APPENDIX D— 27 
Note: This should be done prior to steriliza- 
tion before use. 
4. Use needle-locking (Luer-Lokc® type) 
syringes only, and be sure that the needle Is 
locked securely Into the barrel. A disposable 
syringe-needle unit (where the needle Is an 
integral part of the unit) Is preferred. 
5. Wear surgical or other type rubber glove.s 
for all manipulations with needles and 
syringes. 
6. Fill the syringe caref>illy to minimize air 
bubbles and frothing of the inoculum. 
7. Expel excess air, liquid and bubbles from 
a syringe vertically into a cotton pledget 
moistened with the proper disinfectant, or 
into a small bottle of sterile cotton. 
8. Do not use the syringe to expel force- 
fully a stream of Infectious fluid Into an 
open vial or tube for the purpose of mixing. 
Mixing with a syringe Is condoned only if 
the tip of the needle is' held below the surface 
of the fluid In the tube. 
9. If syringes are fllled from test tubes, take 
care not to contaminate the hub of the 
needle, as this may result In transfer of 
Infectious material to the fingers. 
10. When removing a ssTlnge and needle 
from a rubber -stoppered bottle, wrap the 
needle and stopper In a cotton pledget mois- 
tened with the proper disinfectant. If there 
Is danger of the disinfectant contaminating 
sensitive experiments, a sterile dry pledget 
may be used and discarded Immediately into 
disinfectant solution. 
11. Inoculate animals with the hand "be- 
hind” the needle to avoid punctures. 
12. Be sure the animal Is properly restrained 
prior to the inoculation, and be on the alert 
for any unexpected movements of the ani- 
mal. 
13. Before and after injection of an animal, 
swab the site of Injection with a disinfectant. 
14. Discard syringes Into a pan of disin- 
fectant without removing the needle. The 
syringe first may be fllled with disinfectant 
by immersing the needle and slowly with- 
drawing the plunger, and finally removing 
the plunger and placing It separately Into 
the disinfectant. The filling action clears 
the needle and dilutes the contents of the 
syringe. Autoclave syringes and needles in the 
pan of disinfectant. 
15. Use separate pans of disinfectant for 
disposable and nondisposable syringes and 
needles to eliminate a sorting problem In the 
service area. 
16. Do not discard syringes and needles 
into pans containing pipettes or other glass- 
ware that must be sorted out from the 
syringes and needles. 
C. Opening Culture Plates, Tubes, Bottles, 
and Ampoules 
1. Plates, tubes and bottles of fungi may 
release spores In large numbers when opened. 
Such cultures should be manipulated In a 
Biological Safety Cabinet (6, 16). 
2. In the absence of definite accidents or 
obvious spillage, it Is not certain that open- 
ing of plates, tubes and bottles of other 
microorganisms has caused laboratory Infec- 
tion. However, It Is probable that among the 
highly Infective agents, some Infections have 
occurred by this means and are represented 
in the 80% for which no known act or acci- 
dent Is ascrlbable (3) . 
3. Water of syneresls In petri dish cultures 
Is usually Infected and forms a film between 
the rim and lid of the Inverted plate. Aerosols 
are dispersed when this film Is broken by 
opening the plate. Vented plastic petri dishes 
where the lid touches the rim at only three 
points are less likely to offer this hazard 
(8, 19). 
4. The risk may also be minimized by us- 
ing properly dried plates, but even these 
(when Incubated anaerobically) are likely to 
be wet after removal from an anaerobic Jar. 
Filter papers fitted Into the lids reduce, but 
do not prevent, dispersal. If plates are ob- 
viously wet they should be opened In the 
Biological Safety Cabinet (8) . 
5. Less obvious Is the release of aerosols 
when screw-capped bottles or plugged tubes 
are opened. This happens when a film of 
Infected' liquid which may collect between 
the rim and the liner is broken during re- 
moval of the closure (8) . 
6. Dried, infected culture material may 
also collect at or near the rim or neck of 
culture tubes and may be dispersed into the 
air when disturbed (18). Containers of dry 
powdered hazardous materials (e g.. Class 3 
fungal agents in the spore phase of growth) 
should be opened only in a Biological Safety 
Cabinet (6, 14) . 
7. When the neck of an ampoule contain- 
ing liquid Is broken after nicking with a file, 
the snapping action creates aerosols. The 
following methods have been recpmmended: 
(i) After nicking the ampoule with a file, 
wrap the ampoule in disinfectant- wetted 
cotton before breaking. Wear gloves (2). 
(il) The bottom of the ampoule should be 
held in several layers of tissue paper to pro- 
tect the hands, and a file mark made at the 
neck. A hot glass rod should be carefully ap- 
plied to the mark. The glass will crack, allow- 
ing air to enter the ainpoule and equalize the 
pressures. After a few seconds the ampoule 
should be wrapped In a few layers of tissue 
and broken along the crack. The tissues and 
ampoule neck can then be discarded into dis- 
infectant, and the contents of the ampoule 
removed with a syringe. If the ampoule con- 
tains dried cultures, about 0.5 cm= of broth 
should be added slowly to avoid blowing dried 
material out. The contents may then be 
mixed without bubbling and withdrawn into 
a culture tube (8) . 
