U.S. patent number 3,904,482 [Application Number 05/440,969] was granted by the patent office on 1975-09-09 for method for the cultivation of microorganisms from body fluid.
This patent grant is currently assigned to Becton, Dickinson and Company. Invention is credited to Jack Judson Mehl.
United States Patent |
3,904,482 |
Mehl |
September 9, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Method for the cultivation of microorganisms from body fluid
Abstract
An improved apparatus and method for the collection, cultivation
and identification of microorganisms obtained from body fluids is
disclosed. The apparatus includes an evacuated tube containing a
culture medium, an inert gaseous atmosphere and a vent-cap
assembly. The tube containing the culture medium is fitted with a
stopper for introduction of body fluid by means of a cannula and
after growth of the organisms, transfer of the cultured medium is
completed for sub-culturing or identification procedures.
Inventors: |
Mehl; Jack Judson (Landing,
NJ) |
Assignee: |
Becton, Dickinson and Company
(East Rutherford, NJ)
|
Family
ID: |
46705088 |
Appl.
No.: |
05/440,969 |
Filed: |
February 11, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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342086 |
Mar 16, 1973 |
|
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215730 |
Jan 6, 1972 |
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Current U.S.
Class: |
435/34; 435/801;
600/577; 435/288.1; 435/304.1; 435/309.2; 435/309.1 |
Current CPC
Class: |
A61B
5/150587 (20130101); A61B 5/150755 (20130101); A61B
5/153 (20130101); A61B 5/150351 (20130101); A61B
5/150732 (20130101); B01L 3/5082 (20130101); A61B
5/150221 (20130101); A61B 5/150389 (20130101); A61B
5/150099 (20130101); A61B 5/150396 (20130101); A61B
5/15003 (20130101); A61B 5/150213 (20130101); A61B
5/150496 (20130101); A61B 5/150717 (20130101); A61B
5/154 (20130101); A61B 5/150259 (20130101); Y10S
435/801 (20130101) |
Current International
Class: |
C12M
1/28 (20060101); C12M 1/24 (20060101); A61B
5/15 (20060101); C12M 1/26 (20060101); C12B
001/00 (); C12B 001/24 () |
Field of
Search: |
;195/109,126,13.5R
;128/2F |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Kane, Dalsimer, Kane, Sullivan and
Kurucz
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a division of Ser. No. 342,086 filed Mar. 16,
1973 and is a continuation-in-part of application Ser. No. 215,730
filed on Jan. 6, 1972 now abandoned.
Claims
I claim:
1. The method of culturing microorganisms obtained from a specimen
of body fluid, which comprises:
a. providing an apparatus which comprises,
i. a container having at least one closed end and an open end;
ii. resilient closure means closing said open end;
iii. a nutrient medium partially filling said container and which
is suitable for culturing microorganisms contained in said
specimen;
iv. a gaseous atmosphere filling the space above said nutrient
medium; and
v. a venting assembly mounted on the container and having means for
maintaining fluid communication between the outside atmosphere and
the atmosphere within the container, said venting assembly
comprising a tubular member passing through said resilient closure
to provide fluid communication between said gaseous atmosphere
above the nutrient medium and the atmosphere outside of said
container, said venting assembly including a flexible body portion
having a closed end and an open end so that the closure means
fitted in the open end of the container is capped thereby; the
closed end of the body portion being formed with a hub having an
axial bore therethrough and said tubular member is a cannula
rigidly mounted in the axial bore between the ends of said bore and
wherein a shield is mounted on the hub to enclose the portion of
the cannula extending outwardly from the closed end of the body
portion, the shield being formed with a plurality of ribs which are
radially disposed around the inner surface thereof so that the
shield when mounted on the hub forms a plurality of passageways for
the circulation of microbial gases from the interior of the
container to the outside atmosphere and for diffusion of
atmospheric oxygen from the outside to the inside of the container,
said passageways being of such small cross section as to constitute
a microbial barrier;
b. inoculating said culture medium disposed in said container with
a specimen of body fluid to be cultured; and
incubating said inoculated culture medium under environmental
conditions of temperature, gaseous atmosphere and for a requisite
period of time to facilitate maximum growth of any organisms
present in said inoculated culture, whereby simultaneously any
aerobic microorganisms will grow and multiple in the top portion of
the medium and any anaerobic microorganisms will simultaneously
grow and multiply in the bottom portion of the culture medium while
any facultative and actively motile microorganisms will grow and
multiply throughout the medium.
