Appendix 



IV. BLOOD-FREEZE STORAGE OF BACTERIA 



Some stock cultures should be maintained for checking each new lot of media and conjugate 

 and for staining along with each batch of unknowns. 



These stock cultures can be obtained from positive clinical specimens or from a reference 

 laboratory. The stock cultures can be stored as described by Feeley et al. in this manual or by 

 the blood-freeze method described below. 



A. Supplies 



1 . Freeze tubes 



The freezing tubes should be made of Pyrex glass (e.g.. Corning #9820, size 

 6 X 50 mm). The Pyrex will stand the sudden change in temperature to which it 

 will be subjected. New tubes should be boiled in three changes of distilled water to 

 remove any deleterious substance. These tubes are shaken thoroughly to remove the 

 water, dried, plugged, and sterilized in a hot air oven. The cultures can be identified 

 by using Va- X 1-in strips of waterproof adhesive tape on which the name or number 

 of the culture has been typed or printed in India ink. 



2. Storage boxes 



Arrange for some type of storage boxes that will fit into the available freezer 

 space and in which the cultures can be systematically filed. Storage boxes can be 

 constructed of 3/8-in marine waterproof plywood, plastic, or cardboard. 



B. Procedure 



Transfer the stock cultures to charcoal yeast extract agar slants, and incubate them 

 at 35°C for 48-72 h. or to Feeley-Gorman agar slants, and incubate for 3-5 days at 35°C 

 in air plus 2.5% CO2 . 



1 . Freezing 



After growth is obtained, add 1 to 2 ml of sterile defibrinated rabbit or sheep 

 blood aseptically to each slant. Use capillary pipettes to suspend the growth in the 

 blood, then aspirate and deliver approximately 0.2 ml to each of the previously 

 numbered freezing tubes. If cotton plugged tubes are used, cut off excess cotton 

 and flame the Hp. Transfer the tubes to their respective numerical slots in the 

 storage box. Store at — 50°C. 



2. Recultivation 



Do not thaw and refreeze frozen suspensions. To recover the organisms, remove 

 one tube from the freezer, and thaw the contents rapidly by holding the tube 

 tightly in the palm of the hand or by placing the tube in a 35°C water bath. Draw 

 off the thawed suspension with a capillary pipette and transfer to a suitable culture 

 medium. 



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