aseptic awareness training objectives: to further increase awareness about the microbial...

ASEPTIC AWARENESS Training

Objectives:

• To further increase awareness about the microbial contaminants found on our bodies and in the environment.

• To discuss behaviors to minimize contamination by personnel.

• To perform lab exercises to reinforce the concepts learned in class.


What is Aseptic Processing?

What processes are most vulnerable to contamination?


Types of Contamination


What are SPORES

Why are they a A MAJOR CHALLENGE!!!!

http://www.samedanltd.com/members/archives/PMPS/Spring2003/graphics/f1_p12.gif http://micro.med.harvard.edu/faculty/rudner.html


Sources of Contamination


CONTROLLING

contamination

• Facility design • Personnel AND visitors wear gowns appropriately • Personnel AND visitors behave appropriately


Facility Design: Clean Area Classification

FS209 Cleanroom classification

ISO 14644-1 Cleanroom classification ≥0.5um particles/m3

Microbiological Active Air Action Levels

(cfu/m3)

Microbiological Settling Plates Action Levels (diam.

90mm; cfu/4hours)100,000 8 3,520,000 100 5010,000 7 352,000 10 51000 6 35,200 7 3100 5 3,520 1 1


Facilities: General Cleanroom Design

• HEPA/ULPA filters on ceiling• Exhaust vents on floor• Drains in aseptic processing areas are inappropriate• Airlocks and interlocking doors to control air balance• Seamless and rounded floor to wall junctions• Readily accessible corners• Floors, walls, and ceilings constructed of smooth hard

surfaces that can be easily cleaned• Limited equipment, fixtures and personnel• Layout of equipment to optimize comfort and movement

of operators


Facilities: HEPA Filters

http://people.deas.harvard.edu/~jones/lab_arch/nano_facilities/hepa.gif

High Efficiency Particulate Air

Minimum particle collection efficiency: 99.97% for 0.3µm diameter particles.

Disposable

Filter made of pleated borosilicate glass microfiber


Class 10,000 cleanroom

http://www.americancleanrooms.com/am/photogallery_08.html Class 100 cleanroom

Facilities: Cleanroom Classification


Facilities: Cleanroom Classification

FS209 Cleanroom

classification

ISO 14644-1 Cleanroom

classification≥0.5um

particles/m3

Viable Microbes(cfu/m3)

Ave Airflow Velocity

(fpm)Air

changes/hr

100,000 8 3,520,000 100 5-10 5-48

10,000 7 352,000 10 10-15 60-90

1000 6 35,200 7 25-40 150-240

100 5 3,520 1 40-80 240-480


Facilities: Pressure Differentials

• Used to maintain airflow in the direction of higher cleanliness to adjacent less clean areas

• A minimum of 10-15 Pascals should be maintained between the aseptic area and an adjacent rooms with differing cleanroom classifications (doors open)


http://news.thomasnet.com/images/large/451/451402.jpg

Facilities: Air Lock

Permits the passage of objects and people into a cleanroom.

Consists of two airtight doors in series which do not open simultaneously.

Spray down materials with 70% IPA before placing in the airlock


• Fiber-shedding materials such as cardboard and paper– Cardboard packaging must be removed and items placed into

non-cardboard containers.

• Wood (i.e. wooden pallets)• Undesignated charts

Facilities: Material NOT permitted in a Cleanroom


Facilities: Cleaning

Water should be changed FREQUENTLY

1. Vacuum all accessible surfaces

3. Mop floors using a lint free polyester mops attached to stainless steel handles

2. Wipe surfaces with a cleaning solution


http://www.rit.edu/

http://www.imi.org.uk/

Avoid Cleanrooms When Sick!!!


• Avoid cleanrooms when ill• Frequent bathing and shampooing• Avoid getting sunburned• Avoid cosmetics such as face powder, hair sprays,

perfumes and aftershave• Clothing should be clean, nonfrayed and nonlinting• Avoid smoking

Personnel: Hygiene

ASEPTIC AWARENESS TrainingINCORRECT

3

21

4


FOREHEAD


http://www.engenderhealth.org/IP/surgical/sum4.html

Personnel: GlovesWhen are gloves worn?What compromises gloves?How often should gloves be examined and sanitized?


