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Agar Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Agar and links to relevant pages within the course.

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Case Studies in Clinical Microbiology
The carbohydrate utilization reaction seen in the QuadFerm system shown in the image provides a definitive identification of N. gonorrhoeae.View Page
The patient was admitted to the hospital. The sputum specimen was inoculated to sheep blood agar. Based on the colony morphology and the alpha hemolysis seen in the image to the right, the most likely identification is:View Page
The name of the rapid test seen in the image, often used to differentiate S. pneumoniae from viridans streptococci, in which a drop of 10% deoxycholate was placed on an area of growth, is:View Page
The colonies shown in the blood (BAP) agar (upper) and MacConkey (MAC) agar (lower) biplate are a 24 hour growth from an aerobic blood culture bottle that became positive at 12 hours after inoculation. The appearance of the colonies on MAC agar rules out the following two bacterial species:View Page
The bacterial species shown growing on 5% sheep blood agar was recovered from the spun sediment of a midstream urine specimen after 24 hours incubation at 35C. Each of the following tests would be useful in supporting the presumptive identification of Enterococcus species except: (Choose all that apply)View Page
The spot test that is helpful in separating Enterococcus species (positive as shown in the image) from the viridans streptococci and S. pneumoniae (both negative) is:View Page
In this image is a quadrant plate containing brain heart infusion agar supplemented with 6 ug/mL of vancomycin. The right upper quadrant was inoculated with the test strain of Enterococcus faecium. The presence of growth in the inoculated quadrant indicates resistance to vancomycin.View Page
Colony Morphology

The growth observed on the anaerobic blood agar plate after 48 hours incubation (see upper image), revealed a spreading colony. The spreading nature of the colony is better observed in the lower image. No growth was observed on subcultures incubated aerobically indicating that this isolate is truly an anaerobe (although aerotolerance studies would be needed for confirmation). The spreading nature of the colony and the lack of hemolysis are highly suggestive of Clostridium septicum. However, biochemical confirmation is necessary.

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Staph on Blood Agar

The image of the surface of a 5% sheep blood agar illustrates the colonies that grew out of the foot drainage after 24 hours at 35C. They are entire, convex, smooth, and have a slight yellow pigmentation. Hemolysis is not observed. A Gram stain was prepared from one of the isolated colonies.

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Staph aureus Mannitol

The ability to grow in 6.5% NaCl and to produce acid from mannitol was once considered sufficient to identify S. aureus. Although other Staphylococcus species have these characteristics, mannitol salt agar, as shown here, can still be used for epidemiologic studies in suspected cases of S. aureus outbreaks. As shown in the image, the presence of colonies indicates the ability to grow on 6.5% NaCl; and, the yellow pigment of the colonies indicates acid production from mannitol.

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Methicillin-Resistant Staphylococcus aureus (MRSA) Screen

Perhaps the most efficient means for detecting methicillin-resistant staphylococci in clinical laboratories is the use of the agar dilution screening test. Illustrated in the image is a Mueller-Hinton agar plate containing 6 ug/mL of oxicillin, previously inoculated with a strain of Staphylococcus aureus. Oxacillin is used as a marker for methicillin resistance because it is more stable in the agar medium. Growth on this screening medium is presumptive for methicillin resistance. Thus, in the presence of growth, as shown here, a follow-up minimum inhibitory concentration (MIC) test must be performed to determine the exact level of resistance.

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Methicillin-Resistant Staphylococcus aureus (MRSA) Disk Test

The disk diffusion test can also be used in the detection of methicillin-resistant Staphylococcus aureus. Illustrated in the image is the surface of a Mueller-Hinton agar plate previously inoculated with a strain of S. aureus suspected of being methicillin-resistant. Although the zone of inhibition is at the borderline for resistance (18 mm); the presence of small colonies within the zone of inhibition (yellow arrows) indicates the presence of heteroresistant strains. The interpretation here, therefore, is "methicillin-resistant" staphylococci, even though the zone diameter appears to be adequate. The detection of the heteroresistant strains indicates that minimum inhibitory concentration (MIC) studies are required.

