In previous editions of this book, methods of preparing and using selective culture media as well as identifying specific isolates was described. However, technologic advances in microbiology have largely replaced older methods of identifying bacterial isolates in practice. Furthermore, the diverse array of bacterial pathogens, requirements for unique culture media, the risk of sample contamination, and the need for subjective interpretation of results dictate that even routine bacterial cultures and identification are best reserved for the commercial laboratory equipped to carry out these increasingly complex procedures and experienced in doing so.
What follows are fundamental methods and techniques used to properly collect diagnostic specimens and the appropriate methods for transporting samples to a laboratory in order for the best possible diagnostic result to be obtained.
Direct Microscopic Examination
Before actually collecting and submitting a sample to a laboratory for bacterial culture, it is appropriate (whenever feasible to do so) to prepare, stain, and examine, under direct microscopy, exudates or fluid from the suspect material or tissue. Staining the air-dried sample with a rapid Romanowsky-type stain (e.g., Diff-Quik stain) or a Gram stain may reveal evidence of neutrophilic inflammation (neutrophilia, especially with a left shift) and occasionally degenerative neutrophils with intracellular bacteria visible. These findings greatly facilitate patient management by documenting the immediate need for interventive empiric antimicrobial therapy until definitive culture and antimicrobial susceptibility results are obtained. The absence of cytologic evidence of bacterial infection does not rule out the possibility that the patient is infected or bacteremic (Table Common Bacterial Culture Results).
TABLE Common Bacterial Culture Results
|External ear canal|
|Dog||Malassezia, Clostridium, Staphylococcus (a few), Bacillus (a few); never Streptococcus, Pseudomonas, or Proteus||Many Staphylococcus and Malassezia together; Pseudomonas, Proteus, Streptococcus, Escherichia coli|
|Cat||Not documented||Staphylococcus aureus, β-hemolytic streptococci, Pasteurella, Pseudomonas, Proteus, E. coli, Malassezia|
|Dog||Micrococcus, Clostridium, diphtheroids, Staphylococcus epidermidis, Corynebacterium, Malassezia||S. aureus (coagulase positive), Proteus, Pseudomonas, E. coli|
|Cat||Micrococcus, Streptococcus, S. aureus, S. epidermidis||S. aureus, Pasteurella multocida, Bacteroides, Fusobacterium, hemolytic streptococci|
|Conjunctiva||Staphylococcus, Streptococcus, Bacillus, Corynebacterium, diphtheroids, Neisseria, Pseudomonas||S. aureus, Bacillus, Pseudomonas, E. coli, Aspergillus|
|Vagina||Staphylococcus, Streptococcus, Enterococcus, Corynebacterium, E. coli, Haemophilus, Pseudomonas, Peptostreptococcus, Bacteroides||Brucella canis; pure culture of organism (especially E. coli, Staphylococcus, Pseudomonas) when accompanied by tissue reaction at vaginal cytology|
|Urine||<1000* organisms per milliliter; presence of several organisms suggests contamination||More than 100,000* organisms per milliliter and often pure culture; E. coli, enterobacteria, Klebsiella, Proteus, Pseudomonas aeruginosa, P. multocida, Staphylococcus, Streptococcus|
* Absolute numbers of bacteria depend on the collection technique.
Collecting diagnostic samples for bacterial culture should be attempted as early in the disease process as possible. It is also critical to accomplish the sample collection under aseptic conditions. It is appropriate, therefore, to perform a surgical scrub of the skin or tissue from which the sample is to be collected in advance. This is especially true for tissue biopsies and fluid samples collected by needle aspiration through intact skin. Failing to adequately prepare the collection site can result in significant contamination and complicate diagnostic interpretation of results.
In addition, it is recommended to collect the diagnostic sample before the administration of antibiotics in order to minimize the risk of false-negative culture results. In the event antimicrobials have been administered to a patient with a suspected infection, and that is not responding to treatment, discontinuing treatment for 48 hours before attempting sample collection is generally recommended.
Collection of an adequate amount, or volume (fluid), is equally important in obtaining meaningful result. For example, a single sterile cotton-tipped swab of contaminated tissue should be considered inadequate sampling and inappropriate for any patient. Multiple specimens are always recommended when feasible. Also, biopsy material, surgically removed tissue, and several milliliters of fluid (e.g., urine) should be collected and placed in a sterile container that can be appropriately sealed (leak-proof container) before transport. A “clean catch” of urine in a “clean cup” is not appropriate.
Inexpensive commercial containers specifically designed for the transport of infectious material are readily available today and should be used. Many containers designed to hold bacterial samples contain buffered, nonnutritive transport media to sustain the growth of pathogenic bacteria yet minimize overgrowth of bacterial contaminants during the time required to transport the sample. Most commercial laboratories provide appropriate containment devices for the transport of bacterial samples.
Collection Technique and Sample Transport
Because most diagnostic specimens collected for bacterial culture are submitted to commercial laboratories for bacterial isolation, identification, and antimicrobial susceptibility testing, it is important to prepare the sample properly for shipping.
Special transport media are generally not required for routine aerobic culture specimens as long as the sample can remain moist and relatively cool and the sample can be inoculated onto culture medium within 3 to 4 hours only. For samples that must be shipped overnight to a laboratory, it is imperative that the specimen be kept cool (not frozen) and moist. Elevated temperatures during shipping contribute to bacterial overgrowth of nonpathogenic bacteria, making isolation and identification of disease-producing organisms difficult. Special transport media may be required. Contact the individual laboratory regarding information pertaining to shipping of specimens for bacterial culture.