(lii) The researcher uses an intense, but 
tiny, gas-oxygen flame and heats the tip of 
the hard glass ampoule until the expanding 
internal air pressure blows a bubble. After 
allowing this to cool, he breaks the bubble 
while holding It In a large low temperature 
flame; this immediately Incinerates any in- 
fectious dust which may come from the 
ampoule when the glass is broken (16). Pre- 
liminary practice with a simulant ampoule of 
the same type actually In use is necessary to 
develop a technique that will not cause ex- 
plosion of the ampoule. 
(Iv) A simple device has been recom- 
mended consisting of a sleeve of rubber tub- 
ing Into which the ampoule Is In.serted before 
It Is broken (17, 18). 
D. Centrifuging 
1. A safety centrifuge cabinet or safety 
centrifuge cup (3. 7, 8, 14, 22) may be \ised 
to house or safeguard all centrifuging of in- 
fectious substances. When bench type cen- 
trifuges are used In a Biological Safety Cabi- 
net, the glove panel should be in place with 
the glove ports covered. The centrifuge opera- 
tion creates air currents that may cause 
escape of agent from an open cabinet (2, 3, 
4, 13). 
2. In some situations. In the absence of 
O-rlng cap sealed trunnion cups, specimens 
can be enclosed in sealed plastic bags before 
centrifugation ( 12 ) . 
3. Before centrifuging, Inspect tubes for 
cracks. Inspect the Inside of the trunnion cup 
for rough walls caused by erosion or adhering 
matter, and carefully remove bits of glass 
from the rubber cushion (4, 10). 
4. A germicidal solution should be added 
between the tube and trunnion cup to disin- 
fect the materials In case of accldentlal 
breakage. This practice also provides an ex- 
cellent cushion against shocks that might 
otherwise break the tube (4, 10). 
6. Avoid decanting centrifuge tubes. If you 
must do so, afterwards wipe off the outer rim 
with, a disinfectant; otherwise the Infectious 
fluid will spin off as an aerosol (4, 10) . 
6. Avoid filling the tube to the point that 
the rim, cap or cotton plug ever becomes wet 
with culture (4, 10) . 
7. Screw caps, or caps which fit over the 
rim outside the centrifuge tube are safer than 
plug-in closures. Some fluid usually collects 
between a plug-ln closure and the rim of the 
tube. Even screw-capped bottles are not 
without risk, however; If the rim Is soiled 
.some fluid will escape down the outside of 
the tube. Screw-capped bottles may Jam In 
the bucket, and removing them Is hazardous. 
Propping such bottles higher In the bucket 
w'ith additional rubber buffers Is mechani- 
cally unsound (8) . 
8. Kitchen foU is often used to cap centri- 
fuge tubes. This creates more risk than the 
.screw cap. Foil caps often become detached 
in handling and centrifuging (8). 
9. The balancing of buckets is often mis- 
inaanged. Care must be taken to ensure that 
matched sets of trunnions, buckets and 
plastic inserts do not become mixed. If the 
components are not Inscribed with their 
weights by the manufacturer, colored stains 
can be applied to avoid confusion. When the 
tubes are balanced, the buckets, trunnions 
and inserts should be Included In the pro- 
cedure; and care must be taken to ensure 
tha.t the centers of gravity of the tubes are 
equidistant from the axis of rotation. To 
illustrate the Importance of this, two identi- 
cal tubes containing 20 g of mercury and 20 g 
of water respectively will balance perfectly on 
the scales; but their performance in motion 
Is totally different, leading to violent vibra- 
tion with all Its attendant hazards (5). 
10. Fill and open centrifuge tubes or trun- 
nion cups in a Biological Safety Cabinet (10) . 
E. High-Speed Centrifuges (22) 
1. In (high-speed centrifuges the bowl Is 
connected to a vacuum pump. If there Is a 
breakage or accidental dispersion of Infected 
particles the pump and the oil In It will be- 
come contaminated. A high efficiency filter 
should be placed between the centrifuge and 
the pump (8). 
2. High speed rotor heads are prone to 
metal fatigue, and where there Is a chance 
that they may be used on more than one 
machine each rotor sihould be accompanied 
by its own log book indicating the number 
of hours run at top or de-rated speeds. Fail- 
ure to observe this precaution can result In 
dangerous and expensive disintegration. Fre- 
quent Inspection, cleaning and drying are 
important to ensure absence of corrosion or 
other traumata which may lead to creeping 
cracks. Rubber O-rlngs and tube closures 
must be examined for deterioration and be 
kept lubricated with the material recom- 
mended by the makers. Where tubes of dif- 
ferent materials are provided (e.g., celluloid, 
polypropylene, stainless steel), care must be 
taken that the tube closures designed spe- 
cifically for the type of tube In use are em- 
ployed. These caps are often similar in ap- 
pearance, but are prone to leakage If ap- 
plied to tubes of the wrong material. When 
properly designed tubes and rotors are well 
mabitained and bandied, leaking should 
never occur (6). 
3. Cleaning and disinfection of tubes, ro- 
tors and other components requires consid- 
erable care. It Is unfortunate that no single 
process Is suitable for all Items, and the vari- 
ous manufacturers’ recommendations must 
be followed meticulously If fatigue, distor- 
tion and corrosion are to be avoided. This is 
not the place to catalogue recommended 
methods, but one less well appreciated fact Is 
worthy of mention. Celluloid (cellulose ni- 
trate) centrifuge tubes are not only highly 
Inflammable and prone to shrinkage with 
age and distortion on boiling, but can be- 
