Search: safety levels

Premises

The premises where work with biological agents is carried out should be carefully designedin order to

Avoid endangering the people working there

Avoid endangering the environment

Work cleanly, efficiently and purposefully

The following sections briefly list the most important standards and guidelines that are relevant to the handling of biological agents and provide information on the layout anddesign of microbiological workspaces.

Biological Agents Ordinance (BioStoffV)

The Biological Agents Ordinance [1] forms the legal basis for working with biological agents, in particular microorganisms. It serves to protect employees against hazards caused by biological agents (also referred to as "biological substances") and to protect other persons insofar as they may beat risk due to the use of biological agentsby employees or business ownerswithout employees.

Important information inthe BioStoffV:

The term biological agentis explained in more detail (§ 2)

The terms "specific" and "non-specific" activities in connection with biological agentsare explained in more detail (§ 2). This distinction is important for choosing the appropriate level of protection.

Biological agents are categorised into risk groups based on the risk of infection they pose (§ 3)

The BioStoffV stipulates that a risk assessment must be carried out in accordance with § 5 of the German Occupational Health and Safety Act (Arbeitsschutzgesetz)before starting work (§ 4)

An explanation of activities with and without protection level classification as well as the employer's documentation and record-keeping obligations(§ 6 and § 7)

Basic obligations of the employer with regard to occupational health and safety and general hygiene measures

Further content and obligations can be taken directly from the BioStoffV [1].

Technical rules for biological agents (TRBA)

While the Biological Agents Ordinance provides the legal framework, the TRBAs substantiatethe current status of the respective requirements in the individual areas.

The most important TRBAs for brewery laboratories are:

TRBA 100: Protective measures for activities involving biological agents in laboratories

TRBA 400: Guidelinefor risk assessment and for the instruction ofemployees regardingactivities involving biological agents

A complete list of TRBAs is provided bythe German Federal Institute for Occupational Safety and Health [2].

Classification of microorganisms into risk groups

Section 3 of the German Ordinance on Biological Agents (BioStoffV) defines four risk groups for biological agents [3]:

Risk group 1: Biological agents that are unlikely to cause disease in humans

Risk group 2: Biological agents that may cause disease in humans and could pose a risk to employees; unlikely to spread to the community; effective prevention or treatment is usually possible

Risk group 3: Biological agents that may cause serious disease in humans and pose a serious risk to employees; theymay presenta risk of spreading to the community, but effective prevention or treatment is normally possible

Risk group 4: Biological agents that cause serious disease in humans and pose a serious risk to employees; they may present a high risk of spreading to the community; there is usually no effective prevention or treatment available

Examples of substances fromthe respective groups can be found in the literature [3].

Microbiologylaboratories in the brewing and beverage industry generally work with organisms in risk class 1. Exceptions include, for example, microbiological drinking water analysis (see assignmentof laboratories toprotection levels and safety levels).

Classification of laboratories into protection levels and safety levels [4, 5, 6]

According to the BioStoffV, four protection levels are assigned to the four risk groups. The protection level and risk group are identical for specific activities.

Activities are considered specific according to § 2 BioStoffVif:

1. The activities are directly focused onone or more biological agents

2. The biological agent or agents is/are known at least by species, and

3. The exposure of employees during normal operation is sufficiently known or can be estimated

If one of these conditions does not apply, the activities are non-specific. In the case of a non-specificactivity, the further procedure is regulated by § 5 BioStoffV. Annex II BioStoffV lists the protective measures for protection levels 2, 3 and 4.

Biological safety levels are not to be confused with protection levels. Biosafety levels are used to categorise the hazards of genetic engineering work in genetic engineering facilities. The safety levels are also arranged in a 4-level model, whereby biosafety level 1 is often referred to as "BSL-1".

The terms "BSL-1 laboratory" and "protection level 1 laboratory" are often used interchangeably in everyday life. However, as brewery laboratories do not work with GMOs, the term "protection level 1 laboratory" would be more correct.

Further details on the categorisation of brewery laboratories can be found in TRBA 100, Section4.4.3. (1). This states: Laboratories in which sterility tests, colony count determinations and other biological quality-assurance work iscarried out that doesnot serve to specifically detectbiological agents of risk group 2 and highercan be carried out under the conditions of protection level 1. This includes, for example, samples from the production of foodstuffs, medical devices, pharmaceuticals or cosmetics.

