BIOL 3052 Lecture Notes - Lecture 10: Colistin, Acanthamoeba, Peptide

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27 Jan 2017

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Method of isolation and enumeration of microorganisms in a mixed culture and distributing it evenly. The technique makes it easier to quantify bacteria in a solution. The spread plate technique involves using a sterilized spreader with a smooth surface made of metal or glass to apply a small amount of bacteria suspended in a solution over a plate. The plate needs to be dry and at room temperature so that the agar can absorb the bacteria more readily. A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate. Pipette out 0. 1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. Flame the glass spreader (hockey stick) over a bunsen burner. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the. Incubate the plate at 37 c for 24 hours.

Related Questions

In a quadrant streak, why do you intersect the previous lines and not the ends of the streak?

Select one:

a. More cell amount variability at ends, usually higher cell population

b. Keep streaks in the middle of the plate

c. Prevent cell death

d. To maintain a mixed culture

e. To count CFUs

How should you hold the inoculating loop?

Select one:

a. Maintain it at an angle at all times

b. In a test tube rack at all times

c. Like a pencil

d. Balanced on top of the tube

e. With dish gloves

Which of the following is used in the spread plate and not the streak plate method?

Select one:

a. Glass spreading rod

b. Flame loop

c. Agar plate

d. Bunsen burner/sterile utensil

e. Starting culture to sample

What would likely happen if the inoculum was not spread adequately over the agar surface?

Select one:

a. Contamination

b. Degradation

c. Sterilization

d. Uneven distribution

e. Cells will not grow

You have a mixed culture of unknown bacteria and want to identify one of the species by several subsequent experiments (needing multiple colonies of a pure culture). You do a quadrant streak and transfer to a sterile liquid medium. What next step makes the most sense?

Select one:

a. Perform a Gram stain

b. Note all colony morphology characteristics

c. Use the spread plate method or new streak plate on an isolated colony of the plate

d. Quantify the cells

e. Repeat using the other two methods of isolation

What would likely occur if the flame loop was not sterilized between streaks on the quadrant streak method?

Select one:

a. Bacteria will not stick to the loop

b. Virus will be introduced

c. Flame loop will be too hot

d. The sample will be contaminated

e. Subsequent streaks would have more colonies

Why is the glass rod in the spread plate method put in alcohol and ignited instead of sterilized like a flame loop?

Select one:

a. Glass oxidizes

b. Glass melts

c. Glass takes longer to cool

d. Glass burns

e. Glass is more sterile

Experiment 1: Effect of organic challenge on the efficacy of Milton fluid.

This experiment is designed to demonstrate two concepts. Firstly, that a disinfectant, like Milton Fluid, will kill microbial contaminants on surfaces such as glass. Secondly, to evaluate the effect of organic challenge on the ability of Milton to act as a disinfectant. Milton is 1% Sodium hypochlorite containing 16.5% Sodium Chloride.

Method

i. Label 3 universal bottles A, B and C.

ii. Pour milk into universals A and B until they are 1/3 full.

iii. Let stand for 5 mins.

iv. Pour milk down sink.

v. Clean universal A, but not B with detergent and bottle brush.

vi. Add Milton solution to all universals (A, B and C).

vii. Let stand for 15 mins.

viii. Pour Milton solution down the sink.

ix. Using aseptic technique, divide the nutrient broth provided between the 3 universals A, B and C.

x. Incubate overnight at 35 degrees Celsius.

Experiment 2: Antibiotic disk test (Perform antibiotics test assay).

1. Three test stains are provided with this test 1) E.coli, 2) Staphylococcus "albus" (epidermidis), 3) Bacillus subtilis; antibiotics are unless announced otherwise provided a) Penicillin, b) Cefoxitin, c) Ampicillin. Antibiotics b) and c) are provided as pre-soaked discs while a) is provided as 100mg/1 and 10mg/1 solution.

2. Open sterile swab without touching the cotton tip.

3. Open source culture using aseptic technique.

4. Dip cotton, tip of swab into broth.

5. Carefully 'paint' the entire surface of a sterile agar plate with the cotton bud to cover all areas of the plate. (This will grow up to become a lawn of bacteria.)

6. Label the bottom of the plate with the numbers 1 to 4 evenly spaced out around the table.

7. Sterilize a forceps using alcohol. Danger - check the location the fire blanket in the lab. Keep the alcohol at least 20cm away from Bunsen. If the alcohol container catches fire, cover with a lid and call a demonstrator.

8. Dip a sterile disc into the Penicillin 10mg/1 dilution, shake off excess.

9. Place this carefully onto the surface of the plate at the position marked 1.

10. Repeat for the 100mg/1 Penicillium solution, putting the disc at location 2.

11. Use a pre-soaked disc Cefoxitin. For position 3 and a pre-soaked disc of Ampicillin for position 4.

12. Incubate plates overnight at 35 degrees Celsius.

I need a good lab report for these two experiments with a structure of:

Objectives:
Introduction:
Materials and Methods:
Results:
Discussion/Conclusion:

I can write up the results part. Many thanks in advance.

BIOL 3052 Lecture Notes - Lecture 10: Colistin, Acanthamoeba, Peptide

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