Specimen collection

Urines for routine microscopy culture and sensitivity

The microscopy method used in the laboratory uses an analyser to estimate the number of white and red blood cells, and the number of bacteria in urine. This helps to distinguish infection from contamination. If there is evidence of infection then a culture and sensitivity test will be performed.

The containers used for urine microscopy and culture are changing: 

BHNFT Boric Acid Urine Monovette – points to remember

SARSTEDT Boric Acid Instructions for Use

A midstream urine (MSU) or catheter specimen should be sent to the laboratory in a Sarstedt Monovette tube. These tubes contain boric acid so should be filled to the marked line. Otherwise, results may be unreliable and the sample may be rejected.  

Universal containers are no longer accepted for these tests. Urines received in inappropriate unsterile containers will not be processed.

Please note - red topped universals containing boric acid must be at least half full (10mls) of urine. Samples received containing less than this will be rejected. 

This is due to the fact that unreliable results may be obtained as boric acid can affect the viability of the organisms, if in too high a concentration. If only a small sample can be obtained from the patient a white topped container should be used but sent to the lab ASAP.

Urines should be received within one to two hours of collection. If this is not possible then refrigeration at 4°C for up to 24 hours is possible for most specimens, without much change in bacterial count. However, the white cells may become unrecognisable.

Red blood cells may lyse in dilute urine shortly after the specimen is taken. An on-site “stick” test may give a more accurate indication of the presence of blood.

Suggestions for the collection of a mid-stream urine (MSU) sample

Men and others with a penis

Retract the foreskin if necessary. Then pass the first part of the urine stream into the w.c. pan. Catch the second part into a sterile universal container.

Women and others with an uterus 

If there is a menstrual or vaginal discharge, the use of a vaginal tampon is helpful. 

The patient should be instructed to clean the vulva from the front -backwards - using a cotton-wool swab soaked in sterile water. The patient should separate the labia with two fingers of one hand. 

Antiseptics must be avoided. Keeping the labia separate, the patient passes the first part of the urine stream into the w.c. Then catches the second part into a sterile universal container.

Babies and young children

A clean-catch specimen is preferred as urine held in adhesive bags is frequently contaminated.

Further advice

For more advice on taking urine samples, read on the main NHS website: How should I collect and store a pee (urine) sample?

Urine for TB culture

Three complete, consecutive early morning specimens are usually required. 

These may be refrigerated each day and taken to the laboratory together. The laboratory can supply suitable containers.

Urine for legionella and strep pneumoniae antigen testing

Urine samples should be collected in the normal way, into a white topped plain sterile universal container.

They can be kept at room temperature for up to 24 hours, but ideally should be sent to the laboratory as soon as possible. Alternatively they can be stored at 2-8 C in a refrigerator.

Urine for ova, cysts and parasite testing (schistosoma)

In urinary schistosomiasis, very few ova are present in the urine. The number of ova in the urine varies throughout the day, being highest in urine obtained between 10am and 2pm. 

In patients with haematuria, eggs may be found trapped in the blood. Mucus may be found trapped in the terminal portion of the urine specimen. 

It is therefore preferable to obtain total urine collected over the time period between 10am and 2pm. 

Alternatively, a 24 hour collection of terminal samples of urine may be helpful. Sterile containers without boric acid must be used (white topped universal).

Cannulae

The incidence of infection is related to the length of time the cannula remains in situ.

The catheter tip may be infected secondarily. This can be caused by organisms already infecting the hub or insertion site, that track down the catheter lumen or tunnel. It may also acquire organisms from fluids passing through it or from the bloodstream, itself.

Most central venous line-associated infections are caused by organisms from the skin near the exit site. Access is gained via the intravascular segment of the cannula.

Sample collections

Cannulae

Disinfect the skin around the cannula entry site. 

Remove cannula using aseptic technique. 

Cut off 4cm of the tip into a universal container using sterile scissors, and send this to the laboratory.

Cannulae should only be sent if there is evidence of infection.

Swabs

Cannula associated swabs (e.g. swabs of catheter insertion sites) may be employed as alternative specimens. 

