Option 2 for inoculating the MH‐II agar plate:Prepare a bacterial suspension in saline, tryptic soy broth, or MH broth that contains between 1 × 108 and 2 × 108 CFU/ml of the bacteria in question.Pick three to five isolated colonies from the primary plate.Transfer the colonies into the media.Adjust the turbidity to a 0.5 McFarland Standard.Inoculate the plate with the bacterial suspension.
For both options, use the following procedure. Place the antibiotic disk dispenser over the plate and depress the plunger to dispense the disks and gently tamp the antibiotic disks onto the agar surface.
Place the plate in an incubator, agar side up, for 24 hours at 37°C (99°F).Figure 13.1 Pattern of bacterial streaking on a Mueller Hinton II agar plate and placement of antimicrobial disks.Figure 13.2 Antimicrobial susceptibility test. This bacterial organism was sensitive to two antibiotics, intermediate to one, and resistant to the other five antibiotics.
After the incubation period, use a clear ruler to measure the diameter of the zone of inhibition around each disk (Figure 13.2). Measure on the bottom side of the plate viewing through the plate. If adjacent areas of inhibition overlap, measure the radius and multiple by 2.
Record the results in millimeters. Always round up to the next millimeter.
Compare the results with the standard zone of inhibition for each antibiotic provided by the manufacturer and report the result as resistant, intermediate, or susceptible. The zone of inhibition of bacterial growth is different for each antibiotic due to the differences in migration of the different‐sized antibiotic molecules through the agar, the solubility of the antibiotic, and the diffusion properties.
Further Reading
1 Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, FitzPatrick ES. 2011. Antibacterial agents. In: Veterinary Microbiology and Microbial Disease, 2nd edn. Chichester: Wiley Blackwell, pp. 143–8.
2 Ricketts SW. 2011. Uterine and clitoral cultures. In: McKinnon AO, Squires EL, Vaala WE, Varner DD (eds). Equine Reproduction, 2nd edn. Ames, IA: Wiley Blackwell, pp. 1963–78.
3 Songer J, Post K. 2005. Antimicrobial susceptibility testing and principles of antimicrobial therapy. In: Veterinary Microbiology Bacterial and Fungal Agents of Animal Disease. Amsterdam: Elsevier, pp. 21–31.
14 Microbiology: Microbial Culture
Patrick M. McCue1 and Jillian Bishop2
1 Equine Reproduction Laboratory, Colorado State University, USA
2 University of Kansas Health System, USA
Introduction
A clinical microbiology laboratory can be set up at most veterinary practices to detect and identify common microbial pathogens encountered in equine reproduction. Microbial testing allows for early diagnosis of infectious endometritis and consequently the development and implementation of a therapeutic plan.
Equipment and Supplies
Incubator, refrigerator, biohazard waste container, biohazard bags, sterile cotton tip swabs/applicators, disposable inoculating loops, Vircon® disinfectant, quad plates (with tryptic soy agar (TSA) with 5% sheep blood, MacConkey II/TSA with 5% sheep blood, Gram‐positive chromogenic agar, and Gram‐negative chromogenic agar; Vetlab Supply, Inc., Palmetto Bay, FL, USA).
Technique
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