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 The sample collected from the mare can be applied to either a single plate (i.e., tryptic soy agar (TSA) with 5% sheep blood), two individual plates (i.e., one with TSA with 5% sheep blood and the second with MacConkey II agar), or a “split plate” which has one half with TSA with 5% sheep blood and the other half with MacConkey II agar. MacConkey II agar is specific for Gram‐negative bacteria (Table 14.1).

 In addition, the swab may be applied to a chromogenic agar. Bacterial or fungal colonies that grow on chromogenic agar will turn a specific color, dependent on the organism.

 Another option is to apply the sample onto an agar that is designed to promote growth of fungal organisms and inhibit development of bacterial organisms, such as Sabouraud agar. Incubation time varies widely for fungal organisms, and ranges from 2 days for some yeast organisms to 2–4 weeks for dimorphic fungi.Table 14.1 Agar used in the culture of microbial organisms.Agar typeCharacteristicsTryptic soy agar (TSA)TSA is an all‐purpose medium that supports the growth of bacteria that do not have a specific nutritional need. The addition of 5% sheep red blood cells to the agar allows for a visual differentiation of some bacterial organisms due to various types of hemolysis:α‐hemolysis: partial hemolysis, often appears as a zone of green, gray, or brown discoloration around the colonyβ‐hemolysis: clear, colorless zone caused by complete hemolysis of the red blood cellsγ‐hemolysis: no detectable hemolysisMacConkey II agarMacConkey agar utilizes bile salts and crystal violet to inhibit the growth of most Gram‐positive bacteria and most Gram‐negative cocci, thus selecting for Gram‐negative bacilli (i.e., rods). This agar can also be used as a visual differentiation medium based on the bacteria’s ability or inability to ferment lactose. Organisms that ferment lactose produce acid that turns the acid indicator in the agar (phenol red) a reddish or pink color. Strong lactose fermenters will produce red colonies surrounded by a pink ring of precipitated bile salts. Non‐lactose fermenters will produce colorless or transparent colonies. It is important for these plates to be checked by 24 hours as continued incubation will result in altered results as the bacteria use up the available lactose, resulting in misrepresented color characteristicsChromogenic agarChromogenic agar is an all‐purpose medium. It is used for the presumptive identification of bacterial organisms based on the production of a color compound in the bacteria. Additional chromogenic media 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	Small, light blue colonies
Escherichia coli	Neg.	Rods	Cream‐colored colonies (α‐hemolysis) (2–3 mm)	Medium sized, gray to pink colonies	Medium, pink to red colonies
Klebsiella pneumoniae	Neg.	Rods	Large, gray, mucoid colonies (non‐hemolytic) (2–4 mm)	Large, pink, mucoid colonies	Large, blue with slight pink halo colonies
Pseudomonas aeruginosa	Neg.	Rods	Flat metallic blue colonies (β‐hemolysis) (3–4 mm)	Large, pale greenish colonies	Transparent white to green colonies	Grape‐like odor on blood agar; fluorescence with Wood’s UV light
Staphylococcus aureus	Pos.	Cocci (round, clusters)	Medium, cream to gold colonies (± β‐hemolysis) (2–3 mm)	No growth or limited growth of pink colonies	White to light yellow colonies
Candida albicans			Soft, creamy, raised, glistening colonies			Beer‐like odor; confirm with fungal agar (i.e., Sabouraud agar) for up to 7 days