If, during the course of the activities,biological agents of risk group 2 or 3 are selectively propagated or enriched, the activitiesmust be carried out under the conditions of at least protection level 2 in accordance with the BioStoffV.

Biological protection levels (biosafety level or BSL laboratory) are used to categorise the hazards of biological agents (biological substances). This classification is standardised by EU Directive 200/54/EC on the protection of workers from risks related to exposure to biological agents at work. It is implemented in Germany by the Biological Agents Ordinance (BioStoffV). It categorises laboratories into four protection levels according to the risk groups. These build on each other, i.e. the rules of the lower protection levels also apply to the higher protection levels. A basic distinction must be made between specificand non-specificactivities. If it is a specificactivity, the protection level corresponds to the risk group. For non-specificactivities, the further procedure is also regulated by the BioStoffV (§ 5).

Protection level 1: General hygiene measures must be observed; this applies to structural, technical and organisational requirements; special hygiene measures in accordance with the technical rules for biological agents (TRBA) continue to apply to activities with protection level assignment; for work in the laboratory, the protective measures for activities involving biological agents in the laboratory (TRBA 100) must also be observed.

Protection level 2: The protection level area must be marked as such, including the protection level designation and the biohazard symbol; only named employees are permitted in the laboratory area; activities involving aerosol formation only in the area of a safety cabinet (clean bench) or in technical facilities with an equivalent level of containment; prior to disposal, mandatory inactivation via proven physical or chemical methods (usually autoclave). Suitable equipment for viewing the laboratory from the outside; TRBA 100: in addition to lab coats, protective gloves, face protection if necessary; mandatory eyewash facility; windows and doors must be closed; disinfection after completion of work according to hygiene plan.

Protection level 3: Access restricted todesignated employees; only with access control.

Protection level 4: Laboratory structurally separated; can be sealed for possible fumigation: filters for supply and exhaust air; access via three-chamber airlock.

If work is carried out with genetically modified organisms, the German Genetic Engineering Safety Ordinance (GenTSV) applies in accordance with the German Genetic Engineering Act (GenTG). This defines additional biosafety levels (hazard classification for genetic engineering work in genetic engineering facilities). (biosafety levels 1–4).

General definition: Genetically modified organisms (GMOs) are produced by genetic engineering. Not officiallyrelevant for brewing laboratories.

General information on the layoutof a brewery laboratory [2, 3]

Some general principles apply to the basic design of microbiologylaboratories. These general instructions are specified in TRBA 100 in sections5.2–5.5 for protection levels 1–4.

As an example, only the rules of TRBA 100 for protection level 1 should be mentioned here:

In the case of activities involving biological agents of risk group 1 and non-specificactivities that pose very little or no risk to employees, the occurrence of an infectious disease is unlikely. It is therefore sufficient to ensure that the laboratory is operated as intended:

(1) Protection level 1 laboratories should consist of separate, sufficiently large rooms. Sufficient working space must be provided for each employee in accordance with the activity.

(2) Surfaces (work surfaces, floors) should be easy to clean and must be imperviousand resistant to the substances and cleaning agents used.

(3) A wash basin should be available in the work area.

(4) The basic rules of good microbiological techniquesmust be observed (see Annex: "Basic rules of good microbiological techniques"). Point 9 of the GMT only applies to specificactivities.

(5) Biological agents in risk group 1 can only be disposed of without pre-treatment if the outcomeof the risk assessment or other regulations (e.g. water, waste or genetic engineering legislation) do not prevent this.

(6) When working with biological agents of risk group 1 with a sensitising or toxic effect, measures must be taken to minimise employee exposure. This may involve, for example, the use of a safety cabinet, the use of suitable respiratory protection or the avoidance of spore-forming development phases in fungi or actinomycetes.

The "basic rules of good microbiological techniques" (GMT) are as follows in accordance with TRBA 100:

(1) Windows and doors in the work areas should remainclosed during activities.

(2) It is prohibitedto drink, eat or smoke in the work areas. Food may not be stored in the work area.

(3) Lab coats or other protective clothing must be worn in the work area.

(4) Mouth pipetting is prohibited; pipetting aids must be used.

(5) Syringes and cannulas should only be used if absolutely necessary.

(6) All activities must beperformed as carefully as possible to prevent the formation of aerosols.

(7) After finishing work and before leaving the work area, hands must be carefully washed, disinfected if necessary and re-moisturised(skin protection plan).