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S. anginosus ("milleri") Biochemicals

The combination of decarboxylation of arginine (red color in the 2nd tube from left compared to the yellow color of the control to its left), the hydrolysis of esculin (black pigment in the esculin agar tube) and the reduction of nitrates to nitrites (red color in last tube on the right) are biochemical characteristics confirmatory for S. anginosus ("milleri").

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Listeria motility

Listeria monocytogenes is optimally motile at 25C; and is non-motile at 35-37C. Motility may be directly assessed by observing bacterial cells with a tumbling motion in a direct mount preparation. In soft motility agar, the identification can be made by observing for a thin, umbrella-like lateral extension of growth from the stab line (noted by arrows in the image).

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Colony Morphology

Image of the surface of blood agar after 24 hours incubation at 35C in 10% CO2, on which are growing tiny, translucent, gray colonies surrounded by a narrow zone of "soft" beta hemolysis. There was no growth on the MacConkey plate.

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Shown in theimage are three tubes: (1) motility agar (note subsurface flare shown by arrows); (2) esculin hydrolysis (+), and (3) VP (+). The reactions illustrated here are sufficient to rule out Erysipelothrix rhusiopathiae.View Page
Eikenella BAP

Image of the surface of a 5% sheep blood agar plate after 48 hours incubation at 35 degrees C in 10% CO2. The colonies shown are small, flat, entire, dull gray, and show superficial pitting of the agar (see yellow arrows). A slight discoloration of the agar surrounding the colonies is seen. A bleach-like odor is detected. Similar growth was seen on a chocolate agar plate set up in parallel. Growth was not observed on the MacConkey plate.

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A bacterial isolate that produces pitting of the agar and has a bleach-like odor is probably E. corrodens. What are the closely related species that must be ruled out? (Choose all that apply)View Page

Electrophoresis
Agarose Gel

Agarose gels are chemically purified forms of agar, a polysaccharide extracted from seaweed. The gel pores allow for separation of proteins based on their individual charge and mass. Agarose gel will naturally clear after drying the separated proteins.Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes. The gels come prepackaged with a plastic template to lay over gel for sample application or slots etched in the gel for these samples.

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Microbiology / Serology Question Bank - Review Mode (no CE)
Which of the following media contains the X and V factors necessary for the growth of Haemophilus influenzae:View Page
On sheep blood agar Haemophilus influenzae may exhibit satellite formation around all but which of the following organisms:View Page
Which of the following media is commonly used when performing the Kirby-Bauer disk diffusionView Page
Which of the following is the most suitable specimen for the isolation of Bordetella pertussis:View Page
MacConkey agar contains all of the following except :View Page
Match type of media on the right with media on the left:View Page
Match the organisms on the right with culture medium:View Page
Match the culture media on the right with possible organism on the left:View Page
Match the organism on the right with the most suitable culture media on the left:View Page
Which of the following media would you use to isolate Francisella tularensis:View Page
Which of the following media is a selective medium for Campylobacter jejuni:View Page
Which of the following two media are appropriate for Bordetella pertussis:View Page
Sheep blood agar contains inhibitors to which of the following organisms:View Page
Which one of the following statements about E.coli O157:H7 is false:View Page
What is the best term to describe the clear areas seen around the colonies on this blood agar plate:View Page
The Thayer-Martin agar plate seen in this illustration exhibits marked growth. The most likely organism found here would be:View Page
The red/pink color of the colonies (E. coli) seen on this MacConkey Agar Plate is an indication of:View Page
The most likely organism to be cultured on the Lowenstein-Jensen agar slant illustrated here would be:View Page
The grow seen here on PPLO agar is most likely caused by:View Page

Molecular Methods in Clinical Microbiology
Molecular Versus Culture - Pros and Cons

