Lab 6

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Lab 6: Aseptic Technique

Purpose: To learn an aseptic technique to reduce the minimum contamination, while transferring bacteria from one place to another place by using inoculating loop or a needle. Also correctly label on a agar plate.

Preparation:

  • Work on a clear table.  Put all unnecessary items away.
  • Wear a lab coat, wash hands before transferring bacteria and after.
  • Disinfect the table with disinfectant before and after work.
  • Keep all cultures closed and tubes upright in a rack until ready to use.

Procedure:

- Each student, using aseptic technique, will make three cultures of the assigned species. The first will be in a broth tube, the second a slant tube, and the third in a deep.

– Slants and deeps are broth solid media (contain agar), but are cooled at different angles and used for different purposes.
– A deep can be made with semisolid agar (half the usual concentration) to determine whether a bacterium is motile.
+ Motile bacteria can move away from the inoculation stab line as they grow.
– When bacteria grow in broth, they create a cloudy appearance that is described as turbid.
– Some bacteria will grow in large clumps (flocculent), create a membrane at the top of the tube (pellicle), or settle on the bottom (sediment).
CIMG0223

1. Disinfect the table to prevent cross-contamination.

2. Clear work area.
3. Collect the sterile culture tubes you will need and label them with your initials, the date, and the organism to be cultured. I had Microcccus luteus.
4. Inoculating the nutrient broth
a. Turn on the bunsen burner so that there is a blue flame with an internal blue cone.
b. Sterilize inoculating loop by running it through the tip of the internal blue cone of the flame so that it turns red-hot.
– allow the loop to cool for at least 30 seconds before using it
– work close to the flame to prevent microbes in the air from getting in
– do not put the sterilized loop on the work surface.
c. Pick up the culture tube of your assisned species. Remove the cap and flame the mouth of the tube. Do not set the cap down!
d. Immerse the cooled loop into the culture tube, remove the loop, and continue to hold the loop while flaming and recapping the tube. Replace the tube in your rack.
e. Uncap and flame the new, blank broth tube. Immerse the inoculating loop into the broth and then remove it. Flame the mouth of the tube and recap it. Leave the cap loose fr incubation.
f. Sterilize your loop before setting it anywhere.
5. Inoculating a slant
a. Continue to use aseptic technique when capping and uncapping your tubes.
b. Use your loop to transfer bacteria to the surface of the slant tube; move the loop at a flat angle back and forth across the exposed surface area from the lowest to highest spot.
c. Sterilize your loop when you are finished before transferring the bacteria.
6. Inoculating a deep
a. Use your inoculating needle to transfer bacteria from the culture provided into the sterile deep tube. Plunge the needle straight down the middle of the deep and then pull it straight out again.
b. Sterilize the inoculating needle before setting it down.
7. Incubate all tubes at 35 degrees Celsius until the next lab period.
8. Record observations of all species provided, observing your cultures and those of your lab partners.

(source:  Mission college microbiology copyright 2009 by Melanie O’ Brien)

Observation:

Bacterial species Is the broth turbid? Does the broth have flocculent, pellicle, or sediment? (note which) Pigmentation in broth Appearance on slant (even, beaded, or rhizoid) Pigmentation on slant

Micrococcus luteus

No

Sediment

Pale

Even

Yellow

Pseudomonas aeruginosa

Yes

Pellicle sediment

Pale

Even

Aqua

Proteus vulgaris

Yes

Sediment

Yellow

Even

White

Escherichia coli

Yes

Sediment

pale (dark)

Even

pale

Sketches of Growth in deep.

CIMG0230

Questions

  • Which bacterial species in this exercise are motile?

E.coli

  • What is the purpose of flaming a loop before use?

To kill excess organisms to reduce the chances of contamination

  • What is the purpose of flaming a loop after use?

To kill bacteria that can be harmful

  • Why should the loop be cool before touching it to a culture?

So the hot loop won’t kill the culture, since most of the cultures are sensitive to heat.

  • Why is aseptic technique important?

Aseptic technique is important to reduce the contamination in other for the results to be reasonable.  Also to reduce the chances of risk to humans.

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