However, routine investigation of cannula associated swabs from asymptomatic patients is of dubious value

Please use plain white topped universal containers with screw-on lids. 

Specimens obtained by deep coughing or post-physiotherapy specimens are preferred. 

Saliva and pernasal secretions are not suitable as they will be contaminated with mouth flora and may give mis-leading results.

Ensure lids are tightly closed prior to sample transport to prevent leakage.

Culture of non-purulent material is not helpful as mouth flora inevitably predominates, and will be misleading except when M. tuberculosis is found. 

Inadequate specimens may be discarded and not tested.

For mycobacterium tuberculosis and fungal culture, at least three early morning specimens will give a more reliable result than a single sample

In all cases the Microbiology Department must be informed when a CSF has been taken.

During normal working hours the laboratory must be informed directly via extension 2687.

At all other times, the microbiology biomedical scientist (BMS) on call must be contacted via switchboard.

It is the responsibility of the doctor initiating the request to ensure that CSF samples are expected by the laboratory. If requesting this test out of hours, please only phone the on-call biomedical scientist once you have taken the sample. Please remember to put your bleep or contact details on the request form so the result can be phoned to you.

For routine microbiological investigation at least 1 ml of CSF should be sent in sterile universal containers.

For cases of suspected subarachnoid haemorrhage (SAH) at least 2 samples should be sent to microbiology - ideally the first and last specimen taken - for a cell count.

One sample should be protected from light and sent to biochemistry for xanthochromia (normally this should be sample 2).

Samples will automatically be sent for PCR testing by laboratory staff if:

• microscopy results indicate infection 
• and, or after discussion with the Consultant Microbiologist, but please indicate on the form or order a PCR test on ICE if this is specifically required

For biochemical investigations (such as protein and glucose) further specimens should be sent in accordance with their protocol. Please refer to their web pages.

Use blue topped universal pots with a collecting spoon.

Samples for examination for ova, cyst and parasites should be freshly passed and sent to the laboratory as soon as practical.

Details of any foreign travel is essential.

For detection of ova, cysts and parasites: ideally three faeces samples collected over no more than a 10 day period, should be sent. 

It is usually recommended that specimens are collected every other day and no more than one specimen to be taken on the same day. This is because shedding of cysts and ova tends to be intermittent.

Samples that are formed or solid, are not tested for Cl.difficile. The sample container should be at least a quarter full so the sample consistency can be noted. Please ensure a representative sample is sent to avoid an incorrect result being issued.

Suggestions for collection of faeces specimens

It is of paramount importance not to scoop the specimen from the WC basin as this will be contaminated and may lead to false results.

Pass the motion or part of the motion into a suitable container.

With the spoon attached to the blue lid, scoop some of the motion into the specimen container, taking care not to contaminate the outside of the container.

Do not fill more than half full. For certain tests ( e.g GDH/CDT) the container needs to be at least a quarter full.

Make sure the lid is securely fastened and please ensure that the person’s identity is written clearly on the label of the specimen container. Then place and seal it, in the polythene bag at the back of the request form provided.

Send the specimen to the microbiology laboratory as soon as possible.

For further information about taking a stool sample please read on the main NHS website: How should I collect and store a poo (stool) sample?

Specimen collection guidelines for H.pylori Faecal Antigen Testing

Specimen collection requirements

Solid, semi-solid or liquid cultures are approved for this test and should be transported in an airtight sterile container.

Specimen storage and transport

The specimen should be sent to the laboratory as soon as possible but may be stored in the fridge overnight.

Limitations of the faecal antigen test

Antimicrobials, proton pump inhibitors and bismuth preparations are known to suppress H.pylori. Ingestion of these prior to testing may cause false negative results to occur. 

In such cases, the test should be repeated on a new specimen obtained two weeks after discontinuing treatment. 

A positive test result for a patient ingesting these compounds within two weeks prior to performing the faecal antigen test, should be considered accurate.

Other samples for parasitology investigations

Detection of threadworms

These methods are more reliable than examination of a faeces sample for the detection of threadworm (pinworm) infection.

Sellotape slide

Apply a 6 cm piece of clear sellotape to the perianal region first thing in the morning.

