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Blood Culture

ISOLATOR* 1.5 TUBES
ISOLATOR* 10 TUBES

Intended use
The ISOLATOR 1.5 Tube is intended for the collection of small volume, paediatric blood samples to be used for isolation of micro-organisms. The blood sample is transferred from the tube directly to conventional agar growth media for the purpose of isolation and identification of micro-organisms.

Principles of the test
The ISOLATOR 1.5 Tube contains agents which lyse leucocytes and erythrocytes in blood, and block coagulation.
The specific agents used in the tube are:
Purified Saponin, an effective and rapid cell lysing agent, non-toxic to micro-organisms.
Polypropylene Glycol to block the foaming tendency of Saponin.
Sodium Polyanetholsulphonate (SPS) which acts as an anticoagulant, neutralises the bactericidal properties of blood and inhibits phagocytosis.

Reagents
Each ISOLATOR 1.5 Tube contains the following reagents in aqueous solution (content prior to sterilisation).

Polypropylene Glycol

8 mg/litre

Sodium Polyanetholsulphonate

9.6 g/litre

Purified Saponin

40 g/litre

The internal components of the tube are sterile.

Precautions
Used tubes, syringes and needles contain human body fluids. All materials should be handled as if they are capable of transmitting disease. Handle with appropriate care. Autoclave all used materials before discarding.
Tube reagents can cause transient eye irritation. In the event of contact with the eyes, flush with copious amounts of water and seek medical advice.
Care must be exercised to avoid injury when needles are used.
The ISOLATOR 1.5 Tube is not intended for the transportation of specimens through the mail.
FOR IN VITRO DIAGNOSTIC USE ONLY

Storage Instructions
The tubes can be stored between 2-40°C; room temperature (25°C) is recommended. Turbidity within the solution in the tube is normal. Tubes in use should be at 20-30°C to ensure proper mixing of the blood with the reagents at the time of collection.

Specimen Collection and Preparation
1
Open a needle cartridge. Twist to break the tamper-evident seal. Remove cap, exposing the rear end of the needle and threaded hub. Do not remove front needle cover.
2 Assemble needle and holder. Thread needle into holder until firmly seated. Take care not to touch the needle valve to the holder.
3 Use an appropriate disinfectant (e.g. 10% PVP iodine solution) for disinfecting the stopper of the ISOLATOR 1.5 Tube. Do not allow the iodine solution to pool on the stopper. Pooling could result in the introduction of disinfectant into the tube; this may interfere with the recovery of micro-organisms.
4 Allow the disinfectant to dry completely. Insert the stopper of the ISOLATOR 1.5 Tube into the holder. Advance the tube straight onto the needle but no further than the guideline on the holder.

Blood Drawing Procedure
1
Apply a tourniquet and select a venipuncture site. Loosen the tourniquet, double cleanse and disinfect the site with an appropriate agent (e.g. alcohol and PVP iodine). Allow the disinfectant to dry for at least one minute. Always collect the ISOLATOR specimen before collecting the other specimens to avoid contaminating the blood culture.
2 Reapply the tourniquet. Remove the needle cover. Perform venipuncture with the patient’s arm or other venipuncture site in a downward position. During venipuncture hold the tube/needle assembly so that the needle is elevated relative to the bottom of the tube.
During the collection procedure, do not permit contents of the tube to contact the stopper in order to avoid the possibility of backflow of reagents from the tube with the attendant possibility of adverse patient reaction.
Push the evacuated tube to the end of the tube holder or until blood flow is visible. When blood flows into the tube, remove the tourniquet.
3 Immediately remove the tube when fill is complete and flow has ceased (approximately 1.5 ml).
4 When sampling is completed, remove the needle/holder assembly with the last tube. Apply and hold a dry sterile compress to the venipuncture site. Elevate the arm.
5 Remove the tube from the needle/holder assembly. Immediately mix the collection tube to prevent coagulation and to initiate red blood cell lysis by gently inverting the tube four or five times. Incomplete mixing will result in blood clotting in the tube.
6 Handle and discard used needle in a suitable manner.
7 Label the specimen appropriately.

