Bacteriology

Blood cultures

An adult blood culture set consists of 2 bottles, one blue top (aerobic) and one gold top (anaerobic)). For age 12 and under use Paediatric pink top bottle (add Gold bottle if anaerobes suspected).

  • Positively identify the patient
  • Gain informed consent
  • Thoroughly wash and dry hands using Ayliff 6 stage procedure
  • Prepare the skin by rubbing it in a criss cross back and forth motion for 30 seconds with 70% isopropyl alcohol / 2% Chlorhexidine, Frepp applicator
  • Allow it to dry before piercing the skin. 
  • Flip the plastic caps off the blood culture bottles and disinfect the rubber diaphragms with 70% isopropyl alcohol / 2% Chlorhexidine sanicloth wipe. Allow them to dry. Use a sterile sanicloth wipe for each bottle used.
  • Apply gloves and an apron
  • The specimen must be collected using an aseptic non-touch technique to reduce the chance of contamination. 
  • Blood cultures must be taken before other blood samples.(see order of draw card)
  • A BD eclipse needle and vacutainer or a Safety Lok blood collection butterfly must be used for adults
  • The culture bottles must remain upright when being filled
  • If possible the specimen should be collected before antimicrobials are given.
  • The recommended adult specimen volume is 8-10 ml, volumes as low as 3 ml can be used, however recovery will not be as great as with larger volumes. 
  • As soon as the blood is collected, the bottles must be labeled at the patient’s bedside and a form (either hard copy or electronic, iSOFT completed)
  • The blood culture bottles should be transported to the laboratory without delay. 
  • Blood Culture bottles are now polycarbonate and can be safely sent via the pneumatic Pod system.
  • They are available from the department.

Cerebrospinal fluid 

An adequate amount is essential 

– send at least 2-3 ml. This is particularly important if Mycobacterium infection is suspected where small numbers of organisms may be present. If the specimen is bloodstained send the 1st and 3rd samples so that differential red blood cell counts may be performed. 

The results of microscopy and any positive cultures are always telephoned. If glucose and or protein analysis is required send a separate specimen and request to the Combined Laboratory. 

Faeces  

Send a “plum-sized portion” or 5-10 ml if liquid. Ask the patient to defecate into a clean bedpan or other convenient container if at home. Use the plastic spoon to transfer a portion of faeces into the pot. For liquid faeces use a plastic medicine spoon. Take care not to contaminate the outside of the faeces pot. For all investigations if more than 1 specimen is to be submitted ensure that these are obtained on successive days.

  • Tests performed are based on clinical information given, patient age, any foreign travel, and whether in-patient or GP patient. If tests have been requested that are not indicated, they may not be done (e.g. C difficile on a formed stool)
  • C difficile testing is usually only performed on samples taking the shape of the container, or liquid.
  • For In-Patients of more than 3 days: C. difficile testing is usually the only test done if the patient develops diarrhoea after admission, so culture for other pathogens such as Salmonella and campylobacter would only be done on special request (This is called the 3-day rule).
  • amoebic dysentery: for examination for amoebic trophozoites the specimen must reach the laboratory within 1 hour of its production. It is advisable to arrange this examination with the laboratory in advance. 
  • sellotape slide/perianal swab for E. vermicularis (Threadworm): 

 

Sellotape Slide

For optimal results perianal impressions should be taken between 10p.m. and midnight, or early in the morning before defecation or bathing. Cut a 4-inch strip of sellotape, press the middle 1-2 inches against the perianal skin firmly. Sellotape should be pressed flat (sticky side down) onto a clean microscope slide, placed in a slide box and submitted to the laboratory. Alternatively perianal swab- moistened cotton wool swab rubbed over perianal area, replaced into container and submitted to the Laboratory. (Occasionally an adult worm may be collected from patient – this should be submitted in suitable size container containing sterile saline.) 

It is recommended that samples should be taken for at least 4 to 6 consecutive days. However, in practice it is more usual to receive 1 sample. 

NOTE: When handling samples wear gloves. Eggs can remain viable for several weeks, and are resistant to disinfectants and are not killed by chlorination in swimming pools.

Genital tract swabs

Cervical and high vaginal swabs must be taken with the aid of a speculum. It is important to avoid vulval contamination of the swab. For Trichomonas, swab the posterior fornix. If there are obvious candidal plaques, swab the lesions. If pelvic infection, including gonorrhoea, is suspected, swab the cervical os. If herpes simplex is suspected send an additional swab from the lesion in virus transport medium. 

