1.         General Procedure For Submission Of Specimen

            Place the specimen in the appropriate STERILE container*, close tightly, and label it with the patient's name and registration number.   If the specimen is submitted on a slide, the slide must be labelled with the patient's name and registration number.  A request form that contains information on the patient, the specimen and the required examination(s) must accompany every specimen.   ALWAYS SEND THE SPECIMENS IMMEDIATELY TO THE LABORATORY.

            For despatch, all outstation specimens must be placed in a receptacle.   Seal the receptacle hermetically, otherwise close it securely. To avoid damage during transport, pack the  receptacle with sufficient absorbent material in a strong case and mark the package with the words ‘PATHOLOGICAL SPECIMEN: FRAGILE – HANDLE WITH CARE’

2.         Specimen For Bacteriological Examination: Collection, Media And Transport

2.1        Cerebrospinal fluid (CSF)

3 - 5 ml CSF is sufficient for bacteriological examination.

Specimen Collection:

1.         CSF is obtained by lumbar puncture.

2.         Place CSF in a sterile container /tube for at least 3 separate tube so that the CSF can be analysed biochemically, haematologically and microbiologically.

3.         Submit the most turbid tube to microbiology laboratory.

Transportation:

Send CSF specimen to the laboratory immediately. Do not refrigerate CSF specimen.   Only CSF for viral studies could be refrigerated or frozen.

2.2.       Conjunctival swab

When the conjunctiva is acutely inflamed, the discharge is treated as described for pus with particular attention to Neisseria  gonorrhoeae if pus is from a newborn.

Specimen collection:

1.         Obtain the specimen with a sterile, premoistened cotton or calcium alginate swab.

2.         Roll the swab over the conjunctiva before topical medications are applied.

3.         Use a separate swab for the other eye because culture of swabs from both eyes shall be done separately.

Transportation:

The swab should be transported immediately to the laboratory in Transport Media (e.g. Stuart's Media).

2.3        Ear swab

These swabs are usually taken to diagnose bacterial infection of the skin of the auditory meatus and occasionally infection of the middle ear when the eardrum has ruptured.  P. aeruginosa, S. aureus and many other aerobic bacteria frequently cause otitis externa.  These may be mixed with the normal flora such as diphtheroids and coagulase-negative Staphylococcus.

Specimen collection:

1.         Insert a sterile swab into the external auditory canal carefully until resistance is met.

2.         Rotate the swab against the ear mucosa.

Transportation:

The swab should be sent immediately to the laboratory in Transport Media (eg Stuart's Media)

2.4        Nasal swab

In cases of rhinitis with a purulent nasal discharge, detection of organisms like Neisseria meningitidis, Haemophilus sp, Staphylococcus aureus, Streptococcus pneumoniae, ß-haemolytic streptococcus and Corynebacterium diphtheriae is performed.  However it is difficult to assess the significance of S. aureus, S. pneumoniae and S. pyogenes as some healthy individuals are carriers of these organisms.

Infective granuloma and rhinoscleroma, most often involving the nose is caused by Klebsiella rhinoscleromatis.

When nose swabs are taken to detect carriers, it should be processed according to the type of organisms to be identified.

2.5.       Septic workout  for neonates

This is for the detection of possible pathogen and identification of the organisms isolated.

Specimen collection:

1.         Insert a sterile swab into the nose until resistance is met at the level of the turbinates (approximately 1 inch into the nose).

2.         Rotate the swab against the nasal mucosa.

Repeat the process for the other side.

Transportation:

The swab should be sent immediately to the laboratory in Transport Media (e.g. Stuart's Media).

2.6.       Genitalia Specimen

Infections of the genital tract are caused by a wide variety of microorganisms which are sometimes difficult to culture routinely.  In this laboratory, only the common bacterial pathogens are detected. The common specimens sent are high vaginal swab, endocervical swab, vaginal swab and urethral swab. The specimens are processed according to the clinical history and the disease suspected.

Specimen collection:

i.          High Vagina swab :

Use a speculum without lubricant. Collect secretions from the mucosa  high in the vaginal canal with sterile swab .

ii.          Endocervical swab:

Do not use lubricant during the procedure. Wipe the cervix clean of vagina secretion and mucus.  Rotate a sterile swab, and obtain exudates from the endocervical glands. If no exudates are seen, insert the sterile swab into the endocervical canal and rotate the swab.

iii.         Vaginal swab :

Use normal saline and clean vulva area. Rotate a sterile swab, and obtain exudates from the vaginal mucosa.

iv.         Urethral swab:

Female:   Collect specimens 1 hour or longer after patient has urinated. Stimulate discharge by gently massaging the urethra against  the pubic symphysis through the   vagina.  Collect the discharge with a sterile swab. If discharge cannot be  obtained, wash external urethra with betadine soap and rinse with water. Insert a urethrogenital swab 2-4 cm into the urethra, gently rotate the swab, and leave it in place for 1-2 seconds. Withdraw the swab, and put it in the appropriate transport media for culture.           