(8) Work areas should be kept clean and tidy. Only the equipment and materials actually required should be presenton the work surfaces. Supplies should only be stored in areas and cupboards provided for this purpose.

(9) The identity of the biological agents used must be checked regularly, provided thisis necessary to assess the hazard potential. The intervals depend on the hazard potential.

(10) When working with biological agents, employees must be instructed verbally and at the workplace before starting work and at least once a year thereafter.

(11) Employees who are inexperienced in microbiology, virology, cell biology or parasitology must be given particularly comprehensive instruction, careful guidance and supervision.

(12) If necessary, pests must be controlled regularly and professionally.

In addition, the following general advice applies tothe laboratoryset-up:

Clear separation from other work areas and clear labelling of the area

Self-closing doors with a viewing window

Floors and work surfaces in the sterile area should be seamless and easy to clean

Avoid dust traps (pipework, suspended lamps, cupboards that do not end at ceiling height, open shelves, etc.).

Walls and ceilings must also besmooth and washable

Avoid floor drains, air conditioning and ventilation systems wherever possible

The sterile room should contain the bare minimum of equipment

The number of people in the sterile area must be kept to a minimum

If possible, provide separate rooms or clearly demarcated areas for:

Sample receipt

Sample preparation

Sample analysisincluding incubation

Storage of the reference organisms

Preparationof media and the corresponding equipmentincluding their sterilisation

Sterility check

Decontamination

To prevent accidental cross-contamination, laboratory equipment should not be routinely exchanged between areas.

Further information on laboratory equipment can be found in the literature.

Light microscope with fluorescence

Fluorescence microscopy is a light microscopy method that utilises the physical effect of fluorescence. UV or short-wave visible light of certain wavelengths (excitation light) is absorbed by fluorescent substances and emitted as longer-wave radiation (emitted light) as a result of the Stokes Shift. In contrast to bright-field microscopes, the image is only produced by the emitted light. Fluorescence microscopes are therefore only suitable for samples with inherent fluorescence or for samples into which fluorescent substances can be introduced.

Design and mode of operation:

The desired wavelength is filtered out of a light source using an excitation filter and projected onto a dichroic mirror. Dichroic mirrors only reflect light below a critical wavelength. Above this wavelength, light can pass through the mirror. The excitation light reflected through the objective to the specimen is then absorbed by the electrons of the fluorescent substances. As a result, these reach a higher energy state. However, due to the instability of this state, the electrons revert to their ground state, releasing the energy they have absorbed. The resulting emission light is lower in energy and therefore has a longer wavelength. This longer wavelength emission light reflected by the objective can pass through the dichroic mirror, thereby reaching the eyepiece or detector. An additional optical filter eliminates the remaining excitation light so that, as far as possible, only the emitted light is detected. The image of the specimen then appears in the respective emission colour on a black background.

Fig. 1: Schematic structure of a fluorescence microscope

Source: https://de.wikipedia.org/wiki/Fluoreszenzmikroskopie (accessed on 19.10.2024)
Author and licence: Krzysztof Blachnicki, derivative work: user Dietzel65; https://creativecommons.org/licenses/by-sa/3.0/ (accessed on 19.10.2024)

Stereomicroscope

As a special type of light microscope, stereomicroscopes differ from all other microscopes in that they have two separate optical paths. This special feature allows specimens and, in particular, surface structures to be viewed in three dimensions.

Design and mode of operation:

A separate optical path is provided for each eye, with the optical paths travelling at different angles onto the specimen, thereby creating a stereo effect. Special prisms allow objects to be magnified in the correct direction and in three dimensions. The design-related maximum magnification is around 100:1.

Devices for determining cell counts in liquids

The classic method for determining live cell counts is to plate the suspension on a suitable agar plate. However, this method is labour-intensive and time-consuming, and the preparation of any dilution series and plating as well as the evaluation of the results are potential sources of error. It is quicker and easier to determine cell numbers using counting chambers on the microscope or by means of automated cell counting systems.

A wide variety of counting chambers are available on the market – these differ mainly in terms of the applied counting grids and chamber depths. Counting chambers with a chamber depth of 0.1 mm are used for yeasts, whereas counting chambers with a chamber depth of 0.01 mm are used for bacteria. A reference volume corresponding to the count can be calculated from the chamber depth and the counting grid, which outlines defined areas using grid lines. This allows the results to be given in cells/ml or similar. Handheld counters are used for the actual counting process. By treating the sample with the relevant staining reagents, it is also possible to quantify live/dead cells (viability).

