Blood cultures and bone marrow aspirate

Blood culture is required when bacteremia (septicaemia) is suspected. Collect the blood as the temperature begins to rise. Always collect blood from peripheral vein except when ‘catheter-related bloodstream infection (CRBSI) is suspected, whereby both peripheral and catheter blood should be drawn concurrently with same volume. 

For bone marrow aspirate, 1-2 ml of aspirate is required and to be inoculated directly into the appropriate culture bottles.

Method of collection: 

1. Disinfect the culture bottle with 70% isopropyl alcohol. 

2. Palpate for the vein first. 

3. Before venipuncture, the skin must be carefully disinfected with 70% alcohol. 

4. Swab concentrically starting at the centre with skin disinfectant. Allow the skin disinfectant to dry before blood collection (do not palpate vein at this point). 

5. Perform venipuncture.

6. Remove the cap of culture bottles, wipe the top part with alcohol and allow drying.

7. Inoculate adequate volume of blood into each bottle.

8. Gently invert inoculated blood culture bottle 2 to 3 times.

9. Label each bottle with patient’s name and identification number. Label should not blocked the existing barcode (on the bottle).

10. After venipuncture remove disinfectant from the skin with alcohol.

Note: 

• Acute sepsis: 2-3 sets from separate sites, all within 10 minutes. 

• Endocarditis : 3 sets at 3 intervals at least 30 minutes apart.

• In the suspicion of catheter related bacteraemia, blood must be drawn from both the line and peripheral vein and submitted concurrently.

Blood Film for Malarial Parasite (BFMP) 

1. Clean new glass slides with absolute alcohol.

2. Select the third finger from the thumb (big toe can be used for infants). 

3. Clean the finger with 70% alcohol swab. 

4. Dry the finger with cotton towel. 

5. With a sterile lancet, puncture the ball of the finger using quick rolling action.

6. By applying gentle pressure to the finger, express the first drop of blood and wipe it away with dry cotton wool. 

How to prepare thick blood film?

Apply gentle pressure to the finger and collect a single drop of blood (20μL) on the surface of clean slide.

1. 1. 1. Using the corner of another glass slide as a spreader, quickly spread the blood to make an even, thick film. 

      2. The blood is spread in a circular motion with 3-6 movements and spread over 15mm diameter.

      3. Label the slide with patient’s registration number and date of collection with grease pencil. 

      4. Place the blood film in a slide tray to air dry at room temperature. 

How to prepare thin blood film? 

1. With another gentle pressure to the finger collect a small drop of blood onto a new slide about 5 mm away from the edge of the slide. 

2. Rest the blood slide on a firm, flat surface. 

3. Use another slide as a spreader. 

4. Touch the drop of blood with a spreader and allow the blood to run along its edge. 

5. Keep the spreader at an angle of 30-45˚ and in steady movement, firmly push the spreader forward to prepare a thin smear. 

6. Label the slide with patient’s registration number and date of collection with grease pencil.

7. Place the blood film in a slide tray to air dry at room temperature.

* Thick and thin blood film to be done on the same glass slide (side by side). 

Note: 

• For both thick and thin smear, a good smear is one that on drying still shows the hands of a watch placed beneath it or one can read newsprint through it. 

• Because of the potential for drug resistance in some of the Plasmodium species, particularly P. falciparum, it is important that every positive smear be assessed and the parasitemia reported. 

• In cases where the patient is hospitalized, repeat BFMP should be performed 24, 48, and 72 h after initiating therapy. 

• Blood in EDTA tube is required for Plasmodium knowlesii/malariae PCR confirmation. 

Blood Film for Microfilaria Parasite (BFFP) 

Preparation of smear 

1. Collect a big drop of blood (60μL)  by pricking a finger. Blood collection must be done after 9.00pm. 

2. Make an oval thick blood film on a clean glass slide. 

3. Dry it in a horizontal position, taking care to protect it from dust and pests. 

4. Send immediately to the laboratory after the smear dried off.

Cerebrospinal Fluid (CSF) 

A. Lumbar puncture 

1. Disinfect site with 2% iodine tincture. 

2. Insert needle with a stylet at L3-L4, L4-L5 or L5-S1 interspace. Upon reaching the subarachnoid space, remove the stylet and collect 1-2 ml of fluid into sterile bijou bottle. 

Note: always send the most turbid tube to the lab.

