1. Chapter 41 Culture Techniques
Microbiology Unit 7
Chapter 41
Culture Techniques
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2. Culturing Specimens
A systematic approach is needed to identify pathogenic bacteria
Develop flowcharts for use in the clinic for the most commonly seen bacteria
Include tests to differentiate those bacteria
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3. Flowchart
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4. Typical Sequence of Testing of Microbiology Specimens
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5. Presumptive Identification
Staining and culturing
Veterinarian may start treatment based on results
Plate suspected pathogens on primary medium
Blood agar or MacConkey agar
Incubate for 18 to 24 hours – examine for growth
Follow flow chart for further identification
Definitive identification
Requires additional biochemical testing
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6. Presumptive Identification (cont.)
Blood agar
Grows both gram-positive and gram-negative
Selection of colonies is preferable
MacConkey agar
Gram-positive organisms do not grow
May inhibit growth of potentially pathogenic bacteria
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7. Inoculation of Culture Media
Aseptic technique at all times
Sear surface of cadaver or excised organs
Culture plates kept closed except when removing specimens
Tubes – pass neck of tube through a flame and do not put down cap, hold in pinkie finger
Flame wire – start at near end first and work toward distal end
Could cause splattering of bacteria – aerosolization
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8. Inoculating with a Swab
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9. Streaking Culture Plates
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10. Quadrant Streaking Method
Designed to aid in isolation of pure bacterial cultures
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11. Inoculations of Slant and Butt
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12. Inoculations of Tube Media
Enough bacteria should be on the wire to inoculate the surface even after stabbing the butt
Slant surface inoculated in an “S” shape
Tube cap on loosely
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13. Inoculations of Tube Media (cont.)
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14. Inoculations of Cultures
Pathogens that can invade internal organs
Incubate at 37º C
Some fish, skin pathogens, and environmental organisms
Incubate lower than 37º C
Incubation time depends on the generation time of the species and type of medium
Most are 48 hours with examination at 18 to 24 hours
Incubate plates inverted to prevent condensation on the surface of the plate
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15. Candle Jar For pathogens that require carbon dioxide for growth
Place plates in a large jar
Place a lit candle on top of the plates and seal jar
The candle dies, leaving a decreased amount of oxygen and an increase in CO2 in the jar
Not anaerobic condition
Examine plates for 18 to 24 hours; if no growth, reincubate in jar for another 18 to 24 hours
Large laboratories have incubators that control temperature, CO2, O2, and humidity
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16. Colony Characteristics
Help identify the bacterium
Size
Pigment
Density (opaque, transparent)
Elevation (raised, flat, convex, droplike)
Form (circular, irregular, rhizoid, filamentous, undulate)
Texture (glassy, smooth, mucoid, buttery, brittle, sticky)
Odor (pungent, sweet, etc.)
Any hemolysis (alpha, beta, gamma)
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17. Colony Characteristics (cont.)
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18. Colony Characteristics (cont.)
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19. Culture of Anaerobes Do not use swabs
Preferred blocks of tissue (2-inch cube minimum) in a closed, sterile container and pus and exudate collected in a sterile syringe
Air expelled, needle plugged
Special anaerobic specimen collection systems
Culture as soon as possible after collection
Blood agar and thioglycollate broth
Incubate in an anaerobe jar
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20. Summary
A systemic approach is needed for proper evaluation of cultures
A quadrant streak method is used to isolate a pure culture
Slant tubes can be inoculated on the slant surface, the butt area, or both
High CO2 environments can be created with a candle jar
Cultures incubated at 37º C and initially examined at 18 to 24 hours
Presumptive identification can be achieved by evaluation of the colony morphology
Morphology evaluation includes size, density, pigment, elevation, form, texture, odor, and hemolysis
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