Microbiology

1. Blood Culture

Number and timing of collection
Volume of blood
Culture Medium
Blood Collection Method

2. Central Nervous System Specimens

Materials Required
Method

3. Ear Specimens

Middle Ear
Outer Ear

4. Eye Specimens

Preseptal cellultis - abscess drainage
Acute orbital cellulitis - abscess drainage
Canaliculitis - canalicular material
Acute dacrocystitis - abscess drainage
Blepharitis - lid margin
Conjunctivitis - conjunctiva
Conjunctivitis-gonococcal
Keratitis - cornea
Endophthalmitis - intraocular fluid

5. Gastrointestinal Specimens

Stool
Gastric layage

6. Genital Specimens

Herpes simplex detection
N. gonorrhoeae culture
Chlamydia trachomatis PCR
Gram stain, wet mount or nucleic acid probe test (VP3) for detection of vaginal candidiasis, trichomoniasis or bacterial vaginosis.
Haemophilus ducreyi culture

7. Respiratory Specimens

Lower respiratory tract infections
Upper respiratory tract infections

8. Sterile Body Fluids (Other than CSF and Blood)

9. Urine Specimens

Clean catch, midstream, female
Clean catch, midstream, male
Dipslide collection (after office hours)
Adhesive perineal bags for urine collection in infants. (Paediatric Urine collector)
Straight catheter or in-out catheterization
Indwelling catheter
Ilea conduit urine
Suprapubic aspiration of the urinary bladder

10. Wound Specimens

Abscess
Burns
Bites and traumatic/ surgical wounds
Decubitus ulcers

11. Miscellaneous Specimens

Intravenous catheters
Dental culture

12. For Fungal Culture

13. For Mycobacterial Culture

Specimen	Source	Method of Collection	Comments
BLOOD CULTURE	Blood	Number and timing of collection
The following factors directly influence blood culture results: 1) The volume of blood collected 2) Method of skin disinfection Note: Please cushion the blood culture bottles adequately when sending them to the laboratory via the pneumatic tubes.		Take 2 sets of blood cultures from different sites for acute sepsis. For endocarditis, take 3 sets. Each set comprise one aerobic and one anaerobic bottle which is optional. Volume of blood Adults : 5-10 ml Culture medium Adults: Bactec Aerobic Plus/F Bactec Anaerobic Plus/F Optional) Paeds: Bactec Paeds Plus Bactec Anaerobic Plus/F (optional) Note: Bactec Aerobic Plus/F and Bactec Paeds Plus are suitable for candida. Use fungal culture media only for fungi other than candida. Blood collection method By puncture of peripheral veins or arteries. 1. Disinfect the culture bottles 2. Disinfect venepuncture site Note: Gowns and sterile drapes are not necessary. Care in procedure is more important. - Wash and dry hands. - Palpate for a vein to locate the site for venepuncture. - Wear sterile gloves. Use chlorhexidine 0.5% with 70% alcohol and swab concentrically, starting from the centre. Allow 1 min for dying. (Do not palpate the vein again.) 3. Blood Collection - If available, use a butterfly needle with tubing connected to a luer adapter and tube holder. - The vacuum in the vial will usually exceed 5 ml so the user should monitor the flow by observing the 5 ml mark on the Bactec bottle. - When the desired volume has been drawn, crimping the tubing should stop flow.	The number of temporally spaced cultures and timing of collection may not be as critical as once thought. Blood for culture should not be drawn from an indwelling intravascular catheter. There is a risk in contamination of the catheter. Anaerobic blood cultures may not be necessary unless anaerobes are suspected. Blood culture is usually not helpful for diagnosis of invasive aspergillosis Alternatively, if using a needle and syringe, then transfer the blood into the Bactec bottle using a syringe transfer device, if available. Never inoculate tubes for other tests before inoculating the blood culture bottles.

