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.

MRSA

• Methicillin resistance Staphylococcus aureus • A mutated S.aureus, where it have the Mec a

gene• This is a antibiotic resistant bacteria(AKA

superbug)• this organism is a evolutionary model.• This organism can go septic and kill the host.

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