Aseptic Technique –

sampling and testing

Laura B. Guardi

Aseptic technique – a definition

A set of specific practices and procedures

performed under carefully controlled

conditions with the goal of minimizing

contamination of the sample and/or test

environment

Aseptic does not mean sterile!

Aseptic technique – the challenge

There are more bacterial cells living

on and in the your body than there a

human cells that make up the body

~37 trillion cells in the human

body

~100 trillion microbial cells in and

on the human body - if each cell

was represented by a £5 note,

the resulting stack of money

would reach from the earth to the

moon and back 14 times.

Aseptic technique – the challenge

The presence of humans is intrinsic to the manufacture

and testing of pharmaceutical products

We are constantly shedding microbes from our bodies

into the surrounding environment > majority of EM

recoveries are human commensals

Huge potential for us to contaminate the test

sample/and or test environment > false result

Concern for microbial QC in sterile and non-sterile

GMP facilities

Staff Performing Sampling

Should not enter or work in the

live culture handling section of a

laboratory unless special

precautions are taken (e.g.

protective clothing, gloves, hand

washing on exit).

Staff supporting of aseptic

processing, should not work in

the vicinity of live culture

laboratory operations.

Aseptic Technique for Sampling

Wear sterile gloves, or

gloves sprayed with 70%

alcohol immediately before

sampling (except for TOC)

Do not touch the container

neck, lid or inside of the lid

Hold the lid of the container,

facing down. Do not place it

on a surface. Replace the lid

as soon as sampling is

complete

Additional Considerations for Sampling

TIME – time between sampling & testing/incubation

should be minimised

TEMPERATURE – (for storage) must be specified

CONTAINER – must be inert and not negatively

impact results

A Note on Water Sampling

Sampling should reflect actual use (flushing, use of

hoses tec.)

Analytical samples & TOC first

Bacterial samples (after sanitisation)

See ISPE “Good Practice Guide: Sampling Pharmaceutical Water,

Steam, and Process Gases for further details”

Segregation of Lab Activities

Clean / Aseptic Areas

EM samples handling & incubation

Reading of sterility & media fill samples

Dirty / Live Areas

Bioburden testing

Handling live cultures (e.g. cell banking, ID testing)

Ideally physical segregation

Alternatively organisational controls - protective

clothing, disinfection procedures, dedication of

equipment (e.g. BSCs, pipettes etc.)

Segregation of Activities

All testing should be performed

under controlled conditions.

Sterility tests - preferably be

carried out under an isolator to

prevent false-positives

Bioburden testing of

product/raw material/utilities –

in a BSC

Identification – on the bench?

Proper use of bio-safety cabinets

Designed to protect the user and the sample (not true

for LAF cabinets)

Ensure HEPA filter pressure is in the safe range

Disinfectant with 70% alcohol before/after use

Periodic disinfection with a sporicide (e.g. weekly,

including under the base plate, sporicide may require

rinsing off)

Equipment log book in place (usage and maintenance)

Do not disrupt the airflow (by overloading or blocking

the vents)

Only forearms and hands inside the cabinet

Proper use of Bunsen burners

The updraft/heat from the flame

creates a small clean area

within which you can work

Yellow safety flame (valve

closed) – used for lighting and

to indicate the burner is on

when not being used

Keep the valve fully open to

create the hottest (blue) flame –

the hottest part is the tip of the

inner blue flame

No gloves!

Proper use of Bunsen burners

To sterilise loops/spreaders

Heat all of the wire until

glowing red in the hottest part

of the flame before/after each

use

Angle downwards to prevent

any culture dripping down the

handle

Allow to cool before applying

to culture

Proper use of Bunsen burners

To flame the tops of glass vessels

Pass the neck of the vessel

back/forth through the hottest

part of the flame (1-2s) after

opening/before closing

Heating the mouth of the vessel

creates a convection current

away from the opening helping

to prevent contamination

Hold at a 45°angle

Handling plates and tubes

Always wear gloves (except

when using a Bunsen)

When working in a BSC gloves

should be sprayed with 70%

alcohol between tasks/before

each entry

Follow correct technique for

donning sterile gloves

Wear the right size

Handling plates and tubes

Plates should be opened away from

the user and only as much as

required

Caps and lids of bottles/tubes should

be held facing down, not placed on

the work surface, and replaced as

soon as possible.

Don’t lean or pass over open

plates/bottles/tubes

Don’t touch the mouth of the open

vessel

Handling pipettes

Automatic pipettes – always hold

them upright to prevent culture

from contaminating the barrel

Serological pipettes – peel apart

the wrapper at the top end and

place into the suction device

before removing the wrapper

Pipettes must not come into

contact with the work surfaces or

the outside of the sample

containers/petri dishes

Analyst Training and Qualification

Qualification in aseptic technique should be part of

basic training and performed before training in any test

procedures.

It should reflect actual tasks to be performed and may

include (multiple) performance of:

Transfer of a pure culture e.g. agar>agar,

agar>broth, broth>broth

Transfer of a sterile liquid (via loop and/or pipette)

Periodic re-qualification may be required for critical

tasks (e.g. sterility testing) or after an extended

absence (e.g maternity leave)

Analyst Training and Qualification

Qualification in aseptic technique should be part of

basic training and performed before training in any test

procedures.

It should reflect actual tasks to be performed and may

include performance of:

Transfer of a pure culture e.g. agar>agar,

agar>broth, broth>broth

Transfer of a sterile liquid (via loop and/or pipette)

Summary

Aseptic technique is a skill and learned only by

practice

USP <1117> - Microbiological Laboratory Best

Practices (non-mandatory guidance)