to 10⁻⁶ dilutions |
| Stomacher bags | — | Sterile, with side filter (BagFilter), 400 mL |
Health and safety (OHS)
- Food samples may contain pathogens — wear gloves
- Hot agar (~47°C) — burn risk
- Autoclave — high pressure and temperature, training mandatory
- All waste must be autoclaved before disposal (121°C/20 min)
- Work under clean conditions — laminar flow cabinet or designated area