Traditional culture methods for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) employing mannitol salt and/or blood agar for cultivation, can take up to 72 hours for isolation and identification, depending on the identification procedures utilized. Concurrent with the development of molecular assays, improvements in culture methods have also been achieved. CHROmagar™ media, specific for MRSA, are employed by many laboratories. These media are both selective and differential, containing chromogenic substrates. MRSA strains utilize the substrates to produce colonies of a specific and characteristic color, minimizing the need for additional identification procedures.Initially these agars required 48 hours of incubation; newer formulations require only 24 hours incubation.Given the reduced incubation and identification requirements, what are the pros and cons of the molecular assays? Cost per test will be greater with the molecular assays as compared to culture methods. Will molecular methods provide for a more efficient workflow and significant improvement in availability of results? To some extent, this will depend on how they can be implemented within each different laboratory setting. Both of the previously described molecular assays require manual specimen preparation and extraction before the sample is placed into the instrument. This hands-on work may actually be greater than the effort expended in swabbing and streaking a culture plate. How much an obstacle this is for implementation will depend on both the volume of testing and the staff available. In a high volume setting, this will be a greater factor.Will tests be performed as specimens come in, or will specimens be accumulated and batched? If controls are required with each run, batching is desirable to reduce this cost. If testing will occur in batches, how many batches can be performed in one day? This will be heavily influenced by the capacity of the instrument. (For example, a single Smart Cycler unit can run up to 16 samples; multiple units would be needed in a high volume lab.) Can they be set up on more than one shift? The greater the number and frequency of batches that can be run, the greater improvement in turnaround time can be realized. Given these variables, implementation of a molecular assay for MRSA is not a given in each laboratory.

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Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
Screening Cultures for MRSA

Surveillance is a critical component of any program for controlling multi-drug resistant organisms (MDROs). Many institutions are using active surveillance cultures to identify patients who are colonized with a targeted MDRO. With respect to MRSA, an increasing number of hospitals are screening patients upon admission and on a periodic basis (usually weekly). The anterior nares is the primary site that is swabbed for screening.There are several selective and/or differential media that can be used for this purpose.Baird Parker Agar is a selective medium for the isolation of S. aureus; on this medium S. aureus produces black colonies with a clear halo.Mannitol Salt Agar is also a selective medium; S. aureus produces yellow colonies which contrast with the red color of the medium.Chromogenic agars have been developed for the isolation and presumptive identification of different species of bacteria and yeast. The media are formulated so that as different organisms utilize various substrates in the media, the organism of interest produce colonies with a unique color. Chromogenic agars specifically designed for the detection of MRSA are commercially available.In addition to culture methods, there are now commercially available, FDA approved methodologies for screening for MRSA by PCR. Although equipment and cost factors may not make these a viable option for every laboratory, they may offer greater sensitivity and improved turnaround times.

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Assume you perform microbiology for an institution submitting surveillance cultures for MRSA. Which isolate should receive further workup to rule out methicillin (oxacillin) resistance?View Page
Stool Culture

Stool culture is very effective in detecting C. difficile. Unfortunately, non-toxigenic strains will also grow, requiring strains to be tested for toxin production. The greatest disadvantage to culture is the length of time that is needed before results are available, which may be up to four days. However, antibiotic sensitivity testing following culture is useful for strain-typing that would provide necessary epidemiological information during nosocomial outbreaks.Colonies of C. difficile will appear white, flat, and spreading on blood agar (see top image on the right). Cycloserin-cefoxitin-fructose agar(CCFA) is a selective media that is used for isolation of C. difficile. There is however, no distinction between pathogenic and commensal strains, which all produce yellow colonies with a characteristic "ground glass" appearance. as shown in the bottom image on the right. The characteristic odor of "horse manure" aids in identification of C. difficile. Stool samples are directly inoculated onto CCFA and incubated in an anaerobic atmosphere at 37°C for 48 hours. Large, thin, gram-positive bacilli with spores will be observed on a Gram stain of a typical colony, as shown below.

