October 2020

Diagnostic Bacteriology

By Dr. M.M. Chengappa and Amy Burklund

One of the first major goals of the bacteriology laboratory is to isolate or detect clinically significant microorganisms from an affected site and, if more than one type of microorganism is present, to isolate them in approximately the same ratio as occurs in vivo. Attention must be given to the site cultured as well as to the method of obtaining the sample for culture. The determination of significance is made a great deal easier if the sample is obtained aseptically from a normally sterile site. Obtaining a sample from the alimentary canal and expecting meaningful answers, may be unrealistic unless one is looking for the presence or absence of a particular microorganism, such as Salmonella or certain types of E. coli.

Sample Collection

Attention must be given to how the diagnostic specimens are collected; if not, interpretation of results may be difficult. Most infectious processes arise subsequent to the contamination of a compromised surface or site by microorganisms that are also a part of the flora occurring on a contiguous mucosal surface. In other words, microorganisms isolated from an affected site are often similar (if not identical) to those found as part of the normal flora of the patient.

Transport of Samples

The sooner the specimen is processed in the microbiology laboratory, the better. Realistically, the time between sample collection and processing may range from minutes to hours to days. Drying (all microorganisms) and exposure to a noxious atmosphere (oxygen for obligate anaerobes) are the major factors that compromise specimens and lead to inaccurate diagnosis. For this reason, it is important that the specimen be kept moist and, if conditions warrant (see below), air excluded. Moistness is maintained by placing the sample in a transport (holding) medium composed of a balanced salt solution usually in a gelled matrix. Because this medium does not contain any nutrient material, microorganisms in the sample multiply poorly, if at all, (and thereby relative numbers and ratios are preserved) but remain viable for some time, generally for at least 24-48 h. If swabs are submitted, the preferred swab is the eSwab™. These eSwabs™ are acceptable swabs for aerobic, anaerobic, Mycoplasma and fungal culture and can be used for other laboratory testing as well. Fluids that may contain anaerobic bacteria (e.g., exudate from draining tracts, peritoneal and pleural effusions, abscess material) should be inoculated onto appropriate media immediately. If the specimen is collected by needle aspiration, all the air must be expelled from the barrel of the syringe prior to collection. The contents then must be transferred to a sterile tube with no additives. Be sure to completely fill the tube, so as to leave no oxygen, which might be toxic to the anaerobes during transport.

Demonstration of Bacteria

The presence of bacteria is accomplished by examination of stained smears made from a portion of the clinical sample, culture techniques, molecular/immunological methods, or a combination of these methods.

Direct Smears

Information obtained from examination of a stained smear is valuable because it may be the first indication (and sometimes the only one) that an infectious agent is present. Also, what is seen (shape, staining characteristics) will help guide the choice of therapy 24 h before culture results are available.

As is the case with a sample obtained from a normally sterile site, the presence of bacteria in bladder urine is a significant finding. However, interpretation of the results of analysis of urine samples obtained by catheter or by “catch” is difficult because of the confounding presence of flora flushed from the distal urethra. Finding bacteria by direct smear in concentrated (the preferred) or unconcentrated urine obtained by percutaneous aspiration of bladder urine is a significant finding.

Several types of stains are available. The gram stain is the most common. Each type of stain has advantages and disadvantages. The gram stain is useful in that the shape and the gram-staining characteristics of the agent are seen. The disadvantage of the gram stain is that the cellular content of the sample is not readily discerned.

Culture Techniques

Media are inoculated with a portion of the specimen. Inoculation should be performed in a semiquantitative fashion (especially samples of bladder urine obtained by catheter or catch).

Determination of the relative numbers of microorganisms in a sample greatly helps interpretation of significance. Colonies of microorganisms growing on all four quadrants of a petri plate indicate that there are large numbers of microorganisms in the sample. If a sample yielded one or two colonies growing on the plate, the significance of these colonies and thus the question as to the infectious etiology of the condition would be in doubt. Enrichment prior to plating of a sample obtained from a normally sterile site should be performed in certain cases where the causal agent is believed to be in low number. Note that the enrichment procedures would lead to the proliferation of contaminating microorganisms.

Determination of clinical significance is aided by the cytology of the sample obtained from the affected site. Isolation of numerous microorganisms from a normally sterile site without the presence of inflammatory cells should be suspect. One exception to this rule is cryptococcal infection wherein the sample may contain a large number of yeast cells but very few inflammatory cells (the cryptococcal capsule is immunosuppressive). The isolation or demonstration of a “significant number” of microorganisms from a normally sterile site without evidence of an inflammatory response can be explained by contaminated collection devices; contamination of the medium inoculation device in the microbiology laboratory; or contamination of the medium before inoculation. Collection devices (e.g., catheters) sterilized by liquid disinfectants quite often become contaminated by microorganisms able to survive in such environments, Pseudomonas, for example.

Media plates may be streaked in any fashion as long as individual isolated colonies are produced after incubation. Assessing relative numbers is very subjective. Relative numbers of microorganisms may be reported. Determination of the actual numbers of bacteria present is only important when analyzing urine obtained by “catch” or catheter because of the problem of contamination of the sample by bacteria in the distal urethra. In this instance, disposable calibrated loops containing 0.001 or 0.01 ml of urine are used to inoculate appropriate media. Greater than 10⁵ bacteria/ml of urine obtained by catheter or “catch” is considered significant (i.e., the bacteria are more likely to be coming from the bladder rather than the distal urethra).

Aerobic Bacteria

The standard medium inoculated for the isolation of facultative microorganisms is a blood agar plate. Many laboratories include a MacConkey agar plate as well. MacConkey agar is useful because enteric microorganisms (members of the Family Enterobacteriaceae, e.g., Escherichia coli, Klebsiella, and Enterobacter) grow very well, as does the nonenteric Pseu­domonas. Most other nonenteric gram-negative rods and all gram-positive microorganisms do not grow well on this medium. Assessing growth on MacConkey agar will facilitate detection of enteric organisms.

Anaerobic Bacteria

Anaerobic bacteria grow on blood agar that is specially prepared to eliminate oxygen. After anaerobic plates are inoculated, they should be placed in a closed, anaerobic environment. Processing specimens for anaerobic culture is time-consuming and expensive. The most common sites that contain anaerobic bacteria are deep tissue wounds; draining tracts; abscesses; pleural, pericardial, and peritoneal effusions; pyometra; osteomyelitis; and pneumonic lungs. Anaerobic culture of sites that contain a population of anaerobic bacteria as part of the normal flora is often unrewarding (e.g., feces, vagina, distal urethra, and oral cavity) unless the diagnostician is looking for a specific species or type of anaerobic organism. Anaerobic culture of the urinary tract is not routinely performed because the recovery of these anaerobes from this site is extremely rare. Clinical Conditions suggestive of anaerobic infections are: foul-smelling discharges; deep infections from penetration of cutaneous or mucosal surfaces; necrotic tissue, gangrene, pseudo membrane formation; gas in tissue or exudate; endocarditis with a negative aerobic culture; infection associated with malignancy or other disease causing tissue destruction and impaired circulation; bite wounds; deep abscesses; septic pleuritis; aspiration pneumonia; fractures associated with trauma to soft tissue; infections following surgery of the gastrointestinal tract; and septic processes such as pyometra.

M. Chengappa, DVM, PhD, AVCM Diplomate is the KSVDL Bacteriology Section Head. Amy Burklund is the KSVDL Bacteriology Laboratory Administrator.

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