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Lab 3
Microbiology 2421
Quiz
• On a negative stain, what is stained?
• Why do you heat fix a slide?
Quiz
• On a negative stain, what is stained?
• the slide, or everything except the cell itself.
• Why do you heat fix a slide?
• So the organism are not washed off.
Today
• Lab 2 equation
• There 3 procedures that have to be completed before the end of lab today.
• Hang drop slide
• Negative staining
• Simple staining
Lab 2
• The equation that is used
• Colonies =X
• X/10^-8=_Y____ cfu/(1/10)ml
• Y/.10=cfu/ml
• Turn in lab 2 when finish and begin with lab 3
Calculating Colony forming unites
• Counted 11 colonies on your plate that was at 10^-8 dilution.
• 11/10^-8=1x10^9(cfu/(1/10)ml)
• Since this is at .1ul since you measured this and you need it at 1ml.
• 1x10^9/.1= 1.1x10^10(cfu/ml)
Negative staining
• Procedure II: Negative Staining• 1. Using Bacillus megaterium and Staphylococcus aureus,
transfer a small amount of bacteria to a drop of water to each half of a slide (2 bacteria can be stained on one slide)
• 2. Add 1-2 drops of nigrosin solution to the bacteria and mix with the inoculating loop.
• 3. Air dry and observe.• 4. Draw your observations in the circles provided below.• 5. Repeat this procedure using 1% Congo red.• 6. Remember to stain at least two of the isolated colonies
from last week’s session.
Negative stain
Negative stain
Simple stain
• 1. Using a waxed pencil, label the underside of a microscope slide with the initials of the bacterium you are staining.
• 2. Add a small drop of H20 on each half of the slide. Using a sterile inoculating loop, transfer a small amount of bacteria to the water. Remember, you only need enough to make the water lightly turbid.
• 3. Allow the sample to air dry. When this is ready, you should be able to see a slight smear on the slide.
• 4. Heat fix the preparations by passing the slides through a Bunsen burner flame 3 times. This will “fix” the bacteria to the slide so the preparation will not wash off.
• 5. Choose 2 bacteria for each stain (so, 2 slides each with 2 bacteria, each slide with a different color). Flood one slide with methylene blue and one slide with safranin for 3 minutes.
• 6. Wash the slides gently with distilled water and blot dry gently so you do not wipe the bacteria off the slide.
• 7. Observe the shapes and arrangements of the bacteria using the oil immersion objective.
• 8. Fill in the information below for each bacterium and draw your observations.
Simple stains
Simple stains
Hang Drop slides
• This is to view the microbe ability to move.
• Be able to determine a true motile microbe from Brownian motion.
• Prokaryotic cells have flagella of the ability to glide.
Hang drop slide
• Procedure I: Hanging drop slide• You will notice that special slides will be used for this procedure
that must be returned after the procedure. There is a small indentation in the slide that allows the drop containing the specimen to hang so motility can be detected.
• 1. Using the utensil that has been supplied, apply a small amount of petroleum jelly around the edge of the indentation.
• 2. Sterilize the inoculating loop and apply a small loopful of Pseudomonas aeruginosa to the cover slip.
• 3. Turn the slide so it is facing downwards and carefully align it over the cover slip so the inoculum is within the indentation.
• 4. Carefully invert the slide so that the cover slip is on top and the specimen can be observed under the microscope. Loop for the movement of the different bacteria.
• 5. Repeat with Klebsiella pneumonia.
Bacteria that will be used today
• Part 1:hang drop slide • p. Aeruginosa• k. Pneumonia• Part 2: negative stain • b. Megaterium• s. Aureus• Part 3: simple stain • s. Aureus• b. Megaterium• s. Marcesecns• p. Aruginosa
• What are 3 mistakes that I did in the scientific name?
Scientific names
• 1. The genus should be capitalize
• 2. The species should be lower case
• 3 the name should be italicizes or underline.
Bacteria that will be used today
• How the names should look.
• Part 1:hang drop slide • P. aeruginosa• K.pneumonia• Part 2: negative stain • B. megaterium• S. aureus• Part 3: simple stain • S. aureus• B. megaterium• S. marcescens• P. aruginosa
Next week
• Finish lab 3 turn it in.
• Study for the next weeks quiz