PROCEDURES FOR PREPARATION OF PLASMA 
(These procedures supplement the "Regulations Governing 
Blood Donors, Blood Banks and Plasma Banks" and relate 
to Section 108 of the Sanitary Code of the City of New 
York, and were approved .by the Board of Health on 
December 14, 1943, January 1, 1944) 
1. The method employed for the removal of blood from the donor shall conform to the 
accepted standards of aseptic surgery, 
2. The apparatus used for the removal of the blood and the receiving unit shall be 
chemically clean and sterile, and shall contain as an anticoagulant either 
sodium citrate of known pyrogen-free quality dissolved in a pyrogen-free distilled 
water at least equal to Aqua Injectio, U,S,P, XII, or in a pyrogen-free pre- 
servative solution conforming to any of the following or equally satisfactory 
f ormulae: 
l) Sodium Citrate (dihydric) (3.2%) 2 parts 
Dextrose (anhydric) (§,4%) 3 11 
Blood 10 " 
Reference; "The Survival of Preserved Erythrocytes 
After Transfusion", 
Canadian Medical Assn, Journal, 1943, 
2) Sodium Citrate (dihydric) (3,2%) 2 parts 
Dextrose (anhydric) (5,4%) 13 " 
Blood 10 " 
Reference: "Studies on Preserved Human Blood. Various 
Factors Influencin'?; Hemolysis", Journal of 
the American Medical Association, 1940, 
Percent 
3) Sodium Citrate (dihydric) 0,43 
Dextrose (anhydric) 4,68 
Monabasic sodium phosphate 0,025 
Dibasic " " 0,25 
This Destrose Buffer Citrate solution is used 
in the followin ■ proportions: 
Dextrose Buffer 
Citrate Solution 3 parts 
Blood 2 " 
Reference: "The Use of Stored Blood and Plasma in 
Clinical Practice". Missouri State Medical 
Society. April 27-28. 1942, Grams per liter of solution 
4) Sodium Citrate (dihydric) 8^ 
Dextrose (anhydric) 18.56 
Sodium chloride 4.18 
This solution is used in the following 
proportions: 
Solution 1 part 
Blood 1 part 
Reference: "A New Method for the Preparation of Dilute 
Blood Plasma and the Operation of a Complete 
Transfusion Service." N,Y. State Journal of 
Medicine, January 1941, 
Of these four formulae, formula $1 is preferred. 
3, An acceptable serological test for syphilis shall be made in a qualified laborator 
on a specimen of blood taken from the donor at the time of bleeding and the blood 
shall not be used for the production of normal human plasma unless the result of 
t 
the test is negative* 
4. Each bleeding shall be drawn into its own receiving bottle, which shall be the 
same container used later for separating the plasma from the red cells. Pooling 
of whole blood before separating the plasma from the red cells is not permitted. 
It is advisable that within 1 hour and not later than 5 hours after bleeding the 
blood shall be placed in a cold chamber which shall have a temperature range of 
2° to 5° C, (freezing must be avoided). If transportation of the blood before 
removal of the red cells, from bleeding clinic to processing laboratory, becomes 
necessary it shall be transported in shipping cases provided with refrigeration 
sufficient so that (a) if the blood has alreadv been cooled as specified above, 
the temperature of each individual blood will not be in excess of 10° C. on 
arrival at the processing laboratory, or (b) if the blood is shipped before its 
temperature has been reduced to 2° to 5° C. the refrigeration capacity of the 
shipping case shall be such as to insure continued reduction- (the reduction 
capacity need not extend below 10° C.) of the temperature of each blood during 
the transportation interval. In either event the blood shall be placed in a cold 
chamber having a temperature range of 2° to 5° C,, immediately upon arrival at 
the processing laboratory and until required for further processing. The blood 
may be removed from the cold chamber during the interval required for freeing the plasma of cells and then shall be returned immediately to the cold chamber. The 
plasma shall be separated from the cells within 72 hours by centrifugation find 
within 7 days by sedimentation of the time of drawing the blood from the donor, 
if collected in a citrate solution, or within 14 days if collected in a pyrogen- 
free preservative solution in accordance with one of the formulae stated in 
paragraph 2. 
