microbio lab midterm

Key steps in proper aseptic transfer technique

Flame the inoculating loop until red hot, allow to cool briefly, transfer culture without touching non-sterile surfaces, flame loop again after transfer, and work near a flame to create an updraft.

How to hold culture tubes during aseptic transfer

Hold tubes at an angle (not vertical), remove caps with your pinky finger while holding the loop, and flame the mouth of the tube before and after transfer.

What must always be included on plate labels

Your name/initials, date, organism name, and any relevant treatment or dilution information.

Proper procedure for biohazard waste disposal

Place contaminated materials in designated biohazard bags or containers, never in regular trash. Sharps go in sharps containers. Autoclave materials before final disposal.

Four steps of Gram staining in order

1) Crystal violet (primary stain), 2) Iodine (mordant), 3) Alcohol/acetone (decolorizer), 4) Safranin (counterstain).

Color of Gram-positive bacteria after staining and why

Purple/violet. They retain the crystal violet-iodine complex because their thick peptidoglycan layer prevents decolorization.

Color of Gram-negative bacteria after staining and why

Pink/red. Their thin peptidoglycan layer doesn't retain crystal violet during decolorization, so they take up the safranin counterstain.

Common causes of inconclusive Gram stain results

Over-decolorization, under-decolorization, old cultures, improper heat fixing, incorrect timing of reagents, or using cultures older than 24 hours.

Most critical step in Gram staining

The decolorization step with alcohol/acetone. Too long and all cells appear Gram-negative; too short and all appear Gram-positive.

Purpose of the streak plate method

To isolate individual bacterial cells so they grow into separate, pure colonies that can be identified and studied.

Describe the quadrant streak method

Divide the plate into 4 quadrants. Streak the first quadrant with the inoculum, flame the loop, then streak from quadrant 1 into quadrant 2. Repeat for quadrants 3 and 4, flaming between each.

Why flame the loop between quadrants when streaking

To reduce the number of bacteria on the loop progressively, allowing individual cells to be deposited and grow into isolated colonies.

Signs of contamination on a streak plate

Unexpected colony morphologies, multiple different colony types when you expect one, fuzzy growth (mold), or growth in areas that shouldn't have been inoculated.

Competent bacteria

Competent bacteria have the ability to take up foreign DNA from their environment. This can occur naturally or be induced by chemical treatment (CaCl2) or electroporation.

Bacterial conjugation

The transfer of genetic material between bacterial cells through direct contact via a pilus. Often transfers plasmids carrying antibiotic resistance genes.

Kirby-Bauer test interpretation

Measure the zone of inhibition (clear area) around antibiotic disks. Larger zones indicate greater susceptibility. Compare measurements to standard charts to classify as Sensitive, Intermediate, or Resistant.

Antibiotics targeting the bacterial ribosome

Tetracycline, streptomycin, erythromycin, and chloramphenicol all target the bacterial ribosome to inhibit protein synthesis.

Antibiotics targeting the bacterial cell wall

Penicillin, ampicillin, and vancomycin target cell wall synthesis by inhibiting peptidoglycan formation.

Countable colony range for CFU/mL calculations

30-300 colonies per plate. Below 30 is statistically unreliable; above 300 is too crowded to count accurately.

CFU/mL formula

CFU/mL = (Number of colonies) / (Volume plated × Dilution factor)

CFU/mL calculation example

CFU/mL = 150 / (0.1 × 10⁻⁵) = 150 / 10⁻⁶ = 1.5 × 10⁸ CFU/mL

Choosing dilution plate for counting

Choose the plate with colonies in the countable range (30-300). If multiple plates are in this range, use the one closest to the middle of the range for best accuracy.

Recombinant DNA technology

The process of combining DNA from different sources to create new genetic combinations. In bacteria, this involves inserting foreign genes into plasmids to produce desired proteins.

Engineering bacteria to produce proteins

Insert the gene for the desired protein into a plasmid, transform the plasmid into bacteria, and culture the bacteria. They will express the foreign gene and produce the protein (e.g., insulin, growth hormone).

Applications of recombinant DNA in medicine

Production of human insulin, growth hormone, clotting factors, vaccines, and other therapeutic proteins. Also used in gene therapy and diagnostic tests.