B. Ommaya reservoir fluid 

1. Clean the Ommaya reservoir site with antiseptic solution and alcohol. 

2. Remove Ommaya fluid via the Ommaya reservoir unit and place into bijou bottle. 

Note: 

• CSF for microscopic examination, cell count, Gram stain and Indian ink can be collected into a single screw capped sterile container (Bijou bottle). 

• Cryptococcal antigen test request require different sample in Bijou bottle.

C. Brain abscess and CNS biopsy sample 

1. Aspirate material or biopsy sample collected from lesions must be placed into thioglycollate broth and send immediately to the laboratory for anaerobic culture. 

2. Do not add formalin. 

Mycobacterium: Acid – fast bacilli stains and culture.

1. Acceptable specimens: Respiratory secretions, urine, CSF, body fluids, whole blood and tissue biopsies. 

2. Collect respiratory secretion, urine and tissue biopsies in sterile container.

3. Place whole blood into a commercial TB blood culture bottle.

4. Collect a minimum of 3 early morning sputum or urine specimens in successive 24 hours period for AFB stain. 

Sterile body fluid C&S (excluding CSF, urine and blood) 

1. Clean needle puncture site with skin disinfectant solution.

2. Aseptically perform percutaneous aspiration to obtain pleural, pericardial, peritoneal or synovial fluid.

3. Collect fluid into a sterile screw-cap container.

4. For anaerobic culture send sample in thioglycollate broth immediately to the lab.

Pus / swab / tissue 

1. Send pus if available, in a sterile screw-cap container.

2. Swab is an inferior substitute and should be avoided, if sent, in an Amies Transport Medium. 

3. Send all tissues for culture in sterile screw-cap container or thioglycolate broth (if suspect anaerobic organism). 

4. Do not add formalin to the specimen. 

Note: 

• A dry swab may fail to yield organisms in smear and culture. 

• Surface swabs of deeply infected lesions (e.g. sinus tract from osteomyelitis, pressure sores) usually grow surface contaminants like coliforms and pseudomonas.

Respiratory specimens

Upper Respiratory

1. Nasal swab: Submitted primarily for detection of MRSA carriers. 

1. Wet a sterile swab in sterile Normal Saline and insert the swab into the the nasal cavity until resistance is met at the level of turbinates (approximately 1 inch into the nose). 

2. Gently rotate the swab against the nasal mucosa. 

3. Repeat the process on the other side with the same swab. 

2. Nasopharyngeal swab / aspirate: This is done to isolate Corynebacterium diphteriae for the diagnosis of diphtheria.

1. 1. 1. Carefully insert a flexible wire calcium alginate-tipped swab through the nose into the posterior nasopharynx. 

      2. Rotate the swab (keep the swab near the septum and floor of the nose). 

      3. If Nasopharyngeal aspirate; Gently pass a sterile catheter through one nostril as far as the nasopharynx. 

      4. Attached a sterile syringe to the catheter and aspirate a specimen of mucous. 

      5. Put into sterile container and send immediately to the laboratory. 

3. Throat swab: Submitted primarily for the detection of Group A Streptococci, Corynebacterium diphteriae, and Neisseria gonorrhea. 

1. Depress tongue gently with tongue depressor. 

2. Extend sterile swab between the tonsillar pillars and behind the uvula (avoid touching the cheeks, tongue, uvula, or lips). 

3. Sweep the swab back and forth across the posterior pharynx, tonsillar areas, and any inflamed or ulcerated areas to obtain a sample. 

Lower respiratory tract 

1. Sputum 

1. Collect early morning specimen after rinsing the mouth and gargling with water. 

2. Instruct patient to cough deeply and expectorate sputum only and not saliva into the sterile screw-cap container. 

2. Tracheal aspirate/ bronchial washing / broncho alveolar lavage / lung aspirate / lung biopsy. 

1. Tracheostomy is followed by colonization within 24 hours of insertion of the tube. 

2. Results must be correlated with clinical findings such as fever or infiltrate on chest x-ray.

Stool Culture and Sensitivity 

1. 1. 1. Collect faeces into a sterile / clean wide-mouth screw-capped plastic container. 

      2. If the faeces is liquid, the container may be filled to one-third full (excessive amount will result in spillage when opened). 

Note: 

• For stool clearance culture in cases of typhoid and cholera, stool should be sent upon completion of therapy. 