5, It is recommended that all transfers of plasma from one container to another be 
made in a closed system. A closed system is defined as such an apparatus which 
will permit nothing to be drawn into the system at any point except the liquid 
under transfer and the air required for replacement when negative or positive 
pressure is applied at the proper place. All air for replacement must first pass 
through a suitable antibacterial filter, except that when the transfer is being 
made in a closed "sterility room" equipped with mechanical or physical air- 
sterilizing devices of proven quality, and in operation when the transfer is being 
made, the air for replacement need not be passed through such a filter. 
PREPARATION OF THE PLASMA. POOL 
6, The supernatant plasma shall be drawn from the bleeding bottle after adequate 
centrifugation or sedimentation through a closed system into the pool bottle. 
Only pooled human plasma shall be placed in the final container and for this 
purpose a minimum of 8 individual plasmas shall constitute a pool. The completed 
pooled plasma shall contain not more than 50 mgm. of hemoglobin per 100 cc. (See 
appendix B for a method of determining hemoglobin.) 
7, It is recommended that the end of the closed system which is introduced into the 
plasma be fitted with a glass or metal tube of suitable caliber and that the shafi 
of this tube be enclosed in a suitable flexible sheath of rubber or other equiva- 
lent material. The upper end of the sheath is attached to the rubber tubing where 
it joins ■'with the above mentioned glass or metal tube and the lower end is fitted 
around the neck of a glass bell, the dimensions of which are such that the bell 
will not touch the lip of the bleeding bottle when the rim is resting on the 
shoulder of the bleeding bottle. When the protective sheath is ;fully extended. the above glass or metal tube should reach through the neck of the glass bell and 
slightly beyond. When in operation the glass bell is placed over the mouth of 
the plasma-containing bottle so as to rest on the shoulder of this bottle in 
order that the above glass or metal tube may be inserted into the plasma. The 
exposed end of the glass or metal tube should be protected by a suitable device 
so that the plasma will be drawn into the tube from the sides rather than from 
below. 
8. If plasma is to be processed only to the liquid state and dispensed in this form., 
or if it is intended to be processed to the frozen state without subsequent dry- 
ing, it is recommended there shall be added to the pool prior to taking the 
sterility sample a sufficient amount of sterile 50 per cent dextrose solution so 
as to give a 5 per cent concentration of dextrose in the finished plasma. 
Dextrose shall not be added to the plasma which is to be shell frozen and sub- 
sequently dried, 
9. Immediately after withdrawing the sample for the sterility test a sufficient 
amount of a suitable preservative may be added, except that phenol or a similar 
compound shall not be considered a suitable preservative. (At the present time 
the following preservatives and concentrations are considered suitable: For 
liquid dr frozen plasma phenyl mercuric borate 1:15,000 or sodium ethyl mercuri 
thiosalicylate (Merthiclate) 1:10,000, For dried plasma phenyl mercuric borate 
1:50,000 or sodium ethyl mercuri tbiosalleviate (Merthiolate) 1:35,000.) The 
use of a preservative shall not be relied upon for the maintenance of sterility 
and should not be considered as a substitute for meticulous care in the preven- 
tion of contamination of the plasma. 
TF8TS FOR STERILITY 
10. Tests for sterility are required on the plasma before the additirn of the preser- 
vative. At this point the tests may be made on the plasma from the individual 
bleedings on a sample taken from the plasma pool prior to the addition of the 
preservative. Sterility tests are also required on the finished product as con- 
tained in the finished dispensing unit, 
11, Test culture medium—The standard culture medium for making the sterility test if 
designated as "Fluid Thioglycollate Medium" and may be prepared according to tw* recognized formulae. (Appendix f) However, experience has shewn tv at of these 
the Linden formula is the simpler •and also more i©Gnomical and is therefore • 
recommended, (See appendix 0) 
12* Sterility tests on individual bleedings,—then plasma is to be processed to the 
liquid or frozen state, the sterility test may be made on the individual bleed- 
ings, The following procedure shall be followed: The plasma shall be drawn off 
into separate individual containers through a closed system and retained in 
these containers until the sterility test is completed. The test sample shall 
be withdrawn from these individual plasma containers. The amount of the test 
sample shall be not less than 5 cc, from each bleeding irrespective of the volume 
of the blood drawn. This amount shall be cultured at 37° G, for 7 days in one 
or more portions of thioglycollate medium. If evidence of contamination appears 
the plasma shall be discarded. 
