BIOSAFETY IN LABORATIESBIOSAFETY IN LABORATIES

INTRODUCTIONINTRODUCTION

Name : Owais YaqubFrom : ESCO Singaporeg p

: MSc Microbiology: MSc Microbiology : Accreditation in NSF 49 USA

Guidelines for BioSafety in Lab

C & I t ti ll A t blCommon & Internationally Acceptable

C t f Di C t lCenters for Disease Control USA

WHO Guidelines for BiosafetyWHO Guidelines for Biosafety

Biosafety levels

Biosafety levels Requirements

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Biosafety levels 1

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Biosafety levels 2

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Biosafety levels 3

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Biosafety levels 4

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Biosafety levels 4

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Summary of Biosafety Level

Lethality Medium Cure ExampleLethality Medium Cure Example

1 Safe Liquid Yes B.Subtilisq

2 Some Liquid Some HIV

3 Serious Airborne Some TBC

4 Extreme Airborne None Ebola4 Extreme Airborne None Ebola

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Microbiological lab Standardization Concepts

BSL Levels

Whole lab roomBiosafety cabinets standards

ISO 14644 Air quality standard in side the BSL Levels

1/2/3/4

standards NSF 49 / EN 12469

standard in side the cabinet

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ISO standard for Air quality

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History of HEPA Filter

HEPA: High Efficiency Particulate Air• First designed in the 1940s for radioactive particle control in the US for Manhattan Project

• A major milestone in filtration technologyfiltration technology

- “Classical” definition of 99.97% at 0.3 microns

HEPA & ULPA Filter

HEPA: High Efficiency Particulate Air ULPA: Ultra Low Penetration Air

Important definitions: Modern “American-convention” per IEST-RP-CC001.3 :

• HEPA: 99.99% at 0.3 microns• ULPA: 99 999% at between 0 1 to 0 2 microns• ULPA: 99.999% at between 0.1 to 0.2 microns

Note: The “classical” definition of HEPA filter isNote: The classical definition of HEPA filter is99.97% at 0.3 microns, but nowadays allBSC and LF in US use 99.99% at 0.3 m

HEPA & ULPA Filter at MPPS

Virus Filtration

• Virus has typical size of 4 to 0.003 micron, therefore, some virus lies in the MPPS range and may not be 100% filtered out by ULPA filters

• However, viruses are obligate parasites (attach itself to a cell if they have proper connectors) anditself to a cell if they have proper connectors) and usually not found in the air

• Experience has shown that HEPA filters, when properly installed in supply-air systems, provide

ti f t i t lsatisfactory virus control

LF, Fumehoods & BSC

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Laminar Flow CabinetsDefinition of laminar / unidirectional flow:Air flow straight, parallel pass in enclosed space

Principle of laminar flow cabinet:Sterilize air though filter and blow it across workSterilize air though filter and blow it across work surface as a particle-free laminar air stream

Typical laminar air flow velocity: 0.4 - 0.5 m/s (80 – 100 fpm)

Purpose of a laminar flow cabinet: P d t t ti l (d t t t t )Product protection only (does not protect operator)

Horizontal vs. Vertical• Advantage of Vertical Laminar Flow Cabinet:

- No blocking caused by large objectNot blowing air straight to operator’s face 8hrs/day- Not blowing air straight to operator s face 8hrs/day

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UV Light- To destroy microorganisms in the air or surface- Despite blue-violet glow -> check intensity- Standard: 40 W/cm2, Esco: 120-240 W/cm2

- UV light attract dust -> need periodical cleaning- Eye irritation, burnt skin- Beware of UV reflection from stainless steel table- Close the window -> zero intensity- Laboratory to be vacated when UV light is on,

l bi t h f t / lidi hunless cabinet has front cover / sliding sash- Routine surface decontamination required, since

UV is NOT a replacement for surfaceUV is NOT a replacement for surface decontamination

The Need for BiosafetyThe need for biosafety

- High risk of laboratory-acquired infections- First documented in the US in the 1940s

Purpose for BSC

- Operator protection by inflow (not in LF)- Product protection (like LF ; only BSC Class 2 p ( ; y

and 3, but not Class 1)

Containment and Safety

Containment principles- Primary containment (safety equipment)- Secondary containment (facility design)

Standard microbiological practices

Principle of selecting which class of BSC- Only operator or operator & product protectiony p p p p- Degree of operator protection (Biosafety level)

