Pus Sample: Collection, Processing, Staining and Culture

This post was most recently updated on June 22nd, 2019

Skin is the body’s largest and thinnest organ which serves as an anatomical barrier between the sterile internal organs and the external environment which is teemed with microorganisms. Break in the skin surface may results into skin and soft tissue infections. Wound infection can also occur as a complications of surgery, trauma, and bites or diseases that interrupt a mucosal or skin surfaces.

Wound infections may be caused by one to many organisms depending on the site of the infection. For example, dermatophytes are responsible for infections in the keratinized layer; superficial skin wounds are often caused by aerobes only while anaerobes are commonly isolated from abscesses of the perineal, inguinal, and buttock area, whereas non-perineal infections are caused by mixed facultative aerobic organisms. Similarly, postoperative wound are often infected with a mixture of aerobes and anaerobes, while deep wound infections such as internal body or organ infections can be caused by one or several aerobes and/or anaerobes.

Abscesses are accumulations of pus in tissue and any organism isolated from them may be of significance.

Wounds especially postoperative wounds may become colonized with potential pathogens. A gram stain is a useful diagnostic tool in determining colonization versus infection. A gram stain showing few or no polymorphonuclear cells with relatively large amounts of normal skin flora are consistent with colonization. However, wound gram stains showing moderate to many polymorphonuclear cells usually are indicative of infection.

Possible pathogens in Pus

Gram positive Gram negative
Staphylococcus aureus Pseudomonas aeruginosa
Streptococcus pyogenes Escherichia coli
Enterococcus species Proteus species
Anaerobic streptococci Klebsiella species
Other streptococci Bacteriodes species
Clostridium perfringens and other clostridia Acinetobacter species
Actinomycetes Other enteric bacilli
Mycobacterium tuberculosis  
Others
Fungi: Histoplsama, candida and fungi that cause mycetoma
Parasites: Entamoeba histolytica (in pus aspirated from amoebic liver abscess)
Viruses: Pox viruses and herpes viruses

NOTE: This blog post contains information ONLY about isolation and identification of common bacterial isolates (aerobes and facultative anaerobes) from pus aspirate/swab.

Specimen Collection

As far as possible, collect specimens before antimicrobial therapy and/or before application of antiseptic dressing. The ideal specimen is an aspirate from a previously undrained abscess, or a tissue biopsy. Ideally, a minimum volume of 1mL (up to 5 mL) of pus should be collected. Large volumes of purulent material maintain the viability of anaerobes for longer.

The aspirate should be collected in a sterile syringe – any air bubbles should be expelled. Needle safely and tightly capped (needles should NOT be sent).

A tissue specimen should be placed in a sterile universal bottle (or any sterile leak proof container) and sent to the lab for immediate processing if anaerobes are suspected. If there will be a delay in transporting, the tissue should be placed in an anaerobic transport system.

Comparison of aspirated pus and swab culture from an infected joint site. Image source: Koneman’s Color Atlas and Textbook of Diagnostic Microbiology

Swabs are less desirable because of the smaller amount of specimen that is sampled and the fact that they are often contaminated with normal skin flora, making interpretation of results difficult. When using swabs, the deepest part of the wound should be sampled, avoiding the superficial microflora. Swabs should be well soaked in pus.

Specimen Transport:

Label the specimen and deliver it to the laboratory as soon as possible with a completed request form. The volume of specimen and the nature of the suspected organism influences the acceptable transport time. The recovery of anaerobes is compromised if the transport time exceeds 3hr. If delays in transportation to the laboratory are unavoidable sample should be placed in the transport medium (Amies transport medium or Cary-Blair transport medium) to minimize drying and minimize exposure to oxygen if anaerobes are suspected.

If processing is delayed, refrigeration is preferable to storage at ambient temperature.