Alternative needle/syringe method for specimen collection
1
Assemble a sterile needle onto a 3 ml syringe or use sterile needle/syringe combinations. Loosen but do not remove the needle shield.
2 Use 10% PVP iodine solution for disinfecting the stopper of the tube.
3 Prepare the venipuncture site as previously described.
4 Remove needle shield and perform venipuncture. Collect 1.6 ml of blood.
5 Add 1.5 ml of the blood to the ISOLATOR 1.5 Tube by puncturing the stopper with the needle. Do not force the blood into the tube. This may cause the top to pop off the tube.
6 After removal of the needle from the tube, immediately mix the blood with the reagents in the tube by gently inverting four or five times.
7 Replace the protective cover onto the needle and discard in a suitable manner.

Specimen Processing
1
Specimens should be processed as soon as they are received in the laboratory. Immediate processing of the ISOLATOR 1.5 Tube results in faster isolation, minimises antimicrobial effects of blood, maximises the opportunity for polymicrobial isolation and may provide valuable quantitative information.
Specimens may be held in ISOLATOR 1.5 Tubes for up to 16 hours at room temperature without adverse effect on the recovery of micro-organisms. Specimens obtained from patients on antimicrobial chemotherapy should be processed immediately. Colony counts will not reflect the colony forming units per millilitre of blood if the specimen is held in the tube for more than 4 hours.
Do not refrigerate specimens collected in ISOLATOR 1.5 Tubes. The recovery of cold-sensitive organisms such as Neisseria gonorrhoeae may be dramatically decreased.
2 Vigorously mix the contents of the tube. A Vortex-type mixer (highest setting for 5-10 seconds) is recommended.
3 Disinfect the stopper with an appropriate disinfectant. Allow to dry for one minute.
4 Using a 3 ml syringe enter the upright tube at an angle so that the needle emerges from the bottom of the stopper between the wall of the tube and the side of the stopper. Tilt (do not invert) the tube to a horizontal position and collect the blood. Be sure to remove the blood that may have accumulated in the base of the stopper.
Expel any air in the syringe into the tube and remove the needle/syringe. Discard the tube.
5 Divide the lysate evenly among the primary isolation media, using a maximum of 0.35 ml per plate. Suggested culture media and growth conditions are shown in Table 1.
6 Keeping the lids of the plates as low as possible, position the needle over the medium (don’t touch the agar with the needle).
7 Dispense up to 0.35 ml of inoculum in a straight line across the surface of the plate, avoiding the edge of the agar. Discard the needle and syringe appropriately.
8 Raising the plate cover only far enough to admit a long sterile disposable or wire loop, cross-streak the inoculum starting at the top and proceeding to the bottom of the inoculum line (do not streak to edge of the agar). Rotate the plate 90 degrees and streak parallel to the original inoculum line. Rotate plate 45 degrees and streak a third time to ensure maximum distribution of the inoculum. Do not sterilise the loop between plates.
9 After plating and streaking, either appropriately discard the disposable loop, or sterilise the inoculating loop.
10 Plates should be placed under appropriate incubation conditions as soon as possible after inoculation to optimise the isolation of fastidious and anaerobic micro-organisms.
11 Incubate aerobic plates upright for the first 24 hours, and anaerobic plates upright for the first 48 hours. Thereafter incubate all plates inverted.
12 Examine all plates daily until discarded. Even when growth appears early, the plates should be reincubated to check for a second organism which may grow later. Plates should be examined with lids in place whenever possible. If a lid must be removed due to condensation or to better visualise colonies, do not remove the lid completely; raise it only high enough to examine the area in question.

Table 1.
Suggested culture media and growth conditions.

No Medium

Incubation conditions

Discard

1 Blood Agar

Anaerobic 35-37°C

6 days

1-4 Chocolate Agar

5% CO2, 35-37°C

4 days

1 Sab Dext Agar

Aerobic, 22-30°C

8 days

Plates should be pre-dried at least overnight at room temperature. This enhances absorption of the inoculum and reduces condensation on the plate lid.

Interpretation of Results
1
If a colony appears only within the area inoculated, it should be considered a significant positive culture regardless of genus or species. While colony counts in paediatric blood cultures are generally higher than those found in adults, it is not uncommon for the counts to be low (<10 cfu/ml) during episodes of bacteraemia associated with upper respiratory tract infections or occurring after antimicrobial therapy.
2 If colonies appear on both the inoculated area and outside the inoculated area, consider the colony within the inoculated area as a positive culture and the one outside as a contaminant.
3 If a colony appears only outside the inoculated area, it may be considered a plate contaminant.

