are available for use in fungal cultures. This type of media contains chromogens, or substrates, that release a particular colored compound when degraded by specific microbial enzymes. It is important to have a description of colors produced for a given company’s chromogenic media as they may vary in the chromogens used in production of the media. Also, the color results should be interpreted within 24 hours as the colors may alter with continued incubationSabouraud agarSabouraud agar is a non‐selective medium used for the cultivation of fungal organisms. The acidic pH and addition of chloramphenicol inhibits bacterial growth. The Emmons modification to the original formula has a higher pH and a reduced dextrose level to help in a greater recovery of fungi. It is advisable to utilize the added chloramphenicol if using the Sabouraud dextrose Emmons agar. Incubation time varies widely for fungal organisms, anywhere from 2 days for some yeast up to 2–4 weeks for dimorphic fungus. This difference is incubation time, along with colony morphology, can help in the presumptive identification of the organismMueller Hinton agarMueller Hinton agar is a non‐selective medium used for antibiotic susceptibility testing using paper disks impregnated with a specific concentration of an antibiotic. This medium is low in sulfonamide, trimethoprim, and tetracycline inhibitors. It also provides satisfactory growth of most non‐fastidious pathogens and demonstrates batch‐to‐batch reproducibility for standardized testing. The addition of 5% sheep red blood cells aids in the visualization of β‐hemolytic bacterial growth
Remove a quad plate of TSA with 5% sheep blood, MacConkey II agar, and Gram‐positive and Gram‐negative chromogenic agar from the refrigerator and allow it to equilibrate to room temperature. Label the bottom side of the agar plate with the mare’s name, source of sample, and date.
Streak all four quadrants with the sample swab on the inside of each corner of the plate (primary streak) (see Figure 14.1 as an example).Figure 14.1 Inoculation of a streak plate for cultivation of microbial organisms. This example shows how to streak one entire plate; a slight modification is required to streak each quadrant of a quad plate.
With a new sterile swab, streak through the primary streak one or more times and continue to streak the second third of the plate (secondary streak).
With a new sterile swab, streak through the secondary streak one or more times and continue to streak the final third of the plate (tertiary streak). The concept is to separate the colonies so that individual colonies can be identified.
Perform the same streaking pattern on the other quadrants.Figure 14.2 Culture of Streptococcus equi subspecies zooepidemicus on a quad plate with TSA with 5% sheep blood (upper right), MacConkey II agar (upper left), Gram‐positive chromogenic agar (lower right), and Gram‐negative chromogenic agar (lower left). Note the growth of small white colonies with β‐hemolysis (black arrow) on blood agar, the lack of growth on MacConkey agar, and the small light blue colonies on the Gram‐positive chromogenic agar.
If additional agars are to be used, inoculate in the same manner as previously described.
Incubate the plate(s), bottom side up in a 37°C (99°F) incubator for 20–24 hours.
After incubation, observe the quad plate for the appearance of any colony growth and determine if more than one type of colony is present. Identify hemolytic activity of colonies developing on TSA agar. Determine if contaminants are present (e.g., where are the colonies located on the plate, are they actually touching a streak line, are there more than one type of colony?) (Figures ; Table 14.2).
Observe any additional inoculated agars for presumptive identification or growth of a fungal/yeast organism (i.e., Sabouraud agar).
Characterize the amount of growth as either no growth, very light growth, light growth, moderate growth, or heavy growth.
Plates with mixed growth should be subcultured and individual organisms subsequently identified.
Plates should be cultured for a minimum of 72 hours before being discarded.
Figure 14.3 Culture of Escherichia coli on a quad plate with TSA with 5% sheep blood (upper right), MacConkey II agar (upper left), Gram‐positive chromogenic agar (lower right), and Gram‐negative chromogenic agar (lower left). Note the growth of cream‐colored colonies without hemolysis on blood agar, the medium‐sized pink colonies on MacConkey agar, and the pink to red colonies on Gram‐negative chromogenic agar.
Figure 14.4 Culture of Pseudomonas aeruginosa on a quad plate with TSA with 5% sheep blood (upper right), MacConkey II agar (upper left), Gram‐positive chromogenic agar (lower right), and Gram‐negative chromogenic agar (lower left). Note the growth of flat metallic green‐blue colonies on blood agar, the large pale greenish colonies on MacConkey agar, and the growth of transparent white to green colonies on Gram‐negative chromogenic agar.
Figure 14.5 Culture of Klebsiella pneumoniae on a quad plate with TSA with 5% sheep blood (upper right), MacConkey II agar (upper left), Gram‐positive chromogenic agar (lower right), and Gram‐negative chromogenic agar (lower left). Note the growth of large gray mucoid colonies without hemolysis on blood agar, the large pink mucoid colonies on MacConkey agar, and the growth of large blue colonies with a slight pink halo on Gram‐negative chromogenic agar.
Table 14.2 Culture characteristics for common microbial organisms associated with infectious equine endometritis.
| Organism | Gram Stain | Morphology | TSA/5% Sheep Blood Agar | MacConkey Agar | Chromogenic Agar | Comments |
|---|---|---|---|---|---|---|
| Streptococcus equi subsp. zooepidemicus | Pos. | Cocci (ovoid, chains) | Small, white, round colonies (β‐hemolysis) (0.5–1.0 mm) | No growth |
|