Laboratory examination of Pus sample

  • Describe the appearance of the specimen: Describe presence/absence of sulphur granules (needed only for the suspected cases of mycetoma or actinomycosis, when requested).
  • Preparation of the Smear
  • If Pus swab is sent:
    • Only one aerobic pus swab: Inoculate the culture media first before using the swab to make smears for Gram staining
    • If swabs (one anaerobic and two aerobic) are submitted for culture, use the second swab for making gram stain
  • If tissue sample is submitted: make Gram stain from ground tissue.
  • Is pus aspirate is sent: using a sterile pipette place one drop of pus on to a clean microscope slide. Spread this using a sterile loop to make a thin smear for Gram staining.
  • Gram Staining: Make an evenly spread smear of the specimen on a clean, grease-free slide. Allow the smear to air-dry in a safe place. Heat fix the specimen and stain by Gram staining technique. Examine the smear for the presence of bacteria and pus cells (PMNs) using 100x objective lens and look especially for:
    • Gram negative rods ( Possible pathogens are E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus or Bacteroides species)
    • Gram positive cocci in pairs, chains or clusters (possible pathogens are Staphylococcus aureus, Streptococcus pyogenes, anaerobic streptococci or enterococci).
    • Gram positive large rods with square ends (possible pathogens are Clostridium perfringens or Bacillus anthracis)
    • In the case of anaerobic infections large number of pleomorphic bacteria (streptococci, Gram positive and Gram negative rods of various size and fusiform bacteria) may be seen. Sometimes, Gram positive yeast cells with psuedohyphae may be seen, which can be Candida albicans.
Staphylococcus in Gram Stain

Pus Culture

Culture Media:  Wound specimens collected on aerobic swabs or pus aspirate should be plated on to the following media:

Incubation Condition:

  • Temperature: 35ºC -37ºC
  • Atmosphere: Blood Agar Plate in carbon dioxide enriched atmosphere (e.g. 5% CO2 incubator or in a candle jar) and MacConkey agar plate in ambient air (normal incubator)
  • Time:  Up to 48 hours (Observe the plate after 24 hours of incubation, if growth is seen do further processing, if not re-incubate for additional 24 hours.)

Examination and Reporting the Culture results

If the growth is seen after 24/48 hours of culture, examination of the colony morphology and identification of the isolates should be done.

In the Blood Agar plate look for the hemolysis. Both Staphylococus aureus and Streptococcus pyogenes gives beta-hemolysis in Blood Agar (Some S. aureus isolates may not show hemolysis).

  • S. aureus gives yellow to cream or white colonies. Colonies are slightly raised and easily emulsified.
  • S.pyogenes produces beta-hemolytic colonies. Colonies are usually small, colourless, dry, shiny or mucoid.
  • Enterococci gives non-hemolytic colonies in blood agar. 

We can differentiate between streptococci and staphylococci by a very simple and rapid test-Catalase test (Staphylococcus-positive, streptococcus-negative). For identification of suspected S. aureus colonies perform coagulase test (to differentiate Coagulase negative Staphylococci from S. aureus) and for suspected Group A Streptococci (S.pyogenes) preform bacitracin sensitivity test (can be added in the blood agar plate with other antibiotics). If enterococci is suspected perform bile esculin test.

Look for growth of lactose fermenter colonies (pink) or non-lactose fermenter colonies (pale) in MacConkey Agar plate.  Lactose fermenter colonies can be of Escherichia coli, Klebsiella spps or Enterobacter spps and non-lactose fermenter colonies can be of Psuedomonas aeruginosa, Acinetobacter spp, Proteus spps etc. 

LF and NLF colonies in MacConkey Agar

Member of the family of the Enterobacteriaceae can be differentiated from other Gram negative bacilli by performing two rapid tests (catalase test +ve, and oxidase test –ve). Identifications of the enteric bacteria can be done by using biochemical tests such as Citrate utilization test, Triple Sugar Iron (TSI) Agar test, Sulphite-IndoleMotility (SIM) test, and Urease test.

Pseudomonas aeruginosa gives large, flat, spreading pale colored colonies in MacConkey Agar. It is oxidase positive and can be identified by its pigments and/or distinctive smell (characteristics fruity smell).

Depending on the facilities available in the diagnostic laboratories, organisms can be identified using enterotube test or API-20E test or other newer diagnostics test available for the identification of isolates.

Antimicrobial Sensitivity Testing

For Streptococcus pyogenes and Enterococci antimicrobial sensitivity testing should be done in MHA supplemented with sheep blood. For the S.aureus and other gram-negative bacilli, Mueller-Hinton Agar (MHA) is used. The selection of the antibiotics panel depends on the isolated organism. Unless indicated routinely used (or first line) antibiotics should be used. If the patient is in Intensive Care Unit (SICU, PICU, NICU) or is receiving particular antibiotics, or the isolate is resistant to first-line antibiotics sensitivity testing should include requested antibiotics and/or second line antibiotics.

References and further reading

  • Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
  • Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  • District Laboratory Practice in Tropical Countries Part-2. Monica Cheesbrough, 2nd edition
About tankeshwar 387 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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