Procedure is basically the same as for liquid media. 1. Flame needle. 2.
Remove closure-flame tube. 3. Obtain small sample from stock culture. 4. Flame and close culture tube. 5. Return tube to rack. 6.
Pick up sterile nutrient agar slant in left hand. 7. Remove closure-flame tube. 8. Gently streak sample from base of agar slant to the top in one continuous stroke.
Do not chop up or dig into the agar. 9. Flame the tube and close. 10.
Flame the needle.IV. Pouring agar into Petri dish aseptically. 1.
Place the three tubes of nutrient agar deeps into a water broth. A thermometer is necessary to measure water temperature-do not let it rest on bottom. Be sure that the water level in the container is high enough to cover the agar in the tubes. 2. Bring the water to a boil. Agar liquefies at approximately 98 degrees Celsius.
It solidifies at 42 degrees Celsius. 3. When agar has liquefied, remove the burner and allow the tubes to cool down in the water bath to about 50 degrees Celsius. 4. Remove tube-take off cap and pass mouth of tube through flame of Bunsen burner.
5. Lift top of Petri dish as illustrated and pour contents of the tube into sterile Petri dish. 6. Protect surface from contamination by holding cover above and replacing it immediately after pouring in the agar.
7. Swirl gently so that the agar is distributed evenly in the bottom of the plate. Allow the agar to solidify completely before proceeding.
This should take only a few minutes. Surface should be even and firm-without lumps or cracks.V. Streaking. 1. Using aseptic technique remove a small amount of the stock culture.
2. Lift the lid of the Petri dish-protecting the surface as much as possible from air contamination. Hold life just high enough to insert needle.
3. Place the culture (inoculums) on the agar surface at spot 1. Follow the diagrams of streaking illustrated-one for each three test organisms. Be careful not to dig into agar. 4. Close the Petri dish.
5. INVERT and incubate. Note: Petri dishes are inverted while incubating to prevent water loss from media and to prevent condensation on lid. 6.
Follow the same procedure for other test organisms. Be sure to sterilize your needle between each organism. 7. Label, collect all tubes in one container and incubate.
IV. & V. Daily Diary/ Qualitative Observations: Day 1: (10/13/11) Members of my group included Lyndsey Hensel, Brooke Brown, Lauren Hathaway, and I. We poured the agar into erlinmier flasks and then kept them in the autoclave. Then we poured the agar from the flasks into 3 Petri dishes and 3 slants, tipped the slant tubes at an angle so they would obtain the ???slant??? form, and waited for them all to dry. The agar had a smell that seemed to be like a beefy, sewage type of smell. Mrs.
Bentley then poured our broth into 3 more tubes, while we waited for the agar to dry, and then inoculated (from the bacterial cultures) Bacillus subtilis, Micrococcus luteus, and Rhodospirillum rubrum, into one of each broth, agar slant, and Petri dish. Afterwards we then took broth (3), agar slants (3), and Petri dishes (3), and set them in the incubator to allow culturing of the microorganisms at a constant temperature favourable to its growth and development. Day 2: (24 hour observation) ??“ We observed our agar and broth cultures and began seeing some growth of Bacillus subtilis in the broth and Petri dish, but especially in the agar slant.
I described the dish of the Bacillus subtilis to be as either a circular or punctiform colonial growth, the agar slant to be a beaded type of growth, and the broth to be of a turbitity type of growth. Also seen some growth of Micrococcus luteus in the Petri dish, very few in the agar slant, and some in the broth. Petri dish was circular, agar slant was beaded (just one decent sized dot), and broth was a turbitity type of growth.
Viewing the Rhodospirillum rubrum, we only saw some type of rhizoid growth only in the agar slant, no other growth was seen in the Petri dish or the broth. Day 3: (48 hour observation) ??“ We observed our growing cultures and noticed that most of the types of growth in each culture remained the same but the amount of growth went way up. The Bacillus subtilis went more to a punctiform type of growth in the Petri dish, and the broth turned into more of a flocculent type of growth. Still, no growth remained in the Petri dish and broth of the Rhodospirillum rubrum.
Day 4: (72 hour observation) ??“ Observing our cultures, we saw that the growth has seemed to peak in the Micrococcus luteus and Bacillus subtilis (some additional growth but minimal), and the types of growth remained the same. But, growth of the Rhodospirillum rubrum began to form in the Petri dish and it seemed to be beaded, just like the Micrococcus luteus.VII. Questions: 1. Why are tubes flamed after closures are removed and before they are replaced A: The tubes are flamed after closures are removed and before they are replaced in order to discourage any microorganisms in the air or on the edge of the tube from falling into the culture and contaminating it. Basically, to prevent contamination in order to have a ???pure??? culture, as well as a qualitative lab experience. 2.
If you were handling a pathogen, what other precautions would you observe A: In our lab we wear goggles if we??™re wearing contacts; essentially, we wear some form of eye protection and a lab apron. Depending on airborne or not I would wear some type of respiratory protection, and if it was highly contagious I would recommend wearing gloves. These are the precautions I would observe when handling a pathogen.
3. What application of aseptic technique can you make to nursing or health practices A: Wearing gloves and sterilizing your hands by washing them before and after handling a patient, also doing the same thing to your medical equipment after usage. These would be a great way of application aseptic techniques that you can apply to nursing or health practices. 4. Why are Petri dishes inverted when incubated A: Petri dishes are inverted when incubated to prevent the top from fogging because of condensation. Prevention of this makes it so the condensation does not drip down onto the agar and kill the specimens. 5. Why is platinum wire used as the inoculation needle A: Platinum wire is used as an inoculation needle because it can heat up very rapidly as well as cool down, which makes the sterilizing quick and efficient.
6. Explain the differences you observe after shaking broth cultures. Our group never ended up shaking the broth tubes when observing our cultures or preparing slides, but I??™m guessing it evenly disperses the specimen for better viewing or sample taking.