Pus , Ulcer materials , Skin specimens

Collection and transport of pus , ulcer materials , skin specimens

Specimens ought to be gathered with the aid of a scientific officer or an experienced nurse. Pus from an abscess is high-quality accrued at the time the abscess is incised and drained, or after it has ruptured naturally. When amassing pus from abscesses, wounds, or different sites, unique care ought to be taken to avoid contaminating the specimen with commensal organisms from the skin. As far as possible, a specimen from a wound need to be collected earlier than an antiseptic dressing is carried out.

In a hospital with a microbiology laboratory

1 the usage of a sterile approach, aspirate or acquire from a drainage tube up to 5 ml of pus. transfer to a leak-proof sterile container. Cotton-wool swab to collect a pattern from the infected site. Immerse the swab in a field of Amies transport medium.

2 Label the specimen and as soon as feasible deliver it with a finished request form to the laboratory. Whilst mycetoma is suspected: acquire a specimen from a draining sinus tract the use of a sterile hypodermic needle to lift up the crusty surface over the sinus establishing. This technique of specimen series has the advantages that the pus acquired is commonly free from secondary organisms and the draining granules can generally be visible really and removed for microscopical examination. Switch the pus to a sterile field.

When tuberculosis is suspected: Aspirate a sample of the pus and transfer it to a sterile box. While the tissue is deeply ulcerated and necrotic (full of dead cells). Aspirate a sample of infected material from the side wall of the ulcer using a sterile needle and syringe. Transfer to a sterile box.

Fluid from pustules, buboes, and blisters: Aspirate a specimen using a sterile needle and syringe. Switch to a sterile field. Serous fluid from skin ulcers, papillomas, or papules, that may include treponemes: acquire a drop of the exudate immediately on a smooth cover glass and invert it on a clean slide. At once deliver the specimen to the laboratory for examination via darkish-field microscopy. Skin specimens for ringworm fungi: gather and take a look. Pores and skin smears for M. leprae: collect and look .

Warning: Specimens from sufferers with suspected plague or anthrax are particularly infectious. Label such specimens high chance and handle them with care.

In a health centre for dispatch to a microbiology laboratory

1.Gather the specimen the use of a sterile cotton-wool swab. Insert it in a field of Amies transport

medium , breaking off the swab stick to allow the bottle pinnacle to get replaced tightly. While the cloth is aspirated fluid from a pustule, transfer the fluid to a sterile, leak-proof container. Stopper, and seal in a leak-evidence plastic or metallic box.

Note: It isn't always viable to move exudate from a suspected treponemal ulcer due to the fact the treponemes continue to be motile for simplest a brief time.

2 .Make a smear of the fabric on a clean slide (for Gram staining) and permit to air-dry in a securevicinity. Warmness-restore the smear .

Caution: Do no longer make a smear for transporting whilst the specimen is from an affected person with suspected anthrax or bubonic plague.

3.Send the specimens with a finished request form to reach the microbiology laboratory inside 6 hours. Commands regarding the packaging and transport of specimen.

Lab examination of Pus , ulcer and skin specimen

Day 1

1 Describe the arrival of the specimen when from a patient with suspected mycetoma or actinomycosis, record the arrival of the specimen and whether or not it contains granules.

Detection of granules

White, yellow, brown, crimson, or black granules of various size, shape, and consistency may be found in pus draining from sinuses in mycetoma (actinomycetic or fungal) and in actinomycosis. The granules are colonies of organisms. To free the granules from the pus, shake a portion of the specimen (or dressing) in sterile distilled water. Watch for a few minutes (to permit the granules to settle), get rid of the supernatant fluid, and switch the various granules to a slide. A hand magnifying lens may be required to see really the small granules.

Be aware: Identification of organisms that motive mycetoma and actinomyosi. Examine the specimen microscopically,while a swab has been used to gather the pus, inoculate the culture media first earlier than the use of the swab to make smears.

Gram smear

Make a lightly spread smear of the specimen on a slide. Allow the smear to air-dry in a secure location. Repair as defined and stain by the Gram technique.Study the smear for microorganism most of the pus cells the usage of the 40_ and 100_ objective . Look mainly for:

• Gram-positive cocci that could be S. aureus or streptococci that would be S. pyogenes or different beta-haemolytic streptococci, anaerobic streptococci, or enterococci .
• Gram-negative rods that might be Proteus species, E. coli or different coliforms, P. aeruginosa or Bacteroides species.
• Gram-positive huge rods with rectangular ends that might be C. perfringens or B. anthracis .
• Massive numbers of the pleomorphic microorganism (streptococci, Gram positive and Gram negative rods of diverse size and fusiform microorganism), related with anaerobic infections.
• Gram-positive yeast cells with pseudohyphae, suggestive of Candida albicans
• Vincent’s organisms if a tropical ulcer is suspected. These appear as Gram poor spirochaetesn(B. vincenti) and Gram terrible fusiform rods.