2. The method of claim 1 wherein said gaseous atmosphere is
non-oxidizing.
3. The method of claim 1 including the step of transferring a
portion of said inoculated medium for subculturing to identify said
microorganisms being cultured.
4. The method of claim 1 wherein the specimen being cultured is
blood.
5. A method of culturing microorganisms from a specimen of body
fluid which comprises:
a. providing an apparatus which comprises,
i. a container having at least one closed end and an open end;
ii. resilient closure means closing said open end;
iii. a nutrient medium partially filling said container and which
is suitable for culturing microorganisms contained in said
specimen;
iv. a gaseous atmosphere filling the space above said nutrient
medium; and
v. a venting assembly mounted on the container and having means for
maintaining fluid communication between the outside atmosphere and
the atmosphere within the container; said venting assembly
comprising a tubular member passing through said resilient closure
to provide fluid communication between said gaseous atmosphere
above the nutrient medium and the atmosphere outside of said
container, said venting assembly including a flexible body portion
having a closed end and an open end so that the closure means
fitted in the open end of the container is capped thereby; the
closed end of the body portion being formed with a hub having an
axial bore therethrough and said tubular member is a cannula
rigidly mounted in the axial bore between the ends of said bore
wherein a shield is mounted on the hub to enclose the portion of
the cannula extending outwardly from the closed end of the body
portion, the shield being formed with a plurality of ribs which are
radially disposed around the inner surface thereof so that the
shield when mounted on the hub forms a plurality of passageways for
the circulation of microbial gases from the interior of the
container to the outside atmosphere and for diffusion of
atmospheric oxygen from the outside to the inside of the container,
said passageways being of such small cross section as to constitute
a microbial barrier; wherein a pressure responsive, gas impermeable
barrier is disposed in said cannula, said barrier being removable
by a pre-determined increase in gas pressure within said container,
generated by any microorganisms being cultured within said
container;
b. inoculating said culture medium disposed in said container with
a specimen of body fluid to be cultured; and
incubating said inoculated culture medium under environmental
conditions of temperature, gaseous atmosphere and for a requisite
period of time to facilitate maximum growth of any organisms
present in said inoculated culture, whereby simultaneously any
aerobic microorganisms will grow and multiply in the top portion of
the medium and any anaerobic microorganisms will simultaneously
grow and multiply in the bottom portion of the culture medium while
any facultative and actively motile microorganisms will grow and
multiply throughout the medium.
6. The method of claim 5 wherein the specimen being cultured is
blood.
Description
BACKGROUND OF THE INVENTION
There are various methods and assemblies employed for the culturing
of microorganisms, particularly microorganisms contained in body
fluids, such as blood. The culturing of blood is of great
importance since blood is normally considered sterile and any
microorganisms contained in blood are indicative of an active
infection. Present practice requires that whole blood be diluted so
that a general dilution of blood to media of from 1 to 8 to 1 to 10
is obtained for culturing purposes. Further, where infections in
the blood are caused by either aerobic or anaerobic microorganisms,
heretofore it has been necessary to culture these in separate
containers each having the proper medium and gaseous
atmosphere.