CENTRAL NERVOUS SYSTEM SPECIMENS	Cerebrospinal fluid by lumbar puncture.	Materials Required
		- Skin disinfectant-0.5% chlorhexidine with 70% alcohol - Sterile drapes - Lumbar puncture needle (20-22 gauge) with stylet - Water manometer - Three sterile screw cap tubes

		Method
		1. Ensure that the patient is motionless 2. Explain that some pain will be inevitable when the needle stretches the dura. 3. Have the patient bend his back so that the head almost touches the knees. 4. Disinfect the skin with 0.5% chlorhexidine with 70% alcohol. 5. Allow 1 min for drying. 6. Insert the needle at L3-L4 interspace or lower to avoid spinal cord damage. Draw at L4-L5 in children because the conus medullaris extends lower in children than in adults. 7. When the subarachnoid space is reached, remove the stylet and CSF will appear in the needle hub. 8. Collect the drops of fluid into sterile screw cap tubes.	Collect the specimen using strict aseptic technique. If only 1 specimen is available, the microbiology lab gets it first. If more than 1 specimen is available, send the second specimen to Clinical Lab.

	Cerebrospinal fluid from the Ommaya reservoir	Clean the Ommaya reservoir with 70% alcohol. Allow 1 min for drying. Remove the Ommaya fluid via the Ommaya reservoir unit and place it in a sterile tube.

	Brain abscess	During surgery, aspirate material from the lesion and send immediately in a sterile container. Indicate clearly that material is from a ‘brain abscess’

	CNS biopsy	This is obtained at surgery. Send immediately in a sterile container without formalin.

EAR SPECIMENS	Middle ear	For intact ear drum, clean ear canal with soap solution. Using the syringe aspiration technique, obtain fluid through eardrum. For a ruptured ear drum, collect fluid on a swab via auditory speculum.

	Outer ear	Use a moistened swab to remove debri from ear canal. Obtain sample by firmly rotating swab in outer canal.





EYE SPECIMENS	Preseptal cellultis - abscess drainage	Ophthalmologist’s decision to make an incision for drainage or aspirate.

Obtain samples for viral and chlamydial cultures before instilling topical anesthetics.	Acute orbital cellulitis - abscess drainage	Diagnosis aided by X-ray and CT scan of orbit and paranasal sinuses. Aspirate with needle and syringe by ophthalmologist.	Gram stain may reveal bacterial morphology such as Actinomyces sp.

For virus isolation, use cotton or dacron swabs with non wood shafts.	Canaliculitis - canalicular material	Express purulent material by compressing lid and canaliculus.

Method of collection depends on site of infection.	Acute dacrocystitis - abscess drainage	Transcutaneous aspiration or incision through wall of lacrimal sac.

	Blepharitis - lid margin	Scrub cotton swab premoistened with sterile saline across anterior lid margins and the ulcerated areas.

	Conjunctivitis - conjunctiva	Obtain 2 swabs (for gram stain and culture) from the infected eye. Roll swab (which had been pre moistened with sterile saline) over the inferior tarsal conjunctiva and fornix of the eye.

	Conjunctivitis-gonococcal	N. gonorrhoeae causes copious purulent discharge. Request for gram stain and culture. Use swab in Amies Transport Media (the usual swab provided by KKH)
		Direct bedside inoculation of culture plate is not necessary.

	Keratitis - cornea	Cornea scrapings as performed by ophthalmologist. Direct culture inoculation on blood agar and chocolate plates or special media for fungus or protozoa where indicated.
		Prepare smears.
		To discuss with microbiologist.

	Endophthalmitis - intraocular fluid	Needle aspiration or from vitrectomy by ophthalmologist. Direct culture inoculation and prepare smears as for keratis-cornea.