Male:   It is used primarily to detect Neisseria  gonorrhoeae.  Collect specimens at least 2 hours after the patient has urinated. Insert a thin urethrogenital swab 2-4 cm into the urethra,  gently rotate it, leave it in place for 1-2 sec, and withdraw it .

v.         Cervical swab: 

Take two swabs using a Cisco speculum, the first swab for culture and the  second  one for the preparation of a smear.   For vulvo-vaginitis of childhood,  the swab should be pushed well into the vagina without speculum and withdrawn with some discharge on it.

For Trichomonas infection, the discharge should be collected into a plain container or a swab which is then immersed in 0.5ml of saline. The fresh specimen should be examined immediately as a wet film under the microscope.  A Charcoal swab is a convenient method to collect material for culture in Trichomonas medium.

For gonorrhoea, discharge collected using a charcoal impregnated swab and should be inoculated immediately on GC medium and incubated in 10% carbon dioxide, or transported in Stuart's transport medium if direct inoculation is can not be done.

Transportation:

            The swab should be sent immediately to the laboratory in Transport Medium (e.g. Stuart's Media).

2.7.       Pus and other body fluids

Specimen collection:

1.         Pus or body fluid should be aspirated aseptically using needle aspiration.

2.         Use a sterile swab if the pus is superficial or amount is little.

Procedure for obtaining a swab is as follows:

 i.         Clean the wound with normal saline.

 ii.         Examine the wound. Take a deep sample if sinus is present.

Obtain adequate and fresh material for culture.

Transportation:

Specimen can be sent as a swab or as "liquid" pus collected in a syringe or sterile bottle.    For blood culture, blood is inoculated into a blood culture system in 1 in 10 proportion and send to IMR.

2.8.       Throat swab

The most common cause of bacterial pharyngitis is Streptococcus pyogenes, which can be found in 10 % of healthy adult and children as part of the pharyngeal flora. Diphtheric lesions and thrush  may involve the pharynx, and laboratory studies for these organisms should be performed where there is clinical evidence or suspicion of the disease.  Group C and G streptococci and Arcanobacterium haemolyticum / C. pyogenes has been implicated to cause pharyngitis, therefore they should be identified if they are present in predominant number.

Occasionally, physicians treating leukaemic and other immunosuppressed patients may request for a throat swab culture to find out the type of colonizing bacteria.  In such cases, one should identify and do sensitivity tests on all the gram-negative rods as this may assist the physician in planning emergency empirical antibiotic therapy if a major infection develops.

Gonococcal pharyngitis is now recognised, therefore request for such cultures should be indicated for the swab has to be inoculated to special media for GC culture.

Sample collection:

1.         Do not obtain throat samples if epiglottis is inflamed, as sampling may cause serious respiratory obstruction.

2.         Depress tongue gently with a tongue depressor.

3.         Extend sterile swab between the tonsillar pillas and behind the uvula.  (Avoid touching the cheeks, tongue, uvula or lips).

4.         Sweep the swab back and forth across the posterior pharynx, tonsillar areas, and any inflamed or lacerated areas to obtain clinical sample.

Transportation:

The swab should be sent to the laboratory immediately.

2.9.       Urine

Infection of the urinary tract is one of the most common bacterial diseases, and proper management requires an understanding of the number and type of bacteria involved.   Quantitative or semiquantitative analysis of  bacteria in urine is a valuable aid in the diagnosis and therapy of the disease.   Infections are usually associated with bacterial counts of 100,000 (10⁵) or more organisms per ml of urine (significant bacteriuria).  In contrast, contamination from the external genitalia, in the absence of infection, usually contributes less than  1,000 (10³) organisms per ml in properly collected and transported specimens.

Specimen collection:

Care must be taken  in the collection, storage, and transport of urine specimens for bacteriological examination.   Specimens that cannot be sent to the laboratory  within 2 hours after voiding must be refrigerated.  Bacterial count remains stable for about 24 hours under these conditions.

It is best to obtain early morning specimens.  Urine specimens can be collected by:

a.         diagnostic catheterisation

b.         clean-voided mid-stream technique

c.         suprapubic aspiration

d.         indwelling catheter

Transporation:

1. Specimen should be sent to the laboratory at once and cultured within 2 hours after collection.
2. If delay is unavoidable, specimen should be packed in ice or added with preservative (commercially available).  The  specimen should be plated within 24 hours after collection.
3. Less than 5% of urine which are properly collected and transported contain multiple organisms except  specimen from patients with neurogenic bladder or chronic indwelling catheters, in which polymicrobial bacteriuria may be detected in 30-80%.

2.10.     Examination For Fungal Infection: Collection, Media And Transport

Specimen:  Refer to Table 1 for types of specimen required and transport container.

Table 1: Specimen Required And Transport Container

Reference:

Medical Mycology. Evans, EGV &MD Richardson (eds). IRL Press, Oxford (1989)

Refer to Table 2 for undesirable specimen for fungal isolation.