Clostridium difficile toxin test 

1. 1. 1. Collect at least 3 to 5 ml of faeces. Faeces should be loose/watery to conform to the shape of the container. Formed faeces will be rejected unless the requisition indicates the patient may have pseudomembranous colitis. 

      2. Rectal swabs are not an acceptable alternative and will not be tested for toxin.

      3. Repeated test within 7 days will be rejected.

Stool Ova and Cysts

1. Collect faeces into a clean wide-mouth container with a tight-fitting lid to prevent accidental spillage and maintain moisture. 

2. The specimen should not be contaminated with water and urine. 

Urine

A. Midstream urine

1. Male

   1. 1. Cleanse the glands and penis with soapy and water. 

      2. Rinse area with wet gauze pads 

      3. While holding the foreskin retracted, begin voiding.

      4. After several milliliters has passed, collect a midstream portion without stopping the flow of urine. 

2. Female

   1. 1. Thoroughly cleanse the urethral area with soap and water.

      2. Rinse area with wet gauze.

      3. While holding the labia apart, begin voiding.

      4. After several milliliters has passed, collect a midstream portion without stopping the flow of urine. 

B. Suprapubic aspirate (SPA) 

Note: 

• SPA is useful in determining urinary infection in adults in which the results from routine procedures are equivocal but the diagnosis is critical. 

• Also, useful for paediatric patients when clean-catch urine is difficult to obtain. 

1. Before SPA, the patient should force fluids until bladder is full. 

2. Shave and disinfect the suprapubic skin overlying the urinary bladder. 

3. Aspirate urine from the bladder by using a needle aspiration technique. 

C. Catheter urine 

Catheters are placed in patients who are unable to pass urine 

1. Clean the catheter collection port with 70% alcohol wipe 

2. Using sterile technique, puncture the collection port with a needle attached to a syringe. DO NOT collect the urine from the bag. 

3. Aspirate urine and place it in sterile container. 

4. Send specimen immediately to the laboratory. 

Autopsy material 

Blood 

1. Aspirate 10ml of the right heart blood either through skin and chest wall or (through unopened heart) from right ventricle after removal of sternum into a set of blood culture broths or sterile tube. 

2. Avoid contamination with bacteria from the water faucet and with the enteric bacteria. A block of splenic tissue may be submitted in lieu of a blood culture. 

Tissue 

1. Best collected before the body is being handled at an earlier stage. Decontaminate the skin or sear surface of heart or other organ before inserting needle or cutting out tissue block. 

2. Collect the tissue and placed in sterile container or thioglycollate broth (if suspect anaerobic organism). Large piece is preferred (because aseptic collection is difficult). In the laboratory, 1 cm cube will be aseptically cut from the suspicious area including some normal tissue for processing.

Skin, nails and hair 

1. Clean cutaneous and scalp lesions with 70% alcohol prior to sampling as this will improve the chances of detecting fungus on microscopic examination, as well as reducing the likelihood of bacterial contamination of cultures. 

2. Prior cleaning is essential if ointments, creams or powders have been applied to the lesion. 

3. Skin, nails and hairs specimens should be collected into folded squares of paper (preferably black envelope) or directly onto agar plate. 

Skin 

1. Material should be collected from cutaneous lesions by scraping outwards from the margin of the lesions with the edge of a glass microscope slide or a blunt scalpel. 

Hair 

1. Specimen from the scalp should include hair roots, the contents of plugged follicles and skin scales. 

2. Hairs should be plucked from the scalp with forceps or the scalp is brushed with a plastic hairbrush and collected onto an agar plate. 

Nail 

1. Nail specimens should be taken from any discoloured, dystrophic or brittle parts of the nail. 

2. Specimen should be cut as far back as possible from the edge of the nail and should include the full thickness of the nail. 

Mouth 

1. Swabs from the buccal mucosa should be moistened with sterile water prior to taking the sample and sent in Amies transport media. 

Ear 

1. Scrapings of material from the ear canal are to be preferred, although swabs can also be used. 

Ocular specimens 

1. Material from patients with suspected fungal infection of the cornea (Keratomycosis) should be collected by scraping the ulcer. The entire base of the ulcer, as well as the edges should be scraped. Swabs are not suitable for sampling corneal lesions. 

2. The material is collected directly onto Sabouraud dextrose agar (SDA) plates for culture and to glass slide for microscopic examination.

Blood 

1. Blood culture for fungal is collected aseptically as in culture for aerobic bacterial culture but using the available commercial fungal bottle. 