There are three popular techniques for the collection of blood or
other body fluids. The collection may be accomplished by the use of
a syringe and needle assembly to withdraw blood from a vein, then
inoculation of the blood into two or three sterile containers of
media. A second method is to employ an evacuated tube containing an
anti-coagulant when collecting blood and subsequently to transfer a
portion of the collected blood to the appropriate medium by needle
and syringe or by removal of the stopper from the evacuated tube
and measuring the appropriate amount of blood specimen by use of a
pipette. A third method for the collection of blood employs a piece
of flexible tubing having needle assemblies at each end thereof;
one of the needle assemblies adapted for use for entering a vein
and the other for directly inserting into an evacuated tube with or
without liquid and solid culture media disposed therein. However,
when using this technique it is necessary to clamp the tube to
prevent additional blood from entering the container so as to
maintain the proper blood-to-medium ratio. It also requires the use
of two types of evacuated tubes, one for aerobic and one for
anaerobic organisms.
One of the problems encountered while culturing anaerobic
microorganisms is the possibility of the stopper being forced from
the evacuated tube by the internal gas pressure and, in some
instances, where a screw cap is used and later is removed, the
stopper may explode from the opening thereby causing contamination
of the work area.
In all of the present methods employed strict aseptic techniques
must be maintained to avoid foreign bacteria contaminating the
sample to be cultured.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved
culturing system for the collection, cultivation and identification
of microorganisms obtained from body fluids. It is another object
of the invention to provide a method and apparatus for the
cultivation of either aerobic or anaerobic microorganisms or both
simultaneously in a single culturing assembly. A further object of
the invention is to provide a collection container which
automatically draws a controlled amount of body fluid, particularly
blood, so as to provide a predetermined ratio of body fluid to
medium without the necessity for measuring the quantity of body
fluid required. It is also an object of the invention to provide a
vent unit assembly adapted to maintain sterility of the stopper
after it has been sterilized and prevent contamination of the
culture medium during incubation. Also, an outlet means is provided
for gases generated by the growth of microorganisms and for the
inflow of oxygen to sustain the growth of the aerobic
microorganisms. The outlet and inflow means includes a total path
length and a plurality of passages of such small cross section that
they constitute a microorganism trap or barrier. It is also an
object of the invention to provide a disposable system for
collecting and culturing microorganisms, which system is
inexpensive to manufacture and simple to use and maintain in
aseptic condition when collecting or transferring body fluids to
the culture medium.
It is an object of the invention to provide an assembly to
simultaneously culture aerobic and anaerobic microorganisms in
which the assembly includes means for controlling and limiting the
rate of oxygen diffusion through the culture medium.
In general, my invention provides an improved system for the
collection of a specimen directly into a tube containing a medium
suitable for the growth of microorganisms contained in the
specimen. Various media may be used, as preferred, including agents
which change color, thereby identifying certain classes of
microorganisms. The system comprises means for automatically
collecting the proper proportion of sample so that a predetermined
sample-to-medium ratio is obtained. Means are also provided to
effect a transfer of the sample to the medium under aseptic
conditions without special equipment or procedures being employed.
Means for maintaining a proper atmosphere within the sample tube
for the growth of either aerobic or anaerobic microorganisms in a
single culturing container is also provided.
My improved method employs a venipuncture assembly for collecting
blood from a vein such as an assembly employing an evacuated tube
and a holder or barrel fitted with a sample needle. Such an
assembly is disclosed in U.S. Pat. No. 2,450,641 and the multiple
sample needle is disclosed in U.S. Pat. Nos. 3,469,572 and
3,494,352. An evacuated tube containing a desired culture medium is
employed to draw the blood through the sample needle. The evacuated
tube is provided with a sufficiently reduced pressure to collect a
predetermined amount of blood therein and is also provided with an
inert atmosphere such as carbon dioxide or nitrogen. The tube
stopper is sterilized and then a venting assembly is mounted on the
collection tube so as to maintain a sterile field around the
stopper during incubation. The venting unit comprises a barrel
portion having a diameter slightly larger than the sample tube
stopper and a needle is mounted in the closed end of the barrel.