13, IK/hen plasma is to be processed to the dried state sterility tests may be made on 
the individual bleedings. The following procedure shall be followed: The size 
of the test sample shall be not less than 2,0 cc. from each bleeding irrespective 
of the total volume of blood drawn. This amount shall be cultured at 37° C, for 
4R hours in one or mare porti ons of thioglycoilate medium. If evidence of con- 
tamination appears the plasma shall be discarded. Since pooling, filling of the 
final containers, and freezing is required within 72 hours (paragraph 26), pool- 
ing of the plasma must follow immediately after the 48 hour reading. However, 
plasma cultures should be inbabated at 37° 0, for the full 7 days and a record 
made as a check on the 48 hour readings. 
14. Sterility test on the pool.—When this method of testing for sterility is 
selected the procedure shall be as follows: For liquid or frozen plasma 2 
separate samples shall be withdrawn from the well mixed pool for the sterility 
test. The size of each sample shall be not less than 20 cc. for each liter of 
plasma in the pool under test. For the sterility test the entire volume of one 
of the two samples shall be planted in one or more portions of thioglycollate 
medium. If evidence of contamination appears the test shall be repeated with the second sample and if this tost also shows the presence of contamination the 
po»l shall be discarded. 
15, Plasma intended for drying.--Immediately after pooling 2 separate samples shall, 
be withdrawn from the well mixed pool for sterility testing. The size of each 
sample shall oe not less than 5 cc, for each liter of plasma in the pool under 
test. For the sterility test the entire volume of one of the two samples with- 
drawn shall bo planted in not less than 10 culture tubes containing adequate 
volumes of thioglycollate medium. In case contamination appears in 70 percent 
or more of the tubes on this retest the entire plasma pool shall be discarded. 
If contamination appears in less than 70 percent of the tubes on the retest the 
plasma may be processed to the final dried form and then shall be held for a 
special sterility test as described in paragraph 23, 
16, Sterility test on the final containers.—When liquid plasma is being processed 
a sterility test shall be made on the plasma in a final container, but this 
test shall not be made before the lapse of at least two weeks since filling the 
containers from the plasma pool. During this interval the plasma shall have 
been stored as required under paragraph 40, 
17, For the purpose of the sterility test a sample shall be withdrawn from the pool- 
reservoir at the tirrv’ of filling the final containers. This sample shall be a 
pool made up of the first 25 cc, flowing from the plasma-reservoir and the last 
25 cc,, provided not morn than 25 final containers are involved. If the plasma- 
reservoir is of larger volume, then additional samples shall be prepared on the 
basis of one additional sample for each 25 final containers or fraction, the 
sample to be withdrawn from the plasma-reservoir just prior to and immediately 
following each group of 25 final containers. The sample shall be drawn direct- 
ly into a separate, empty, final container which has been selected at random 
from the stock of empty sterile containers. 
18. After the lapse of the 2 week: storage period (paragraph 16) and after allowing 
the sample to come in contact with the entire inside surface of the container 
not less than a 20 cc, portion shall be withdrawn for planting for the sterility test. The dilution of the plasma in the culture medium shall be such that the 
preservative contained in the plasma will no longer prevent bacterial growth, 
(See appendix C) In ca-se contaminations appear in any tube planted, the test 
may be repeated from the unused portion of the sample, but no lot shall be passed 
until the final tosi shows no growth. 
19. However, if the lot fails because of growth in the above tests, a retest may lie 
made by sele cting at random one of the filled final containers represented by the 
original sample tested and by carrying out similar sterility tests. The absence 
of growth on this retest shall negate the previous unsatisfactory sterility tests 
and the final containers filled between t; e two portions af the sample showing 
contamination shall be released as satisfactory. 
€0, Frozen plasms,--Proceed exactly as directed for liquid plasma (paragraphs 17, 18 
end 19) except that the test sample shall be held for the 2 week period as direct- 
ed in paragraph IS but in the frozen s ta+e as directed in paragraph 29, along witl 
its cool mates, before thawing as directed in paragraph 44. The test sample 
should be held at room temperature after thawing for 24-48 hours before the cul- 
tures are taken. 
