Sterile Technique: Delivery of Liquids by Pipette

Sterile Technique: Delivery of Liquids by Pipette

Pasteur observed that once a medium is sterilized, it remains sterile unless contaminated from an external source. He used this observation to reject spontaneous generation but it also lays the foundation for sterile (or aseptic) technique, a technique crucial to microbiology and medicine. Several practices are important to the success of this technique:

  1. Sterilize the instruments and keep them that way: keep instruments off possibly contaminated surfaces, and flame them to incinerate loose material which might fall off. Never lie pipets down on the surface of a workbench.
  2. Sterilize the field where work is to be performed with 70% EtOH.
  3. Exclude airborne contamination: keep containers covered as much as possible
  4. Minimize drafts: no breathing, coughing, etc over sterile materials.

Mastering sterile technique is one of the most important prerequisites to working in a microbiological lab. While the steps may seem overly detailed in the following narrative, early care in learning proper technique establishes good technique for the rest of your life. Compare these detailed steps with the demonstration given by the professor. Patience pays off. Go slowly at the beginning, and verbally (not physically) assist your fellow students as they work through the steps.

PREPARATION:

  1. Write out the procedure which you plan to perform in your notebook. Indicate how the sample was prepared and volumes to be transferred. Construct a table of plates to be spread, with plate number, specimen, plate type, dilution factor, aliquot, and two empty columns for colonies counted and final calculated numbers. Label tubes or plates appropriately.
  2. Loosen all caps, mentally review the steps you will be performing and arrange materials to ensure they are available and easily accessible.
  3. Select the appropriate pipet for volume to be delivered, and set the volume to be transferred by rotating the plunger. Pick up a sterile tip, reclose the lid on the box of tips immediately.
  4. Hold displacement pipetter so that the thumb operates the plunger and you keep your little finger on the pipetting hand free for grasping the cap of the vessel.
  5. Pick up specimen vessel with other hand, grip cap of vessel with little finger of pipet hand and twist the vessel and pull it down away from the cap. Do not lay the cap down.
  6. Flame lip of vessel.

USING THE PIPET:

  1. Pass sterile pipet through flame. (Very briefly so that the plastic tip is not melted…)
  2. DRAW UP SPECIMEN: Immerse tip of pipet 1/2 cm below surface of liquid, brace in place by gently pressing pipet against the lip of the tube while drawing the fluid up. To avoid bubbles, draw the fluid slowly into the tip by slowly raising your thumb.
  3. TRANSFER ALIQUOT: Touch off excess fluid from tip of pipet, tilt pipet horizontally to prevent dribbling during transfer. (The fluid will draw up slightly into the tip pipet.)
  4. Reflame the lip of specimen vessel, screw it back into the cap, set vessel down.
  5. DELIVER ALIQUOT: Pick up receiving vessel. Remove its cap and flame its lip (as in steps 5 and 6). Insert pipet, return to vertical, deliver desired volume, remove pipet, flame lip, replace cap, discard tip into used pipet container for sterilization.
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Yeast Plate Count Protocol

Yeast Plate Count Protocol

The plate count is one of the most accurate means of enumeration of viable microbes because you get a visual indicator for every cell in the specimen.  The technique stems from Robert Koch’s insight gained from viewing colonies growing on the surface of a spoiling slice of potato.  In practice, a small aliquot of a liquid suspension of microbes is spread on the surface of  solidified nutrient medium, which when incubated, leads to each cell ‘developing’ into a visible colony through repeated fission.  Many samples may have so many microbes that even a small aliquot would have more than the 30-300 colony forming units per aliquot.  In that case, the sample must be diluted to roughly 300-3,000 CFU per mL of which 0.1 mL can then be plated out.

This exercise employs all of these techniques to assay the number of yeast CFUs in a package of yeast.  Here are the steps and the experimental plan:

1) Suspension of the entire contents of a package of yeast in 100 mL dH2O
2) Dilution of this suspension by serial dilution to a dilution factor of 10^6 (a million fold dilution)
3) plating out aliquots of the 10^6 dilution on nutrient agar + 4% glucose
4) Incubation at 35 (or 37) C for 48 hours to develop the colonies
5) Counting all colonies formed, and calculating the number of CFUs in the original package

REQUIRED SUPPLIES:

fresh package of baker’s yeast
250 mL beaker with 100 mL dH2O
FOR EACH STUDENT:
two nutrient agar plus 4% glucose plates

THREE PROGRESSIVE WORK STATIONS ARE SET UP.