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Various methods have been employed for detection of C. difficile disease: cultivation of the organism, cell cytotoxin neutralization assays, and enzyme immunoassays have all been among the staples of diagnostic approaches. Which statements are accurate characterizations of these assays?View Page
Identification of Enterococcus Species From Clinical Cultures

Gram stain: Gram-positive cocci in singles, pairs, or chains; cells can be ovoid to coccobacillaryColony morphology: On blood agar after 24 hours of incubation, colonies are nonhemolytic or alpha hemolytic (rare strains may be beta hemolytic), and approximately 1 to 2 mm in diameter.Catalase: NegativePresumptive identification: Growth on bile esculin agar and in 6.5% salt broth are two characteristics that have commonly been used to identify enterococcus species to the genus level. A positive esculin in combination with a positive PYR reaction is another approach to presumptive identification.Species identification:E. faecalis and E. faecium are usually easily identified by most commercial systems. Successful identification of the other species on these systems may vary. With respect to vancomycin intermediate or resistant strains, two key characteristics are motility and pigment. E. casseliflavus is both motile and possesses a yellow pigment; E. gallinarum is also motile but non-pigmented. E. faecalis and E. faecium demonstrate neither characteristic.

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Screening Cultures for Vancomycin-Resistant Enterococci

Many hospitals have chosen to implement screening programs to identify patients who are colonized with vancomycin-resistant enterococci (VRE). Screening patients provides information about potential source of illness and allows staff to implement appropriate infection control measures. These measures decrease transmission and reduce the number of patients who become infected with VRE.Peri-rectal or anal swabs, as well as stool specimens, are inoculated onto selective media. One medium utilized is bile esculin azide agar containing 6µg/mL of vancomycin. Black colonies that grow on this medium are identified as Enterococcus to the species level and further confirmed as vancomycin-resistant by an appropriate susceptibility testing method.Chromogenic agars specific for VRE are also commercially available.

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Detecting Vancomycin Resistance

Interpretive standards for Enterococcus species against vancomycinMethodSusceptibleIntermediateResistantDisk diffusion> 17 mm15-16 mm< 14 mmBroth dilution< 4 µg/mL 8-16 µg/mL > 32 µg/mLScreening methodologiesBHI agar with 6 µg/mL vancomycin can be employed as a screen for vancomycin resistance. 1-10 µL of a 0.5 McFarland standard suspension is spotted onto the agar surface and incubated at 35 + 2° C for a full 24 hours. The presence of >1 colony equates to presumptive vancomycin resistance; these isolates would require species identification and a vancomycin MIC.A motility test and documentation of the presence or absence of a yellow pigment can distinguish between the species with intrinsic (VanC) resistance (E. gallinarum and E. casseliflavus) and those with acquired (VanA and VanB) resistance (E. faecium and E. faecalis).

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High Level Aminoglycoside Resistance (HLAR) Screening

HLAR is another significant acquired resistance factor. Since the standard approach for treating systemic infections is a combination of a cell wall targeted antibiotic with an aminoglycoside, assessment of resistance to both classes of antibiotics is important. High level resistance to aminoglycosides will negate the synergistic effect of combined therapy with either penicillin or vancomycin.Standard susceptibility methods (either disk diffusion or broth dilution) will not detect HLAR patterns, unless the protocol incorporates testing at increased concentrations of gentamicin and/or streptomycin. CLSI document M100-S23 outlines recommended protocols for screening for HLAR.Gentamicin HLARDisk diffusion MHA agar; 120 µg gentamicin disk; standard inoculum; standard incubation temperature (35 +/- 2° C; ambient air), 16-18 hours incubation durationInterpretationResistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution BHI broth; 500 µg/mL gentamicin; standard inoculum; standard incubation conditions, incubation duration 24 hours.Interpretation Any growth equates to resistant.Streptomycin HLARDisk diffusion MHA agar; 300 µg streptomycin disk; standard inoculum; standard incubation conditions, incubation duration16 - 18 hours.InterpretationResistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution BHI broth; 1000 µg/mL streptomycin; standard inoculum; standard incubation conditions; incubation duration 24-48 hours. If susceptible at 24 hours, reincubate and re-read at 48 hours.Interpretation Any growth equates to resistant.Clinical correlation A resistant result indicates that synergistic effects will not be achieved between the indicated aminoglyocside and the cell wall active agent (eg, ampicillin, penicillin, or vancomycin).A susceptible result indicates that synergistic effects are possible.