High vaginal swabs 

Introduce the speculum, roll the swab firmly over the surface of the vaginal vault and place the swab in the transport media. 

Cervical Swabs 

Bacteriology – introduce the speculum and roll the swab in the endocervix, place the swab in the plastic transport sheath.

For Chlamydia - take the Chlamydia specimen after the bacteriology one. If there is discharge, mucus or pus in the cervix wipe it off. Insert the Chlamydia swab into the endocervical canal. Rotate firmly around the surface of the canal for 5-10 seconds, withdraw the swab without touching any vaginal surface. Place the swab in Chlamydia transport medium, snip off the shaft and screw the cap on. Avoid contact with water-based lubricants, which may cause false positive results. Put both urethral and vaginal swabs in the same bottle of Chlamydial transport medium

Urethral swabs 

Avoid contamination with microorganisms from the vulva or the foreskin. Small swabs are available for this purpose.

Bacteriology - The patient should not have passed urine for at least 1 hour. For males, if discharge is not apparent attempt to “milk” it out of the penis. Pass the swab gently through the urethral meatus and roll around. Place the swab in the plastic transport sheath.

Chlamydia - Take this specimen after the bacteriology swab. Pass the swab through the urethral meatus and gently but firmly roll it over the surfaces of the urethral epithelium for 1-2 seconds then withdraw. Snip off the swab into Chlamydia transport medium. ‘1st catch’ urine (rich in epithelial cells) is acceptable for Chlamydia testing on males. 

IUCDs

Intrauterine contraceptive devices (IUCDs) 

Send the entire device in a sterile container (Sputum pot). 

Lines and Tips

Line tips should only be cultured if line infection is suspected.

Line infection is confirmed by semi-quantitative culture of a removed line. After removing a line suspected of being infected, from a patient, cut off the intravascular portion using sterile scissors, and place in a sterile universal container. 

Mouth swabs

Sample pus if present otherwise sample any lesions or inflamed areas. A tongue depressor or spatula may be helpful to aid vision and avoid contamination from other parts of the mouth. Place the swab in plastic transport medium. 

Nasal & Pernasal Swabs

Nasal swabs: are usually taken to detect staphylococcal carriage. Moisten the swab before swabbing with sterile saline. Swab the anterior nares by gently rotating the swab in each nostril. Place the swab in the plastic transport sheath 

Pernasal swabs: are used to diagnose whooping cough. Pass the swab gently along the floor of the nose. Best results are obtained by plating out at the bedside. Contact Microbiology if plates are required. In the community place the swab in pertussis transport medium, snip the wire and screw the cap on. 

Taking these samples in patients with whooping cough may precipitate a paroxysm of coughing and cause obstruction of the airways. Resuscitation equipment must be available if whooping cough is suspected. The specimen collector should avoid direct coughs from the patient 

Postnasal swabs: these are taken to investigate meningococcal carriage. The procedure is the same as for throat swabs but rub the swab over the posterior wall of the pharynx only, not the tonsils.

Nasopharyngeal aspirate 

Try to take the specimen first thing in the morning. Pass the tip of the fine feeding catheter along the floor of the nose for about 7 cm. Apply suction. If only a scanty amount of mucus is obtained flush this into the trap by aspirating 1 ml of virus transport medium through the tubing. Replace the mucus extraction top with a screw cap. Transport immediately to the laboratory. 

Serology

See Serology Test profiles below 

Skin, nail and hair for mycology

Skin scrapings - should be taken by gently shaving off material from the active edges of the lesion using a scalpel blade. Send material to the laboratory in a wide mouth container. At least 5 mm2 of skin scrapings are required. 

Nails and hair - clippings should include the full thickness of the nail and extend as far back from the edge as possible. Hairs should be plucked from affected areas together with skin scrapings from associated scalp lesions. As with skin, these specimens may be sent in a wide mouth container. 

Sputum

Expectorated sputum and not saliva is required. Do not collect shortly after the patient has been eating, drinking or cleaning their teeth. If tuberculosis is suspected send 3 early morning specimens of sputum taken on consecutive days. Ask a physiotherapist to assist if a patient has difficulty in producing satisfactory specimens. Collect sputum sample in a sterile plastic wide-mouth container and always send it in its own specimen bag not mixed with other samples, even if they are all from the same patient.

Bronchial washings - after collection remove the cap and the tubing of the sterile suction container and apply the screw cap to the container. 