GASTROINTESTINAL SPECIMENS	Stool	Pass stool into a clean and dry bedpan and transfer to a leak-proof container,
		Send to the laboratory within 2 hours or refrigerate at 40C for less than 24 hrs.
Stool samples are sent primarily for isolation of Campylobacter, Salmonella, Shigella species and to detect Yersinia, Vibrio and Clostridium difficile where indicated.
	Gastric lavage	Primarily for detection of Mycobacterium tuberculosis in patients (most frequently children) who are unable to produce quality sputum. Do procedure first thing after patient wakes up in the morning.
For C. difficile enterocolitis, patient should be passing soft or liquid stools > 5 times in 24 hrs.
		- Pass a well lubricated gastric tube orally or nasally into the stomach and lavage with 25-50 ml of sterile distilled water.
Do not send stool for routine cultures if patient has been admitted for > 3days.		- Recover sample and place into a sterile container.
		- Release suction and clamp before removing tube to prevent mucosal trauma.
Do not send gastric aspirate for culture as sampling pass through areas of heavy bacterial contamination.
GENITAL SPECIMENS	Bartholin’s gland	Pus from gland abscesses can be collected by digital palpation or aspirated by needle and syringe.

Genital specimens are submitted primarily for the detection of sexually transmitted diseases.	Endometrium	Collect transcervical aspirate via a telescoping catheter. Should not be collected through the cervix using a swab because contamination by cervical and vaginal flora would occur.

Routine vaginal specimens for bacterial culture are discouraged because the presence of high numbers of commensal flora makes them difficult to interpret	IUD	Preferably a surgically removed sample to prevent cervical and vaginal contamination	Endogenous cervicovaginal flora often isolated from asymptomatic women. May be associated with PID due to endogenous flora in the 1st 4 months post IUD insertion. However, PID occurring more than 4 months after insertion is often due to sexually transmitted organisms. Presence of Actinomyces does not necessarily indicate disease

	Low vaginal swab	Obtain swabs of the vaginal introitus and anorectum. Cervical swabs are not acceptable and a speculum should not be used.	For group B streptococcus screening. Universal prenatal screening for vaginal and rectal Group B streptococcal (GBS) colonization of all pregnant women at 35-37 weeks gestation is recommended.

	Pelvic specimens (for pelvic inflammatory disease) Cul-de-sac, fallopian tube, ovaries	All collected by invasive techniques or surgically.

	1. Amniotic fluid	Obtained during amniocentesis (uncontaminated) or intrauterine catheter during labour (maybe contaminated).

	2. Chorioamnion swab	If amniotic fluid cannot be obtained, take a swab between the chorion and amnion after stripping the fetal membranes. Contamination can be reduced if care is taken not to touch the edge of the membranes.

	3. Placenta	Placentas obtained during LSCS

For Herpes simplex detection	Penile or vulval vesicles endocervical or vaginal wall specimens.	1. Visually locate the vesicles and aspirate with tuberculin syringe or, unroof lesion with a sterile needle and sample the base of the lesion vigorously with a swab.	Herpes simplex virus culture is more sensitive than immunoflorescence (IF). Do not request for IF if there is no lesion.
		2. Swab cervix clean of mucus and discard. Insert another swab into endocervix, rotate gently and leave for 2 seconds.
		3. Swab the vaginal walls.
		4. Insert the swab into viral transport medium (Hank’s media), break off the swab stick and screw cap tightly.


For N. gonorrhoeae culture	Urethral specimens	1. Express exudate from urethra and collect on a swab.	Swabs for gonococcus can be sent in the usual swab provided as the organism will be preserved in the transport medium.
		2. If there is no exudate, insert a urethrogenital swab 2 cm into the urethra, gently rotate, leave for 2 seconds.
		3. Remove and insert into swab transport medium.


	Cervical or endocervical specimens	1. Moisten the speculum with warm water.
		2. Visualise the cervix.
		3. Remove from the os any vaginal material. 4. Gently compress cervix with blades of the speculum and collect endocervical discharge with a swab.
		5. Alternatively, insert the swab into the os, rotate gently and leave for 2 seconds.
		6. Remove and insert into swab transport medium.