Press the adhesive side of the tape firmly against the left and right perianal folds several times; the tape can be wrapped around a tongue depressor to aid specimen collection. 

Smooth the tape back on the slide, adhesive side down. This should then be transported to the laboratory in a slide carrier box ( available from the laboratory).

Perianal swab

Alternatively a Perianal swab can be taken first thing in a morning before bathing or defecation. A cotton-wool swab in a dry container should be used for collection.

Spread buttocks apart, and rub the moistened cotton wool swab over the area around the anus, but do not insert into the anus. Place cotton wool swab back in its container (no transport medium required). If only charcoal transport swabs are available, place the dry swab into a white topped universal container and cut off the swab shaft so the top of the universal can be screwed on.

Occasionally, an adult worm may be collected from a patient and sent in saline or water for identification.

Please be aware that threadworm ova are highly infectious, therefore hand-washing after this procedure is essential.

Sellotape slides and perianal swabs for E. vermicularis ova – it is recommended that samples should be taken for at least four to six consecutive days. If the results of all these are negative the patient can be considered free from infection.

Urine (for S. haematobium)

In urinary schistosomiasis, very few ova are present in the urine. The number of ova in the urine varies throughout the day, being highest in urine obtained between 10am and 2pm. 

In patients with haematuria, eggs may be found trapped in the blood and mucus in the terminal portion of the urine specimen. It is therefore preferable to obtain total urine collected over the time period between 10am and 2pm. 

Alternatively, a 24 hour collection of terminal samples of urine may be helpful. Sterile containers without boric acid must be used (white topped universal).

Blood culture bottles are available in packs, in blue bags.

These can be obtained from the laboratory during “normal working hours.” Please ensure that the ward areas are adequately stocked during the day.

Please store the packs at room temperature (15 to 30 degrees) away from direct sunlight or heat (for example near radiators). 

Do not use out of date bottles. Always check expiry dates on bottles before use and return any out of date bottles to the laboratory. After blood has been collected please send it to the laboratory as soon as possible. 

To optimise the clinical utility of blood culture results, the interval between sample collection and being incubated in the laboratory should ideally be within 4 hours.

Bottles and procedure pack

A standard set of adult bottles consists of:

• two bottles

• a blue cap aerobic bottle

• a purple capped anaerobic bottle

For paediatrics there is a single aerobic bottle for low-volume culture, (not more than 5 ml). These bottles have a yellow cap.

The laboratory provides the blood culture bottles in a blue bag which contains:

• a blood culture bottle adaptor cap

• safety-multifly needle

• antiseptic skin wipes

• a sticker to record collection to be put in the patient's notes

Full instructions on taking the blood culture are printed on the bag, together with some of the clinical indications for collecting cultures on the reverse.

Patient ID stickers must not cover all of the barcode on the bottles, or cover the green detector pad on the bottom of the bottle. Please do not remove the peel off section of the bar-code sticker from the bottle and place it in patients notes as this is required in the laboratory. However, it can be placed on the request form to assist in sample identification.

If possible take cultures before starting antibiotics, when the patient is already receiving antimicrobials, blood cultures should be collected just before the next dose is due when antimicrobial concentration in the blood is at its lowest.

Blood cultures should be taken once or twice during each clinical episode. A second set taken from a different site not only increases yield but also allows recognition of contamination, however it is recommended three times for endocarditis from different venepuncture sites. 

In most conditions other than endocarditis, bacteraemia is intermittent. Given it is related to the fevers and rigors which occur 30 to 60 minutes after the entry of organisms into the bloodstream, samples should be taken as soon as possible after a spike of fever.

Up to 20 ml of blood can be cultured per two-bottle set (minimum of 5 ml in each bottle, maximum of 10 mls). There is a direct relationship between blood volume and yield; false negatives may occur if inadequate blood culture volumes are submitted.

If the patient is difficult to bleed, a paediatric bottle can be used which is designed for a low level of blood.

Taking the blood

Please see the current guide to taking a blood culture on the Trust’s Intranet under ” B” in nursing procedures.

Label the bottles and send them to the laboratory with a microbiology request card as soon as possible.

