Microbiology sampling: A guide for veterinarians who want reliable results

Microbiological diagnostics rests on one fundamental principle that is frequently underestimated in everyday clinical practice: a result is only as reliable as the sample was good. Even the most advanced laboratory equipment cannot compensate for a poorly collected, contaminated or improperly transported sample. It is precisely for this reason that VetPro wishes to share concrete, practical guidelines that will help ensure every sample reaching our analysts carries genuine diagnostic potential.

This is not a formal list of instructions. This is a practical guide written by colleagues, for colleagues.

General sampling rules: what always applies

Before we address individual sample types, there are three principles that must be met regardless of which material you are sampling:

1. Sample as early in the course of illness as possible

The early stage of an infectious process means a higher bacterial or fungal burden in the sample, which translates into a greater chance of successful isolation and identification of the causative agent. The longer you wait, the more difficult it becomes to distinguish between pathogens and commensal flora.

1. Sample before antibiotic therapy or allow an interval

This is probably the most common cause of false-negative findings in microbiology. Even brief antibiotic use of just 24 to 48 hours can suppress growth of the causative agent to such a degree that the culture remains sterile, even though the infection is clinically apparent. If the patient is already on therapy, collect the sample only after a break of 7 to 10 days. If the clinical situation does not allow therapy to be interrupted, state this clearly on the request form, as it helps us in interpreting the findings.

1. Minimise contamination

Any sample containing flora from the environment or from skin carries a risk of false-positive findings or masking the true causative agent. Aseptic technique is not just a rule — it is a prerequisite for meaningful diagnostics.

Aerobic culture: swabs and surface material

For aerobic bacterial culture, samples are collected using sterile swabs. The sampling technique varies depending on the nature of the lesion:

• For dry skin lesions (ulcers, crusts, excoriations), it is necessary to first moisten the affected area with sterile saline. A dry surface yields fewer bacteria and often does not reflect the microbiome of the active lesion.
• After sampling, the swab is stored at refrigerator temperature (2–8°C). The sample is stable for up to 48 hours.
• If there is a delay of more than 2 hours before submission, moisten the tip of the applicator with two or three drops of sterile saline. This prevents desiccation and loss of viability of bacteria that depend on a moist environment.

One practical detail that is often overlooked: do not sample the swab only from the surface of the visible lesion. If there is secretion from the depth, or the edges of the lesion show active inflammation, these zones carry the greatest diagnostic yield.

Anaerobic culture: tissues and body fluids

Anaerobic causative agents require a special approach, for one fundamental reason — oxygen is lethal to them. Any contact of the sample with air reduces the chance of successful isolation.

Several key points:

• Samples are primarily tissues and body fluids, not swabs. Swabs are acceptable only if immediately immersed in an appropriate anaerobic transport medium.
• Refrigerator temperature is contraindicated for anaerobic samples. Unlike aerobic samples, anaerobic organisms deteriorate at low temperatures. The sample is stored and transported at room temperature.
• Minimise contact with air: use hermetically sealed containers, anaerobic transport containers, or submit the sample in a syringe with the tip sealed.

Blood culture: strict asepsis as a sine qua non

Blood culture is a diagnostic tool that demands the highest level of care during sampling. Contamination of the sample with skin flora is one of the most common causes of false-positive findings and often leads to unnecessary or incorrect antibiotic therapy.

Key guidelines:

• Sampling strictly before antibiotic therapy. This is an absolute priority. After therapy has begun, the chance of a positive blood culture falls dramatically.
• Thoroughly clean and disinfect the skin at the venepuncture site before sampling.
• Transfer blood directly into the bottle with special blood culture medium, changing the needle.
• Disinfect the rubber cap of the bottle with alcohol before use.
• Clotted blood is an unacceptable sample as it cannot be processed.
• Never add anticoagulants to blood for blood culture. Many anticoagulants have antibacterial activity (particularly EDTA) and can inhibit the growth of causative agents.

Respiratory tract samples: sputum and lavage

Diagnosis of respiratory infections by microbiological methods requires an understanding of one anatomical limitation: animals cannot cough up sputum on command.