  1. Repipet Station :
    sterile capped 16 x 150 mm test tubes, three per student
    test tube rack, one per student
    2 repipets with sterile dH2O, set for 9.9 mL
    flame
  2. Two Serial dilution stations (2x two adjacent, sharing equipment):
    displacement pipetters, 20-200uL, and sterile tips
    flame
    yeast suspension on Magnet-o-stir to keep suspended
    vortex
    plastic used pipet container
  3. Two Plating out stations with sterilized field on which to work, (2x two adjacent, sharing equipment):
    2 turntables
    vortex
    2 spreaders in:
    2 250 mL beakers,1/2 filled with 95% EtOH
    displacement pipetters, 20-200uL, and sterile tips
    flame
    used pipet container with 1 inch deep diluted Lysol

PROTOCOL:  Here is a chalkboard diagram of the protocol

Prepare yeast suspension:
First weigh full package of yeast, then suspend contents in 100 mL water. Mix thoroughly (magnet-o-stir works well) for 5-10 minutes . Weigh empty package, determine the dry weight of the yeast added.
Create a data table in your notebook in which you list the plate number, the specimen, its dilution factor, the aliquot plated, a space for the number of CFUs on each plate, and room for calculation of total CFUs in the original sample.

Prepare dilution tubes:
Repipet 9.9 mL sterile dH2O into each of three sterile 16 x 150 mm capped tubes.
Here is a close up of a repipet set for 9.9 mL . Note the calibration stem with moveable clamp, the plunger (which is raised up all the way and then depressed all the way to deliver the volume), and the spout.
Label the tubes 2, 4, 6 (for Yeast Plate Count 10 2, 104 and 106 dilutions).

Perform a 106 serial dilution of the suspension as follows, using a fresh tip on a displacement pipetter for each stage:
a. Deliver 0.1 mL of original yeast suspension into first tube (#2) vortex to mix
b. Deliver 0.1 from #2 into #4 tube, vortex to mix
c. Deliver 0.1 from #4 into #6 tube, vortex to mix

Fankhauser_at_spectroph_P1010005

Determine the A660 of the 10 2 dilution , record in your book and on the computer.

Label two 4% glucose nutrient agar plates on their bottoms with four bits of data:
date
your initials
mL aliquot plated (0.1 or 0.2), and
dilution factor (106 )

Plate out samples:
Measure out 0.1 mL (and 0.2 mL) aliquot of the 106 dilution
Insert the tip just below to surface and release the thumb plunger to draw up the aliquot.
Apply to the surface of the appropriate plates (do not gouge the agar surface)
Spread evenly . (Do not press too hard into the agar. When the sample has been absorbed, the spreader will drag slightly over the agar.)

Incubate inverted at 37C for 48 hours. (Here are plates which have been incubated, two sets of which were incubated upside down. Can you see why the agar side should be up?)

Count the number of colonies . Here are sample plates: a 0.1 mL plate (41 colonies), and a 0.2 mL plate (89 colonies).
(Here are two plates, one with 0.1 mL the other with 0.2 mL aliquots spread. 0.1 = 37 CFU, 0.2 = 97 CFU)

Calculate the original number of colony forming units (CFU) in the package:
CFU/package = CFU/plate x dil factor x 1/aliquot x 100 mL/pkg
For instance, for the 0.1 mL plate:
41 CFU/plate x 10^6 x 1/0.1 mL x 100 mL/pkg = 4.1 x 10^10/package

Smear and stain examples of a yeast colony (Note how the colonies appear “waxy,” from where baker’s yeast gets its specific name, cerevisiae) and a contaminate, staining with methylene blue, illustrate at 1000x . Note in this closer view the staining of the organelles in the yeast cytoplasm . Here is yeast stained with the Gram stain , a closer view , both of which show budding. This view of yeast plus E. coli contrasts their different sizes and staining properties.
In Summer of 2011, two students got tiny colonies on one of their two yeast plates. A gram stain showed them to be more spherical than normal, and the presence of numerous ‘ghosts‘. Here is another image of these affected yeast. Could they be infected with a Killer” virus which infects Saccharomyces?