MCB2010L Midterm exam review

Level - BSL 1

Risk and typical protection

1. Low risk organisms don’t usually cause disease in healthy adults

2. Standard lab practices ; usually an open bench

Level - BSL 2

Risk and typical protection

1. Agents that can cause human disease

2.  Restricted access, PPE, and a biological safety cabinet for procedures that may create infectious aerosols or splashes

Level - BSL 3

Risk and typical protection

1. Agents that can cause serious or potentially lethal disease through inhalation

2. Controlled access, inward airflow, and work in biological safety cabinets.

Level - BSL 4

Risk and typical protection

1. Dangerous agents posing high risk of life threatening disease, often without available treatments or vaccines.

2. Maximum containment using a Class III cabinet or positive pressure suit facility

Proper steps to glove removal

1. Pinch the outside of one glove near the wrist without touching bare skin

2. Peel it off inside out and hold it in the still gloved hand.

3. Slide bare fingers under the remaining glove at the wrist; avoid its contaminated outside.

4. Peel it off over the first glove, enclosing both. Discard appropriately.

5. Perform hand hygiene.

Ways to prevent contamination

1. Keep lids and caps off only as long as necessary: airborne microbes can enter sterile media, and exposure can also release organisms from a culture.

2. Disinfect the bench before and after work

3. wash hands

4. use sterile tools

5. avoid touching sterile surfaces

6. keep containers partly covered

7. do not talk or cough over open media

8. and follow your lab’s instructions for sterilizing loops and handling tube mouths.

9. Let a sterilized loop cool before touching a culture.

10. Keep personal items, food, and drinks out of the work area.

How to identify it and common use (Broth)

1. Liquid in a tube or flask; no solid agar surface

2. Growing many cells; growth may cause cloudiness, sediment, or a surface film

How to identify it and common use ( Agar slant )

1. Solid agar in a tube with an angled surface

2. Growing or maintaining a culture on the sloped surface

How to identify it and common use (Agar deep)

1. Solid or semisolid agar upright in a tube

2. Stab inoculation; certain deeps assess motility or oxygen related growth

How to identify it and common use (Agar Petri dish)

1. A shallow round dish containing a flat agar surface

2. Separating colonies and examining colony appearance

Shape and what to recognize (coccus)

Round cell; plural cocci

Shape and what to recognize (Bacillus)

Rod shaped cell; plural bacilli. Lowercase describes shape; Bacillus is a genus

Shape and what to recognize (Coccobacillus)

Short oval Rod

Shape and what to recognize (Vibrio)

Curved comma shaped rod

Shape and what to recognize (Spirillum)

Rigid spiral cell

Shape and what to recognize (Spirochete)

Thin flexible corkscrew shaped cell

Shape and what to recognize (Diplo)

Pairs, as in diplococci

Shape and what to recognize (Strepto)

Chains, as in streptococci

Shape and what to recognize (Staphylo)

Grape like clusters, as in staphylococci

Shape and what to recognize (Tetrads and sarcinae)

Groups of four and cubical packets, respectively

Cell morphology

describes individual cells and their arrangement

Colony morphology

describes the visible growth on agar, such as colony size, color, edge, and elevation.

1. Charge and results

2. examples (Basic Dye)

1. Positively charged colored portion binds to usually negatively charged cells; cells become colored

2. Crystal violet, methylene blue, safranin, basic fuchsin, malachite green

1. Charge and results

2. Examples (Acidic dye)

1. Negatively charged colored portion is repelled by cells; the background is stained in a negative stain

2. Nigrosin, eosin, Congo red. India ink is also used for negative staining but is a particulate suspension

Gram stain procedure steps

1. Prepare a thin smear, air dry, and heat fix as instructed.

2. Apply crystal violet, the primary stain; rinse gently with water.

3. Apply Gram’s iodine, the mordant; rinse with water.

4. Apply alcohol or acetone alcohol decolorizer briefly; immediately rinse with water.

5. Apply safranin, the counterstain; rinse, blot dry, and examine.

Reagent order

crystal violet → iodine → decolorizer → safranin

Cell type, final color, and why (Gram positive)

1. Gram positive

2. Purple

3. Thick peptidoglycan retains the crystal violet iodine complex during correct decolorization

Cell type, final color, and why (Gram Negative)

1. Gram negative

2. Pink or red

3. Decolorization removes the primary stain; the cells then take up safranin

Decolorization errors (Over decolorization)

Gram positive cells can lose the purple stain and appear pink, giving a false Gram negative result

Decolorization errors (Under decolorization)

Gram negative cells may retain purple and appear falsely Gram positive. Thick smears and old or damaged cells can also distort results

Negative stain recognition

Look for light or clear cells against a dark background. Negative stains generally do not require heat fixation, so cell size and shape are preserved more accurately.