This page covers the core equipment of a clinical and research microbiology laboratory: what each instrument does at a physical and biological level, how it works, what the key operational parameters are, how to recognise when it is not working correctly, and what the safety implications are. It is the knowledge base every laboratory scientist needs before touching the equipment, and the reference every trainer and safety officer should be confident in.
The Autoclave: Steam Under Pressure
The autoclave sterilises materials using saturated steam under pressure. Inside the chamber, steam replaces air, raising the temperature above the boiling point of water (100 degrees Celsius at atmospheric pressure) to achieve temperatures of 121 or 134 degrees Celsius. At these temperatures, bacterial spores, vegetative cells, viruses, and fungi are killed within minutes by irreversible protein denaturation.
Key parameters: temperature (monitored by a calibrated thermocouple in the drain or chamber), pressure (corresponding to the saturated steam pressure for the set temperature), exposure time (the hold period after equilibration is reached throughout the load), and for porous loads, the pre-vacuum cycle parameters.
What can go wrong: if air is not completely evacuated from the chamber or load (as in gravity displacement cycles for porous loads), pockets of air prevent steam from reaching parts of the load and those areas remain below the sterilisation temperature despite the chamber pressure reading correctly. This is why biological indicator placement at the most challenging load position is the definitive validation check.
Operator check before each run: chemical indicator (Class 1 or Class 5) on or in the load should be observed after the cycle for correct colour change. For porous loads, the Bowie-Dick test weekly or more frequently confirms steam penetration.
The Biological Safety Cabinet (BSC): Primary Containment
The BSC protects the operator from exposure to biological aerosols generated during work inside the cabinet, and (for Class II) also protects the work from environmental contamination.
A Class II Type A2 BSC draws room air in through the front opening (inward airflow protecting the operator from splashes and aerosols moving outward). Inside the cabinet, HEPA-filtered, vertically downflowing clean air creates a sterile work environment (protecting the sample). Air from the work zone is drawn through HEPA filters before being partly recirculated over the work surface and partly exhausted (through another HEPA filter) to the room or to ducting. The downflow velocity is typically 0.25 to 0.5 m/s and inflow velocity 0.4 to 0.5 m/s.
What can go wrong: damaged or expired HEPA filters (must be certified annually), working outside the sash opening height marked by the alarm indicator, placing materials at the front grille (blocks inflow, disrupts airflow patterns), using open flames inside the cabinet (disrupts laminar airflow and can damage HEPA filters), working too rapidly (vigorous movement disrupts the airflow curtain and aerosols can escape the work zone).
Decontamination before servicing: BSCs are decontaminated with vaporised hydrogen peroxide (VHP) or formaldehyde gas before HEPA filter changes or maintenance. This is performed by a certified engineer, not by routine laboratory staff.
The Incubator: Controlled Temperature for Bacterial Growth
Incubators maintain a stable temperature for bacterial culture. Standard microbiology incubators operate at 35 to 37 degrees Celsius for most clinical isolates. CO2 incubators (5 to 10 per cent CO2 in air) are used for capnophilic organisms (Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus pneumoniae) that require elevated CO2 for growth. Anaerobic incubators or anaerobic jars maintain an atmosphere of hydrogen and CO2 with catalysts that reduce oxygen to water, creating conditions below 1 per cent O2 for obligate anaerobes.
Temperature monitoring: calibrated thermometers should be placed inside incubators with readings recorded daily (or continuously logged in automated systems). Incubator temperature variation greater than plus or minus 0.5 degrees Celsius from set point should trigger investigation and maintenance.
The Centrifuge: Separation by Density
Centrifuges separate components of a mixture by density using centrifugal force. In clinical microbiology, centrifugation concentrates organisms in CSF (CSF is centrifuged and the pellet is used for culture and Gram stain), concentrates organisms in urine for microscopy, and pellets bacteria from broth for DNA extraction.
The key parameter is relative centrifugal force (RCF, also expressed as g-force): RCF = 1.12 x radius (cm) x (RPM / 1000)^2. Most routine clinical microbiology uses speeds of 1,500 to 3,000 x g.
Critical safety requirement: rotors must be balanced. Each rotor position should have a tube of identical volume and density on the directly opposite position. An unbalanced rotor at high speed generates extreme vibration, can strip the rotor bearing, and can cause catastrophic rotor failure. Centrifuge lids must be closed during operation. For BSL-2 and above materials, sealed rotors or sealed safety centrifuge buckets are required to prevent aerosol generation if a tube breaks during centrifugation.
The Spectrophotometer and Densitometer: Measuring Turbidity
The spectrophotometer measures absorbance of light at a specific wavelength through a liquid sample. In microbiology, it is most commonly used to measure bacterial growth: OD600 (absorbance at 600 nm) is proportional to bacterial cell density in a culture.
A densitometer or nephelometer measures turbidity by light scattering (rather than absorbance) and is the instrument used for comparing bacterial suspensions to McFarland standards. In a clinical laboratory setting, the densitometer is used daily for preparing inocula for disc diffusion and MIC testing to the 0.5 McFarland standard (approximately 1 to 2 x 10^8 CFU/mL).