One pointed end of the needle extends into the barrel portion a
distance sufficient to penetrate the stopper so as to provide
communication between the interior of the tube and the atmosphere.
The other end of the needle extends away from the stopper and is
fitted with a venting shield including minute passageways so that
atmospheric air can enter the culture tube while gases generated
from the growth of microorganisms may flow out of the tube.
After the culture tube fitted with the venting unit has been placed
in the proper atmosphere and thermal environment such as an
incubator at 35.degree.C. and microorganism growth is evident a
sample of the culture medium may be taken for subculture purposes
and for further growth and subsequent identification. The culture
assembly is so arranged that aerobic organisms will grow at or near
the top surface of the culture medium. The anaerobic organisms will
grow at or near the bottom of the culture tube, while facultative
and actively motile organisms may be found throughout the culture
medium.
Samples of the culture medium are removed from the culture tube
employing aseptic techniques to avoid outside contamination.
Various procedures may be employed for removal of culture medium.
The stopper may be removed together with the venting unit intact.
Then, a sample from the topmost portion of the culture medium is
collected either by use of a sterile pipette or a sterile syringe
and needle. The sample is transferred to a sub-culture assembly
such as a Petri dish containing the proper solid culture medium for
identification of the suspected aerobic organisms.
A second sample may be taken from the culture tube by insertion of
a sterile pipette or sterile needle and syringe assembly so that
the tip thereof is disposed near the bottom of the tube. The sample
to be cultured is transferred to a sub-culture assembly as before.
A third sample may be taken from the middle of the culture by the
technique described above. The sub-cultures are cultured in the
proper thermal environment and in the proper gaseous atmosphere,
aerobic in the presence of atmospheric air and anaerobic in the
presence of either nitrogen or carbon dioxide or both or other
means generally known to the art. The facultative organisms will
grow in either the presence of oxygen or in the absence of
oxygen.
The venting unit is so arranged that a flexible tube may be mounted
over the exterior end of the needle as shown in FIG. 10. The other
end of the flexible tube may be connected to an instrument or
alternative means for determining the presence of a particular gas
generated by the organisms.
DESCRIPTION OF THE DRAWING
FIG. 1 is an exploded view in elevation of the culturing assembly
of the invention ready for use.
FIG. 2 is a fragmentary view of the venting unit mounted in
position on the culturing tube shown in FIG. 1.
FIG. 3 is an enlarged fragmentary sectional view taken along the
lines 3--3 of FIG. 2.
FIG. 4 is a sectional view in elevation of the venting unit and the
stopper removed from the culture tube of FIG. 2.
FIG. 5 is an elevational view of the culture tube in a holder shown
in situ on a patient's arm and ready to receive a predetermined
volume of blood.
FIG. 6 is an elevational view showing the culture tube being
removed from the holder in situ after the sample of blood is
obtained.
FIGS. 7 through 9 illustrate various procedures for removing
culture samples from the culture tube.
FIG. 10 is a side elevation showing how gases generated by the
microorganisms being cultured may be conducted to a means for
analyzing these gases.
FIG. 11 is an enlarged fragmentary sectional view in elevation of
the venting unit of FIG. 4 having removable plug means mounted in
the cannula.
FIG. 12 is a side elevation, partly in section, of another form of
the container for the culture medium, with the venting unit mounted
in place.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The improved culture system for culturing microorganisms either
strictly aerobic or anaerobic is shown in FIG. 1 in exploded detail
and is referred to by the numeral 10. An evacuated tube or
container 12 is employed which may be formed of glass or a
transparent plastic material, and is non-toxic to and inert to the
culture medium and organisms to be cultured. A stopper 14 is
removably mounted in the open end 15 of the culture tube 12.
A venting unit 16 is mounted on tube 12 as illustrated in FIG. 2.