Do not refrigerate bottles, or warm them on radiators before transportation to the laboratory.

If the patient is “high risk” then ensure bottles and request cards are labelled with danger of infection stickers.

Minimum volume required for microbiological investigation is 1ml. Place the fluid into a sterile universal container, and tighten the top securely.

Fluid may be inoculated directly into a blood culture bottle, but if a cell count and/or Gram Stain, or microscopy for crystals is required, fluid in a universal container is required. 

Please note the examination for crystals is only performed on joint fluids if specifically requested, or if clinical details mention gout.

Inform the on-call microbiology BMS via switchboard if an urgent cell count, gram stain or examination for crystals (joint fluids only) is required out of hours.

Skin, nail and hair samples can be tested for fungal infection (mycology).

Clean the area with a 70% alcohol wipe before sampling, this minimises contamination. It is also an aid to microscopy, if greasy ointments or powders have been applied.

Skin scrapes, nail clippings and small hair pieces should be taken and placed in a sterile universal or dermapak, and sent to the laboratory as soon as possible. Samples can be stored at room temperature before transportation.

Please send as much material as possible to allow a full investigation to be completed to assist with a correct diagnosis.

Suggestions for taking samples

Skin samples: using a sterile scalpel blade, scrape the edge of the lesion and collect the skin scrapes into a dermapak, fold up and seal or alternatively onto a clean piece of paper. Then transfer this into a sterile universal.

Nail: if possible, collect the subungual debris in addition to nail clippings. Sample the discoloured, dystrophic or brittle parts of the nail only, sampling as far back as possible from the distal part of the nail.

Hair: pluck hairs from the affected area with forceps (infected hairs come out easily) and scrape the scalp with a scalpel. Preferably, the sample should include hair roots, the contents of plugged follicles and skin scales. Hair cut with scissors is unsatisfactory as the focus of infection is usually below or near the surface of the scalp.

Nucleic Acid Amplification Test ( NAAT) – Patient self take instructions (testing is no longer done for chlamydia only).

The laboratory issues specific collection kits for Chlamydia/GC testing using the Aptima Hologic Panther.

The swab to use is an orange Aptima multitest swab specimen collection kit for patient-collected specimens.

Urine (all genders) should be collected using the yellow urine Aptima collection kits.

General Instructions for all collection methods

1. Wash hands before starting.
2. In the privacy of the examination room or toilet, undress as necessary. Comfortably position yourself to maintain balance during the collection procedure.
3. Open the kit package, remove the swab and tube.
4. Warning: If at any time the contents of the tube are spilled on your skin, wash the affected area with soap and water. If the contents are splashed into your eyes, immediately flush your eyes with water. Notify the clinic, a nurse or your GP, if irritation develops.
5. If the contents of the tube are spilled, request a new multi-test swab kit.

Do not take the contents of the tube internally.

Vaginal swab specimen collection

If you are pregnant ensure you inform your healthcare provider.

1. Partially peel open the swab package and remove the swab – do not touch the soft swab tip or lay the swab down on a surface.
2. Hold the swab in your hand, placing your thumb and forefinger in the middle of the swab covering the score line (black line).
3. Carefully insert the swab into the vagina about 2 inches ( 5 cm) inside the opening. Gently rotate the swab for about 10 to 30 seconds, making sure the swab touches the side of the vagina. Remove the swab.
4. While holding the swab, unscrew the top of the tube and place the swab into the liquid in the tube so that the score ( black) line is at the top of the tube.
5. Carefully break off the swab at the score line by pressing the shaft of the swab against the side of the tube and discard the top part of the swab shaft.
6. Screw the cap back onto the tube and ensure it is tightly fastened.
7. Do not pierce the foil lid.
8. Ensure your name and date of birth is on the tube, and hand back to the nurse.