MALDI-TOF Mass Spectrometer: Rapid Bacterial Identification
MALDI-TOF (Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight) mass spectrometry identifies bacteria by the unique pattern of ribosomal and other abundant proteins in the 2,000 to 20,000 Da range. A small amount of colony is spotted onto a stainless steel target plate, overlaid with a UV-absorbing matrix (alpha-cyano-4-hydroxycinnamic acid), and dried. A laser pulse ionises the sample, proteins are propelled through a flight tube under vacuum, and the time to reach the detector corresponds to the mass-to-charge ratio. The resulting mass spectrum is compared to a reference database and the closest match gives the identification.
MALDI-TOF has replaced most conventional biochemical identification in modern clinical laboratories. Identification of the majority of common clinical isolates takes less than 5 minutes with accuracy exceeding 95 per cent at genus level and 90 per cent or above at species level for organisms in the database.
The Anaerobic Chamber and Anaerobic Jar
Anaerobic bacteria die within minutes of oxygen exposure. Working with obligate anaerobes requires maintaining an oxygen-free environment from the moment the sample arrives.
Anaerobic jar or pouch: the simplest approach. An AnaeroGen sachet inside a sealed jar generates hydrogen and CO2 through a chemical reaction; a palladium catalyst within the jar converts residual O2 to water. An anaerobic indicator (resazurin) confirms an anaerobic environment has been achieved. Plates are placed in the jar and incubated without opening until ready to read.
Anaerobic workstation or chamber: a flexible vinyl enclosure maintained under an anaerobic gas atmosphere (typically 80 per cent N2, 10 per cent CO2, 10 per cent H2). Entry is through an interlock chamber that allows materials to be introduced while maintaining the internal anaerobic environment. Operators work through glove ports built into the chamber wall. This allows continuous manipulation of anaerobic cultures without exposure to air.
Frequently Asked Questions
What is a biological safety cabinet?
A BSC is a ventilated enclosure that provides primary containment for biological materials. Class II Type A2 BSCs, the most widely used, protect the operator (through inward airflow), the sample (through HEPA-filtered downward airflow), and the environment (through HEPA-filtered exhaust). They are required for all work with Risk Group 2 and above pathogens.
Why does a centrifuge need to be balanced?
An unbalanced centrifuge rotor creates extreme vibration at high rotational speeds. This stresses the rotor bearings, can cause the rotor to shift on the shaft, and in severe cases can cause catastrophic rotor failure, ejecting the rotor or its contents. For biological materials, rotor failure can also create an infectious aerosol. Balancing places equal volumes on directly opposite rotor positions.
What is MALDI-TOF used for in clinical microbiology?
MALDI-TOF identifies bacteria (and yeast) by their protein mass spectrum, compared to a reference database. It provides genus and species identification from a colony within 5 minutes, replacing many hours of biochemical testing. It is now the primary identification method in most clinical microbiology laboratories for organisms that grow in culture.
What organisms require anaerobic culture conditions?
Obligate anaerobes die in the presence of oxygen. Clinically important obligate anaerobes include Bacteroides fragilis, Prevotella species, Fusobacterium species, Peptostreptococcus species, Clostridioides difficile, Clostridium perfringens, Clostridium botulinum, and Actinomyces species. Samples from deep wounds, peritoneal fluid, abscess contents, and dental infections routinely require anaerobic culture.
What temperature do most clinical incubators run at?
Standard clinical microbiology incubators run at 35 to 37 degrees Celsius, the optimal growth temperature for most human pathogens. CO2 incubators add 5 to 10 per cent CO2 for capnophilic organisms. Incubators for fungal culture often run at 25 to 30 degrees Celsius to support growth of environmental fungi and dermatophytes.
What is a McFarland standard?
A McFarland standard is a turbidity reference used to standardise bacterial suspension density. The 0.5 McFarland standard corresponds to approximately 1 to 2 x 10^8 CFU/mL and is used to prepare inocula for antibiotic susceptibility testing by disc diffusion or broth dilution. Comparison to the standard ensures the inoculum density is within the acceptable range for reproducible results.
What is OD600?
OD600 is optical density measured at 600 nanometres wavelength using a spectrophotometer. It measures how much light is absorbed (and scattered) by a bacterial suspension. It is commonly used to monitor bacterial growth in liquid culture: as cell density increases, OD600 increases proportionally within the linear range (typically OD600 0.1 to 0.8 for most gram-negative bacteria). It must be converted to CFU/mL using a species-specific calibration curve.
What is a loop steriliser (inoculation loop steriliser)?
An electronic loop steriliser (or incinerator) uses an electrically heated coil or wire that reaches 825 degrees Celsius to incinerate the tip of a platinum or nichrome inoculation loop within 5 to 8 seconds. It replaces the open Bunsen burner flame for sterilising loops, eliminating the aerosol and spray risk that occurs when a hot loop contacts liquid culture. It is mandatory in BSCs where open flames are not permitted.
How often should a BSC be certified?
BSCs must be certified by a qualified engineer at least annually, and after any relocation, HEPA filter change, or repair. Certification includes testing airflow velocity at the work surface, HEPA filter integrity (by DOP or PSL particle challenge test), and containment performance. An out-of-certification BSC should not be used for work with infectious material.
What is the AnaeroGen sachet?
An AnaeroGen sachet is a self-contained chemical anaerobic atmosphere generator. When the sachet is opened and placed in a sealed container (jar, bag, or box) with culture plates, it generates CO2 and hydrogen through reaction with water vapour in the air. A palladium catalyst on the sachet converts residual oxygen to water. The result is an anaerobic atmosphere (below 1 per cent O2) within 30 minutes. An anaerobic indicator strip changes from pink to colourless when the O2 level is adequate for anaerobic culture.