Sputum Collection,Processing and Culture

Sputum for microbiological investigation is accumulated and transported as follows

In a clinic with a microbiology laboratory

1 Provide the patient an easy (need not be sterile), dry, huge-necked, leak-proof container and request him or her to cough deeply to provide a sputum specimen. Accrued, adequate safety precautions must be taken to prevent the spread of infectious organisms and to avoid contaminating the outdoor of the field. Use a phenol-containing disinfectant to wipe the out of doors of the container after gathering the specimen. The specimen ought to be sputum, now not saliva. Sputum is first-class accrued the morning,soon after the patient wakes and before any mouth-wash is used. Whilst pulmonary tuberculosis is suspected, up to 3 specimens may additionally need to be examined to come across AFB.

2 Label the container, and fill a request form.

3 Whilst pneumonia or bronchopneumonia is suspected, deliver the sputum to the laboratory with as little delay as feasible due to the fact organisms such as S. pneumoniae and H. influenzae require culturing as soon as possible. When pneumonic plague is suspected:,deliver the sputum to the laboratory as soon as possible. Make certain the specimen is marked excessive hazard.

In a health center for dispatch to a microbiology laboratory

1.Accumulate the sputum in a container provided by means of the microbiology laboratory. Follow the approach and examine the precautions referred to beneath the health facility series of sputum.

2.To make certain the survival of pathogens such as S. pneumoniae and H. influenzae, transfer a purulent a part of the sputum to a cotton-wool swab, and insert it in a box of Amies medium. Label the container using a lead pencil.

3.Send the sputum specimen and swab with a request form to attain the microbiology laboratory inside 6 hours.

Laboratory examination of sputum

On the first day

Specimens for immediate interest:

Whilst pneumonia or bronchopneumonia is suspected, the sputum must be cultured as quickly as feasible due to the fact organisms inclusive of H. influenzae and S. pneumoniae do no longer survive nicely in specimens. Warning: whenever viable, sputum specimens must be examined in a biological safety cupboard.

Specimen

Describe whether the sputum is: Purulent: inexperienced-looking, basically pus Mucopurulent: green-searching with pus and mucus Mucoid: primarily mucus Mucosalivary: Mucus with a small quantity of saliva while the sputum consists of blood, this ought to additionally be stated.

Examine the specimen microscopically

Gram smear

The use of a chunk of the stick, switch a purulent part of the sputum to a glass slide and make a skinny smear. permit the smear to air-dry in a safe location. repair and stain through the Gram technique. Observe the smear for pus cells and fundamental bacteria. appearance especially many of the pus cells for:

• Gram-positive diplococci (capsulated) that would be S. pneumonia. The be hard to distinguish from the ordinary microbial plant life.
• Gram positive cocci in groups that would be S. aureus , but now not often seen.
• Gram negative rods and cocco-bacilli that would be H. influenza . specially when these are the principal organisms.
• Gram-negative capsulated rods that might be ok. pneumoniae, but no longer frequently visible.
• Gram negative diplococci in and between pus

Cells that might be M. catarrhalis .Gram stained smears of sputum must be said with the warning. Cocci, diplococci, streptococci, and rods may be visible in ordinary sputum because those organisms are part of the normal microbial flora of the above respiration tract. Note: while pus cells are present however no bacteria are visible in a Gram-stained smear, this can suggest the presence of microorganisms inclusive of M. tuberculosis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophilia or viruses.

Ziehl-Neelsen smears to detect AFB

Research have proven that the chances of detecting AFB in sputum smears are significantly expanded when sputum is first dealt with five% vv sodium hypochlorite (NaOC1), i.e. bleach, accompanied by centrifugation or in a single day sedimentation . Because NaOC1 kills M. tuberculosis, the NaOC1 awareness approach is likewise safer for laboratory staff. NaOC1 handled sputum can not be used for subculture. Sodium hypochlorite centrifugation technique to concentrate AFB.

1. Transfer 1–2 ml of sputum (particularly that which incorporates any yellow caseous cloth) to a screw-cap prevalent bottle or another field of 15–20 ml capacity. Caution: Open specimen bins with care and at hands period to keep away from inhaling infectious aerosols. while to be had, take care of the specimen inner a protection cabinet.

2. Upload an equal quantity of concentrated sodium hypochlorite (bleach) answer and blend nicely.

3 .Depart at room temperature for 10–15 minutes, shaking at intervals to interrupt down the mucus in the sputum.

4..Add about 8 ml of distilled water, or while unavailable use boiled filtered rain water. Blend well.

5.Centrifuge at 3000 g for 15 minutes or at 250–one thousand g for 20 minutes. notice: while a centrifuge isn't always geared up to take common boxes, divide the specimen between two conical tubes (which may be capped). when centrifugation is not feasible, depart the NaOC1 handled sputum to sediment in a single day.

6.The usage of a tumbler Pasteur pipette or plastic bulb pipette, eliminate and discard the supernatant fluid. Blend the sediment,when tubes have been used, combine the 2 sediments. switch a drop of the well-combined sediment to a smooth scratch-loose glass slide. Spread the sediment to make a thin instruction and permit to air-dry.

7.Heat-fix the smear and stain it the use of the Ziehl- Neelsen method. Study it microscopically for AFB. M. tuberculosis in Ziehl-Neelsen stained sputum smears . Incubate the blood agar plate aerobically and the chocolate agar plate in a carbon dioxide enriched surroundings .

Something extra

Culture of sputum for M. tuberculosis

Culturing and susceptibility checking out of M. tuberculosis are typically undertaken in a Tuberculosis Reference Laboratory, especially for surveillance functions, to decide ranges of drug resistance, and to control remedy screw ups and relapses .

Culture of sputum when pneumonic plague is suspected

For study and document, the cultures ,Blood agar, and chocolate agar cultures appearance specially for a substantial culture of Streptococcus pneumoniae sensitive to optochin,Haemophilus influenza,Staphylococcus aureus,

Much less often isolated pathogens

Klebsiella pneumonia., Pseudomonas aeruginosa,Streptococcus pyogenes,Proteus species,Candida albicans.

Antimicrobial susceptibility test

Susceptibility assessments must be carried out only whilst the amount of cultural growth of a pathogen is tremendous. Lines of S. pneumoniae ought to be examined on blood agar for susceptibility to penicillin, tetracycline, and erythromycin. Penicillin susceptibility is high-quality decided the use of an oxacillin 1 _g disc. A region size less than 20 mm shows decreased susceptibility. H. influenzae lines ought to be examined for beta-lactamase production . and susceptibility to ampicillin, tetracycline, and co-trimoxazole. Susceptibility test of S. aureus strains.

Possible pathogens in Sputum

Gram positive bacteria

• Streptococcus pneumonia .
• Staphylococcus aureus .
• Streptococcus pyogenes .

Gram-negative bacteria

• Haemophilus influenza
• Klebsillae pneumonia
• Proteus species
• Yersina species
• Moraxella catarrhalis

Also Mycobacterium tuberculosis, Mycoplasma pneumoniae, and Legionella pneumophila.

References:

D greenwood, Slack RCB and J Peutherer.Medical microbiology.2001.

JG College, AG Fraser and BP Marmion.Practical Medical microbiology.Fourteenth Edition. Churchill Livingstone, 1996.

JP Micheal, ECS Chan and NR Krieg.Microbiology.Fifth Edition. Delhi: Mcgraw-hill, 1993.

M Cheesbrugh.Medical laboratory manual for tropical countries.London, 2007.