Pre-Lab assignment #2

Question 1

What is the low to high rule?

Answer 1

a. Always start with the lowest objective and then work your way up to the highest objective lens.

Question 2

What part of the microscope helps you to move the microscopic image/specimen left, right, forward and backward?

Answer 2

a. The stage controls

Question 3

What part of the microscope is responsible for making the image crisp or sharp?

Answer 3

a. Fine Focus

Question 4

What is the oil immersion lens?

Answer 4

a. Highest power of 100x and this allows you to see very small specimens

Question 5

What are the steps to conduct the oil immersion technique? (Hint: During and after oil immersion technique)

Answer 5

a. Turn the nose piece so it is between the 40x and the 100x objectives
b. Put a drop of oil on the slide
c. Slowly and gently rotate the nose piece to the oil immersion lens (100X)
d. Look at the microscope from the side and carefully use the fine focus control to make contact between the lens and the oil
i. The lens should never touch the slide
e. Once done, lower the stage to separate the oil immersion lens from the oil and remove the slide
f. Clean the lens with lens paper, and the eyepieces and stage with alcohol

Question 6

What can you change to zoom into an image and observe the specimen at a higher magnification?

Answer 6

a. Objective lenses

Question 7

Define what the stage and the base of a microscope are?

Answer 7

a. Stage: This area is the main, flat plate that holds the slides for observation
b. Base: The base is the bottom support structure of the microscope

Question 8

What is the difference between the course adjustment knob and the fine adjustment knob?

Answer 8

a. Coarse Focus: The larger of two adjustment knobs that moves the objective lenses closer or farther away from the specimen in large steps.
b. Fine Focus: The smaller of two adjustment knobs moves the objective lenses closer or farther away from the specimen in very small steps. It can be used to fine tune the focus on various parts of a specimen after first using the Coarse Focus to get close.

Question 9

Eyepiece lenses are typically what magnification?

Answer 9

a. Typically 10x

Question 10

How do you calculate the total magnification of an image you see under a compound microscope?

Answer 10

a. Multiply the power of the eyepiece (10x) by the power of the objective lens

Question 11

What 3 protocols can you follow to take proper care of your microscope?

Answer 11

a. Remove the microscope from the cabinet by gripping the arm firmly with one hand while placing the other hand underneath the base of the device for support. Do not lift on the stage. Do not remove the dust cover until the microscope is placed and positioned on the table where it will be used.
b. Never touch the lenses with your fingers. Oil from your hands can smudge the lens and in some cases even leave scratches
c. When finished rotate the nosepiece back to the lowest power objective. Lower the stage to its lowest position. Make sure the power cord is neatly wrapped up and replace the dust cover before returning the microscope to the cabinet.

Question 12

What two characteristics does a simple stain reveal about a microorganism?

Answer 12

a. The size, shape and arrangement of a microorganism

Question 13

How can we sterilize an inoculation loop and why is this an important step?

Answer 13

a. Sterilize it by passing it through the flame of the Bunsen burner.

Question 13

What is the best objective to observe the details of bacterial microorganism shape?

Answer 13

a. Oil Immersion

Question 14

How do you prepare and fix a bacterial smear?

Answer 14

a. Get a clean microscope slide
b. Make it with the name and draw a circle nickel size
c. Flip it over, marker size down
d. Add a drop of distilled water to slide
e. Sterilize an inoculating loop through a Bunsen burner
f. Once the loop is cool touch it to the bacteria in the slide and use the loop to mix the cells and the water (Completely fill the circle)
g. Allow the slide to air dry at room temp
h. You know when dry when a thin white haze appears
i. Heat fix the slide by passing it through a flame three times
j. Flood the slide with Safranin (dye) (fill the whole circle with the dye)
k. Leave stain on for 30 seconds then gentle wash slide with distilled water
l. Use bibulus paper to gently blot the slide dry

Question 15

What is the bacterial cell wall composed of? What is the role of the cell wall?

Answer 15

a. Made up of polysaccharides, it gives the cell wall strength and rigidity.
b. The cell wall prevents the cell from shrinking or swelling

Question 16

Describe the differences in the cell wall and cell membranes of Gram-positive and Gram-negative microbes.

Answer 16

a. Gram Positive:
i. Thick cell wall (30 layers of peptidoglycan)
ii. Cell wall surrounds monoderm
b. Gram Negative:
i. Thin cell wall (1 layer of peptidoglycan)
ii. Impenetrable cell wall

Question 17

Gram stain is considered a differential stain. What does this term mean?

Answer 17

a. Means it uses multiple different stains

Question 18

Briefly bullet point the steps and times of a gram stain. Also indicate the role of each step. (Consider a table to organize this information.)

Answer 18

a. Take the crystal violet and add several drops to the smear
i. Allow the Crystal Violet to remain for 30 seconds
b. After 30 seconds, take the deionized water and rinse the slide
c. Next use the grams iodine reagent
i. All the grams iodine to remain on the slide for 60 seconds then rinse
d. Take a full dropper of Ethanol and slowly add the ethanol and let it drip off
i. Wait for the falling off the slide to be a very light purple color
ii. Then rinse with water
e. Take several drops of the red dye to the smear
i. Allow it to sit on the slide for 30 seconds
ii. Rinse with deionized water
f. Place slide on paper towels and blot dry with bibulous paper

Question 19

What color will a Gram-positive bacteria turn after a Gram stain and what color will a Gram-negative bacteria turn following Gram staining?

Answer 19

a. Gram Positive will turn a purple color and Gram Negative will turn a pink color