Chlamydia trachomatis PCR	Urethral specimens	Express exudate from urethra and collect on a swab labeled “Chlamydia PCR” from Laboratory Reception. If there is no exudate, collect urine specimen as below. If for any reason a urethral swab is preferred, contact the microbiologist. A special thin-wire swab is required for the urethra. Insert a urethrogenital swab 2 cm into the urethra, gently rotate, leave for 2 seconds and remove.

	Endocervical specimens	Before obtaining a specimen, remove all secretions and discharge from the cervical os.
		1. Insert the swab 1-2 cm into the endocervical canal, and rotate against the wall of the endocervical canal > = 2 times. 2. Remove and place in specimen container.
	Urine specimens	Patient should collect the first 15-20 ml of voided urine. Patient must not have urinated within 2 hours prior to collection. Do not send urine collected by catheterization.

	High vaginal swab	Use speculum. Wipe away excessive secretions. Obtain secretions from mucosal membrane of vaginal vault with a sterile swab.
Gram stain, wet mount or nucleic acid probe test (VP3) for detection of vaginal candidiasis, trichomoniasis or bacterial vaginosis.
	Chancre	If a chancre is suspected, please request for syphilis serology.

Haemophilus ducreyi culture	Suspect genital lesions of chancroid (Haemophilus ducreyi)	Request for special culture plates from laboratory reception (6394 1352/ 1354). Swab vigorously the base of the ulcer, aspirate fluctuant bubos or collect a cervical swab and directly inoculate the culture plates


RESPIRATORY SPECIMENS
Lower respiratory tract infections	Sputum	Do not collect if patient has a dry cough or unable to expectorate (children <5 years) Rinse mouth with water Produce a deep cough-collect into a sterile screw cap container. Assisted by physiotherapy if necessary.	It is easy to contaminate sputum specimens with oropharyngeal flora. Consider blood culture for bacterial pneumonia.
Bronchial washing is not recommended as it is a poor predictor for agents of pneumonia.
Nasopharyngeal aspirate is not recommended for culture. Sampling passes through areas of heavy bacterial contamination. Collect sputum if patient is able to cough.	ETT aspirates	Estimate the distance needed to insert the catheter into the trachea. Pass the catheter through the ETT. Do not use the same catheter to suction oral secretions Aspirate material using an intermittent suction device into a sputum trap.	Same problems of contamination as nasopharygeal aspirates. The suction catheter must pass through densely colonized areas. The ETT is often colonised with gram negative bacteria. The presence of these organisms in culture may or may not indicate the etiology of pneumonia. Culture results must be correlated with clinical findings.

	Bronchial brushings	1. After inserting bronchoscope, insert the cytology brush unit into the channel opening of the scope. 2. Push the brush out of its sheath and obtain brushings 3. Pull the brush back into the unit and withdraw the entire brush unit. Brushings dry rapidly and drying can be detrimental to culture. 4. Snip the brush. 5. Drop into sterile saline.

	Bronchial alveolar lavage (specimen of choice)	A lavage would wash cells out of small airways that bronchoscopy could not reach.
		1. Insert and wedge bronchoscope in the subsegmental bronchus. 2. Attach a specimen trap to the bronchoscope. 3. Instill non-bacteriostatic sterile saline through the channel in 20 ml increments. 4. Gently suction the saline before administering the next aliquot.
	Oral lesions	Sampling of superficial tissues would reflect commensal oral flora. Tissue biopsies and needle aspirates are specimens of choice. Remove oral secretions or debri from surface of lesion with a swab, discard.
	Nasal swabs	Using a second swab, vigorously sample lesion, avoiding any normal tissues. 1. Use a swab pre-moistened with saline. 2. Carefully insert at least 1 cm into the nares. 3. Rotate against nasal mucosa.	Anterior nose cultures are reserved for detecting staphylococcal and streptococcal carriers if there is no indication of the presence of a lesion. Nasal cultures do not predict the etiological agent of sinus or middle ear infections and should not be submitted in lieu of specimens from these sites.
Upper respiratory tract infections		Lesions in the nose require samples from the advancing margins of the lesion.