The venting unit includes a body portion 18 which is made of a
flexible plastic, for example, polyethylene. The body portion 18 is
cylindrically shaped having a diameter slightly larger than stopper
14 of culture tube 12 and is open at one end so that the stoppered
end of the tube will be capped by venting unit 16 as shown in FIG.
2. The other end of the body portion is closed by an end wall 19. A
hub or tip 20 is formed at the center of wall 19 and has an axial
bore 21 formed therethrough so that cannula 24 will be positioned
axially of body portion 18. Cannula 24 is rigidly mounted in bore
21 by any suitable means, such as by epoxy. Cannula 24 is mounted
in tip 20 between ends 25 and 26. End 25 is pointed to facilitate
piercing stopper 24 so that fluid communication is maintained
between the interior of tube 12 and the atmosphere.
As shown in FIG. 2 the venting unit is mounted in position with the
pointed end 25 of the cannula 24 extending through the stopper 14
so that the pointed end 25 is spaced from the top of the culture
medium 30. When the assembly 10 is placed in the proper thermal
environment an exchange of atmosphere is accomplished by cannula 24
by passing gases contained in the space above the culture medium 30
through cannula 24 to the atmosphere and passing the proper
atmosphere into the space above the culture media.
Venting unit 16 also includes a plastic shield 28 which is formed
having internal ridges 29, as shown in FIG. 3, disposed around the
interior thereof so that when it is removably mounted on top 20,
the ridges contact the outer surface of tip 20 to form passageways
31 for conducting the atmosphere contained within the culture unit
through the cannula 24 and subsequently may be expelled to the
outside atmosphere. At the same time, the outside atmosphere will
pass through the passageways 31 between ridges 29, and through
cannula 24, so that the aerobic microorganisms contained in the
culture unit will grow at an optimum rate.
In FIG. 11 which illustrates another form of the venting unit, the
passageway through cannula 24 may be plugged initially with a
viscous material 52 such as a mixture of petroleum jelly and
paraffin. This may be used when one desires that all oxygen be
excluded from the medium initially when culturing anaerobic
microorganisms. Then, as the gas pressure generated by these
microorganisms, for example, CO.sub.2, methane, or gas other than
oxygen increases, the plug 52 of viscous material is pushed out of
cannula 24, thereby establishing an open continuous passageway from
the surface of the medium to the outside atmosphere, and thereby
eliminating any possibility of gas pressure blowing out the
stopper. At this advanced stage of growth, diffusion of atmospheric
oxygen through the passageways to the surface of the medium would
have no deleterious or inhibiting effect on the growth of the
anaerobes.
In FIG. 4 the venting unit 16 is depicted illustrating one of the
techniques employed to remove stopper 14 from culture tube 12 to
obtain a sample of culture medium while maintaining aseptic
conditions. Body 18 of the venting unit 16 is squeezed and by
pulling upwardly the stopper is removed from tube 12. Thus, as
illustrated, stopper 14 is shown pierced by cannula 24 with the
pointed bevelled end 25 extending therethrough.
FIG. 7 illustrates the culture tube 12 with stopper 14 removed. A
pipette 40 fitted with a resilient bulb 42 at its outer end, is
disposed into the tube so that the lower pointed end of the pipette
is immersed in the culture medium. Any desired amount of inoculated
culture medium may be removed for sub-culturing purposes.
FIG. 8 illustrates still another method of removing a sample from
culture tube 12 for sub-culturing purposes. The venting unit 16 is
removed and a sterile syringe 44 and needle assembly is used to
withdraw culture medium 30 from tube 12 by piercing stopper 14 with
the needle 45. The needle is of sufficient length so that its
pointed bevelled end 46 is immersed in the culture medium.