Bronchial lavage - the specimen will be collected by a specialist according to local protocol in a sterile container.

Surface swabs and skin swabs

Rotate the swab on or in the required site. Place the swab in the plastic transport sheath. 

Under aseptic conditions transfer material to a sterile universal container that does not contain formalin as this inactivates pathogens very rapidly. 

Throat swabs 

Diagnosis of viral pharyngitis and streptococcal pharyngitis depends on the culture of a throat swab. 

Bacteriology - sample the posterior portion of the pharynx, tonsillar areas and areas of ulceration, exudation or membrane formation. Depress the tongue with a spatula. Try not to touch the lips, tongue, mouth or saliva. Place the swab in the plastic transport sheath.

Virology - moisten the swab in virus transport medium before taking the specimen. Follow procedures as for the bacteriology throat swab. Snap off the swab into transport medium. If a respiratory virus is suspected a nose swab can also be taken 

Urine

The laboratory utilises a flow cytometry analyser which rapidly screens urine, selecting only those likely to be positive for culture. If ordering urine tests electronically in iCM or ICE, please ensure that the correct category of urine test is selected (There are several options including catheter urine screen, screen for child/ pregnant patient etc), as this ensures the laboratory’s resources are used effectively.

Clean-voided midstream urine is preferred for bacterial and fungal cultures. The reliability of microscopy and culture results depends on the avoidance of contamination and prompt transport. 

It is recommended that in females the hands and the perineal area be washed with soap and water prior to specimen collection. Part the labia and clean the area around the urethral meatus from front to back. Spread the labia with the fingers of one hand. 

In males retract the foreskin, if present, and clean the skin surrounding the urethral meatus. To avoid contamination with urethral organisms the patient must be instructed not to collect the first part of the urine. Start passing urine into the toilet, bed pan or urinal. When the urine is flowing freely collect urine in a clean sterile container. 

If a preservative (boric acid) is used pour urine up to the 20 ml mark on the label.

Catheter specimens
should be obtained aseptically with a sterile syringe and needle following disinfection of the catheter specimen port with alcohol. Clamp tubing below the sampling cuff. Clean the sampling cuff with a mediswab.

Aspirate urine using a syringe and transfer to a sterile universal container. Unclamp the tubing. Patients with long-term catheters are invariably colonised with 1 or more micro-organisms. 

Urine - Investigation for mycobacterial infection
Send 3 early morning urine specimens (when the urine is most concentrated) taken on consecutive days. 

Urine – ileal conduit - open the dressing pack. Remove the stoma appliance. Clean the area around the stoma. Dry thoroughly. Gently insert a urinary catheter into the stoma to a depth of 2.5-5 cm. Drain sufficient urine into a receiver. Remove the catheter and pour urine into a sterile universal container. Attend to the stoma. 

Urine for Schistosomiasis collect a midday urine specimen or a 24-hour collection in a sterile container. Preservatives must not be used. Peak egg excretion occurs between noon and 3 p.m. In patients with haematuria, eggs may be found trapped in the blood and mucus in the terminal portion of the urine specimen. 

Wounds and ulcers


Always state the site and nature of the wound. This is essential, as the laboratory may need to interpret findings against a background of normal flora present in a given part of the body. Swabs of superficial wounds and ulcers are rarely helpful, in the absence of surrounding cellulitis. Pus is always preferable to a wound swab. 

If copious pus or exudate is present, aspirate with a sterile syringe and transfer to a sterile universal container. If insufficient to aspirate rotate a swab on the advancing edge of the lesion firmly, having first removed any superficial slough and debris, and place the swab in transport media.                    

MRSA Screens

For information on sampling of multiple sites, read the ESwab - Sample Collection Guide.

Reading MRSA CulturesScreening swabs/ broths should be collected as per current hospital guidelines, please ensure that the request form or electronic order clearly states if the request is for MRSA  screening only, or is for full culture plus MRSA.

PVL Screens

Infections caused by PVL strains of S.aureus normally cause pus-producing skin infections (e.g. abscesses, boils, carbuncles) and cellulitis. However on rare occasions , they can lead to more invasive infections. Locally patients and families suffering recurrent boils/abscesses without prior skin trauma is the most common presentation. Black top charcoal swabs or Eswabs should be used for sampling and the request form should clearly state if PVL screen is required.

 

Review Status

Reviewed June 2018 
This page will be updated September 2018

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