Guide to Specimen Collection for Microbiology

Transfer pleural, pericardial and synovial fluids aspirated aseptically into a sterile universal container and send immediately to the laboratory.

a) Samples should be collected before the initiation of therapy wherever possible.

b) Repeat specimen collection if the initial examination is negative. If possible, collect and examine three specimens at 2-3 day intervals. No more than one specimen should be examined within a 4-hour period, as the shedding of cysts and ova tends to be intermittent.

c) If E. histolytica or G. lamblia are suspected and the first three specimens are negative, it is recommended to submit three additional specimens at weekly intervals.

d) Ensure the sample is free from contamination by urine or water.

e) The container should not be overfilled. It should be no more than half full (approximately up to 10 grams of stool).

f) Samples should be transported promptly to the laboratory, accompanied by the completed requisition form.

1. The external meatus is cleaned with a dry swab moistened with sterile saline. Let the site dry before sampling.

2. Pass a swab gently into the external canal and collect whatever exudate that is found there.

3. Replace the swab in its carrier-tube (Amies Transport Medium) and send immediately to the laboratory.

1. Collect the exudate with a sterile swab.

2. Inoculate the swab into Amies Transport Medium and send to the laboratory

1. Using a scoop, collect a small amount of stool (1/4 volume of container), taking care to include materials containing pus, mucus or blood if present.

2. Place the stool into a sterile container, screw the cap tightly and send it immediately to the laboratory.

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

Notes: 

• Do not contaminate stool with urine 

• For stool clearance culture in case of typhoid, stool should only be sent upon completion of therapy.

Insert a sterile swab deep into the anus so that the swab may come into contact with some faecal material. A satisfactory rectal swab is one which show some faecal staining. Inoculate the swab into Amies Transport Medium and send to the laboratory.

Note:

• This sampling method is only for CRE screening. It is a less satisfactory specimen than stool

For bone marrow aspirate, a sufficient amount of aspirate is required and to be inoculated directly into the blood culture bottles. If unable to get bone marrow, trephine is acceptable and to be collected in a sterile container (Do not put formalin if the sample is sent for culture and susceptibility). If using a commercial blood culture bottle, the procedure for inoculation of the sample into the bottle is the same as Blood sample for C&S. (Please refer to the test list to view more regarding the volume etc).

Method of collection:

1. Before venipuncture, the skin must be carefully disinfected with 2% chlorhexidine in 70% alcohol.

2. Clean the tops of the bottle with 70% alcohol.

3. Inoculate the specified volume of bone marrow aspirate into each bottle.

4. Do not store the specimens in the refrigerator.

This is only done for screening of MRSA carriers.

1. The swab needs to be moistened with sterile saline before sampling. 

2. Swab both the anterior nares (same swab) and insert the swab into the nasal cavity until resistance is met at the level of turbinate (approximately 1 inch into the nose) and gently rotate against the nasal mucosa. Replace the swab in its carrier-tube and send the specimen to the laboratory immediately.

Throat swabs are obtained to isolate organisms for example Group A Streptococcus and Corynebacterium diphtheria.

1. Insert swab carefully through the mouth with the tongue depressed.

2. Rub the swab over each tonsillar area and the posterior pharynx. Any area with exudate should be touched.

3. Do not touch tongue or lips.

4. Place the swab in its carrier-tube (Amies Transport Medium) and send the swab to the laboratory immediately.

5. If diphtheria is suspected:

   • Lift edge of the membrane and swab under it to search for deeply located diphtheria organisms.

   • Insert the swab into Amies Transport Medium and transport immediately. 

   • Please alert Microbiology team upon sending sample suspected of diphtheria.

1. Collect the sputum early in the morning. 

2. Ask the patient to rinse the mouth, then cough deeply and spit directly into a sterile container. Ensure that the expectorate is sputum and not saliva. 

3. Send the specimen immediately to the laboratory. 

Notes:  

• Send nasopharyngeal aspirate, transtracheal aspirate or lung aspirate whenever possible or indicated. These specimens are more representative of the lower respiratory tract and are devoid of contaminants from the mouth.

• Salivary sputum is unsuitable to proceed with culture testing.

Male patients

1. Withdraw the prepuce and clean thoroughly the glans penis with water.

2. Pass the first part of voided urine to flush out the bacteria from urethra, then collect the midstream portion in a sterile boric acid container according to required volume level or sterile container and close it tightly. Insufficient volume of specimen collected may cause rejection by laboratory.

3. Send specimen immediately to the laboratory within 2 hours of collection. Transport urine immediately to the laboratory after collection. If urine cannot be delivered to the laboratory within 2 h after collection, refrigerater for up to 24 h both during the holding period and during transport. Do not freeze. 

Female patients

1. Clean the periurethral area and perineum thoroughly with water.

2. Hold the labia apart and pass the first part of voided urine.

3. Collect the midstream portion in a sterile boric acid container according to required volume level or sterile container and close it tightly. Insufficient volume of specimen collected may cause rejection by laboratory.

4. Send specimen immediately to the laboratory within 2 hours of collection. Transport urine immediately to the laboratory after collection. If urine cannot be delivered to the laboratory within 2 h after collection, refrigerater for up to 24 h both during the holding period and during transport. Do not freeze.