In FIG. 9 still another method for removel of culture medium 30 is
illustrated. A micro pipette having cylindrical body 47 with a
diameter sufficient to be slidably disposed within cannula 24 of
the venting unit 16 is employed. The micro pipette is illustrated
having a flexible bulb 48 mounted on the cylindrical body. It
should be understood that any other means for withdrawing the
sample may be employed, for example a hypodermic syringe with a
long needle inserted through the cannula 24. After the sample is
removed the shield 28 is replaced over tip 20 to maintain aseptic
conditions.
FIG. 10 shows another use of the invention herein. The venting unit
16 is shown with the shield 28 removed. A flexible tube 50 is
coupled to the outer end 26 of cannula 24 so that the gases
contained within the culture tube 12 will be conducted through
flexible tube 50 and analyzed by any suitable means so that the
presence of a gas peculiar to certain types of organisms can be
detected. Also, the volume of gas being generated by the organisms
will be indicative of the growth rate of the culture within the
tube.
In the embodiment illustrated in FIG. 12 container 55 may be shaped
approximately as shown. Tubular neck portion 54 is approximately
the size and shape of the upper portion of container 12, so as to
cooperate with the venting unit 16, in the manner as previously
described and shown in FIG. 4. The primary advantage for having the
container 55 in the form illustrated in FIG. 12 is to provide for a
much larger blood sample. If only a few of a given type of
microorganism are present in the blood stream, statistically there
is a much better chance to detecting this type of microorganism if
the size of the blood sample can be substantially increased while
maintaining the 1 to 8 to 1 to 10 dilution ratio for culturing.
Another advantage for having container 55 as illustrated in FIG. 12
is to provide a base which will permit container 55 to stand
upright without having to be placed in a tube rack or holder.
It is contemplated when employing the invention herein that any
suitable culture medium may be used to culture microorganisms that
may be contained in body fluids. An example of a culturing medium
that may be used is Supplemented Peptone Broth blood culture
medium, sold by Becton, Dickinson and Company. Many culture mediums
are available commercially for culturing specific microorganisms
and many are modified for culturing several types of
microorganisms. The body fluids that may be employed for culturing
purposes when using the invention of the assembly herein are, for
example, synovial fluid, spinal fluid, peritoneal fluid, pleural
fluid, urine, blood, etc.
The reason the very desirable culturing of both aerobic and
anaerobic microorganisms simultaneously is possible with the
assembly of the invention herein is that design factors in
combination are utilized providing a very large diminishing oxygen
gradient. Even though oxygen at normal atmospheric partial pressure
is present outside the device of the invention, the design factors
thereof are so proportioned and combined that the amount of oxygen
diffusing toward the bottom of the column of the culture medium is
less than that which will adversely affect the growth and
multiplication of anaerobic microorganisms contained therein. The
significant design factors are the following stated in terms of
diminishing the rate of oxygen diffusion in respect to:
1. the total cross section of the microbial trap, i.e. passageways
31 formed between ribs 29, the smaller the cross section the less
the diffusion rate;
2. the longer the passageway 31 of the microbial trap the smaller
the diffusion;
3. the smaller the cross sectional diameter of the lumen of the
cannula 24 of the venting unit 16, the smaller the oxygen
diffusion;
4. the longer the lumen of the cannula 24 of the venting unit 16,
the smaller the oxygen diffusion;
5. the smaller the cross sectional area of the top of the column of
culture medium 30 the smaller the oxygen diffusion; and
6. the longer the column of culture medium 30 (from top to bottom)
the smaller the oxygen diffusion.
The ranges of dimensions of these factors in combination, in a
preferred form of the invention, are substantially as follows:
1. the total cross sectional area of the passageways 31 of the
microbial trap ranging from 0.000025 square inch to 0.002500 square
inch in combination with:
2. the length of the passageways 31 of the microbial trap ranging
from 0.200 inches to 0.625 inches, in combination with;
3. the diameter of the lumen of the cannula of the venting unit
ranging from 0.016 to 0.052 inches, in combination with;
4. the length of the lumen of the cannula of the venting unit
ranging from 0.50 to 2.000 inches, in combination with;
5. the total cross sectional area of the top of the column of
culture medium equivalent to a disc ranging from a diameter of 10
millimeters to 20 millimeters, in combination with;
6. a length of culture medium column ranging from 50 millimeters to
200 millimeters.
