Basic Bacterial Culture and Identification

• Pure Culture Techniques
• Antibiotic Sensitivity Testing
• Gram Staining
• Acid-Fast and Methylene Blue Staining
• Urinary Tract Bacterial Culture
Culture of Normal Flora Organisms
MATERIALS:
• Sterile cotton swab
• Bacteriological loop
• Nutrient Agar plates
• Biohazard discard container
DIRECTIONS:
1. Carefully swab the selected skin surface site in an area approximately 4 cm in diameter. Swab thoroughly, rotating the swab to ensure that a good inoculum has been obtained.
2. Transfer the bacteria to the agar culture dish by touching the swab to the agar surface in a single spot. Once you feel confident that a good inoculum has been transferred, the swab can be discarded in the biohazard container.

3. With the inoculating loop, proceed to streak in a second and third streak pattern (see illustration). Briefly, isolate bacterial colonies by pulling several streaks out from the original swab inoculum site. Use the method shown above to avoid tearing the agar.

4. Incubate the plate at 37oC for 24-48 hours.

5. Test the colonies that have grown by Gram staining; identify Staphylococcus aureus colonies by testing Gram-positive cocci using a specific latex agglutination reagent that binds to Staphylococcus aureus.
Pure Culture Techniques
Microbiologists have developed special techniques and equipment to isolate and grow pure cultures of microorganisms that are free from contaminating forms. This exercise is designed to give the student an introduction to these special techniques. It is important that techniques be practiced until some degree of skill is developed. Pure cultures are essential when identifying unknowns. During the course of the semester, each student will be expected to develop independently a patient-oriented case to correlate with each of 4 separate bacterial isolates. It is important, therefore, to maintain unique isolates in pure culture to be submitted with each case summary. Further instruction will be given during later labs.
THE STREAK PLATE TECHNIQUE
The procedure of streaking a plate with an inoculating loop is used to spread millions of cells over the surface of a solid medium so that some individual cells are deposited at a distance from all others. These cells grow and reproduce, forming an isolated colony. One or more colonies will be well separated from all others and represent a source of a pure culture. The procedure is similar to the one used for streaking from the collection swab.
MATERIALS:
• Streak plates from the previous lab session
• Nutrient agar plates (NA)
• Bunsen burner
• Bacteriological loop
DIRECTIONS:
Examine the streak plates prepared during the previous lab period and locate a number of well-isolated colonies. Now you must transfer a portion of each colony to a separate agar slant. To "pick" a colony you will be using an inoculating loop. Sterilize the loop in the burner flame, let cool 3-5 seconds then touch the end of the loop to the isolated colony, picking up the microorganisms from the colony. Now re-cover the streak plate and pick up one NA plate. You will now be holding the inoculating loop in your right hand and the fresh NA plate in your left (lefties reversed).
Remove the lid from the plate, place the inoculating loop at one edge of the plate and with a sweeping stroke, inoculate the agar using the same tri-streak method as used for the initial isolation. Replace the lid. Flame the loop and proceed to inoculate another plate from different colonies. Try to use colonies that are visibly different in morphology. Incubate the plates in the 37oC incubator until the next class period.
There is nothing difficult about picking colonies and inoculating slants, but you must avoid contamination.

Lectures on applied clinical microbiology