Section 3 Medical Laboratory Biosafety Cabinets

A biosafety cabinet is designed to protect the operator, the laboratory environment, and the experimental materials from exposure to infectious aerosols and splashes that may be generated when handling infectious experimental materials such as primary cultures, bacterial and viral strains, and diagnostic specimens.

Biosafety cabinets can be divided into three major classes—Class I, Class II, and Class III—to meet different biological research and epidemic prevention requirements. See Table 7-4.

Table 7-4 Classification of Biosafety Cabinets

Class Type Exhaust Recirculated Air (%) Cabinet Airflow Inflow Velocity (m/s) Protection Target
Class I May exhaust into room — Turbulent flow ≥ 0.40 Operator
Class II Type A1 May exhaust into room 70 Unidirectional flow ≥ 0.38 Operator and sample
Type A2 May exhaust into room 70 Unidirectional flow ≥ 0.50
Type B1 Must not exhaust into room 30 Unidirectional flow ≥ 0.50
Type B2 Must not exhaust into room 0 Unidirectional flow ≥ 0.50
Class III Must not exhaust into room 0 Turbulent flow No inflow opening; when one glove is removed, glove port velocity ≥ 0.70 Primarily the operator, sometimes also the sample

Class I and Class II biosafety cabinets are essentially sealed enclosures with a working front opening and an exhaust opening at the top. Contaminated aerosols generated in the work zone are contained within the cabinet by the airflow drawn in through the front opening. In addition, contaminated aerosols in the exhaust air are captured by the HEPA filter.

Two points should be noted about the local isolation provided by Class I and II cabinets:

  1. The defensive means of drawing air inward from the opening by negative pressure is not absolute and is unreliable when handling highly dangerous pathogens and chemical substances. This is because once the cabinet stops operating, the isolating effect of the airflow no longer exists; and for pathogens attached to hands and instruments passing through the opening, this airflow isolation is completely ineffective. Furthermore, if the cabinet interior becomes contaminated, microorganisms may multiply inside the cabinet.

  2. Gaseous components in the air can pass through the HEPA filter, so great care must be taken when using such gaseous substances. It takes about 20–30 s for a gas in the cabinet to be diluted to 1/10 of its concentration; when a biosafety cabinet is used for chemical experiments, appropriate measures must be taken according to the nature and quantity of the substances handled.

I. Class I Biosafety Cabinet

A Class I biosafety cabinet draws air in through its front opening and exhausts air that has been treated by a HEPA filter through the exhaust outlet, as shown in Fig. 7-24.

[Figure: Schematics of a Class I biosafety cabinet, four panels — (a) airflow diagram with air entering at the front opening and exhausting upward after HEPA filtration at the top; (b) side-view schematic of the exhaust, with leader lines marking the exhaust duct and exhaust HEPA filter; (c) side view showing the operator's arms reaching into the cabinet, marked "5"; (d) front view with four circular working ports, marked "1", "2", "3", "4"]

Fig. 7-24 Class I Biosafety Cabinet

In general, the average velocity at the opening is 0.38 m/s or above; when handling hazardous substances, an appropriate baffle can halve the opening area, raising the average opening velocity to 0.76 m/s or above. Considered purely from the biological standpoint, the exhaust air may be discharged into the room.

The Class I biosafety cabinet supplies the work zone with air from the room, so it cannot be used for operations requiring sterile, clean conditions. However, this type of cabinet is most suitable for general examination work such as biochemical and serological testing in hospitals and similar settings. It can also house equipment that generates large amounts of aerosol (such as centrifuges and ultrasonic cleaners), provided the construction is suitably adapted. For a centrifuge, for example, the centrifugation chamber must be isolated from the motor so that the wind generated by the spinning centrifuge does not affect the cabinet's performance.

The front of a Class I biosafety cabinet may also have 2–4 circular openings fitted with long-sleeve gloves, as shown in Fig. 7-24 (c) and (d).

The airflow pattern inside a Class I biosafety cabinet is shown in Fig. 7-25.