Clinical Significance
The clinical significance of a micro-organism isolated from a patient’s blood should be determined by the Physician, taking into consideration the patient’s history, clinical status, repetitive cultures and other pertinent laboratory findings.

ISOLATOR* 10 TUBES

Intended use
The ISOLATOR 10 Tube is intended for the collection and concentration of micro-organisms from blood and other body fluids. The tube is used by clinical laboratories to concentrate micro-organisms before transfer to conventional agar media for isolation and identification.

Principles of the test
The ISOLATOR 10 Tube contains agents which lyse leucocytes and erythrocytes in blood, and block coagulation.
The specific agents used in the tube are:
Purified Saponin, an effective and rapid cell lysing agent, non-toxic to micro-organisms.
Polypropylene Glycol to block the foaming tendency of Saponin.
Sodium Polyanetholsulphonate (SPS) which acts as an anticoagulant, neutralises the bactericidal properties of blood and inhibits phagocytosis.

Reagents
Each ISOLATOR 10 Tube contains the following reagents in aqueous solution (content prior to sterilisation).

Polypropylene Glycol

8 mg/litre

Sodium Polyanetholsulphonate

15.3 g/litre

Purified Saponin

28 g/litre

The internal components of the tube are sterile.

Precautions
Used tubes, syringes and needles contain human body fluids. All materials should be handled as if they are capable of transmitting disease. Handle with appropriate care. Autoclave all used materials before discarding.
Tube reagents can cause transient eye irritation. In the event of contact with the eyes, flush with copious amounts of water and seek medical advice.
Care must be exercised to avoid injury when needles are used.
The ISOLATOR 10 Tube is not intended for the transportation of specimens through the mail.
FOR IN VITRO DIAGNOSTIC USE ONLY

Storage Instructions
The tubes can be stored between 4-40°C; room temperature (25°C) is recommended. Turbidity within the solution in the tube is normal. Tubes in use should be at 20-30°C to ensure proper mixing of the blood with the reagents at the time of collection.

Specimen Collection and Preparation
1
Open a needle cartridge. Twist to break the tamper-evident seal. Remove cap, exposing the rear end of the needle and threaded hub. Do not remove front needle cover.
2 Assemble needle and holder. Thread needle into holder until firmly seated. Take care not to touch the needle valve to the holder.
3 Use an appropriate disinfectant (e.g. 10% PVP iodine solution) for disinfecting the stopper of the ISOLATOR 10 Tube. Do not allow the iodine solution to pool on the stopper. Pooling could result in the introduction of disinfectant into the tube; this may interfere with the recovery of micro-organisms.
4 Allow the disinfectant to dry completely. Insert the stopper of the ISOLATOR 10 Tube into the holder. Advance the tube straight onto the needle but no further than the guideline on the holder.

Blood Drawing Procedure
1
Apply a tourniquet and select a venipuncture site. Loosen the tourniquet, double cleanse and disinfect the site with an appropriate agent (e.g. alcohol and PVP iodine). Allow the disinfectant to dry for at least one minute. Always collect the ISOLATOR specimen before collecting the other specimens to avoid contaminating the blood culture.
2 Reapply the tourniquet. Remove the needle cover. Perform venipuncture with the patient’s arm or other venipuncture site in a downward position. During venipuncture hold the tube/needle assembly so that the needle is elevated relative to the bottom of the tube.
During the collection procedure, do not permit contents of the tube to contact the stopper in order to avoid the possibility of backflow of reagents from the tube with the attendant possibility of adverse patient reaction.
Push the evacuated tube to the end of the tube holder or until blood flow is visible. When blood flows into the tube, remove the tourniquet.
3 Immediately remove the tube when fill is complete and flow has ceased (approximately 10 ml).
4 When sampling is completed, remove the needle/holder assembly with the last tube. Apply and hold a dry sterile compress to the venipuncture site. Elevate the arm.
5 Remove the tube from the needle/holder assembly. Immediately mix the collection tube to prevent coagulation and to initiate red blood cell lysis by gently inverting the tube four or five times. Incomplete mixing will result in blood clotting in the tube.
6 Handle and discard used needle in a suitable manner.
7 Label the specimen appropriately.