21, Dried plasma.—The plasma sample for sterility test shall be collected at the time 
of pooling in the same manner as directed for liquid plasma under paragraph 17, 
After collection of the sample it shall be subjected to shell freezing and drying 
under the same conditions of exposure as to time and temperature as is used for 
final containers. The dried sample shall then be restored to its original liquid 
■volume by the audition of diluent taken from a completed container of diluent as 
described under paragraph 36, 
22. Sampling and testing of this restored plasma shall be carried out exactly as de- 
scribed under paragraphs I7 and*18, except that not less than a 10 cc, portion 
shall oe tested instead of "not less than 20 cc," as required for liquid or 
frozpn plasma. 
23. However, if the entire pool fall.? into the special category of showing contamina- 
tion, but less than 70 per cent, on the basis of the pool sterility test as 
described in paragraph 14, a more rigid final sterility test must be made. For this test a final container of dried plasma shall be taken at random from 
the lot and dissolved in the prescribed way (paragraph 21), using the amount 
of diluent necessary to return the plasma to its original volume. Of this 
restored plasma not less than 2 samples of not less than 50 cc, each shall be 
cultured in suitable quantities of culture medium (see appendix C). If growth 
appears in either or both portions the test may be repeated, using for this 
test a new bottle of plasma. If evidence of contamination is confirmed the 
entire pool shall be discarded. 
THE FINAL CONTAINER 
24. Type to be used.--In the processing of plasma either to the liquid, frozen or 
dried state, the final container shall be filled directly from the pool-reservoir 
The final container shall be of good quality clear glass closed after filling 
with an air, moisture and bacteria impermeable seal am1 shall be constructed to 
withstand all ordinary handling and shipping hazards without danger of breaking 
or otherwise impairing the original state. The container shall be equipped 
so that the necessary connections for the injection of the plasma into the 
recipient may be attached easily, directly and aseptically to the closure of 
the plasma container. 
25, The final dried product shall contain not more than 1 percent of moisture at 
any time during the dating period (paragraph 4b). "Moisture" is defined as 
that weight which is lost when the dried plasma is exposed over PpOg in a 
vacuum chamber, (See appendix E for the method of determining moisture.) 
26* Method of filling,--Filling of the final containers when processing to liquid 
•r frozen plasma shall be done not later than 24 hours after completion of the 
sterility tests on the individual containers, or on the pool. Filling of the 
final containers, when processing to the dried state, shall be accomplished 
within such time after the blood is removed from the donor so that the plasma 
in the container can be brought to the frozen state within 72 hours of the 
removal #f the blood from the donor. 27. The entire pool shall be filled into containers during a single filling opera- 
tion and this shall be accomplished through a closed system (paragraph 5). The 
lumen of the filling tube leading to the final container shall be fitted with a 
filter adequate for the removal of all particles of such coarseness as to be 
dangerous for intravenous administration, (See appendix D for a description of 
a suitable filter.) 
28. The container into which the plasma is filled from the pool-reservoir shall also 
be the final container used for dispensing the finished product. At no subse- 
quent stage, as for example during the drying, restoration, or otherwise, shall 
the plasma be removed from this container except for injection into the re- 
cipient. 
