Microbiology Laboratory Tips and Tricks: An Organism Isolation and Identification Survival Guide

Jan. 27, 2020

While clinical microbiologists adjust to new technologies that identify organisms with granular specificity, it is important that we retain simple identification techniques and tools in our microbial identification arsenal. This is particularly relevant for laboratories that cannot afford expensive diagnostic platforms but is also applicable to well-equipped teams: in many cases, obtaining a presumptive identification of an organism using simple microbiology techniques is helpful for therapeutic decisions. This article includes unique methods that can be used to identify pathogens in the absence of modern technology.

Culturing Streptobacillus monoliformis (Rat Bite Fever) and Other Fastidious Organisms

Rat bite fever is most commonly associated with the exposure of, you guessed it, a rat bite. This condition, most often caused by the organism Streptobacillus monoliformis, can be observed in people who have pet rats or exposure to rodents, particularly children who like to offer kisses to their furry friend and sustain bites on the lips or other parts of the face.

Patients often present with symptoms that include fever, chills, joint pain and a rash that spreads across the hands and feet. While the differential diagnosis of this disease may seem straightforward, culturing this organism in the laboratory can be tricky. Culturing the blood is the best way to isolate the organism. However, there is a small but important caveat to this process that must be noted. S. monoliformis is extremely fastidious and is inhibited by the anticoagulant sodium polyanethol sulphonate (SPS), which is used in commercial blood culture bottles.

A number of methods can be used to enhance growth of S. monoliformis in blood culture, including the use of broth with low levels of SPS (anaerobic blood culture bottles often have reduced SPS) or use of resin-containing bottles. Additionally, the supplement FOS (fastidious organism supplement) contains NAD and Hemin, which help support the growth of fastidious organisms and can be added to improve growth. Additionally, this supplement helps to neutralize the SPS in the blood culture bottle, which many fastidious organisms have been shown to be susceptible to. What’s the major take home point here? If the clinical team does not tell the microbiology laboratory that Rat Bite Fever is suspected, culture methods may be insensitive and the organism may not grow. It is imperative that the clinical team informs the microbiology laboratory that S. monoliformis is on the differential diagnosis so that effective steps can be taken to encourage organism growth.

Why does it matter that the organism grows in the blood culture bottle? There are 3 important reasons:

  1. The gram stain of S. monoliformis is incredibly characteristic. The Gram stain from solid media will show tangled Gram-negative rods that often have bulbar swellings and are variable in shape and size. If given the patient history, it is possible to make a presumptive diagnosis of Rat Bite Fever from the Gram stain and history alone. If the organism does not grow in the blood culture, then a Gram stain cannot be made. Additionally, this organism may not always Gram stain successfully and alternative methods like the Acridine Orange stain may need to be considered.
    Gram stain of S. monoliformis. Notice the faint, pleomorphic rods with bulbar swellings (highlighted by the arrow).
    Gram stain of S. monoliformis. Notice the faint, pleomorphic rods with bulbar swellings (highlighted by the arrow).
    Source: Photo courtesy of A. Prinzi.
  2. Culturing S. monoliformis on solid media is equally challenging. Once it is determined from the Gram stain that the organism is most likely S. monoliformis, additional culture steps must be taken to ensure growth. Special culture media could be used, but most importantly, a microaerophilic growth environment should be created. While a variety of systems can be used to do this, any version of the candle jar system is acceptable and has been found to be cost-effective and useful, particularly in resource-limited settings. In its simplest form, the candle jar is any container with a lid that can hold culture plates and a small candle. When the candle is lit, the flame burns until the majority of oxygen is used up, creating an oxygen-poor but carbon dioxide-rich environment (typically containing about 5% CO2).
    A simple candle jar used to create a microaerophilic environment.
    A simple candle jar used to create a microaerophilic environment. Culture plates are placed inside the jar with a lit candle and the lid is sealed tight.
  3. Confirming the identification of the organism can ensure that the appropriate treatment is administered. Additionally, it is important to ensure that the disease does not progress as S. monoliformis infection has been shown to cause endocarditis, meningitis and pneumonia among other conditions. If a MALDI-TOF instrument is available, this technology also serves as a reliable way to identify all known species of Streptobacillus.

The Immunocompromised Patient and Malassezia furfur: Olive Oil Isn’t Just for Salads

Infection with Malassezia furfur may be more common than infection with Candida in patients with complicated underlying disease. This is particularly true in patients receiving total parenteral nutrition (TPN) who are at an increased risk of developing a blood infection with lipid-dependent Malassezia.

Because most species of Malassezia require lipids to grow in vitro, solid culture media should be supplemented with a natural oil or other fatty substance in order to encourage growth of this organism. If a fungal blood culture is ordered on a patient with severe underlying disease or someone who is TPN dependent, it would be advantageous to add a set of plates with lipid supplementation. What is the easiest way to do this? Pour olive oil on the plate.