Penile meatal swab specimen collection

1. Partially peel open the swab package and remove the swab – do not touch the soft swab tip or lay the swab down on a  surface
2. Hold the swab in your hand, placing your thumb and forefinger in the middle of the swab covering the score line (black line).
3. Uncircumcised people will have to roll the foreskin down before starting collection.
4. Hold your penis with your free hand (the hand not holding the swab). Using your other hand (with the swab), roll the swab at the tip or outside the opening to the penis through which you pass urine. Be sure to roll the swab completely around the opening to ensure you get a good sample. It is not necessary to insert the swab deep inside the penis.
5. While holding the swab, unscrew the top of the tube and place the swab into the liquid in the tube so that the score (black) line is at the top of the tube.
6. Carefully break off the swab at the score line by pressing the shaft of the swab against the side of the tube and discard the top part of the swab shaft.
7. Screw the cap back onto the tube and ensure it is tightly fastened.
8. Ensure your name and date of birth is on the tube, and hand back to the nurse.

Rectal swab

1. Partially peel open the swab package and remove the swab – do not touch the soft swab tip or lay the swab down on a surface.
2. Hold the swab in your hand, placing your thumb and forefinger in the middle of the swab covering the score line (black line).
3. Carefully insert the swab into your rectum about 1 to 2 inches (3 to 5 cm) past the anal margin (outside of the anus), and gently rotate for 5 to10 seconds. Withdraw the swab without touching your skin.
4. While holding the swab, unscrew the top of the tube. Then place the swab into the liquid in the tube so that the score (black) line is at the top of the tube.
5. Carefully break off the swab at the score line by pressing the shaft of the swab against the side of the tube. Discard the top part of the swab shaft.
6. Screw the cap back onto the tube and ensure it is tightly fastened.
7. Do not pierce the foil lid.
8. Ensure your name and date of birth is on the tube, and hand back to the nurse.

Throat swab

1. Partially peel open the swab package and remove the swab – do not touch the soft swab tip or lay the swab down on a surface.
2. Hold the swab in your hand, placing your thumb and forefinger in the middle of the swab covering the score line (black line).
3. Carefully insert the swab into your mouth, ensuring contact with both of your tonsils and the back of the throat. Then withdraw the swab without touching the inside of your cheeks or tongue.
4. While holding the swab, unscrew the top of the tube and place the swab into the liquid in the tube so that the score (black) line is at the top of the tube.
5. Carefully break off the swab at the score line by pressing the shaft of the swab against the side of the tube and discard the top part of the swab shaft.
6. Screw the cap back onto the tube and ensure it is tightly fastened
7. Do not pierce the foil lid.
8. Ensure your name and date of birth is on the tube, and hand back to the nurse.

Urine collection

The patient should not have urinated for at least one hour prior to collection.

1. Collect the sample into a sterile preservative free collection cup (this should be the first 20 to 60 ml of the urine, not midstream).
2. Remove the cap from the tube and transfer 2 ml of the urine using the disposable pipette provided. Ensure the tube is filled to a level between the 2 black collection lines and label with patient information.
3. Do not pierce the foil lid.

Specimen storage and Transport

After collection, the sample should be stored between 2C and 30C until tested.

Samples should be assayed within 30 days of collection. If longer storage is needed samples may be frozen at -20 to -70 for up to 12 months.

Urine samples not in the Aptima tubes (i.e in primary universals) must be transported to the lab between 2C and 30C.

Urines should be transferred to the Aptima tube within 24 hours of collection.

Swabs in viral transport media eg. green topped virocult, are provided via the laboratory (extension 2726).

Do not use swabs beyond the expiry date on the packaging. Always label the specimen with the patient identifiers and the site the swab was taken from, for example, throat swab.

To use - carefully remove the swab from the packaging at the end indicated ‘peel here’. Use the swab to collect material from the affected area, unscrew the green container lid and place the swab in the liquid transport media in the tube. Carefully break off the swab against the neck of the tube and re-cap the tube securely. 

Swabs once taken, should be returned to the laboratory ASAP. If this is not possible they can be stored in a fridge at 2C to 8 C overnight.

Vesicle fluid, nose, throat ,eye, skin and genital sites: use a green topped viral swab. Use the swab to collect material from the affected area. Place the swab in the liquid media in the transport tube and re-cap the tube securely. Store in the fridge if transport to the laboratory is to be delayed.

NPA: collect the sample into a sterile universal container.

Faeces: send the sample in a plain universal or blue topped universal with a spoon.