Specimens submitted for anaerobic culture need to be inoculated onto culture media within minutes after collection. Although special anaerobic transport media are available, they may not be well suited for extended shipping times (>4 hours).
Among the most frequently tested fluids for bacteria, urine supports the growth of several types of bacteria. Therefore it is necessary that the genitalia be cleaned before collection of urine (free-catch specimen) or cystocentesis (preferred). Use of a urinary catheter to collect urine is likely to introduce urethral bacteria and may result in false-positive culture results. Bacteria will survive for only a limited time in urine. Samples collected must be sealed, and unless processed within 2 hours the sample must be refrigerated. Samples held for longer than 8 hours may not contain viable bacteria. If extended transportation times are required to reach a laboratory, a urine reservation tube (Vacutainer Brand Urine Transportation System, BD, Franklin Lakes, New Jersey) will allow storage for up to 48 hours at room temperature (Table Interpretation of Quantitative Urine Cultures in Dogs and Cats).
TABLE Interpretation of Quantitative Urine Cultures in Dogs and Cats*
|Colony-Forming Units per Milliliter of Urine|
|Voluntary voiding||>100,000^||> 10,000||10,000-90,000||1000-10,000||< 10,000||<1000|
|Manual compression||>100,000^||> 10,000||10,000-90,000||1000-10,000||< 10,000||<1000|
*The data represent generalities. On occasion, bacterial urinary tract infections may be detected in dogs and cats with the fewer organisms (i.e., false-negative results).
^Caution: Because contamination of midstream samples may result in colony counts of 10,000/mL or more in some dogs (i.e., false-positive results), they should not be used for routine diagnostic culture of urine from dogs.
Exudates and Transudates
Collection from fluid filled compartments (e.g., abscesses, seromas) requires collection with a needle and syringe. The maximum quantity possible should be collected and submitted. The skin or tissue overlying the area from which the sample is to be collected should be surgically prepared. If it becomes necessary to flush an open lesion (or perform trachea-transtracheal aspiration or bronchoalveolar lavage [BAL]), it is recommended that a buffered solution of sterile Ringers lactate be used. Use of fluids that contain preservative may actually inhibit the growth of bacteria.
If it is necessary to submit fecal material for specific bacterial isolation, at least 2 to 3 g of feces should be submitted. A single cotton-tipped swab inserted rectally is unlikely to yield meaningful results. Multiple (up to three) samples are recommended when attempting to isolate specific pathogens (e.g., Salmonella). Samples should be submitted in a sealed, leak-proof container (always appreciated by the lab). The containerized sample should refrigerated if there is a significant delay (several hours) involved in submission to the laboratory.
Confirmation of the presence of bacteria in the blood (bacteremia) can be difficult and requires some patient preparation before collection of a series of samples. In addition, samples should be collected only in vials clearly marked for the collection of blood. Furthermore, there are several reasons for an infected patient to have a negative blood culture result — for example, prior or concurrent antimicrobial therapy, chronic (low-grade) infection, and intermittent shedding of bacteria into blood. Sample volume, numbers of samples submitted, skin preparation, and timing of collections are variables that can directly affect results.
Clip and surgically prepare the skin over the cephalic, recurrent tarsal, and/or jugular veins. Do not draw blood for culture through an indwelling intravenous or intraarte-rial catheter. Collection vials are available for aerobic and anaerobic bacterial culture. It is generally recommended that three blood samples be collected from separate veins over a 24-hour period. There is no advantage to collecting arterial blood. It has been suggested that samples collected during times when the patient is febrile may improve the likelihood of isolating bacteria. The volume of blood collected is determined by the size of the patient, the collection vials (adult, pediatric, infant) used, as well as the laboratory equipment used to propagate the culture. In addition to adult human blood culture collection vials (10 ml), pediatric blood collection vials (5 to 10 mL) and infant collection vials (0.5 to 1.0 mL) are available.
It is appropriate that sterile technique be adhered to during collection of all samples. This includes the use of sterile gloves by the individual collecting the sample. Once blood has been collected, air should not be allowed to enter the collection vial. The vial should be gently inverted (never shaken) two to four times. Vials may be maintained at room temperature (the laboratory maintains samples at 37° C).
The opportunity to submit complementary cultures (e.g., from urine) from patients in which blood cultures are being collected can help to confirm the infecting bacteria and may lead to identification of a likely source (Boxes Indications for performing a blood culture and Indications for submitting specimens for anaerobic culture).
|BOX Indications for performing a blood culture|
|Any acute illness with fever (fever of unknown origin)||Undiagnosed anuria or oliguria|
|Leukocytosis, particularly with a left shift||Thrombocytopenia|
|Neutropenia||Disseminated intravascular coagulation|
|Unexplained tachycardia||Intermittent shifting leg lameness|
|Undiagnosed hypoglycemia||Sudden development of or change in, a murmur|
|Unexplained tachypnea or dyspnea|
BOX Indications for submitting specimens for anaerobic culture
- Any focal pain and swelling with fever
- Nonhealing bite or puncture wound
- Foul-smelling wounds with persistent discharge
- Presence of gas in tissue, especially if associated with a penetrating injury
- Abscess, especially if recurrent
- Necrotic or devitalized tissue
- Dark, discolored discharge from the site of a penetrating injury
- Visible sulfur granules in any discharge
- Identification of filamentous bacteria during routine microscopy of exudates
- Failure to obtain bacterial growth using aerobic techniques