STANDARD OPERATING PROCEDURE

Procedure: Freeze drying (Martin Christ machine)

School/Department: School of Molecular Bioscience
SOP prepared by: Gordon Stevenson, Nick Coleman, Leona Campbell
Version: SMB015.3

Section 1 - Personal Protective Equipment

1. Lab Coat or Lab Gown
2. Hair tied back if long
3. Proper enclosed footwear
4. Safety glasses or goggles
5. Nitrile or latex gloves
Section 2 – Potential Hazards + Safety precautions
1. Freeze dryer generates a high vacuum which can cause implosion of glass ampoules – this
poses a threat especially to eyes – wear safety glasses at all times when using the freeze
dryer.
2. Glass ampoules can break if mishandled, and the sharps generated pose a risk of cuts – such
cuts are especially dangerous if hazardous microbes are present in the ampoules. Handle
ampoules with care at all times, and know the risks and properties of the microbes you are
working on.
3. Freeze dryer generates low temperatures. Ampoules and other parts of the machine may cause
cold burns if touched to exposed skin. Wear gloves when handling these cold parts, or avoid
contact.
4. Freeze dryer is an electrical device and thus poses electrical shock hazard if misused or if faulty.
Inspect machine for any problems before use (e.g. problems with power supply, sparks, burning
smell etc). If any problems present, do not use machine, notify service centre and/or
manufacturer. Do not allow water to enter the internal parts of the machine. Keep flammables
away from the machine.
5. Do not use freeze drier for evaporating samples containing organic solvents!

Section 3 – Procedure (Specific for Martin Christ Alpha 1-2LD machine)

1. Do not use the freeze dryer unless an experienced user has shown you in detail how to use it.
The use of the propane torch to constrict the ampoules is hazardous, and should be done under
supervision the first time for new users.
2. Switch ON vacuum pump and allow it to operate (warm-up for 15 minutes) before starting any
procedures. The lever opening the pump to the chamber remains closed during this time.
CLOSED position is when the lever is pointing to the left and horizontal. OPEN is when pointing
upward and vertical. The drain valve knob (lower left-hand side of machine) on the chamber
should be screwed in to the CLOSED position
3. Turn ON freeze dryer machine (right-hand side). Press the left-hand (MODE) button to exit the
tutorial (unless you want to read it!). Press the MODE button on the left of the display. Three
options appear on screen (1) Freezing mode; (2) Warm up pump; (3) Main Drying. If the pump
has been pre-warmed for 15 mins then scroll to "Freezing" mode and press right hand button to
<ENTER>. The display will start counting Time and also show the condenser coil temperature,
which should go down to - 550C (this takes about 11 mins).
4. Once coil is chilled, you can (a) place your samples inside the chamber; (b) place your frozen
flask samples onto the ports around the chamber or (c) add your small ampoule samples onto
the "Christmas tree" if drying bacteria etc.
5. OPEN the lever on the vacuum pump to the vertical position. This will create a vacuum in the
chamber. Open the required ports to your flasks if using those. This "Freezing" mode phase can
be allowed to continue for 20-30 mins. to effect the initial freezing of small samples if they
haven't already been frozen, or to maintain a frozen larger sample in a frozen state until the
initial drying has occurred.
6. After the initial freezing phase, press the left-hand (MODE) button and a message will come up
saying "Continue with phase", press right hand button to <ENTER>. The menu will show the

Creation date: 25/10/2012 Next review due: 9/4/2016 Page 1 of 3

Last review date: 9/4/2014
 next phase - scroll to Main Drying and press <ENTER> again to confirm. Drying can be
continued until you are satisfied your sample is completely dry - see p21-22 of the manual for
advice.
7. At the end of the run, turn the lever on the pump off (return to left facing horizontal position)
Note - it is very important that the pump is not turned off whilst a vacuum still exists in the
chamber as it will suck the oil out of the pump!
8. Press the left-hand (MODE) button to bring up Standby mode - press <ENTER> to confirm. The
machine will turn off the vacuum-freezing coil. Open the drain valve (lower left-hand side of
machine) to vent the vacuum. A tube should be connected to allow the defrosted liquid to drain
any condensate into a beaker.

9. Procedure specific to Small samples

Pre-frozen small samples can be freeze dried in containers on shelves inside the chamber
although the shelves should be pre-cooled to prevent samples which are pre-frozen from melting
before they can be freeze dried.

10. Procedure specific to Large Sample

Larger liquid samples can be dried in flasks attached to the main head fitting. The freeze dryer
should be pre-prepared and cooled ready for operation. Pre-freeze the sample as a "shell" inside
your flask and then attach to a vacuum port. Open the port and allow for the sample to be dried.
Drying can be continued until you are satisfied your sample is completely dry - see p21-22 of the
manual for advice. With larger samples that may take longer to effect the initial Freeze/Dry
phase, you could consider freezing it at lower temperatures e.g. in liquid nitrogen or in a dry-
ice/ethanol bath.

11. Procedures using Ampoule Tree

As long as samples are very small inside ampoules i.e. 0.2 ml or less then pre-freezing is
probably unnecessary. Carefully, so as not to damage the base, set up the tree on top of
chamber and add your samples to the tree. Close off any un-used ports and then open the
vacuum pump lever to allow vacuum drying to commence.
12. When drying is complete, CAREFULLY use the propane burner to constrict the ampoules
completely. First turn gas on at cylinder, light flame, adjust air/gas ratio to obtain a hot, stable
flame. Keep hands clear of the flame and use flame to constrict ampoules. Avoid contact with
hot glass (this stays hot for a long time!). Turn off gas at cylinder when you are finished.

Section 4 – Disposal / Spills / Incidents

1. Spills of samples that are biohazards must be done according to the biohazard spills SOP – see
this document.
2. Incidents must be reported immediately to your supervisor and with 24 hours on the online
incident reporting system
Section 5 – Repairs / Certification / Validation
1. N/A
Section 6 – Relevant safety data sheets
1. All users should be familiar with the risks of the microorganisms or other biological samples used
in the machine – consult SDS if these are available
Section 7 - References
1. See also risk assessment and SOP for Biohazard Spill (SMB004), Working with Risk group 2
microorganisms (SMB026), and Gas cylinders (SMB017).

Creation date: 25/10/2012 Next review due: 9/4/2016 Page 2 of 3

Last review date: 9/4/2014
 SOP Consultation, Training and Approval
Print names and enter signatures and dates to certify that the persons named in this
section have been consulted/trained in relation to the development and implementation
of this Standard Operating Procedure. WHS Representative (WHS Committee) certifies
that consultation has taken place.

Position Name Signature Date

Supervisor
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student
employee / student

Name Authorising (Printed): DIANNE FISHER .......................................................

Signature: ..............................................................Date: 11/7/14 ............................

WHS Committee Representative Name (Printed): MARKUS HOFER ...................

Signature: ..............................................................Date: 18/7/14 ............................

Creation date: 25/10/2012 Next review due: 9/4/2016 Page 3 of 3

Last review date: 9/4/2014