Table 2: Undesirable Specimen

Reference:

Practical Lab Mycology. Koneman EW and Roberts GD (eds). Williams and Wilkins, Baltimore (1985)

2.11      Examination For Anaerobic Bacterial Infection: Collection, Media  And Transport

Anaerobic bacteria cause a variety of infections in humans, including appendicitis, endocarditis, meningitis, osteomyelitis, septic arthritis, bacteremia, and wound infection. Fifty to sixty percent of important infections is associated with anaerobic bacteria.

Anaerobes vary in their sensitivity to oxygen: some anaerobes are killed after a brief exposure (10 min) to atmospheric oxygen. They also vary in their nutritional requirements. So, proper collection, media, and incubation are vital to the recovery of anaerobes. Knowing which anaerobes have been isolated helps the physician to decide on treatment of patients with anaerobic infection.

This manual outlines practical procedure that can be used to isolate and identify the clinically important anaerobes from properly collected specimens.

Specimen Collection

Principle

Specimens should be collected properly to avoid contaminating them with normal flora.

Abscess:         

            Aspirate material with needle and syringe after the surface of intact tissue is disinfected with a povidone-iodine wash that remains on the surface for at least 1 min.    When needle use is contraindicated, aspirate material through a plastic catheter or directly into the syringe.

Sinus tract or deep wound drainage:

Aspirate material with a small flexible plastic catheter and syringe after proper disinfection of skin surface, or  collect curettings of material from deep within the tract or wound.

Decubiti and other surface ulcers:

Aspirate material by needle and syringe after thorough disinfection of the surface area, collect small curettings of materials from deep tissue at wound margin.

Other situations:

In some case, when aspiration or biopsy is not feasible, anaerobic swab could be used and accepted for anaerobic culture.

Note:    Anaerobic swab are the least desirable specimen because:

- small volume

- greater chance of contamination with normal flora

- excessive dryness

- bacterial adherence to cotton fibres

- poor gram stain quality.

Reference:      

Clinical Microbiology Handbook. American Soc. Microbiology    (Isenberg, H.D. ed. in chief). 1992.

Specimen transport and preservation

Principle

Anaerobic bacteria are sensitive to exposure to oxygen. Thus, anaerobic specimen should be transported in air-free container. Anaerobic transport media, if used, will avoid direct contact of air on the specimen and keeps the specimens under low redox potential condition.

Specimens aspirated by needle and syringe:

May be transported directly to the laboratory provided that all air is expelled and the needle is inserted into a rubber stopper.

Fill completely a small sterile tube/vial with screw cap thus displacing all air before the cap is screwed on tightly.

Inject the specimen immediately after collection into either evacuated sterile bottle such as ‘Vacutainer’ or bottle containing transport media.

For tissues:

Drop quickly into bottle/tube containing an anaerobic transport medium. Big tissue should be placed in sterile container and transported to the lab. in an anaerobic jar.

All swabs must be transported in anaerobic transport media.

All specimens should be transported as soon after collection as possible. When delay is unavoidable, leave the specimen between 15^o C to room temperature. Suggested optimum transport time for certain specimen volume are as follows:

Reference

Clinical Microbiology Handbook. American Soc. Microbiology.(Isenberg, H.D. ed. in cheif). 1992

3.         Detection of DNA of Mycobacterium tuberculosis Complex by Polymerase Chain Reaction(PCR)

The detection of DNA of Mycobacterium tuberculosis complex in clinical specimen is one of the ways to diagnose M. tuberculosis infection.  By using the polymerase chain reaction (PCR) and DNA probes, the genes encoding the IS 986 of Mycobacterium tuberculosis complex can be detected.

Specimen Collection and Transportation

The test can be performed on tissue, sputum, CSF, pleural fluid, gastric lavage, pus, ascitic fluid, urine, and bile aspirate.   About 1 to 2 ml of the specimen is required for the test. All specimens must be collected in sterile containers.  The number of specimens required depends on the type of infection, quality of the specimen and host factors such as previous or concomitant use of antibacterial drugs. 

Specimen must be placed in a sterile tube.  Swabs should be used only if the specimen cannot be collected by any other method.  When swabs are used, they must be placed in a sterile container without transport media.  All specimens must be sent immediately to the laboratory.   The physicians/hospitals will be notified if the specimen sent is not suitable for the test.   All tissue samples must be transported to the laboratory in ice. Other samples may be sent at room temperature. 

i.          Abscess

Ideally, an abscess should be sampled before it ruptures or is drained.  The skin is disinfected with 70% alcohol followed by an iodine preparation. It is then aspirated with a needle and syringe.  An additional sample is taken from the wall of the abscess.  The specimens collected during surgery are obtained in the same way.  Abscess material should never be collected on a swab because the amount of specimen will be inadequate and the swab itself will inhibit the activity of  Taq polymerase.

ii.          CSF and other