🧫 Total Viable Count at 30°C (TVC)

Microbiology PN-EN ISO 4833-1

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

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).

   ⏱ Time: 30 min • 🌡 Temperature: 121°C → 44–47°C

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.

   ⏱ Time: 5 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.

   ⏱ Time: 10 min

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!

   ⏱ Time: 15 min

7. Incubation

   Invert the plates (lid down, to prevent condensation dripping onto colonies). Incubate at 30±1°C for 72±3 hours.

   ⏱ Time: 72 h • 🌡 Temperature: 30°C

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

Reagents, media and consumables

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

Training and video materials

• Serial Dilutions and Plating — JoVE (Source: JoVE)

Frequently asked questions