Automated counting systems work on the basis of various detection and counting mechanisms.

Cell counting systems with automated image recognition work in a similar way to counting chambers with a defined reference volume and corresponding evaluation algorithms. Depending on the system, the total cell count of the evaluation field is determined. When using staining reagents (e.g. methylene blue), the number of stained cells can also be measured to determine viability. Other systems work with fluorescent dyes that penetrate the cells of the sample and can be identified and counted by an integrated detector. As the corresponding fluorescent dyes can only penetrate and bind to dead cells, only the dead part of the cells is determined without further reagents. However, by using a lysis buffer or similar, the total cell count can also be determined and the viability calculated from both measurements.

Measuring devices based on the principle of Coulter particle counting are suitable for determining the total cell count as well as the size of the measured cells. Coulter counting measures the change in electrical resistance (impedance) between two electrodes arranged individually in reaction chambers. A small volume of the sample to be measured, diluted with a conductive electrolyte solution, is introduced into one chamber and then automatically sucked into the second reaction chamber through a capillary or opening of a suitable size. Each cell displaces a volume of electrolyte corresponding to its own volume and changes the electrical resistance between the electrodes as it passes through the opening. The current voltage or current pulse is measured. A short increase in resistance is detected as a cell. The height of the current pulse is proportional to the volume of the detected cell.

The cell counts of a suspension can also be determined with a flow cytometer, in this case using a laser-based measurement. The sample is passed through the laser beam in a stream of liquid and the light scattering (forward and sideways scattered light) is measured with detectors.

Microbiological safety cabinet

There are basically three classes of microbiological safety cabinets. Class I safety cabinets only offer protection against contamination for the person using the cabinet, but not for the material they are working with. Class II safety cabinets are suitable for use in brewery microbiology labs – they provide protection for both the operator and the sample material. Class III safety cabinets are fully enclosed systems for increased protection of the operator. This is achieved by permanently installed gloves, airlocks, constant negative pressure and supply and exhaust air filtration. However, the increased protection level also leads to more complicated work processes.

Design and function of a Class II safety cabinet:

Room air is drawn in through the front opening and is fed through a HEPA filter together with aerosols and particles. Part of the filtered exhaust air is channelled in a laminar flow from top to bottom along the open front screen and is then extracted back down to the filter together with room air. This circulation principle prevents contamination of the environment and cleans the fresh air drawn in before it comes into contact with the sample material. The safety cabinets are equipped with sensor-controlled function monitoring with an alarm function.

Spectrophotometer

Spectrophotometers are used in brewery analysis to measure concentrations, for example. For this purpose, a beam of light of a specific wavelength is passed through the sample and the light attenuation caused by the sample contents is recorded. Various ready-made cuvette test kits and reagent test systems are available for other applications, e.g. in water analysis or for enzymatic analyses.

A distinction is made between single-beam and dual-beam photometers. While single-beam systems have a better signal-to-noise ratio, dual-beam photometers are characterised by greater measurement stability. The devices commonly used are UV-Vis photometers, which utilise the ultraviolet (UV) range as well as the light visible to the human eye (Vis).

Structure and mode of operation:

A light source emits polychromatic light, which is broken down in the monochromator so that it only leaves at a specific wavelength. The monochromatic light passing through the exit slit then passes through the sample in a cuvette. Part of the light is absorbed here. The downstream detector measures the intensity of the light passing through the sample.

Fig. 2: Measuring principle of a single-beam absorption spectrometer

Set-up of a simple photometer to measure the absorption of individual light frequencies in liquids

Source: https://de.wikipedia.org/wiki/Spektralphotometer#/media/Datei:Photometer_mit_Monochromator.png (accessed on 19.10.2024)
Author and licence: Autor Sciencia58; https://commons.wikimedia.org/wiki/File:Photometer_mit_Monochromator.png

Centrifuge

Laboratory centrifuges support the separation of substances and are used in sample preparation, for example. Samples are placed in suitable containers and then set in a uniform circular motion in a rotor. The resulting centrifugal force, which depends on the speed and rotor geometry, acts on the sample contents, which are separated according to their inertia.

PCR thermocycler

PCR (polymerase chain reaction) has become an established alternative detection method in the brewery and beverage sector and enables the precise and rapid detection of brewery-relevant bacteria and yeasts. PCR is an in-vitro technique that can be used to specifically amplify deoxyribonucleic acid segments (DNA segments).