The following example is illustrative of the use of the invention
herein for culturing microorganisms which may be either strict
anaerobic or strict aerobes in the same culturing tube employing
the same culturing medium where the body fluid is blood.
EXAMPLE
A blood collecting assembly is employed, such as is disclosed in
U.S. Pat. Nos. 3,469,572 and 3,494,352, where a culturing specimen
is to be collected through a multiple sample needle along with
other blood specimens for hematology or other determinations in the
laboratory, utilizing the same venipuncture. Where only a culture
specimen is required the blood collecting assembly may be employed
such as is disclosed in U.S. Pat. No. 2,460,641. The blood
collecting assembly of these patents includes a blood sampling
needle "N", a holder "H" for mounting the needle therein and for
receiving and directing an evacuated tube into the assembly with
the needle. Initially a venipuncture is made in the usual manner. A
tube 16 mm in diameter containing a predetermined volume of gaseous
carbon dioxide and nitrogen maintained at a predetermined pressure
less than atmospheric pressure and containing a liquid blood
culturing medium the length of which is 120 mm is placed in the
holder "H". The culturing medium used is Supplemented Peptone Broth
blood culturing medium sold by Becton, Dickinson and Company. As
illustrated in FIG. 5, the tube 12 is moved into the holder "H" so
that the stopper 14 is penetrated by one end of the blood sampling
needle "N". Only a predetermined amount of blood will enter the
tube so that the blood-to-culture medium ratio ranges from between
1 to 8 to 1 to 10. The culturing tube is withdrawn from the holder
"H" as illustrated in FIG. 6 and is ready to be sent to the
laboratory. Before placing the tube in the proper thermal
environment the contents are thoroughly mixed by repeated inversion
and a venting unit is mounted on the culturing tube such that the
stopper is punctured by a venting cannula mounted in the venting
unit. The venting cannula has a length of 1.45 inches and a lumen
diameter of 0.040 inches. The venting unit is fitted with a venting
cap to maintain sterility and to provide a flow of gaseous
atmosphere into and microbial gaseous by-products out of the
culture tube. The venting cap provides a plurality of passageways
forming a microbial trap so that the diffusion of gaseous
atmosphere into the culture tube is maintained at a controlled
rate. The passageways have a cross sectional area of 0.00025 square
inches and a length of 0.625 inches. The rubber stopper of the
culturing tube is maintained in aseptic condition to prevent
outside contamination by bacteria. Where aerobic or both anaerobic
and aerobic culturing is to take place, the atmosphere within the
incubator is air, or air plus added carbon dioxide.
The culturing assembly is maintained in an upright position in an
incubator in the proper thermal environment and atmosphere for a
predetermined period such as for 12 to 24 hours. A sample is then
removed for sub-culturing or staining. Any of the techniques for
removing specimens from the culturing tube for sub-culturing as set
forth in FIGS. 7 through 9 may be employed.
Also, another method for removing a sample of incubated culture
medium and blood is to remove the venting unit; mix the cultured
medium and blood sample by repeated inversion; replace the venting
unit; remove the shield 28; and invert the container so that the
venting unit is downward and shake gently to expel one or more
drops of liquid through cannula 24 onto a receiving surface, such
as a sub-culture medium or slide. It should be remembered, however,
that any of the procedures followed must be under aseptic
conditions to prevent outside contamination.
It is obvious where body fluids other than blood are to be employed
the culturing medium may be modified accordingly particularly where
a specific type of organism may be suspected of being present. Many
modifications and changes may be made without departing from the
teachings herein and as defined in the appended claims.
* * * * *