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Which of the following statements reflect accurate identifications of Enterococcus species?View Page
With regards to identifying resistance in enterococci, which general statements are true?View Page
Testing for Vancomycin Susceptibility

The current CLSI recommendation is that MIC tests should be performed to determine the susceptibility of staphylococci to vancomycin. The disk test does not differentiate vancomycin-susceptible isolates of S. aureus from vancomycin-intermediate strains.Disk diffusion will detect S. aureus isolates containing the VanA vancomycin-resistance gene (VRSA). These isolates will show no zone of inhibition around the disk (zone = 6mm); their identification should be confirmed. Isolates producing vancomycin zones > 7mm should not be reported as susceptible without performing a vancomycin MIC test.Recommended methods are CLSI Broth Microdilution, Agar Dilution, and Etest® with inoculum prepared to match McFarland 0.5 turbidity standard. The Etest® is considered the most discriminatory of these methods as it allows for visualization of small colonies around zones of inhibition. A pure culture MUST be used. Repeat test for confirmation.The CLSI recommends that the inoculum should be prepared using the direct suspension method and plates incubated for a full 24 hours in ambient air at 35° C. Screening for vancomycin resistance in Staphylococci (MIC's > 8 ug/ml) can be performed utilizing a vancomycin agar screening plate – BHI (brain heart infusion) agar containing 6 mg/mL vancomycin. However testing on BHI screening agar does not reliably detect all vancomycin intermediate S. aureus strains.

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Interpretation of Oxacillin and Cefoxitin Disk Diffusion Tests

Oxacillin is the agent of choice for standardized MIC methods (broth & agar dilution). However, since 2006 the Clinical Laboratory Standards Institute (CLSI) has recommended the use of 30 µg cefoxitin disk rather than the oxacillin disk to detect mecA-mediated resistance in the disk diffusion test because the cefoxitin disk test is easier to read and is as sensitive and specific as MIC methods. Results are still reported as "oxacillin-resistant" or "oxacillin-sensitive." Cefoxitin is a better inducer of the mecA gene and gives clearer, easier to read endpoints in disk diffusion tests.The oxacillin disk is read for light growth within the zone of inhibition using transmitted light (plate held up to light), ANY discernible growth within zone of inhibition is indicative of resistance. The cefoxitin disk is read using reflected light.Interpretive Critieria for Cefoxitin Disk Diffusion TestResisitantIntermediateSusceptibleS.aureus/MRSA<21 mmN/A> 22 mm

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Which of the following scenarios represents appropriate detection of MRSA by the Kirby Bauer method?View Page