Primary Containment

• All manipulations inside Biohazard Safety Cabinets• All manipulations inside Biohazard Safety Cabinets• Clothing: wrap-around gowns, scrub suit, or coverall• Gloves: infectious materials / contaminated G o es ec ous a e a s / co a a ed

equipment• Frequent changing of gloves and hand wash• If manipulations can not be done inside BSC:

- Personal protective equipment (respirator, face, shield)shield)

- Physical containment devices (centrifuge cup)

Secondary Containment

• All windows are closed and sealed• Interior surfaces (walls floor ceiling): easy forInterior surfaces (walls, floor, ceiling): easy for

cleaning and decontamination• Bench top: resist moderate heat & chemicalsp• Chairs: non-fabric material-> easy decontamination• Waste decon: autoclave, chemical treat, incineration

D t d h t i til ti HEPA filt d• Ducted exhaust air ventilation - HEPA filtered• Exhaust: away from occupied area and air intakes• Directional room airflow:clean to contaminated areaDirectional room airflow:clean to contaminated area• Audible alarm for HVAC failure• Hands-free sink for hand washing - near exit door• Eyewash station• Personnel shower

Std. Microbiological Practice• Wash hand:

- Handling hazardous materialsCh l it f l b- Change glove, exit from lab

• No eating, drinking, smoking, handling contact lens

• Safe handling of sharp objects• Minimize aerosol generationMinimize aerosol generation• Work surfaces decon per day or after spill • Waste decon inside lab | outside with leak proof cont

Section 4BTypes of Biosafety Cabinets

LF, Fumehoods & BSC• Laminar Flow (LF):

- Product protection (no personnel protection)- Not for biohazard agents or chemical fumes

Fume Hood:• Fume Hood:- Removes toxic chemical (ducting sys./ductless)- No HEPA filter -> not for biohazard agentsNo HEPA filter > not for biohazard agents

• Biohazard Safety Cabinet (BSC)- Product and personnel protection (not Class 1)- HEPA can’t filter chemicals -> ducting or

bcarbon

Types of BSC

Class Inflowv (m/s)

Recirc.Air

Exhaust Air

Contd. PlenumSurrounded by

ExhaustAlternatives

BiosafetyLevel

US 0 38 I id /1 US:0.38EN:0.70 0 % 100 % Outside air Inside room /

Hard duct 1, 2, 3

2 A1 US:0.38EN:0.40 70 % 30 % Outside air Inside room /

Thimble duct 1, 2, 3

2 A2 US:0.50EN:0.40 70 % 30 % Negative

plenumInside room /Thimble duct 1, 2, 3

2 B1 US:0.50EN:0.40 30 % 70 % Negative

plenumHard duct

only 1, 2, 3

2 B2 US:0.50EN: NA 0 % 100 % Negative

plenumHard duct

only 1, 2, 3

3 Closed>0.5”WC 0 % 100 % Negative

plenumInside room /

Hard duct 1, 2, 3, 4p

Note:Small amount Toxic Chemicals (carcinogen) & Radionuclides must Duct

Class II BSC: Basics

• Both operator and product protection• Biosafety level 1, 2, 3• Inflow away from operatory p• HEPA filtered exhaust to environment

HEPA filt d l i d fl• HEPA filtered laminar downflow• Volatile Toxic Chemical in cell culture:

biological & chemical -> need ducting

Class I BSC• Only operator protection (no product protection).

• Biosafety level 1,2,3

• Inflow away from operator.operator.

• HEPA filtered exhaust to environment.

C t t d S it h t31

• Current trend: Switch to Class II The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

Class II A1 BSC: Airflow

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Class II Type A2 BSC Airflow

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Class II B1 BSC: Airflow

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Class II B2 BSC Airflow

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Class III BSC

36The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

Risk Assessment

The customer and distributor (you) must do risk assessment of the challenge agents first before

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g gjumping down and choosing the cabinet

Field Production Production Type Type

Testing List No Test Field

EN & NSF Production

Per EN Production

Per NSF Type

Per EN Type

Per NSF 1 Inflow velocity √ √ √ √ √ 2 Downflow velocity √ √ √ √ √ 3 HEPA / ULPA filter leak test √ √ √ √ √ 4 S k tt / i fl i li ti √ √ √ √ √4 Smoke pattern / airflow visualization √ √ √ √ √5 Site installation assessment (ex: alarm) √ √ √ √ √ 6 Light intensity √ √ √ √ 7 Noise level √ √ √ √ 8 Vibration level √ √ √ √8 Vibration level √ √ √ √9 Electrical safety testing to IEC 61010 √ √ √ √