Urine: send the urine in the sterile universal container.

Any other specimen type – (such as a biopsy), please contact the consultant microbiologist for advice.

Paired sera are not now usually required. However, we need to know enough clinical details to decide which viruses to screen for. We need to know a date of onset to decide whether collection of a second serum is appropriate.

Only a limited range of viruses can be tested for, and often serology is unhelpful.

Viral serology is helpful when a specific virus is suspected (e.g. rubella, CMV), or with particular clinical scenarios such as rash, flu-like illness, and  respiratory tract infections.

Patients with vague or long-standing problems (“lassitude” etc) almost never produce diagnostic results.

In an attempt to guide you to request the correct serology tests when requesting using ICE, use the microbiology tab and under virology tests, there are clinical symptoms boxes present. So if the patient has jaundice,  tick the jaundice section and all the relevant laboratory tests will be selected for you.

Blood samples can also be sent to virology for immunity status checking, e.g after routine vaccinations for Hepatitis B. Or as part of the antenatal screening for infectious disease, or when a pregnant person has been in contact with varicella zoster (VZV or chickenpox).

Please see SOP-MC-VIR-D-46- List of Serology tests for details of the specific blood tubes required, for specific tests. Please supply as much clinical information as possible to allow us to perform the correct tests, to interpret the result correctly and to add appropriate comments.

Please be aware that if serology and virology tests are requested a separate sample should be sent to Virology. The request should not be added onto a blood sample already sent to blood sciences except in exceptional circumstances e.g a difficult to bleed patient. This is due to potential contamination from the biochemistry analysers. In these instances please ring the virology laboratory to discuss.

Swabs for routine bacterial culture

For instructions on how to take swabs, please refer to the Royal Marsden nursing procedures manual from the Intranet (under nursing procedures in links).

Please ensure swabs used have not passed the expiry date and are intact with the seal unbroken.

Place the swab in the tube containing Amies charcoal transport medium, and send it to the laboratory as soon as possible. If same day transport is not available then they can be stored in a refrigerator overnight but do not freeze. 

Fine wire swabs (orange topped) are available for use when taking ear swabs.

For the diagnosis of pertussis, fine flexible wire pernasal swabs (blue top)  are available.

Provisional results will be available after 24 hours if urgently required. Please telephone the microbiology laboratory (extension 2687), with final results being available on ICE after 48 hrs. However, some results could take up to 10 days, depending on examinations required.

Pus and swabs

Abscess pus, abscess swab, deep-seated pus swab, post-operative wound swab, wound exudates

Collect specimens before antimicrobial therapy where possible.

The specimen will usually be collected by a medical practitioner.

Samples of pus are preferred to swabs. However, pus swabs are often received (when using swabs, the deepest part of the wound should be sampled, avoiding the superficial microflora). Swabs should be well soaked in pus.

Swabs for bacterial and fungal culture should be placed in the transport medium provided in the tube.

If possible a few ml of pus in a sterile universal bottle or even a few drops still in a syringe is much better than a swab (note - the syringe must be safely capped and needles should not be sent),

Ideally, a minimum volume of 1 ml of pus is required.

The volume of specimens influences the transport time that is acceptable. Large volumes of purulent material maintain the viability of anaerobes for longer.

Numbers and frequency of specimen collection are dependent on the clinical condition of the patient.

If delays in transportation to the laboratory are unavoidable then the samples should be kept in a refrigerator.

The recovery of anaerobes is compromised if the transport time exceeds 3 hours.

Surgical Specimens and tissue/bone samples

Specimens must not be put in formol saline for microbiology. 

For orthopaedic samples, please use the universals containing beads in saline.

Please put small bits of tissue into these containers ( no more than 1 cm sized), and attach a patient identification label to the container.

For all other tissue samples - use dry sterile universal containers.

Make sure specimens are sent to the laboratory ASAP. If this is not possible, refrigeration is preferable to storage at ambient temperature. However the recovery of anaerobes is compromised, if the transport time exceeds 3 hours.

The laboratory (or on-call biomedical scientist) must be informed if the specimen is urgent or requires processing out of hours.