	Nasopharyngeal swabs	1. Collect specimen with a small nasopharyngeal swab inserted through the nose or mouth. 2. Remove excess secretions from the anterior nares. 3. Gently pass the swab through the nose and into the nasopharynx. 4. Rotate the swab and leave for 1—15 sec. 5. Remove carefully and place in transport medium. 6. Or, bend the wire at an angle and insert the swab into the throat	These specimens are cultured primarily to detect N. meningitidis carriers or to diagnose B. pertussis infection. Routine nasopharyngeal swabs for bacterial culture are not recommended. Not to be used to detect the agent causing sinus infections.


	Throat specimens	1. Using a tongue blade to hold the tongue down, look for localised areas of inflammation and exudate at the back of the throat and tonsillar areas. These are the most productive areas for culturing the aetiologic agents of acute pharyngitis. 2. Firmly rub the affected areas with the swab. Do not touch the cheeks, gums or teeth as the swab is withdrawn.	The area of the inflammed throat must be firmly and completely sampled. Beta haemolytic streptococci groups A, C and G are routinely identified and reported.




STERILE BODY FLUIDS (OTHER THAN CSF and BLOOD)	Eg. pleural, ascitic fluid.	Disinfect needle puncture site using 0.5% chlorhexidine with 70% alcohol.
		Allow 1 min for drying.
		Aseptically perform percutaneous aspiration.
		Expel bubbles from syringe and inoculate into sterile screw cap tube.

		Volumes required for the following cultures:

		- Bacteria including anaerobes- 1-5 ml
		- Fungi and mycobacteria - >10mls if possible

URINE SPECIMENS	Clean catch, midstream, female	Supply the patient with clear verbal or written instructions as follows:
		1. Wash hands
Never collect urine from bedpan or urinal.		2. Clean urethral opening and vaginal vestibule with soapy water.
Thoroughly clean urethral opening with soap rather than disinfectants.		3. Rinse area with water.
Urine at room temperature supports the growth of pathogens as well as contaminants.		4. While holding labia apart, began voiding.
Transport within 2 hours. If not, refrigerate for not longer than 24 hours.		5. Collect midstream urine in wide mouth container.
Transport urine for viral cultures on ice.
	Clean catch, midstream, male	Supply the patient with clear verbal or written instructions as follows:
		1. Wash hands. 2. Retract the foreskin. 3. Clean glands with soap and water and rinse with water. 4. While holding foreskin retracted, begin voiding. 5. Collect midstream portion of urine.

	Dipslide collection (after office hours)	1. Check the dipslide for contamination and ensure that media is not dried out or fallen off. 2. Collect midstream urine as above. 3. Dip the dipslide into the urine for 2 seconds. 4. Drain off excess urine and replace the dipslide in its container. 5. If there is not enough urine to dip, syringe the urine using a sterile syringe and flood both sides of the dipslide. 6. Ensure that media is completely flooded.


	Adhesive perineal bags for urine collection in infants. (Paediatric Urine collector)	Clean urethral area with saline. Apply the adhesive perineal bag. As soon as urine is passed, transfer the urine to a sterile container using a sterile syringe. Avoid contact with the bag.	Culture must be correlated with microscopy result.

	Straight catheter or in-out catheterization	Not recommended for routine urine cultures. Procedure may introduce urethral flora into bladder. Risk of contamination is not completely eliminated by this procedure as organisms from cathetertip may still contaminate the specimen. Use when results from a clean catch is equivocal and a diagnosis is critical.
		1. Clean urethral area with soap and water. 2. Rinse with water or wet gauze pads. 3. Aseptically insert catheter into bladder. 4. Discard first 15-30 ml. 5. Collect the middle and late flow into a sterile container

	Indwelling catheter	The specimen of choice is urine collected through the sampling port. Clean catheter collection port with 70% alcohol. Use a needle and syringe to aseptically collect 5-10 ml of urine.	Routine processing of urine from patients with chronic indwelling catheters may be of no value. Large numbers of potential pathogens or colonizers are common.