Alternative needle/syringe method for specimen collection
1
Assemble a sterile needle onto a 20 ml syringe or use sterile needle/syringe combinations. Loosen but do not remove the needle shield.
2 Use 10% PVP iodine solution for disinfecting the stopper of the tube.
3 Prepare the venipuncture site as previously described.
4 Remove needle shield and perform venipuncture. Collect 11 ml of blood.
5 Add 10 ml of the blood to the ISOLATOR 10 Tube by puncturing the stopper with the needle. Do not force the blood into the tube. This may cause the top to pop off the tube.
6 After removal of the needle from the tube, immediately mix the the blood with the reagents in the tube by gently inverting four or five times.
7 Replace the protective cover onto the needle and discard in a suitable manner.

Specimen Processing
1
Specimens should be processed as soon as they are received in the laboratory. Immediate processing of the ISOLATOR 10 Tube results in faster isolation, minimises antimicrobial effects of blood, maximises the opportunity for polymicrobial isolation and may provide valuable quantitative information.
Specimens may be held in ISOLATOR 10 Tubes for up to 16 hours at room temperature without adverse effect on the recovery of micro-organisms. Specimens obtained from patients on antimicrobial chemotherapy should be processed immediately. Colony counts will not reflect the colony forming units per millilitre of blood if the specimen is held in the tube for more than 4 hours.
Do not refrigerate specimens collected in ISOLATOR 10 Tubes. The recovery of cold-sensitive organisms such as Neisseria gonorrhoeae may be dramatically decreased.
2 Place the tube into an adaptor in a fixed angle rotor (35 degree) in a suitable centrifuge. Use only the unique adaptors intended for the ISOLATOR 10 Tubes. Centrifuge at 3000xg for 30 minutes. The centrifuge brake should not be used; this could disturb the concentrate and decrease recovery of micro-organisms. Be careful when removing the tube from the centrifuge to avoid mixing the supernatant fluid and the microbial concentrate.
3 The proper orientation of the adaptor is critical for the centrifugation of ISOLATOR 10 Tubes without breakage or leakage. Putting the adaptor in upside down or spinning it empty may cause it to deform. It is therefore important to properly orientate the adaptor in the rotor and to remove adaptors that are not being used before centrifugation. Periodic application of a light lubricant inside the adaptor will make tube insertion and removal easier.
4 Following centrifugation, carefully remove each ISOLATOR 10 Tube from its adaptor and place in the ISOSTAT rack. A slight clockwise twist will facilitate insertion of the tube into the rack. Be sure that tubes are firmly seated and vertically aligned, to avoid breakage while applying the cap.
5 Disinfect the stopper with an appropriate disinfectant. Do not allow the disinfectant to pool in the stopper cavity. Allow to dry for one minute.
6 Place the rack on the base of the ISOSTAT Press.
7 Remove an ISOSTAT Cap by peeling the back off the sterile pack. To avoid contamination, handle by the sides only. Do not touch the top of the cap or the tip of the internal spike.
Place a cap over the stopper of each ISOLATOR 10 Tube. If more than one tube is being processed, position caps on all tubes in the rack before proceeding to the next step.
8 Position a tube with its cap under the press head. Gently pull the handle of the press down as far as possible and hold down for five seconds. The spike will penetrate the stopper and the cap will be firmly seated on top of the tube. Return the handle to the upright position.
If more than one tube is being processed, rotate the rack to position the next tube. Press the cap onto this tube, and continue until caps have been pressed onto each tube in the rack. Carefully move the rack of tubes from the press to the work area.
9 Remove a supernatant pipette from the pack. To avoid contamination, handle pipettes by the bulb only. Do not touch the pipette stem.
10 Squeeze the bulb of the ISOSTAT supernatant pipette to collapse it and to provide a vacuum for supernatant withdrawal. Do this before inserting the stem of the pipette into the ISOLATOR 10 Tube.
Do not squeeze the pipette bulb after insertion of the pipette stem into the tube. Bubbling may disturb the microbial concentrate and result in the decreased recovery of micro-organisms. If this occurs the ISOLATOR 10 Tube should be centrifuged again. The ISOSTAT cap must be removed prior to centrifugation.
