Biotechnology: Microbiology & Cell culture

Microbiology Overview:

  1. What is Microbiology:

  • Microbiology is the study of microorganisms and their effects on other living organisms. The name micro comes from that they have to be viewed at a microscopic level, such as bacteria, yeast, algae, protozoa, fungi, and viruses.

  1. Why is it important to understand microbes in biotech?:

  • It is important to understand that microbes are harmless and are used to benefit mankind. However, harmless microorganisms cause many types of disease affecting people, animals, and plants.

 Domains of Life/Cells/Postulates:

  1. List the 3 domains of life:

  • Life on Earth is organized into Archaea, Bacteria, and Eukarya

  1. Which domain is the most diverse:

  • Bacteria — found everywhere and have the most species variety

  1. Which domain is most related to us(Eukarya) 

Archaea — even though they look like bacteria, they share more genetic similarities with eukaryotes (humans, plants, etc.).

  1. What are the properties of each domain?

Bacteria: Prokaryotic (no nucleus), cell walls with peptidoglycan, found everywhere

Archaea: Prokaryotic, live in extreme environments (hot springs, salt lakes), no peptidoglycan in walls. Eukarya: Eukaryotic (have nucleus + organelles), includes animals, plants, fungi, and protists

  1. Compare Plant and Animal Cells

  • Plant cells: Have a cell wall, chloroplasts, and a large central vacuole.

  • Animal cells: Have centrioles and lysosomes, no cell wall or chloroplasts.
    ➡ Both have a nucleus, mitochondria, a cell membrane, cytoplasm, ribosomes, and ER

  1. What are the functions of organelles?

  • Nucleus: Controls cell; holds DNA. Mitochondria: Powerhouse—makes energy (ATP). Ribosomes: Make proteins. Endoplasmic Reticulum (ER):

    • Rough ER: Has ribosomes; makes proteins.

    • Smooth ER: Makes lipids.

  • Golgi Apparatus: Packages and ships proteins. Lysosome: Breaks down waste (mainly in animal cells). Chloroplast: Does photosynthesis (only in plants).Cell wall: Supports and protects (plants only).

  • Cell membrane: Controls what enters and leaves.

  1. Describe at least 3 different examples of how microorganisms have changed the course of history.

  • Disease: Caused major outbreaks like the Black Plague or COVID-19. Medicine: Discovered antibiotics like Penicillin from fungi. Food: Used in making yogurt, cheese, and bread (fermentation).

  • (Bonus) Biotech: Used in making insulin and vaccines.

  1. What is the purpose of Koch’s postulates?

To prove that a specific microbe causes a specific disease.

  1. What are the four postulates? How did the yogurt lab model the postulates?

  1. The same microbe must be found in all cases of the disease.

  2. The microbe must be isolated and grown in a pure culture.

  3. When the pure culture is introduced into a healthy host, it should cause the same disease.

  4. The same microbe must be re-isolated from the new host.

  • LAB: Bacteria (like Lactobacillus) were isolated and used to transform milk → yogurt, showing how microbes cause specific reactions (similar to Koch’s idea).

Bacteria:

  1. Label the structures of bacteria. Sketch a bacterium and indicate the DNA, pili, ribosomes, cell wall, cell membrane, and plasmids.

  • DNA: Circular chromosome in nucleoid region (no nucleus).

  • Plasmids: Small extra rings of DNA that carry extra genes (like antibiotic resistance).

  • Ribosomes: Make proteins.

  • Cell membrane: Controls what enters/leaves.

  • Cell wall: Protects and gives shape.

  • Capsule: Outer coating for extra protection (not all bacteria have it).

  • Pili (Fimbriae): Tiny hairs that help bacteria stick to surfaces or share DNA.

  • Flagella: Tail-like structure used for movement.

  1. What is Gram staining, and how does it work to differentiate among bacterial species?

Gram staining helps scientists tell if bacteria have thick or thin cell walls.

  • Gram-positive: Thick cell wall → purple (holds crystal violet dye).

  • Gram-negative: Thin cell wall + extra membrane → pink/red (doesn’t hold violet, stains with safranin).

Steps:

  1. Stain with crystal violet.

  2. Add iodine.

  3. Alcohol wash.

  4. Counterstain with safranin.

  1. How are bacteria named?

  • Greek words

  1. What are the three types of bacterial morphologies?

  1. Coccus (Cocci): Spherical (round).

  2. Bacillus (Bacilli): Rod-shaped.

  3. Spirillum (Spirilla): Spiral or corkscrew-shaped.

  1. What is the difference between aerobic and anaerobic bacteria?

  • Aerobic bacteria: Need oxygen to live.

  • Anaerobic bacteria: Die or don’t grow in oxygen.

  • Facultative anaerobes: Can survive with or without oxygen

  1. What does it mean to be a facultative aerobe? Facultative anaerobe?

  • Facultative anaerobe: Grows with or without oxygen (but prefers oxygen).

  • Facultative aerobe: Also means the same — can live both ways, switching energy sources when oxygen is low

  1. How are phototrophic and chemotrophic microbes used in food production?

  • Phototrophic microbes (use sunlight) help make oxygen and support ecosystems.

  • Chemotrophic microbes (use chemicals for energy) are used in making yogurt, cheese, bread, pickles, and fermented foods because they break down sugars and produce useful acids or alcohols.

Example:

  • Lactobacillus → Yogurt

  • Yeast → Bread

  1. List and describe the three main types of solid media used to culture bacteria.

  • Nutrient agar: General-purpose medium for growing many bacteria.

  • Selective media: Contains chemicals that only let certain bacteria grow (e.g., MacConkey agar).