	Ilea conduit urine	1. Remove the external urinary appliance and discard the contained urine. 2. Gently swab the stomal opening with 70% alcohol pad and then with an iodine solution (10% povidone iodine). Using sterile technique, insert a double catheter into the stoma. 3. Catheterise the ilea conduit to a depth beyond the fascial level. Collect the urine drained into a sterile container.

	Suprapubic aspiration of the urinary bladder	1. Wipe the skin with 0.5% chlorhexine and 70% alcohol. Allow 1 minute for drying. Apply local anesthetic if necessary. 2. Introduce a 22 gauge needle into the full bladder at the midline between the symphysis pubis and the umbilicus, 2 cm above the symphysis. 3. Aspirate the urine from the bladder and place sample in a sterile container.	Used when routine cultures are equivocal and results critical. Useful in paediatric patients when clean catch samples are difficult to obtain.


WOUND SPECIMENS	Abscess	Always remove surface exudate (with superficial micro flora) by wiping with sterile saline or wipe with 70% alcohol to decontaminate intact skin.

The name of a specific anatomic site is required. Eg. ‘conjunctiva’ or ‘cornea’ instead of ‘eye’	Open	Aspirate if possible or pass swab deep into lesion and firmly sample lesion’s advancing edge.

Distinguish between surface wounds and deep or surgical wounds where anaerobic infection is likely for the latter.	Close	Aspirate with needle and syringe. If excision done, submit a portion of abscess wall for culture.

Skin decontamination is critical.	Burns	De bride the area. Sample firmly with a swab the exudate which appears. Specimen of choice is a biopsy tissue.	Cultures of surface specimens usually represent only colonization and may be misleading. Additionally, organisms may not be distributed evenly over the burn wound, so sampling of different areas is recommended

Send a second swab for gram stain to assess the quality of the specimen for culture. Presence of leucocytes in the absence of epithelial cells represent an appropriate specimen.

The representative swab specimen is taken from the advancing margin of the lesion. Ensure that lesion margins and abscess walls are firmly sampled with the swab.	Bites and traumatic/ surgical wounds	As for abscess (open).	Do not culture fresh bites or traumatic wounds.


	Decubitus ulcers	Cleanse surface with sterile saline. If biopsy specimen is not available, vigorously swab base of lesion.


MISCELLANEOUS SPECIMENS	Intravenous catheter	1. Clean the skin around the catheter site with 0.5% chlorhexidine with 70% alcohol. 2. Allow 1 min for drying. 3. Aseptically remove the catheter and clip the distal 5 cm of the tip into a sterile container.	Acceptable intravenous catheters for semi quantitative culture (Maki or roll plate method):
			Portacath, Hickman, Broviac, umbilical, central, CVP, peripheral, arterial, hyperalimentation and Swan-Ganz.
	Dental culture	1. Cleanse gingival margin and supragingival tooth surface to remove saliva and debri. 2. Using a periodontal scaler, remove subgingival lesion material and transfer to a sterile container.




For Fungal culture	Tissue	Cover specimen with minimal sterile saline without preservative. Minute tissues such as a corneal specimen should be inoculated by the ophthalmologist into culture media before transport to laboratory.	Specimens for fungal cultures are best sent in a sterile container.


For Mycobacterial culture			When collecting specimens, avoid contamination with tap water that may contain viable or non viable saprophytic mycobacteria. This can produce false positive smear results

In general, swabs are not recommended for isolation of mycobacteria. They provide only a limited amount of material and the hydrophobicity of the mycobacteria often compromises their isolation onto solid or broth media

Microbiology Specimen Collection List