1. Catheter urine specimens should be taken by aseptic puncture of the catheter conduit (clean the catheter collection port with 70% alcohol wipe) and syringe out into a sterile container. 

2. Urine from collection bag is unsuitable for culture.

3. Send specimen immediately to the laboratory within 2 hours of collection. Transport urine immediately to the laboratory after collection. If urine cannot be delivered to the laboratory within 2 h after collection, refrigerater for up to 24 h both during the holding period and during transport. Do not freeze. 

Note:

1. Reject: Urinary catheter tip is unsuitable for culturing as it is invariably contaminated with urethral organisms.

1. This is obtained via suprapubic aspiration (aseptic technique - disinfect the skin with 2% chlorhexidine in 70% alcohol.) or cystoscopically.

2. Urine is collected in a sterile container.

3. Send specimen immediately to the laboratory within 2 hours of collection. Transport urine immediately to the laboratory after collection. If urine cannot be delivered to the laboratory within 2 h after collection, refrigerater for up to 24 h both during the holding period and during transport. Do not freeze. 

1. Cleanse the superficial area thoroughly with sterile saline, changing sponges/gauze with each application. Remove all superficial exudates and necrotic tissues. 

2. Collect biopsy (3-4mm) or curette sample from base or advancing margin of lesion (leading edge of the lesion) - where pathogen should be present, and colonizing organisms are less likely to occur. Do not send slough/necrotic tissue for culture.

3. Collect tissue specimens and aseptically transfer into a sterile container.

4. Do not add formalin solution, if not, the specimen will be rejected.

5. Send immediately to the laboratory.

Note: 

• Slough/ necrotic tissue are unsuitable for culturing and will be rejected.

1. Disinfect the skin over the inflamed area with 2% chlorhexidine in 70% alcohol.

2. With a sterile syringe, aspirate the pus or exudates and transfer the pus into a sterile container.

3. Send the specimen immediately to the laboratory.

1. Using a sterile speculum lubricated with sterile normal saline and not antiseptic cream, swab either from the posterior fornix or the lateral wall of the vagina.

2. Inoculate the swab into amies/ charcoal transport media and send the specimen to the laboratory as soon as possible.

1. Collect fresh stool (approximately 5g) in a sterile container.

2.  Send to the laboratory immediately.

1. Collect fresh stool (approximately 5g) in a sterile container.

2. Send to the laboratory immediately.

1. Collect fresh stool (approximately 5g) in a sterile container.

2. Send to the laboratory immediately.

Acceptable specimen: Sputum, ETT aspirate, Gastric lavage (paediatric only), respiratory secretions,  urine, CSF and body fluids. Swab specimens and Stool are NOT acceptable.

1. For sputum: Collect a minimum of 3 early morning sputum / spot specimen (1 specimen per day). 

2. Collect in a sterile container.

3. If blood specimen: Withdraw 1-5 ml of blood and put into Myco F Lytic Blood Bottle under aseptic technique.

A sample from the bag is obtained as follows:

1. Disinfect the port of the bag with 70% alcohol.

2. Collect at least 30ml of fluid through the disinfected area using a needle and syringe.

3. Place the sample into a sterile container / and inject 8-10 ml in each Blood culture bottle (Aerobic and Anaerobic).

4. Send to the laboratory immediately.

1. Remove superficial debris by thorough irrigation and cleansing with non-bacteriostatic sterile saline

2. Gently roll swab over the surface of the wound approximately 5 times, focusing on area where there is evidence of pus or inflamed tissue

3. Surface swabs of deeply infected lesions (e.g: sinus tracks from osteomyelitis, pressure sores) usually grow surface contaminants like Coliforms and Pseudomonas.

Notes: 

Wound swab specimens are generally discouraged. Whenever possible please sent tissue specimen

Microfilariae exhibit a marked periodicity depending on the species involved, therefore the time of specimen collection is critical. If a filarial infection is suspected, the optimal collection time for demonstrating microfilariae is at night, 6 pm to 12 am.

Preparation of smear

1. Collect a big drop of blood by pricking a finger. Blood collection must be taken after 6 pm to 12 am.

2. Make an oval thick blood film size ~ size 2x3 inches on a clean glass slide.

3. Dry it in a horizontal position and protect it from dust and pests.

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

Notes

Reject if smear was sent out of recommended time frame.

1. Send BI test tubes that has gone through the autoclaving cycle together with untreated BI (Control) to the laboratory.

2. Ensure both the BI TEST and BI CONTROL are from the same batch.

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. Prior cleaning is essential if ointments, creams or powders have been applied to the lesion. Skin, nails and hair specimen should be collected into folded squares of paper or directly onto agar plate.

1. Skin

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

2. Hair

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

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

3. Nails

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

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

Scrapping of materials from the ear are to be preferred, although swabs can also be used. 

1. Material from patients with suspected fungal infections of the cornea (keratomycosis) should be collected by scrapping the ulcer. The entire base of the ulcer, as well as the edges, should be scrapped. (Swab is not suitable for sampling corneal lesions).

2. The material is collected directly onto agar plates for culture and to a glass slide for microscopic examinations (Please refer to list of test for more information).