  • Differential media: Shows differences between bacteria using color changes (e.g., blood agar).

  1. Describe how to make LB broth and LB agar.

  • LB Broth: A liquid nutrient solution for bacteria to grow in.

    • Mix LB powder with water → sterilize → pour into test tubes.

  • LB agar: Same ingredients plus agar powder (makes it solid).

    • Melt, sterilize, and pour into petri dishes.

  1. What is antibiotic selection, and why is it used in molecular biology?

It’s a method used to find bacteria that successfully received a new gene (like in cloning).

  • Only bacteria that take in the plasmid with the antibiotic resistance gene survive on antibiotic plates.
    ➡ Helps scientists identify which colonies have the desired DNA

Lab Specific Questions:

  1. Describe the Kirby-Bauer disk diffusion test and how it works.

It tests which antibiotics work best against bacteria.
Steps:

  1. Spread bacteria evenly on an agar plate.

  2. Place small paper disks soaked with antibiotics.

  3. Incubate (let bacteria grow).

  4. Measure the zone of inhibition — clear area around disk shows bacteria were killed or stopped growing.
    ➡ Bigger zone = more effective antibiotic.

  1. How can you read a petri dish for the zone of inhibition?

  • Look for clear circles (zones) around antibiotic disks.

  • Measure the diameter in millimeters.

  • Compare to a chart to see if bacteria are resistant, intermediate, or sensitive to that antibiotic.

  1. List at least three tools and how they’re used in microbiology.

  1. Inoculating loop/needle: Transfers bacteria safely.

  2. Micropipette: Measures and transfers small liquid amounts.

  3. Incubator: Keeps bacteria warm so they grow.

  4. Autoclave: Sterilizes equipment with steam.

  5. Petri dish: Holds agar for bacterial cultures.

  1. Describe specifically how aseptic technique is used to transfer bacteria.

Aseptic technique = keeping everything clean and sterile so you don’t contaminate your samples or yourself.

Steps:

  1. Wash your hands and clean your area with disinfectant.

  2. Flame your loop (inoculating loop) until it glows red → this kills all bacteria.

  3. Let the loop cool a few seconds (so you don’t kill the bacteria you’re transferring).

  4. Open your test tube or petri dish lid slightly (don’t leave it open too long—air can carry bacteria).

  5. Dip the loop into the bacterial culture and then streak or transfer it to new media (like an agar plate).

  6. Flame the loop again after use to sterilize it.

  7. Close the containers immediately.

  8. Label your plate and place it lid-side down in the incubator.

Purpose: Keeps unwanted bacteria from contaminating your experiment and keeps you safe from exposure.

  1. In general, explain aseptic technique.

Aseptic technique is the practice of preventing contamination of sterile materials, cultures, and the environment during lab work.

  • Keep everything clean and closed.

  • Sterilize tools before and after use.

  • Don’t talk, sneeze, or wave your hands over open plates.

  • Always work near a flame or sterile area (the heat makes an “updraft” that keeps dust away).

  • It protects samples (so you only grow the bacteria you want).

  • Protects you from infection.

  1. List the major labeling criteria that are used for labeling media, like a petri dish.

When labeling a petri dish, you should always write on the bottom (agar side), not the lid, because lids can get mixed up.

Label should include:

  1. Your name or initials

  2. Date

  3. Name of bacteria or sample (ex, E. coli)

  4. Type of media (ex, LB agar, nutrient agar)

  5. Temperature or conditions if required (ex, 37°C)

Extra tip: Plates are stored upside down (lid on the bottom) to stop moisture from dripping onto your colonies.

  1. Describe in detail the two methods commonly used to quantify bacteria.

Quantify = count bacteria.
There are two main methods:

Direct counting:

  • Use serial dilution to make the bacteria less concentrated.

  • Plate a small amount (usually 0.1 mL) of the diluted sample on agar.

  • After incubation, count the colonies that grow.

  • Each colony = one colony-forming unit (CFU).

  • Then multiply by the dilution factor to find the number of bacteria in the original sample.

    \text{CFU/mL} = \frac{\text{# of colonies}}{\text{amount plated (mL)} \times \text{dilution factor}}

Turbity:

  • Measures how cloudy (turbid) the bacterial liquid is using a spectrophotometer.

  • The more bacteria, the more light gets blocked.

  • Gives a quick estimate but not exact colony numbers

  1. What is a colony-forming unit (CFU)?

CFU is a single bacterial cell (or small group) that grows and forms one visible colony on an agar plate.

Since bacteria are too tiny to count one by one, we assume each colony = one starting cell.
So CFU tells us the number of viable (living) bacteria in a sample.

  1. A biotech student performs a serial dilution of a bacterial culture six times to achieve a 1 million-fold dilution, and if 10 ”L of that final dilution yielded 45 colony-forming units (CFU) when plated, what is the concentration of bacteria in the original culture (CFU/mL)?

  2. Final dilution = 1,000,000-fold = 10⁻⁶

  3. Volume plated = 10 ”L = 0.01 mL

  4. Colonies counted = 45 CFU

Step 2: Plug into the CFU formula.

CFU/mL = (Number of colonies x Dilution Factor) / Volume of culture plated

Step 3: Substitute the values.

CFU/mL=450.01×106\text{CFU/mL} = \frac{45}{0.01} \times 10^6CFU/mL=0.0145​×106CFU/mL=4,500×106\text{CFU/mL} = 4,500 \times 10^6CFU/mL=4,500×106CFU/mL=4.5×109\text{CFU/mL} = 4.5 \times 10^9CFU/mL=4.5×109

✅ Final Answer:
The original culture had 4.5 × 10âč CFU/mL (4.5 billion bacteria per milliliter).