29, Plasma to be processed to the frozen state shall be placed in the freezing 
compartment immediately after filling. In order to satisfactorily bring the 
plasma to the frozen state the properly stoppered containers are placed in a 
mechanical deep-freeze type of compartment or its equivalent. The temperature 
of the freezing compartment or device, and its freezing capacity, shall be such 
that each bottle of liquid plasma placed therein will be brought in its entirety 
to the frozen state within ('. hours# 
30, When plasma is to be processed to the dried state the drying may be begun 
immediately after shell freezing or the frozen plasma may be stored at a suffi- 
ciently low temperature (preferably -18° 0, or lower) to satisfactorily hold 
it in the frozen state to await the outcome of the sterility test or to await 
a more convenient time for carrying out the drying process. In either event, 
the final disposition of the entire lot shall depend upon the results of the 
tests for sterility referred to in paragraphs 21, 22 and 23, Drying shall be 
accompli shed by a method which is not deleterious to the plasma constituents 
and which will result in a readily soluble product. In general, the finished 
dried product is of a finer quality if the drying is completed relatively soon 
after being brought to the frozen state. 51, General instructions,--The final container for the liquid or frozen plasma shall 
bear a label showing the official name of the product, the amount of citrated 
plasma present, the amount and kind of preservative present, the amount of 
diluent added, if any, with its composition, the lot number and if the container 
label is the outside label it shall also show the expiration date and the name 
and address of the manufacturer* 
32. The final container of dried plasma shall bear a label indicating all of the 
points in paragraph 31 and in addition the amount of diluent needed to restore 
the dried material to its original liquid volume. In addition, the label shall 
carry a statement recommending that the plasma should be used promptly after 
restoration. 
33. It is recommended that either the label or an accompanying circular of instruc- 
tions contain a statement warning against the danger of overheating the liquid 
plasma before administration and that when safe warming facilities are not 
available, or when an emergency exists, it is safe to administer the plasma 
without preliminary warming, 
34. It is recommended that either the label or an accompanying circular of instruc- 
tions contain a warning as* to the danger of injecting the plasma intravenously 
without the use of a filter in t>e lumen of the tube leading from the plasma- 
reservoir to the recipient. (See appendix D.) 
35. It is recommended that a sterile and suitable apparatus for injecting the plasma 
into the recipient accompany each final container of plasma, 
3S. In the case «f dried plasma the label or an accompanying circular shall give 
full directions for re-dissolving the dried plasma as applicable to the par- 
ticular type of container used and full directions for getting the restored 
plasma from the container into the recipient in an aseptic manner. 
THE DILUENT FOP DRIED PLASMA. 
37, A suitable container holding the necessary amount of pyrogen-free, sterile, and 
•therwise suitable diluent containing 0,1* citric-acid shall accompany each container of dried plasma. This container shall be closed after filling with an 
air, moisture and bacteria impermeable seal. The quantity of diluent in the con- 
tainer shall be enough to restore the plasma to its original volume. The con- 
tainer shall be equipped so that the necessary connections for the transfer of 
the diluent to the dried plasma may be attached easily, directly, and aseptically, 
(See appendix A, pyrogen test.) 
38, However, the above requirement shall not be effective if specific requests for 
the dried plasma without the diluent are made bv hospitals, clinics, etc. ' When 
dried plasma is dispensed in this manner, the accompanying circular shall give, in 
addition to other directions, explicit directions and warnings as to the kind of 
diluent required, the quantity to be used, and any other essential information in 
order to safeguard the recipient. 
DATING AND STORAGE OF PLASMA 
39. Liquid plasma.—The expiration date for liouid plasma shall, not exceed 2 years 
from the date of manufacture. The date of manufacture is calculated as the date 
of bleeding the donor. 
40, Liquid plasma shall be stored as near as possible at a constant temperature within 
the range 15 to 30° C. A statement to this effect shall appear on the label. 
This temperature range does not necessarily insure the preservation for the entire 
dating period of all immune bodies which may bq present in normal plasma. If 
these 'substances are desired i4 is recommended that recourse be had to dried nor- 
mal plasma, either liquid or fried normal human serum, or to liquid plasma 
within 2 months of processing or thawing, 
41. Frozen plasma.--The expiration date shall not exceed 3 years from the date of 
manufacture when kept continuously in the frozen state as specified in paragraph 
45, The dat- of manufacture is calculated as the date of bleeding tke donor, 
42, Frozen plasma which has been thawed as in paragraph 44 and stored as described 
in paragraph 40 should be used as soon as possible after thawing. 43, Frozen plasma shall be stored continuously at minus 18° 0, or lower until needed. 
44, Frozen plasma shall be thawed in the following manner: The bottle of frozen 
plasma is placed immediately in a constant temperature water bath provided with 
circulating water, or its equivalent, and maintained at 37° C, As soon as all 
of the plasma is melted, and its temperature has reached the specified storage 
range for liquid plasma, the bottle is removed and stored until used as recom- 
mended for liquid plasma, 
45, It is recommended that frozen plasma be kept in the processing laboratory or 
other suitable depot and that shipments be made only after thawing as directed 
in paragraph 44, Provided, however, where either the processing laboratory or 
the purchaser can assure satisfactory shipment in the frozen state and eventual 
thawing as described in paragraph 44, this may be done. 
