Biosafety Requirements in the LPA Lab: Refrigerated Centrifuge Use

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The refrigerated centrifuge is used in the mycobacteriology laboratory for concentrating bacterial cells, following the processing of sputum or other specimens. It is also used for spinning down reagents from walls of containers. The temperature maintained within the refrigerator centrifuge is 4°C to reduce the percentage of mycobacteria killed due to heat generated during centrifugation. The required G force is 3000 x g (not RPM).

Essential Features of the Refrigerated Centrifuge

Swinging Bucket Rotor: The component that is rotated by the drive system and holds the containers being centrifuged. It is air and liquid-tight. It allows for longer distance of travel which allows better separation, and it is easier to withdraw or decant the supernatant without disturbing the pellet

Figure 1: Advantage of the swing bucket in the centrifuge

Sealed Safety Centrifuge Cups:
- Safety cups protect laboratory staff by providing containment of any aerosols that are produced during centrifugation, especially if tubes break (see Figure 2)
- Safety cups must be opened inside the biosafety cabinet (BSC)
- Safety cups have a silicone- coated rubber O-ring that provides an air-tight seal
- These seals must remain coated with silicone so that seals stays air-tight

Before use, it is important to inspect all the components.

Figure 2: Refrigerated Centrifuge

Figure 3: Rotor and Centrifuge Cup (indicated by arrow) in a Refrigerated Centrifuge

Figure 4: Safety bucket in use

Principle of Procedure

A centrifuge is intended to separate particles in a liquid by sedimentation. Dense particles sediment first, followed by lighter particles.
In a TB laboratory, centrifuges are used for the sedimentation and concentration of tubercle bacilli within liquefied sputa or body fluids (usually urine).
To obtain a high sedimentation efficiency of 95%, i.e. 95% of bacilli recovered in the sediment and only 5% of bacilli still in suspension to be discarded with the supernatant, it is necessary to maintain 3,000 g for 15-20 minutes.
The high speeds and time used to achieve effective sedimentation efficiency leads to heat build-up in the centrifuge and the specimen.
To prevent cell death due to overheat, the use of a refrigerated centrifuge model, operated at 18-20°C, is recommended.

Refrigerated Microcentrifuge
Provides high-speed centrifugation up to 13,000 rpm Use microcentrifuge tubes with a capacity of 1.5 to 2.0 ml Fixed angle rotor with a lid Rotor can be removed from the centrifuge chamber