Qualifying Personnel

• Assess after gowning/gloving

• Microbiological surface sampling of several locations– Glove fingers– Facemask– Forearm– Chest– Periodic requalification is necessary


Biological Safety Cabinets

Small benchtop sized

class 100 clean area


Vertical Laminar Flow Area

http://www.terrauniversal.com/products/hoods/vlfstation.php


ISOPROPYL ALCOHOL

• Powerful disinfectant and antiseptic

• Mode of action: denatures proteins, dissolves lipids and can lead to cell membrane disintegration

• Effectively kills bacteria and fungi


Particles >= 0.3µm emitted per minute

PersonnelActivity

SnapSmock

MembraneCoverall

No Movement 100,000 10

Light Movement 500,000 50

Heavy Movement 1,000,000 100

Change Position 2,500,000 250

Slow Walk 5,000,000 500

Personnel: Behaviour

Austin Contamination IndexSource: Encyclopedia of Clean Rooms, Bio-Cleanrooms and Aseptic Areas, Dr. Philip Austin,

PE, 2000

Minimize movement: Work slowly and purposefully

Note: Light/heavy movement refer to partial body movements (motioning with arm, tapping toes, etc.).Change of position refers to whole body motion (standing up, sitting down, etc.).

ASEPTIC AWARENESS TrainingCORRECT

ASEPTIC AWARENESS TrainingINCORRECTASEPTIC AWARENESS Training


ENVIRONMENTAL MONITORING

“In aseptic processing, one of the most important laboratory controls is the environmental monitoring program”

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice,FDA, September 2004


Microbial Monitoring MethodsTouch or Contact plates

– RODAC Plates

Swabs

www.esa.int

Environmental Monitoring: Surface Monitoring

http://www.blood.co.uk/hospitals/services/Micro/Bact2.htm


Environmental Monitoring: Particulate Air Monitoring


Active Air MonitoringRCS Plus

Passive Air MonitoringSettling Plates

RCS: Rotor Centrifugal Sampler

Environmental Monitoring: Viable Microbial Air Monitoring


Environmental Monitoring: Trending Data

• Why is it important?• Evaluate the disinfection efficiency• Are certain microbes migrating into the aseptic processing area from

a lesser controller area?• Long and short term• Data by:

– Shift– Room – Shift– Operator – Product– Container type– Filling line– Sampling– Testing personnel


http://www.arches.uga.edu/~kristenc/cellwall.html

QC Micro: Identifying Microbes


Gram Stain



Metabolic Based Assays

Vitek



QC Micro: Identifying Microbes Metabolic Based Assays


Staphylococcus xylosus

Reduction of Tetrazolium Violet



QC Micro: Identifying Microbes Genotype Based Assay: MicroSeq

ASEPTIC AWARENESS TrainingQC Micro: Identifying Microbes

Genotype Based Assay: MicroSeq

PCR: Polymerase Chain Reaction


SEQUENCE THE DATA

QC Micro: Identifying Microbes Genotype Based Assay: MicroSeq


Sepsis: Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood Clotting Organ FailureDeath

http://www.emedicinehealth.com/images/4453/4453-4482-12996-21147.jpg


http://pathmicro.med.sc.edu/fox/lps.jpg

QC Micro: LAL Assay


Endotoxin MACROPHAGE

Tumor Necrosis Factor

Interleukins (1,6,8)

QC Micro: LAL Assay


ENDOTOXIN LIMIT FOR WFI IS 0.25EU/ml

QC Micro: LAL Assay


The Lysate

QC Micro: LAL Assay


Associates of Cape Cod, Inc.

Reading the Results: Gel-clot Test

Lysate clots when endotoxin is present.

Turbidimetric - increase in turbidity. Chromogenic - addition of a peptide that when cleaved during the assay turns a distinct color.

Endpoint - Measure turbidity/absorbance after a definitive time period.Kinetic - Measure rate of increased turbidity/absorbance.

Gel Clot – a clot forms and stays intact at the bottom of the assay tube when it is inverted

QC Micro: LAL Assay

TYPES of LAL ASSAYS


This product was funded by a grant awarded under the President’s High Growth Job Training Initiative as implemented by the U.S. Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee organization and does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental companies or organizations, their services, products, or resources are offered for informational purposes and should not be construed as an endorsement by the Department of Labor. This product is copyrighted by the institution that created it and is intended for individual organizational, non-commercial use only.

aseptic awareness training objectives: to further increase awareness about the microbial...

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cleanroom slide

aseptic areas

increase awareness

necessary slide

hygiene slide

airlock slide

cleanroom classification

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