46, Dried plasma,--The expiration date for dried plasma shall not exceed 5 years from 
the date of manufacture provided the moisture at all times is less than 1 per- 
cent as determined by the method given in appendix E. The date of manufacture 
is calculated as the late*of bleeding the donor, 
47, Dried, plasma may be stored at the prevailing temperature provided this does not 
exceed that designated as excessive heat (over 49° C,) by U,S, Pharmacopoeia XII. 
However, it is recommended that whenever possible it should be kept in a cool 
place, A statement with regard to storage shall appear on the label. 
.APPENDIX A 
Pyrogen '• st (Paragraph 2) 
Test animal.—Use healthy rabbits weighing 1000 gm, or more which have been main- 
■i 
tained for at least 1 week on a uniform diet and have not lost weight. Test the 
thermometer to determine the time required to reach maximum temperature. If the 
animals have not been previously used for such tests, take four rectal temperature 
readings on each of the animals at 2-h«ur intervals 1 to 3 days before use. Insert 
the thermometer beyond the internal sphincter, and allow it to remain sufficient 
time t« reach maximum temperature, but in no case less than 90 seconds,before the reading is recorded. Discard those animals with a temperature in excess 39,8 C. 
On the day of the test take a control temperature reading before the injection of the 
test material. Animals may be used for the test and in subsequent tests after a rest 
period of not less than 2 days, provided the control temperature reading taken on the 
day of the test does not exceed 59,8° C. The reading taken on the tes4' day consti- 
tutes the normal temperature of the test animal from which a subsequent rise due to 
the injection of the test material is calculated. Keep test animals in individual 
cages protected from disturbances likely to cause excitement, Exercise particular 
care t# avoid exciting the animals on the day of taking the control temperatures and 
on the test day. Withhold food from, any animal used, beginning 1 hour before the 
first temperature reading, and oerirdt no food until the day's record is completed. 
Free access to water is allowed. Keep the animals at uniform temperatures (i 5° C«) 
during the control and test period. They should preferably be housed in quarters 
maintained at constant temperature and humidity. 
Conduct of the Test,--Warm the product to be tested to approximately 37° 0, and inject 
10 cc, per kgm. of rabbit, intravenously through the marginal ear vein within 15 
minutes subsequent to the control temperature reading on the day of the test. Record 
the temperature 1 hour subsequent to the injection and each hour thereafter until 
three recordings have been made. Syringes and needles used for these injections must 
have been treated to render them pyrogen-free and ihen sterilized. Not less than 5 
rabbits shall be used for each test and the test shall be considered positive if three 
or more of the 5 animals show an individual rise in temperature of 0o6° C, or more 
above the normal established for each of these animals. If only 1 or 2 of the 5 
animals show a positive response the test must be repeated on a second group of 5 
additional animals. The test shall be considered positive if 2 of the second group 
•f 5 animals show an ind.lvi.dual rise in temperature of 0,6° C, or more ab#ve the 
mormal established for these animals. APPENDIX B 
Methods for the Determination of Hemoglobin (Paragraph 6) 
1, The determination shall be considered sufficiently accurate if a colorimetric 
comparison be made with a standard prepared from hemolyzed blood from a person 
whose blood hemoglobin content has been determined previously in terms of grams 
of hemoglobin. The standard is prepared as a dilution of hemolyzed blood in 
plasma which is free of all visible trace of hemoglobin color. Hemolysis of the 
blood is effected by first diluting the blood 1 to 20 with distilled water and 
waiting until hemolysis is complete, 
2, The blood hemoglobin in the person serving as the source of hemoglobin supply 
shall be determined by a method at least as accurate as can be made by a Sahli 
apparatus equipped with permanent color standards. These color standards shall 
have been calibrated during the course of preparation by the manufacturer, using 
the Van Slyke oxygen capacity method, the Wong iron method, or a method recog- 
nized as the equivalent. (Ref,--Am, I, Clin. Path., v. 3, p. 85; v, 4, p,354) 
3, F«r an accurate quantitative determination of the hemoglobin content of the 
proled plasma either of the following methods may be employed, or one equally 
accurate; 
Arch, Int. Med., v, G4, p0 1271 (Dec,, 1959) 
J, Biol, Chem., v. 92, p. 589, 1931), 
APPENDIX C 
Fluid Thioglycollate Medium for the Sterility Test (Paragraph 11) 
1, This culture medium will be acceptable if prepared by either of the two methods 
given below, provided that if Method B or the designated dehydrated variations 
•f Methods A or B are used the growth promoting qualities for the contaminating 
•rganisms are no less than is the case if cultured in a medium prepared by 
Method A, Methods A and B are recognized as equally satisfactory but Method B 
is recommended as easier to prepare and more economical. The completely prepared media which is available is preferred since that product has already had cultur- 
al tests and is otherwi.se known to be satisfactory. 