Culture the specimen

Blood agar MacConkey agar, cooked meat medium (or thioglycollate broth) Inoculate the specimen:

• On blood, agar to isolate S. aureus streptococci. upload a bacitracin disc if streptococci are visible in the Gram smear.
• On MacConkey agar to isolate gram-negative rods.
• Into cooked meat medium or thioglycollate broth . Cooked meat medium: this is an enrichment medium for aerobes and anaerobes. The glucose in the medium allows providing a fast boom of anaerobes .

Incubate the inoculated blood agar plate at 35–37_C in a carbon dioxide environment (candle jar) and the MacConkey agar plate aerobically. Incubate the inoculated cooked meat medium at 35–37 _C for up to seventy-two hours. subculture at 24 h, and if indicated at forty-eight hours and seventy-two hours.

Anaerobic culture

Whilst an anaerobic contamination is suspected (specimen is frequently foul-smelling), or the Gram smear indicates an ‘anaerobic mixed flora’, inoculate a second blood agar plate and incubate it anaerobically for as much as 48 hours. The anaerobic blood agar plate can be made selective by way of adding neomycin to it. At a very last neomycin concentration of 50–70 _g/ml, the majority of facultative anaerobic gram-negative rods could be inhibited. To aid detection of anaerobes, a metronidazole disc (5 μg) can also be added to the anaerobic blood plates as the majority of anaerobes display a sector of inhibition, while aerobes grow up to the disc.

Day 2 and Onwards

Study and report the cultures of Blood agar and MacConkey agar cultures look mainly for colonies that could be:

• Staphylococcus aureus
• Streptococcus pyogenes
• Pseudomonas aeruginosa
• Proteus species
• Escherichia coli
• Enterococcus species
• Klebsiella species

Anaerobic blood agar subculture and cooked meat culture search for growth that might be Clostridium perfringens, Bacteroides fragilis group or Peptostreptococcus species. C. perfringens: Grows unexpectedly in cooked meat medium with hydrogen sulphide gasoline production (gas bubbles in turbid medium) and reddening but no decomposition of the beef (saccharolytic response). On anaerobic blood agar, colonies are usually visible after 48 h incubation. Most lines produce a double sector of haemolysis (inner sector of clean haemolysis, the outer area of partial haemolysis) as proven. B. fragilis: Grows in cooked meat medium generating decomposition with blackening of the meat (foul-smelling proteolytic reaction). On anaerobic blood agar, non-haemolytic gray colonies (Gram-negative pleomorphic rods) are seen, usually within forty-eight hours. B. fragilis organization. Peptostreptococcus: Grows in cooked meat medium with the manufacturing of large amounts of hydrogen sulphide fuel. On anaerobic blood agar, Peptostreptococcus produces small nonhaemolytic

white colonies (Gram tremendous cocci) after 48 h incubation. They're resistant to metronidazole (5 _g disc). Confirming organisms are anaerobes whilst there may be a blended boom and colonies appear on the anaerobic plate that is not present on the cardio plate, affirm that the organisms are anaerobes via subinoculating the colonies on three plates of blood agar and incubating one aerobically, one anaerobically, and the third in a carbon dioxide ecosystem (candle jar). The culture of cooked meat medium lifestyle the cooked meat broth after overnight incubation and whilst indicated also at 48 h and seventy-two h, whilst the habitual plate cultures are sterile or the organisms remoted do now not correspond to the ones seen on the unique Gram smear.Culture as defined formerly.

Antimicrobial susceptibility test

Susceptibility testing can be required for S. aureus, enterobacteria, and non-fermentative Gram-negative rods. Only automatically used antibiotics must be tested. New and steeply-priced antibiotics have to best be tested on special request or whilst the isolate are proof against other capsules.

Anaerobic pathogens: Susceptibility exams need to no longer mechanically be completed on anaerobic bacteria by means of the disc diffusion method. Maximum anaerobic infections are due to the penicillin-inclined microorganism with the exception of infections originating in the intestinal tract or vagina. Such infections typically comprise B. fragilis which produces beta-lactamase and is proof against penicillins, ampicillins, and maximum cephalosporins. Such infections can be treated with clindamycin, metronidazole or chloramphenicol. Aminoglycosides don't have any pastime towards anaerobes however they may be regularly used for the remedy of sufferers who have blended infections.

Possible bacteria

Gram-positive

• Staphylococcus aureus
• Streptococcus pyogenes
• Enterococcus species
• Anaerobic streptococci
• Other streptococci
• Clostridium perfringens
• and other clostridia
• Actinomycetes
• Actinomyces israeli

Gram negative

• Pseudonomas aeruginosa
• Proteus species
• Escherichia coli
• Bacteriodes species
• Klebsiella species
• Pasteurella species

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.