Mycology: Hyaline and Dematiaceous Fungi
Several saprophytic, hyaline molds have microscopic characteristics that mimic the mold forms of the dimorphic fungi (Histoplasma capsulatum, Blastomyces dermatitidis, etc.). Each of the following can be used to differentiate the saprophytic from the dimorphic fungi EXCEPT:View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
The colonies shown in the upper image were obtained on blood agar from a sputum specimen after 10 days incubation at 30°C. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The diagnosis is:View Page
The growth of the colonies shown in the upper image was obtained on blood agar from a sputum specimen after 8 days of incubation at 30°C. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The diagnosis is:View Page
The colonies shown in the upper image, obtained from a biopsy of an ulcerating skin lesion of the arm, are growing on agar slants of Sabouraud's dextrose agar. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony growing in the left slant. The diagnosis is:View Page
One of the characteristics common to the dimorphic molds is the ability to convert the mold forms to the yeast forms by incubating subcultures in enriched media at 35°-37°C. The upper image illustrates a subculture of a mold colony suspected of being a dimorphic fungus inoculated to the surface of blood agar and incubated for 3 days at 37°C. Note that the colonies have a prickly appearance, suggesting an intermediate stage of conversion. The lower image is a lactophenol blue mount of a portion of one of the prickly colonies. This fungus can be identified as:View Page
The colonies growing on the surface of this brain-heart infusion with blood agar plate were "converted" from a mold colony suspected of being Histoplasma capsulatum by incubating a subculture at 37°C for 5 days. The yeast forms that must be identified in mounts made from one of these colonies to confirm the identification are:View Page
Match the names of each of the yeast species with its most likely colony morphology as seen in the images on the right.View Page
Match the names of each of the yeast species listed with its corresponding appearance when grown in cornmeal agar, as seen in the images.View Page
Match the names of each of the species of yeast listed with its associated phenotypic property that is helpful in establishing a species identification.View Page
Match each of the microscopic structures listed in the drop-down box with the name of its corresponding yeast species on the right.View Page
Arrange in sequence the steps that should be taken to make a definitive identification of Cryptococcus neoformans.View Page
Match each of the fungal species listed below with the appropriate category, indicating whether or not it has the capability of producing pseudohyphae on cornmeal agar.View Page
The colonies shown in this photograph were grown on Guizotia abyssinica (bird seed) agar at 30°C for 72 hours. The most likely identification is:View Page
The growth of the yeast-like colonies shown in the upper image was obtained on blood agar from a skin culture only in the area overlaid by virgin olive oil. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The disease associated with this fungus is:View Page
This photomicrograph is an acid-fast stained smear prepared from a yeast colony growing on ascospore agar. A helmet-shaped, red-staining, acid fast yeast cell is seen in the center of view at the tip of the arrow, against the background, blue-staining blastoconidia. The presumptive identification of Hansenula anomala was made. Predisposing conditions that may indicate that this isolate is more than a contaminant include:View Page
Illustrated in this photomicrograph of a lactophenol blue preparation of a urine sediment is a cluster of yeast cells that were presumptively identified as Cryptococcus species. Further characteristics that may assist in confirming this identification are:View Page
A yeast identification system gave a biotype number for an unknown isolate that did not differentiate between Candida tropicalis and Candida parapsilosis. This isolate could be identified as C. parapsilosis in a cornmeal agar preparation if it produced:View Page

Parasitology Question Bank - Review Mode (no CE)
A specimen suspected of containing which organism may be cultured by placing it on an agar plate seeded with gram negative rods?View Page

Preliminary Identification of the Primary Select Agents of Bioterrorism
Bacillus anthracis

Culture Characteristics:Growth may be noted as soon as eight hours after inoculation and occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), and routine blood culture media Does not grow on MacConkey (MAC) agarColony Morphology on SBA at 35°C, 18-24 hours:Flat or slightly raised, gray to white with a "ground glass" appearance Described as "tenacious" or "sticky" like petroleum jelly, shown in the top image B. anthracis is NOT hemolytic, while B. cereus is hemolyticCharacteristic Features:After 18 hours of incubation on SBA at 35°C, the slightly undulate margin may show curling, displaying a so-called "Medusa head" or described as comma-shaped protrusions, shown in the lower image

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Yersinia pestis

Culture Characteristics: Growth at 22-25oC and at 35oC Organism prefers 25oC, so the colonies will grow faster at this temperature Growth occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), MacConkey (MAC) agar, and routine blood culture media Non-lactose fermenter on MAC agar Growth is slower than other EnterobacteriaceaeColony Morphology on SBA at 35oC: At 24 hours, colonies are pinpoint and translucent with a gray-white color Colonies take on a yellow tint as they age, after 48-72 hours, referred to as a "hammered copper" appearance An irregular or "fried egg" appearance, shown in the lower image, can also be seen at 48-72 hours Characeristic Features: Growth at 22-25oC is a hallmark feature of this organism