2, a. Method A. - Fluid Thioglycollate Medium (Brewer); The broth base for this 
medium is prepared as follows (Changes in the order of compounding the broth or 
in the meat base are permitted provided the growth-promoting qualities remain 
at least equal to th»se obtained by the method as described herein); 
i 
Ground fresh beef (freed of fat) 500 grams 
Sodium chloride 5 " 
Dipotassium phosphate (X2HPO4) 2 ” 
Peptone 10 ” 
Distilled water 1000 cc. 
Put the ground meat into the distilled water, mix 
thoroughly and allow to stand at 5*C, for 24 hours. 
Collect the liquid by straining through cloth, then heat 
for 1 hour in streaming steam followed by 30 minutes at 
15 pounds pressure (121°C.) in the autoclave. Filter 
while hot through moistened filter paper and make up to 
the- original volume. Add the remaining ingredients and 
stir until solution is completed. Adjust the reaction 
with sodium hydroxide to such a point as experience 
shows will result in a pH of 7,5 +• 0,1, in the completed 
broth. Heat in streaming steam for 30 minutes, clear by 
filtration, fill into suitable containers and sterilize 
in the autoclave at 15 to 17 pounds pressure 0-21° to 12300.) 
for 20 minutes. 
To 1000 cc, of this stock broth add the following 
ingredients: 
Grams 
Dextrose (anhydrous) 5.0 
Sodium thioglycollate 1.0 
Agar (Less than 15% moisture by weight) 0,5 
Methylene blue (Use 1 cc, of a 1:500 solution) 0,002 
Add the agar to the cold broth, mix well and heat 
gradually to the boiling point* Cool to approximately 
80°C, and add the remaining ingredients. Stir until 
solution is completed and the ingredients are uniformly 
distributed. Adjust the reaction with sodium hydroxide 
to such a point as experience shows will result in a pH 
of 7,5, -f 0*1, in the completed and sterile medium. 
Distribute in final containers of the desired size and 
sterilize in the autoclave for IS to 20 minutes at 15 to 
17 pounds pressure (121°C, to 123°C,), 
2, b. A dehydrated medium consisting of the essential materials contained in the 
above formula is commercially available. Such, a medium is acceptable provided 
it meets the various requirements herein specified for a thioglycollate medium. 3. a. Method £ - Fluid Thioglycollate Medium (Linden); 
Peptone 20,0 grains 
Dextrose (anhydrous) 5,0 11 
Yeast extract 2,0 " 
Sodium thioglycollate 1,0 ” 
Sodium chloride 5.0 " 
Agar (Less than 15% moisture by weight) 0,5 11 
Dipotassium phosphate 2,5 " 
Distilled water 1000.0 cc. 
0,2/0 solution of Methylene blue (cert,) 1,0 " 
Dissolve the agar in half the volume of distilled water 
by boiling or heating in the Arnold, Dissolve the remaining 
ingredients, except the methylene blue, in the remaining 
water with the aid of heat. Now mix the two portions, adjust 
the reaction with sodium hydroxide to such a point as experi- 
ence shows will result in a pH of 7.5, -f- 0,1, in the completed 
and sterilized medium. Filter clear while hot and add the 
methylene blue solution. Distribute into final containers of 
the desired size and sterilize in the autoclave for 18-20 
minutes at 15-17 pounds pressure (121°C, to 123°C.), 
3. b, A medium may be prepared as a pre-mixed, dehydrated stock of the essential 
ingredients contained in method B, formula. Such a medium is acceptable provided 
it meets the various requirements herein specified for a thioglycollate medium 
as defined under Method A. 