While there are additional (and more expensive) options available that provide lipid supplementation, adding olive oil is simple, cheap and the most commonly used method.

A small amount of olive oil can be poured over a fungal culture plate (like Sabouraud’s Dextrose agar) after inoculation and incubated to encourage the growth of this yeast-like mold.

Culturing Nutritionally Variant Streptococci: The “Sick Strep”

I shared a case study detailing the isolation of the organism Abiotrophia on Twitter a few months ago that generated a surprising amount of interest. The majority of people stated that they were either unfamiliar with the organism or the simple culture methods used to improve isolation of it. The methods used to isolate and identify these organisms are both unique and simple.

Abiotrophia defectiva, Granulicatella adiacens and Granulicatella elegans are all fastidious organisms that were previously called “nutritionally variant streptococci (NVS).” While these organisms are famously fastidious, we should not be fooled by their finicky nature. NVS are important organisms to consider in bacteremia and endocarditis, and have been associated with significant morbidity and mortality.

There are various types of solid media that are supplemented with additional nutrients that may help these organisms grow, but it may not be cost-effective to add these plates to every blood culture in order to capture a NVS. Inoculated blood culture bottles allow for the growth of NVS, which will appear as Gram-positive cocci in chains on the initial Gram stain.

If the blood is plated to routine culture media (TSA with blood, chocolate agar and MacConkey agar), it is likely that growth will only be seen on the chocolate media. If the blood is plated to a TSA with blood plate only, it is unlikely that any growth will be seen. This will be the first hint that the organism in question belongs to the NVS group. These Streptococci require specific growth factors which chocolate agar provides. NVS organisms may be seen on blood agar media growing around a “helper” organism such as Staphylococcus aureus. The beta hemolysis of the S. aureus lyses the red blood cells in the media and releases growth factors such as hemin and NADH, which supports growth of NVS in any part of the plate that has been hemolyzed. This is known as the satellite phenomenon. This satelliting activity is most commonly seen with Haemophilus influenzae. A simple test that can be performed to presumptively identify an organism as a member of the NVS group involves the following steps:

  1. Visually confirm that there is growth on the chocolate agar plate, but not the blood agar plate or any other un-supplemented media (TSA, for example).
  2. Confirm that the Gram stain of the organism growing on the chocolate plate is Gram-positive cocci in chains. These cocci may appear to be a little “sick” looking. They may be variable in size or shape.
  3. Make a 0.5 Mcfarland dilution of the organism growing on the chocolate plate and streak a lawn of this suspension to a fresh blood agar plate using a swab.
  4. Using a new swab, select one colony from a pure culture of Staphylococcus aureus. Draw a vertical line down the blood agar plate inoculated with the strep in question.
  5. After incubating this plate for 18-24 hours, small colonies should grow around the zone of beta hemolysis caused by S. aureus.
  6. If the Gram stain of the pinpoint colonies growing around the zone of beta hemolysis shows Gram-positive cocci in chains, it is likely that you have isolated a nutritionally variant Strep!

This method is known as the “Staphylococcal-streak” method and is an easy way to presumptively identify a NVS.

It's important to always refer back to the original Gram stain. If an organism is seen in a significant quantity but is not growing in culture, it is important to consider fastidious organisms that may require additional assistance from the microbiology team.

Gram stain of Abiotrophia defectiva in shunt fluid. Take note of the faint staining and “sickly-looking” Gram positive cocci in chains.
Gram stain of Abiotrophia defectiva in shunt fluid. Take note of the faint staining and “sickly-looking” Gram positive cocci in chains.
Source: Photo courtesy of A. Prinzi.
A plate inoculated with a lawn of Abiotrophia defectiva and a streak of Staphylococcus aureus at 48 hours of incubation.
A plate inoculated with a lawn of Abiotrophia defectiva and a streak of Staphylococcus aureus at 48 hours of incubation. Note that the small gray colonies (A. defectiva) only grow well near the zone of beta hemolysis produced S. aureus. No growth is seen farther away from the S. aureus streak.
Source: Photo courtesy of A. Prinzi.

The Vancomycin Disk (Leuconostoc, Pediococcus, Vanc-Susceptible GNRS)

While antimicrobial disks are used in the clinical microbiology laboratory as screening methods for resistance and to determine minimum inhibitory concentrations, there are many other uses for the vancomycin disk that may be overlooked. In the lab I have worked in for many years, we have always dropped a vancomycin disk on the blood agar plate of a positive blood culture before it is incubated. In doing this, the disk serves as a quick screening method for vancomycin-resistant organisms.