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Francisella tularensis

Culture Characteristics: Slow growing and fastidious Growth of visible colonies on agar may require two to five days Growth is stimulated by CO2 Will grow initially on sheep's blood agar (SBA), but growth is poor or absent on subculture Prefers cystein-enriched media such as chocolate (CHOC), Thayer-Martin (TM), buffered charcoal-yeast extract (BCYE), and thioglycollate (THIO) Growth is slow in broth, so blood cultures should be held up to three weeks if F. tularensis is suspectedColony Morphology on SBA at 35oC: Can grow poorly or not at all on SBA Tiny, pin-point, translucent colonies after 18-24 hours Difficult to see individual colonies in growth that is less than 24 hours Gray-white, opaque colonies less than 1 mm after 48 hours No hemolysisColony Morphology on CHOC at 35oC: Pin-point, gray-white, opaque growth after 24 hours 1-2 mm, gray-white, smooth, shiny growth after 48 hours Individual colonies exhibit mature growth at 3 days in CO2 environment and have a greenish appearance

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Brucella species

Culture Characteristics: Slow growth on sheep blood agar (SBA) and chocolate (CHOC) Growth in commercial blood culture systems, but may require extended incubation Enriched atmosphere with CO2may enhance growth of some strains If Brucella is expected or suspected, extend incubation up to seven days Colony Morphology on SBA at 35oC: Visible growth may take 48-72 hours Small, convex, and glistening Non-hemolytic

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Burkholderia species

Culture Characteristics: B. pseudomallei grows on sheep blood agar (SBA), chocolate (CHOC) agar, and MacConkey (MAC) agar B. mallei grows on very slowly on SBA and CHOC, but little or no growth on MAC Colony Morphology on SBA at 35oC: B. mallei: Smooth, gray, translucent colonies at 48 hours as displayed in the top, right image B. pseudomallei: Smooth, creamy, white colonies at 24 hours as displayed in the lower image on the right B. pseudomallei: May become dry and wrinkled as shown in the image below, often with a purplish hue at 48-72 hours Colony Morphology on MAC at 35oC: B. pseudomallei: Pink colonies at 24-48 hours (may be colorless at 48 hours) B.mallei: No growth or light pink colonies at 72 hours B. pseudomallei on CHOC at 72h image courtesy of CDC

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Which of the following organisms display the characteristic "Medusa head" on sheep blood agar (SBA) after 18 hours of incubation at 35°C?View Page
Bacillus anthrasis

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows large, gram-positive rods with sub-terminal or central spores (if present) Gray colonies with a ground glass appearance Non-hemolytic on sheep blood agar (SBA) Tenacious or "sticky" colonies like petroleum jelly Catalase positive Non-motile

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Yersinia pestis

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows fat, gram-negative rods in single or short chains that may demonstrate bipolar staining Faster growth at 25oC Gray-white, translucent colonies on sheep blood agar (SBA) at 24 hours that turn slightly yellow and opaque at 48 hours Irregular colonies that have a "fried egg" and/or "hammered copper" appearance after 48-72 hours Catalase positive Oxidase negative Urea negative Indole negative

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Francisella tularensis

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows tiny, weak staining, gram-negative coccobacilli Gray-white, opaque colonies on sheep blood agar (SBA) and chocolate (CHOC) agar a at 48 hours Slow growth in broth (up to three weeks) Oxidase negative Urea negative

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Burkholderia species

Any isolate with the following features should be immediately referred to your LRN reference laboratory:B. mallei: Gram stain that reveals pale staining straight or slightly curved gram-negative coccobacilli Cells arranged in end-to-end pairs, parallel bundles, or Chinese letter form Smooth, gray, translucent colonies on sheep blood agar (SBA) at 48 hours Catalase positive Oxidase variable Indole negative Non-motileB. pseudomallei: Gram stain shows slender gram-negative rods with bipolar staining Smooth form appears in Gram stain as long parallel bundles Rough form appears in Gram stain in an irregular arrangement Smooth, creamy, white colonies on SBA at 24 hours Dry, wrinkled colonies at 48-72 hours Catalase positive Oxidase positive Indole negative Motility positive

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