4, CAUTION: Use distilled water throughout and avoid the addition of any calcium 
salt. 
Sodium thioglycollate is a white or faintly yellow crystalline powder 
with slight odor. Deterioration is indicated by a change of color to 
yellow or yellowish-brown, and is accompanied by a stronger odor which 
ultimately becomes definitely that of carbon disulfide. Sodium 
thioglycollate which shows deterioration must not be used. 
5, After removal of the final containers from the autoclave, cool to below 25°C, in 
order to set the agar. Store at 15°C, to 30°C., avoiding excessive light 
(storage at low temperatures increases absorption of atmospheric oxygen). If 
more than ZQffo of the uppermost portion of the medium has changed to a green 
color, it is not suitable for use. Under such circumstance one reheating in the 
Arnold or water bath is permissible in order to drive off the absorbed oxygen, 
A final reaction of pH 7,5 is interpreted to allow a p reaction of pH 0.1 at the time of manufacture. 
6, Each lot of medium shall be tested for its growth-promoting qualities by a trial 
run, using as the inoculum one or more delicately growing bacteria. 
At the end of the incubation period used for the sterility test less than 50% of 
the medium in each tube shall have changed from the color of the fresh medium to 
a green color. 
7, This medium permits the growth of both aerobic and anaerobic organisms in open 
containers. The type of container and the amount of medium used is dependent 
upon the use to which it is to be put. Tall, slender containers are recommended 
and in each instance a sufficient amount of medium shall be used so as to pro- 
vide ample dilution of any preservative contained in the substance to be tested 
for sterility. This medium is particularly effective in neutralizing the growth- 
inhibiting action of mercurials. In each instance the inoculum should be mixed 
thoroughly into the medium. 
8, When smears are to be made from cultures in this medium it is recommended that 
they be fixed with methyl alcohol instead of heat since this gives a clearer 
background. 
A Filter Adequate for Removal of Particulate Matter (Paragraph 2?) 
1, A filter adequate for the removal of all particles of such coarseness as to be 
dangerous for intravenous administration must be placed in the tube through 
which the plasma flows from the plasma-oool reservoir to the final container 
(paragraph 27), In addition, it must be placed in the lumen of the tube pro- 
vided with each unit of either finished liquid, frozen or dried plasma (35) for 
the purpose of transferring the plasma from the container to the blood stream 
of the recipient. Further, if such equipment is not provided the label on the 
final container (paragraphs 34 and 38), or the accompanying direction circular 
for administration of the plasma, must bear a warning that such filter must be 
used. 
APPENDIX D 2, A metal filter shall be considered adequate provided it is no less effective than 
' 
is obtained by a stainless steel filter when prepared as follows; The metal filter 
consists of a cylindrical tube 1 to lh inches in length, made of stainless steel 
screen or monel metal screen of 160-200 mesh. It should be 3-y to 4 mm. in diameter 
closed at one end. The other end inserts into a stainless steel metal ring 1 mm, 
in thickness. This is inserted in a glass tube with the metal ring uppermost 
towards the dispensing bottle. 
APPENDIX E 
A method for the Determination of Residual Moisture (Paragraph 25) 
1. The amount of residual moisture remaining in plasma which is labeled as a dried, 
•r desiccated, product shall contain not more than 1 per cent moisture when determined 
by the following method; "Expose a 1 to 2 gram sample of the plasma, evenly distribu- 
ted in a weighingbottle not less than 60 mm, in diameter, in a vacuum desiccator at 
less than 1 mm, pressure and over fresh phosphorus pentoxid at room temperature until 
the weight remains constant to the third decimal," In removing the sample to the 
weighing bottle, it is important to avoid unnecessary exposure of the dried plasma to 
the air, particularly if the moisture content of the atmosphere is relatively high*