Vancomycin is a broad spectrum drug that is used to treat infections caused by Gram-positive organisms. Although resistance to this drug is on the rise, we can safely say that the majority of Gram-positive organisms we grow in the microbiology laboratory are still susceptible to this antimicrobial compound. Because of this, it may be easy to preliminarily identify several important organisms based on the growth around this disk. The following organisms should be considered based on Gram-stain morphology and vancomycin resistance:

  • Gram-positive cocci in chains, not inhibited by the Vancomycin disk
    • Pediococcus and Leuconostoc: These bacteria are intrinsically resistant to vancomycin and are opportunistic pathogens. They have been known to cause serious disease, particularly in children, if left untreated. Since these organisms tend to be resistant to penicillin as well, combination therapy is typically warranted. Early identification of these organisms is important so that correct treatment can be initiated. If the original Gram stain shows Gram-positive cocci in pairs and chains and small gray colonies with no zone of inhibition around the vancomycin disk, consider Pediococcus or Leuconostoc.
      Gram stain of Leuconostoc species.
      Gram stain of Leuconostoc species.
      Source: Photo courtesy of A. Prinzi.
    • Vancomycin resistant enterococci: There are some species of Enterococcus that were just “born to be bad” and are intrinsically resistant to vancomycin. These species include E. gallinarum and E. casseliflavus. Although these species are naturally resistant to vancomycin, they are not necessarily resistant to several other drugs and are not considered “super-bugs.” On the other hand, E. faecium and E. faecalis are not normally resistant to vancomycin, so when they become resistant it is very concerning. This type of resistance is acquired over time, and then the organisms can pass this scary mechanism to fellow organisms like a baton in a relay. Concerns about transmission to other patients and difficulty treating are plenty with these organisms, so it is important to identify them as soon as possible. Additionally, infection control methods may vary greatly between vancomycin resistant organisms, so informing the clinical team about the organism identification rapidly is imperative. Enterococci can be quickly differentiated from Pediococcus and Leuconostoc using simple biochemical tests.
  • Gram-positive rods, not inhibited by the Vancomycin disk.
  • Gram-negative rods, inhibited by the Vancomycin disk. Remember when I said that vancomycin is used to treat infections with Gram-positive organisms? This is because the drug typically does not have activity against Gram-negative rods. However, there are a few exceptions to this rule and knowing these organisms can help make a speedy preliminary identification if the culture shows growth of a Gram-negative rod that is inhibited by the Vancomycin disk.
    • Elizabethkingia meningosepticum: This organism is a non-fermenting, oxidase positive Gram-negative rod. Laboratory identification of this organism can be difficult, but matching the colony morphology with the Gram stain and susceptibility to vancomycin can certainly point a microbiologist in the right direction. This Gram-negative rod produces a large wet colony on standard laboratory media. While identifying this organism will take additional steps either through the use of biochemicals, MALDI-TOF or other automated systems, susceptibility to the vancomycin disk and a large slimy colony morphology are clues that Elizabethkingia meningosepticum might be the culprit.
      Elizabethkingia meningosepticum growing on blood agar media. Note the large mucoid colonies.
      Elizabethkingia meningosepticum growing on blood agar media. Note the large mucoid colonies.
    • Porphyromonas species are anaerobic Gram-negative rods that may also be susceptible to vancomycin in culture. This is important to note because laked kanamycin-vancomycin agar (LKV) is often used to culture anaerobic organisms. If an additional anaerobic plate (such as Brucella anaerobic agar) is not used, the organism may be missed in culture due to its natural susceptibility to the vancomycin present in the LKV media.

The table below summarizes vancomycin resistant and susceptible organisms (according to disk diffusion). This table provides a quick reference guide that brings together relevant gram stains, morphologies and basic biochemical tests.

Bringing It All Together

As highlighted in my previous article discussing the importance of a patient history in obtaining a microbiological diagnosis, there are many simple tools in the microbiology lab arsenal that can be used to successfully culture and identify organisms without the use of expensive technology. While advanced technology is a wonderful luxury and great improvement to patient care overall, the use of this technology is not always affordable or within reach. Additionally, many of these new platforms require the growth of an organism which may be difficult if the organism is fastidious and supplemental methods are not used.

This is why a good clinical microbiologist will pay attention to the clues that microorganisms provide. Microbes may be identified successfully if we encourage their growth and observe their natural patterns, performing a few small tricks that reveal their identity along the way.

The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

Author: Andrea Prinzi, Ph.D., MPH, SM(ASCP)

Andrea Prinzi, Ph.D., MPH, SM(ASCP)
Andrea Prinzi, Ph.D., MPH, SM(ASCP) is a field medical director of U.S. medical affairs and works to bridge the gap between clinical diagnostics and clinical practice.