[Figure: Schematic of the airflow pattern in a Class I biosafety cabinet; the legend distinguishes room air, contaminated air, and HEPA-filtered air; labels: 1 front opening, 2 viewing window, 3 exhaust HEPA filter, 4 exhaust duct]

Fig. 7-25 Schematic of Airflow in a Class I Biosafety Cabinet

1—front opening; 2—viewing window; 3—exhaust HEPA filter; 4—exhaust duct

[Figure: Class II-A biosafety cabinet, views (a) and (b); the legend uses dotted areas for negative-pressure contaminated zones and hatched areas for positive-pressure contaminated zones]

Fig. 7-26 Class II-A Biosafety Cabinet

II. Class II-A Biosafety Cabinet

This is the type of safety cabinet most used in microbiology. Like the Class I cabinet, it draws air in through the front opening and treats the exhaust air with a HEPA filter to prevent aerosols from escaping. In the work zone, however, clean air flowing vertically downward is supplied through a HEPA filter—called vertical unidirectional flow in cleanroom air technology, also commonly called vertical laminar flow.

Due to the suction effect of the inflow opening, it is difficult to achieve vertical parallel flow in the lower part of the opening compared with the upper part. As shown in Fig. 7-26 (a), part of the streamlines over the work surface curve toward the front opening and part curve inward.

This gives rise to two concepts—the negative-pressure contaminated zone and the positive-pressure contaminated zone.

In Fig. 7-26 (a), the work surface and a certain height above it are at the negative pressure of the fan's suction side, while within a certain distance below the HEPA filter's discharge face the air is still under the discharge dynamic pressure; therefore, contaminants during operation cannot rise and can only be drawn downward, with the negative pressure reaching its maximum at the fan's suction inlet. Thus the contaminated zone from a certain distance below the discharge face to the fan suction inlet is the negative-pressure contaminated zone.

Contamination in the negative-pressure contaminated zone cannot leak outward.

Contaminants drawn in by the fan are discharged from the fan outlet, where the positive pressure is greatest, and conveyed along the duct to the intake face of the HEPA filter, where there is positive pressure sufficient to overcome the filter resistance and discharge the air at a certain velocity. This section is both full of contaminants and at positive pressure, and is called the positive-pressure contaminated zone.

Contamination in the positive-pressure contaminated zone may leak outward.

In Fig. 7-26 (b), what the fan draws in is clean air that has passed through the HEPA filter; only at the filter's intake face—i.e., the work surface and a certain space above it—is the zone both contaminated and at negative pressure, belonging to the negative-pressure contaminated zone. Therefore this construction has no positive-pressure contaminated zone and is safer than the construction in Fig. 7-26 (a).

Positive-pressure contaminated zones should be avoided in biosafety cabinet construction.

To guarantee unidirectional flow, some designs draw air across the entire opening of the work zone; however, because the lower part of the work zone is occupied during operation with little free space, there is little performance difference from the former design.

Roughly half of the unidirectional flow and all of the air drawn in at the front opening are taken in through the slotted intake at the lower end of the front opening. Therefore, the proper ratio between the vertical parallel flow and the inflow at the opening determines the performance of the safety cabinet. If the suction is too strong, room air penetrates deep into the work zone, causing contamination, as shown in Fig. 7-27 (c); conversely, if the vertical unidirectional flow is too strong, contaminated aerosols escape into the room, as shown in Fig. 7-27 (a); only when both situations are avoided is the cabinet suitable, as shown in Fig. 7-27 (b).

[Figure: Fig. 7-27 Balance of the opening airflow of a Class II-A biosafety cabinet, cases (a), (b), and (c)]

The opening height is no more than 20 cm, allowing ordinary materials and instruments to pass in and out, but care must be taken with large items; a vertically sliding glass window can be used. However, to keep the variation of the inflow velocity at the opening within ±10%, the opening area must be strictly specified for different conditions; therefore, for cabinets equipped with a

sliding window, operation must be at the specified sash height.

Because this class of safety cabinet uses about 70% recirculated air, it is unsuitable for handling highly hazardous chemical substances; when hazardous chemicals are not being handled in the cabinet, it may exhaust directly into the laboratory—an indoor recirculation arrangement that is advantageous in terms of air-conditioning load and duct layout.

Fig. 7-28 is a schematic of the canopy (thimble) connection between a Class II-A biosafety cabinet and the exhaust duct. Class II-A biosafety cabinets are subdivided into Type A1 and Type A2, as shown in Fig. 7-29 and Fig. 7-30.