In addition to the corresponding PCR chemicals (PCR kits), a PCR thermocycler is a basic requirement for carrying out a PCR. A thermocycler is a device that can carry out the temperature cycling of a PCR independently. A PCR thermocycler must be able to heat up and cool down the reaction vessels used as quickly and precisely as possible. Typically, the temperatures it cycles through are between 95 °C and 60 °C or 72 °C.

Depending on the manufacturer, PCR thermocyclers differ in the reaction tubes used and in the way in which the tubes are heated and cooled. Another key difference is the way in which the PCR result is analysed. Standard thermocyclers in their original form can only heat and cool. This type of PCR is analysed in various ways (e.g. electrophoresis) after the actual PCR reaction.

In contrast, real-time PCR thermocyclers interpret the results during the reaction. These devices also have an evaluation unit that can measure and evaluate the fluorescence signals of a dye added to the reaction. Without this optical evaluation unit, PCR analysis would have to be performed in an additional reaction after the actual PCR reaction (e.g. by gel electrophoresis). Technical development in the field of real-time PCR analysers has accelerated and simplified the analysis and contributed greatly to establishing the method in the brewery sector.

Flow cytometer

Flow cytometry enables the automated measurement of particle or cell properties in suspension through simultaneous, multi-parametric analysis of the sample. In addition to cell counting in the liquid flow, it is possible to differentiate the particles or cells based on physical and molecular properties as well as the use of one or more fluorescent dye markers.

Design and mode of operation:

The functional principle of flow cytometric measurements is based on the emission of optical signals by the particles contained in the sample. These are usually cells. In the fluidics system of the flow cytometer, the particles are recorded in real time, enabling absolute cell counts based on volume. For this purpose, the sample material is surrounded by an enveloping flow of isotonic buffer solution and sucked through a cross-sectional constriction in a laminar flow. This process, known as hydrodynamic focussing, separates the cells.

The separated sample passes through one or more lasers with specific excitation wavelengths in the measuring cell; in some cases, xenon or argon lamps are also used. The resulting scattered light and fluorescence emissions can be used to generate electrical signals using appropriate detectors (photomultipliers), which are proportional to the intensity of the originally incident light.

A distinction is made between scattering signals and fluorescence signals. Scattering signals from forward light scattering allow conclusions to be drawn about the relative size of the particles, whereas signals from side-scattered light allow conclusions to be drawn about the structural properties and granularity. The fluorescence signals are dependent on the contained or bound fluorochromes. The type of fluorescent dyes used depends on a variety of factors, e.g. equipment (laser, filters and detectors), interactions with other dyes and the parameter to be determined.

The following cell analysis parameters can be determined using flow cytometry, whereby several properties can be recorded simultaneously depending on the assay:

Physiological parameters:

• Cell growth

• Metabolic activity

• Membrane potential

• Membrane integrity

Binding sites for fluorochromes or fluorescence parameters are:

• Nucleic acids (DNA, RNA)

• Proteins

• Lipids

• Intracellular pH value

• Fluorescent substrates

• Membrane potential

• Cations (Ca²⁺)

• Antibodies

• Autofluorescent proteins

etc.

MALDI-TOF mass spectrometry

Mass analysis using MALDI (matrix-assisted laser desorption ionisation) and subsequent time-of-flight analysis (TOF) is suitable for the rapid identification of microorganisms. The spectra for microbiological identification usually show specific peptide profiles or protein profiles of ribosomal proteins.

Structure and function:

A sample is mixed with a protective liquid matrix and applied to a sample carrier and fixed there by crystallisation. At the start of measurement, the matrix is vaporised using a pulsed laser and the bound biomolecules are dissolved (desorption) and ionised. The ions are then accelerated in an electric field and detected using a time-of-flight mass spectrometer.

The spectra generated can be compared with reference spectra from a database, thereby resulting in the identification of the sample. It is important to use pure cultures; prior isolation of the organisim is absolutely essential.

FT-IR spectrometer

A Fourier transform infrared spectrometer enables the fast and cost-effective identification of bacteria and yeasts in brewery microbiology. An interferogram measured using an FT-IR spectrometer is used to calculate a specific spectrum via Fourier transformation, which can then be compared with a corresponding database.

A prerequisite for successful identification is the use of pure cultures, which requires prior isolation. Standardised growth conditions are also required, as deviations have a direct effect on the final spectrum.