• Tracheal and bronchial lavage (BAL — bronchoalveolar lavage) is the only acceptable substitute for sputum. It is performed under general anaesthesia, and aerobic bacteria including Mycoplasma (PCR) can be isolated from the wash fluid obtained.
• Throat swabs and saliva are unacceptable samples when pneumonia is suspected. They reflect the flora of the upper respiratory tract and commensal microorganisms of the oropharynx, not the causative agents in the lung parenchyma.
• For details about the sampling procedure, contact us before submission — we are happy to walk you through the protocol.

Pleural and peritoneal fluids, abscesses and CSF

Samples from sterile body cavities are collected aseptically, with a needle and syringe. Such material is acceptable for both aerobic and anaerobic bacterial culture, as well as mycological analyses.

A practical note for transport: you can submit the sample directly in the syringe used for puncturing, but without the needle and with the syringe tip sealed over a flame. This ensures a hermetic seal and prevents contamination and loss of anaerobic organisms.

We would particularly emphasise: material from abscesses is often extremely rich in anaerobic causative agents. It is precisely these samples that require the fastest transport and the most careful attention to maintaining anaerobic conditions.

Skin and hair samples

For isolation of pathogenic bacteria from skin, a valid sample is a swab from the surface of the affected area. Always collect some hair from the same zone. Dermatophytes and certain bacterial pathogens colonise follicles and the hair root, so a swab without hair may be false-negative.

For chronic skin lesions, it is important to sample the edge of the active lesion, not the centre of a crust or area of necrosis. The centre of an old lesion generally has a low yield of viable causative agents.

Faeces

Faeces is a valid sample for isolation of enteropathogenic bacteria, particularly when Salmonella or Clostridium species are clinically suspected. In our laboratory, every faecal culture sample is routinely examined for the presence of Salmonella spp.

Important: a rectal swab is an unacceptable sample for this indication. A swab samples the mucosa and a small amount of faeces, which is insufficient material for a reliable culture.

Use fresh faeces, collected immediately before submission. The fresher the sample, the better — enterobacteria, and Clostridium in particular, lose viability relatively quickly outside the host.

Synovial fluid: rapid transport is critical

Sampling of synovial fluid is performed under aseptic, operative conditions. Immediately after sampling, the priority is transport to the laboratory.

A particular reason for urgency: if Mycoplasma is clinically suspected (characterised by arthritis without the presence of typical bacteriological parameters), the sample must reach the laboratory in the shortest possible time from collection. Mycoplasma are extremely sensitive to temperature changes and deteriorate rapidly — do not refrigerate this sample.

Urine

Microbiological examination of urine is particularly sensitive to prior therapy. If the patient has been on antibiotics, the urine result will almost certainly be negative — even when an active infection is present.

Rule: collect urine before starting therapy, or ensure a therapy break of 7 to 10 days so that the result has diagnostic validity.

The recommended collection method is cystocentesis, which minimises contamination from flora of the lower urinary tract and skin.

Tissue biopsies

Tissue biopsies intended for microbiological examination must not be placed in formalin. Formalin fixes tissue and kills all microorganisms — the sample becomes unusable for culture and PCR.

Submit fresh tissue material in a sterile container, preferably without additives. If transport takes more than an hour or two, contact us for a recommendation on the appropriate transport medium.

Anaerobic causative agents: when to consider them

Anaerobic infections are frequently underestimated in veterinary practice, partly because they are diagnostically demanding. Nevertheless, anaerobic causative agents are common or predominant in the following clinical presentations:

• Abscesses of all locations
• Inflammation of anal sacs (especially with closure and secondary infections)
• Bacterial peritonitis
• Septicaemia
• Inflammatory conditions of the digestive tract (particularly necrotising enterocolitis)
• Endometritis
• Post-traumatic soft tissue inflammation (bite wounds, road traffic accident injuries)
• Osteomyelitis
• Post-operative complications
• Pyothorax
• Chronic wounds that fail to respond to standard antibiotic therapy
• Periodontitis

Whenever a patient fails to respond as expected to empirical antibiotic therapy targeting aerobic causative agents — consider anaerobes. And, equally importantly, collect the appropriate sample before (once again) changing the therapy.

VetPro laboratory: Partner in microbiological diagnostics

Correct sampling is a joint project. Both you and we have a role in the final outcome. Our team is always available for consultation before sampling, particularly for specific or atypical cases.

If you are unsure which sample to collect, in which order, in what container or at what temperature to transport it — get in touch with us. It is better to spend five minutes on a consultation than to repeat the sampling.