[Figure: Fig. 7-28 Schematic of the canopy (thimble) connection between a Class II-A biosafety cabinet and the exhaust duct]

1—air volume damper; 2—flexible connector; connected to the exhaust system; 3—housing of the biosafety cabinet exhaust HEPA filter; 4—exhaust canopy (thimble); 5—biosafety cabinet

Note: There is a 25 mm gap between the exhaust canopy 4 and the exhaust HEPA filter housing 3; this gap allows air to be drawn from the room.

The Class II Type A1 biosafety cabinet uses only one blower, which circulates the air inside the cabinet between the return air filter and the exhaust filter. The air between the fan and the two filters is contaminated and at positive pressure. See Fig. 7-29.

Fig. 7-30 is a schematic of the airflow in a bench-top Class II Type A2 biosafety cabinet. A Class II Type A2 cabinet recirculates 70% of the air; the other 30% is exhausted outdoors after filtration through the exhaust filter. Contaminated aerosols generated by operations in the bench work space mix with air drawn in from the room and enter the return air duct. Unlike the Class II Type A1 biosafety cabinet, the return air duct of the Class II Type A2 cabinet (6 in Fig. 7-30) is always at negative pressure, making it safer than Type A1.

[Figure: Fig. 7-29 Schematic of airflow in a Class II Type A1 biosafety cabinet; 1—front opening; 2—front viewing window; 3—exhaust HEPA filter; 4—supply HEPA filter; 5—rear air duct; 6—blower]

[Figure: Fig. 7-30 Schematic of airflow in a Class II Type A2 bench-top biosafety cabinet (a) side; (b) front; 1—front opening; 2—work viewing window; 3—exhaust HEPA filter; 4—supply HEPA filter; 5—positive-pressure duct; 6—negative-pressure duct; legend: room air / contaminated air / HEPA-filtered air; work surface]

III. Class II-B Biosafety Cabinet

Class II-B biosafety cabinets achieve a higher degree of safety than Class II-A cabinets for the following reasons:

  1. The average velocity at the front opening reaches 0.5 m/s or above;

  2. There is no positive-pressure contaminated zone;

  3. The proportion of recirculated air is reduced to 30%, or even to zero.

The glass window at the front opening of a Class II-B biosafety cabinet is a vertically sliding type, but during operation it must, as with Class II-A, be kept at the specified sash height. Because the average opening velocity is high, the opening height is mostly 20 cm; when the window is not at the specified height, an alarm device emits a signal.

Because more dangerous pathogens and chemical substances can be handled in a Class II-B biosafety cabinet, the exhaust must be discharged outdoors, with a sealed exhaust duct connection under negative pressure.

When multiple safety cabinets are installed, each should use an independent duct rather than a common duct.

Class II-B biosafety cabinets are further divided into Type B1 and Type B2, as shown in Figs. 7-31–7-34.

[Figure: Fig. 7-31 Class II Type B1 biosafety cabinet (a) side; (b) front; 1—front opening; 2—work viewing window; 3—exhaust HEPA filter; 4—supply HEPA filter; 5—positive-pressure exhaust duct; 6—blower; 7—additional supply HEPA filter; legend: room air / contaminated air / HEPA-filtered air]

Of the air discharged by the lower fan, part is exhausted through the exhaust HEPA filter and part is returned to the cabinet through the supply filter. Suction openings are provided at the middle and lower rear of the work space; under the action of the system exhaust fan, some air is also discharged there to avoid dead zones.

Fig. 7-32 shows another construction of Class II Type B1, with one fewer HEPA filter.

[Figure: Fig. 7-32 Class II-B safety cabinet with recirculated air (a) side section; (b) front and side; labels: exhaust duct, exhaust HEPA filter, glass sliding window, supply HEPA filter]

Fig. 7-33 is clearly a total-exhaust design: by the suction of the exhaust fan (not built in), contaminated air from the work space and room air are drawn into the negative-pressure exhaust duct 5. The built-in supply fan takes in room air and delivers it to the work space after HEPA filtration.