11 Carefully insert the stem of the supernatant pipette into the ISOLATOR 10 Tube through the membrane of the ISOSTAT cap while maintaining pressure on the bulb.
Insert the pipette into the tube as far as possible; the base of the bulb must rest on the cap.
Release the bulb and allow the supernatant fluid to be drawn into the pipette. Repeat this procedure with the remaining tubes in the rack, using a new pipette for each tube. Confirm that air has entered the pipettes indicating that all the supernatant fluid has been withdrawn.
12 When the supernatant fluid has been withdrawn from all ISOLATOR 10 Tubes, remove and discard the pipettes into an appropriate receptacle for contaminated waste.
13 Remove a concentrate pipette from the pack. To avoid contamination, handle pipettes by the bulb only. Do not touch the pipette stem.
14 Remove the first ISOLATOR 10 Tube from the rack and vigorously mix the contents for 5-10 seconds in order to achieve a homogeneous emulsion. A vortex-type mixer (highest setting) is recommended.
15 Squeeze the bulb of the concentrate pipette to collapse it and to provide a vacuum for concentrate withdrawal. Do this before inserting the stem of the pipette into the tube.
Carefully insert the stem of the concentrate pipette into the ISOLATOR 10 Tube through the membrane in the ISOSTAT cap while maintaining pressure on the bulb.
Insert the pipette into the tube so that the tip reaches the bottom. It may be necessary to manipulate both pipette and tube to properly orientate the pipette tip.
Gradually release pressure on the bulb and allow the concentrate to be drawn into the pipette. A slow controlled release of the bulb is necessary to achieve maximum recovery of concentrate.
16 Immediately remove the pipette and use it to distribute the concentrate evenly onto the selected agar media. Keeping the lids of the plates as low as possible, dispense the concentrate in a straight line across the surface of the agar. Keep the inoculum away from the edge of the plate. For suggested culture media and growth conditions see Table 2.
17 Using the tip of the concentrate pipette, streak through the concentrate, making about 15 to 20 passes perpendicular to the original inoculum line. Streak lines should be kept away from the edges of the plate.
18 Discard used pipettes and ISOLATOR 10 Tubes into an appropriate receptacle for contaminated waste.
19 Plates should be placed under appropriate incubation conditions as soon as possible after inoculation to optimise the isolation of fastidious and anaerobic micro-organisms.
20 Incubate aerobic plates upright for the first 24 hours, and anaerobic plates upright for the first 48 hours. Thereafter incubate all plates inverted.
21 Examine all plates daily until discarded. Even when growth appears early, the plates should be reincubated to check for a second organism which may grow later. Plates should be examined with lids in place whenever possible. If a lid must be removed due to condensation or to better visualise colonies, do not remove the lid completely; raise it only high enough to examine the area in question.

Table 2.
Suggested culture media and growth conditions.

No Medium

Incubation conditions

Discard

1 Blood Agar

Anaerobic 35-37°C

6 days

2 Chocolate Agar

5% CO2, 35-37°C

4 days

1 Sab Dext Agar

Aerobic, 22-30°C

8 days

Plates should be pre-dried at least overnight at room temperature. This enhances absorption of the inoculum and reduces condensation on the plate lid.

Interpretation of Results
1
If a colony appears only within the area inoculated, it should be considered a significant positive culture regardless of genus or species. In adults, bacteraemia at a level of one colony forming unit or less per millilitre of blood is common. Therefore the recovery of a single colony on the streak with the ISOLATOR system can be significant.
2 If colonies appear on both the inoculated area and outside the inoculated area, consider the colony within the inoculated area as a positive culture and the one outside as a contaminant.
3 If a colony appears only outside the inoculated area, it may be considered a plate contaminant.

Clinical Significance
The clinical significance of a micro-organism isolated from a patient’s blood should be determined by the physician, taking into consideration the patient’s history, clinical status, repetitive cultures and other pertinent laboratory findings.

* Isolator is a trademark of Carter-Wallace, Inc., New York, N.Y. 10105 USA

 
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