Lab Quiz #1 MCB2010C

PPE when working with biosafety level 2 organisms in the lab

gloves
lab coat
goggles (no need if you have glasses)

clothing that covers legs and torso. Closed toe shoes.
note: always wipe the desk area before and after coming to lab. Keep long hair tied back and do not bring any food or drinks.

procedure when handling a microbial spill and proper disposal of broken glass

flood the area around the spill with disinfectant solution. Add paper towels on top and keep it there for 20 minutes. Then use paper towels to clean it up. If broken glass was involved after decontamination discard it in the broken glass container.

Biohazordus waste disposal

Trash cans are for uncontaminated non sharp waste
Biohazard bins are for contaminated disposable items (petri dishes, gloves)

Sharps or the broken glass container is for broken slides, coverslips, or glass

Biosafety levels

BS1 (low risk microbe) -BS 4 (high risk microbe)

resolution and contrast in microscopy

resolution: ability of a lens to separate or distinguish small objects that are close together.
Contrast: The difference in light intensity between an object and its background.

Calculate total magnification

multiple magnification by 10

which magnifications use fine focus only

40x and 100x (with oil immersion)

oil immersion theory

reduces distortion caused by different refractive indexes of glass and air. Oil immersion improves resolution.

Brightfield microscope

produces a dark image against a brighter background

Know how to estimate the length/width of a specimen when visible under a calibrated ocular micrometer.

count the spaces when specimen is compared to the ruler. multiply however many spaces to the value of ocular division. 40x objective is 2.5, 100x objective lens is 1. Same applies for both width and length.

how is darkfield achieved?

The light reaches the specimen from an angle with the help of the star diaphragm disk. The condenser blocks all light passing in a straight line from the condenser to the specimen.

When should darkfield be used?

If you are trying to view a unstained, transparent or low contrast slide. It is mainly used to see spirochetes such as:Treponema pallidum (syphilis) Borrelia burgdorferi (lyme disease) Leptospira interrogan (leptospirosis). It is also used to see microbial motility.

In darkfield, what does the specimen and background look like?

The specimen appears light against a dark background

Clinical connection: syphilis

male presents with 4 day old painless penile lesions ( 3 non tender ulcers). Tissue examination was taken for darkfield examination. Darkfield was able to present Treponema pallidum spirochetes which is what syphilis is caused by.

Advantages over brightfield microscope

1. Resolution in darkfield is better than bright field
   
   

2. Improves image contrast without the use of stain (does not kill cells) so you can see living specimens

3. Direct detection of nonculturable bacteria

4. requires no special set up

What are disadvantages of Darkfield?

Specimen must be strongly illuminated which can cause damage to the sample.
along with the sample shining bright, dust particles also appear.
Sample must be spread thinly

Goal of aseptic technique

Prevent contamination of pure cultures and sterile laboratory equipment

Key steps of aseptic routine

1. sterilize all materials before starting

2. Disinfect work area

3. clear work area

4. keep objects covered until use

5. flame inoculation tool

6. remove lids/covers

7. flame mouth of tube and remove inoculum

8. Transfer to a new tube

9. Flame inoculation tool

inoculating bacteria on agar slant

gently graze the surface

Inoculating the bacteria on agar deep

Stab the needle containing bacteria directly into the deep agar