▨ room air　■ contaminated air　□ HEPA-filtered air

[Figure: Fig. 7-33 Class II Type B2 biosafety cabinet (a) side; (b) front; 1—front opening; 2—work viewing window; 3—exhaust HEPA filter; 4—supply HEPA filter; 5—negative-pressure exhaust duct; 6—blower; 7—filter screen]

[Figure: Fig. 7-34 Total-exhaust Class II-B biosafety cabinet (schematic of the negative-pressure contaminated zone)]

Fig. 7-34 is a schematic of the contaminated zones of a total-exhaust, fan-equipped Class II Type B2 cabinet.

The similarities and differences between Class II-A and Class II-B safety cabinets are compared in Table 7-5.

Table 7-5 Comparison of Class II-A and Class II-B Biosafety Cabinets

Item Class II-A Class II-B
Positive-pressure contaminated zone A1 yes, A2 no None
Front shielding structure Fixed-open or vertically sliding window (same as left)
Front opening height (mm) 200 (same as left)
Supply air velocity in work zone (m/s) ≥ 0.23 (same as left)
Recirculated air (%) 70 30–0
Exhaust A1 indoors / A2 outdoors Outdoors

Continued table

Item Class II-A Class II-B
Applicable agents General pathogens Levels 1–3 Levels 1–3
Recombinant genetic material P1–P3 levels P1–P3 levels
Chemical carcinogens A1 not allowed / A2 allowed (at low concentrations) Allowed (at low concentrations)
Radioactive substances A1 not allowed / A2 allowed (at low concentrations) Allowed (at low concentrations)
Volatile solvents A1 not allowed / A2 allowed (at low concentrations) Allowed

Note: The construction of the sliding window must be able to fix its position or be provided with a device that alarms when it is not in the specified position.

IV. Class III Biosafety Cabinet

This class of biosafety cabinet is suitable for operations with the highest degree of hazard in experiments involving viruses, bacteria, parasites, recombinant genetic material, and the like. Through long rubber gloves fitted to a completely sealed negative-pressure cabinet, the operator performs all experimental work in the cabinet's sealed work zone—breeding and dissecting infected animals, handling tissue materials, culturing pathogens, microscopic observation, and centrifugation.

Some national standards require the HEPA filters of a Class III safety cabinet to have an efficiency of 99.997% or more for 0.3 μm particles—one grade higher than ordinary HEPA filters, equivalent to a Class C filter in the national standard.

In a Class III biosafety cabinet (Fig. 7-35), supply air enters the cabinet from top to bottom through a HEPA filter, while the exhaust air is treated by two sets of HEPA filters in series before discharge. The negative pressure inside the cabinet is generally maintained at about 125 Pa. All operations must be performed through gauntlet gloves, and all materials entering or leaving must pass through a double-door autoclave or a dunk tank filled with disinfectant.

[Figure: Fig. 7-35 Principle of the Class III biosafety cabinet (a) side; (b) front; legend: room air / contaminated air / HEPA-filtered air; 1—gloves and glove ports; 2—work viewing window; 3—exhaust HEPA filter; 4—supply HEPA filter; 5—double-door autoclave or pass-box HEPA filter; 5—positive-pressure exhaust duct; 6—blower; 7—additional supply HEPA filter (the original figure has two labels "5", apparently a printing/numbering error)]

Class III biosafety cabinets are further divided into the following two types:

  1. Standalone type: used when only one biosafety cabinet is needed, as shown in Fig. 7-36.

[Figure: Fig. 7-36 Construction of a standalone Class III safety cabinet (a) supply filter at the bottom; (b) supply and exhaust filters on opposite sides; labels: exhaust HEPA filter (2nd stage), exhaust HEPA filter (1st stage), intake HEPA filter, autoclave, chemical dunk tank (pass box), exhaust fan, exhaust to outdoors]

  1. Line (series) type: used when multiple biosafety cabinets need to be connected, as shown in Fig. 7-37.

The line-type biosafety cabinet is abbreviated GBL; at its end is an autoclave with doors on both sides. Experimental materials cannot be taken out of the cabinet directly; they must be sterilized in the autoclave before removal.

With this arrangement, because the pathogens are completely sealed within the glove-box apparatus, the laboratory remains uncontaminated, and special protective clothing, gloves, masks, and similar equipment are not required. See Fig. 7-37.

[Figure: Fig. 7-37 Line-type Class III safety cabinets (multiple units connected; labels: connection section, exhaust fan, exhaust to outdoors, autoclave)]