10 Pressure retention / soap bubble √ √ √ 11 Microbiological personnel protection √ √ 12 Microbiological product protection √ √ 13 Microbiological cross-contamination √ √14 Microbiological performance envelope √ 15 Motor / blower performance √ 16 Drain spillage trough leakage √ 17 Resistance to overturning √17 Resistance to overturning √18 Resistance to distortion √ 19 Resistance to deflection √ 20 Resistance to tipping √ 21 Secondary inflow velocity correlation to DIM √

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y y22 Powder coating chemical resistance √ 23 Powder coating abrasive resistance √ 24 Cabinet design evaluation √

Bad Installation: Location• Cabinet is positioned too high

for comfort. Chair is too tall. F t t t hi flFeet not touching floor.

• No knee or leg room• Heat from lamp too close Heat from lamp too close

above cabinet can affect airflow sensor

• Too close to door - Door swings can impact cabinet’s ability to provideability to provide containment

• High traffic area – Peoplewalking in front of cabinet can disturb airflow

On-Site Field Tests

When / on what circumstances the field testing must be performed:- New installation

Relocation of cabinet- Relocation of cabinet- Airflow alarm cause not determined- When performance is suspectedWhen performance is suspected- At least annually

Recommended tests for laminar flow cabinets(air velocity, filter integrity, particle counting,

d k f t t t )secondary worker comfort tests)

On-Site Field Tests

NSF49 requirement:- Downflow velocityy- Inflow velocity- Airflow smoke patterns

HEPA/ULPA filter leak- HEPA/ULPA filter leak- Site installation assessment tests- Cabinet leak test (only for A1 cabinets)

NSF49 recommendations:- Electrical safety- Electrical safety - Light intensity- Noise level- Vibration

On-Site Field Tests

Si i ll iSite installation assessment test:

- Airflow Alarm

- Sash alarm

Bl i t l k (B2 l )- Blower interlock (B2 only)

- Airflow smoke pattern on exhaust duct

connection

Safer Alternative to BunsenManufacturer: Integra Bioscience

Product: Fireboy plus

Web page: www.integra-biosciences.com/ fireboy_plus_1_e.html

Comments:

The flame is automatically cut off by either a time delay motionby either a time delay, motion-proximity sensor, or when the footswitch is not activated.

Thi th t th bThis ensures that the burner operates for a minimum duration, therefore minimizes the effect the burner has on the cabinet airflowburner has on the cabinet airflow.

Safer Alternative to BunsenManufacturer: Waterpik

Product: TOUCH-O-MATIC® Bunsen Burner

Web page: http://professional.waterpik.com/ViewCoreProductNow.cfm?ProductID=76&Appl=4

Comments:

The flame is automatically reduced as long th t hi / h h d f ththe operator removes his / her hand from the platform. The flame will be increased when the operator places his / her hand on the platform (during this time the flame is largeplatform (during this time, the flame is large enough for sterilisation).

Therefore, the effect on cabinet airflow is i i i dminimized

Safer Alternative to BunsenManufacturer: Kendall Healthcare, Oxford brand

Product: BacticineratorProduct: Bacticinerator

Web page: http://www.kendallhq.com/catalog/ifu/Bacti1.pdf

Comments:

E bl t ili ti f i l tiEnables sterilization of inoculating loops and other small tools by electric process

This means there is no disruption to the cabinet airflow by thermal currents.

Safer Alternative to Bunsen

Product: Glass bead / hot bead electric sterilizers

M f t D t E Fi S i T lManufacturers: Dent-Eq, Fine Science Tool

http://www.dent-eq.com/glassbead.htmlhttp://www.finescience.com/fst/ResEqpGrp/18000-45.html

These sterilizers are compact enough to be placed inside LF and BSC. Since no flame is used, the thermal currents generated are minimal.

Using BSCs: Operation

- Work from “clean to dirty”- Biohazard collection bags should be placed

inside the cabinet instead of outside

Using BSCs: Operation

48Always think about safetyAgain…..if you are not careful…..

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Thank Youa ou

Any Questions ?

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