2. The request for fungal culture should be indicated clearly on the request form and a total of two weeks incubation will be carried out. 

Cerebrospinal fluid 

1. CSF specimens (3-5mls) should be collected in a sterile Bijou bottle for microscopy and culture. 

Bone marrow 

1. This specimen is helpful for making the diagnosis in a number of deep fungal infection, including histoplasmosis and cryptococcosis. 

2. 3 – 5 ml of aspirated material should be collected and transferred into the commercial fungal culture bottle. 

Pus 

1. Pus from undrained subcutaneous abscesses or sinus tract should be collected into a sterile container with a sterile needle and syringe.

2. If grains are visible in the pus (as in mycetoma), these must be collected. 

3. In mycetoma, if the crusts at the opening of the sinus tracts are lifted, grains can often be found in the pus underneath.

Tissue 

1. 1. If possible, material should be obtained from both the middle and edge of the lesions.

   2. Small cutaneous, subcutaneous of mucosal lesions can often be excised completely.

   3. Tissue specimens should be placed in a sterile container without formalin.

Vagina

1. For vaginal infections, swabs should be taken from discharge in the vagina and from the lateral vaginal walls. 

2. Swabs to be sent to the laboratory in Amies transport media.

A. Blood collection, storage and transportation 

1. Draw 3 – 5 ml of blood into a plain tube with/without gel (No anticoagulants or preservative).

2. Clot at ambient temperature.

3. Despatch to the laboratory within 4 hours of collection for serum separation by centrifugation.

4. Outstation specimen; despatch the blood to the respective pathology laboratory for within 4 hours of collection.

5. The respective laboratory shall centrifuge the collected blood at 3,000 RPM for 10 minutes.

6. After centrifugation, separate serum from the cells and transfer to the plain test tube.

7. Screw the test tube tightly and seal with parafilm.

8. Pack serum properly with dry ice/ wet ice and send as soon as possible to lab via courier service or hospital transportation.

Note: 

• Haemolysed, icteric or lipaemic specimens invalidate certain tests. If such specimens are received, the samples will be rejected to ensure that results are of clinical value.

• Clear significant clinical history shall be written on the form.

• Repeated and unnecessary request will be rejected.

B. Other specimens' (Urine & CSF) special requirement for sample.

1. 1. 1. Urine specimens for Legionella Ag testing must be sent in sterile container and transport in 2-8oC.

      2. CSF VDRL  must be sent in a Bijou bottle. 

      3. Sample must be sent immediately to the lab within 24 hours of collection. 

A. Blood collection, storage and transportation for HIV Viral Load, HBV Viral Load, and HCV Viral Load 

1. Collect 6ml of blood into 2 tube of EDTA. Each tube 3 ml of blood.

2. Do not exceed the marked blood level on the EDTA tube.

3. Despatch the blood immediately to Queen Elizabeth Hospital Pathology Department or hospital / health-facilitated laboratories managing specimen delivery to Queen Elizabeth Hospital Pathology Department for plasma separation by centrifugation.

4. The respective laboratory shall centrifuge the collected blood within 4 hours after phlebotomy. 

5. Centrifuge the blood at 3,000 rpm for 10 minutes.

6. After centrifugation separate plasma from the cells and transfer to the plain test tube (polypropylene tube) DO NOT USE plain tube with CLOT ACTIVATOR.

7. Screw the test tube tightly and seal with Parafilm.

8. Separated plasma should be stored at 2 - 8⁰C at no more than 24 hours before delivery.

9. Pack serum properly with dry ice/ wet ice and send as soon as possible to lab via courier service or hospital transportation.

10.  Address the package to:

MAKMAL SEROLOGI/VIROLOGI

UNIT MIKROBIOLOGI 

JABATAN PATOLOGI HOSPITAL QUEEN ELIZABETH

HOSPITAL QUEEN ELIZABETH

KARUNG BERKUNCI N0. 2029

88586 KOTA KINABALU 

SABAH

Note: 

• Specialised test such as molecular test requires Specialist's name, signature and stamp.

• Clear significant clinical history shall be written on the form.

• Please use HQE Viral Load Request Form (download form) or Per-Pat 301 form.

• Repeated and unnecessary request will be rejected.

B. For COVID-19 molecular samples please refer “Polisi dan Prosedur Pengendalian Sample COVID-19 HQE/PATH/GP-03(2022)”