Primary Stain Crystal Violet Stains all cells purple.

3. Mordant Iodine Forms a crystal violet-iodine complex, helping dye stick to cell wall.

4. Alcohol or Removes stain from Gram-negative cells (not thick

Decolorizer Acetone enough to retain it).

5. Safranin Stains Gram-negative cells pink/red (Gram-positives stay

Counterstain purple).
Gram-positive bacteria have thick peptidoglycan walls → retain crystal violet-iodine complex
→ stay purple.​

Gram-negative bacteria have thin peptidoglycan and outer membrane → lose crystal violet
during alcohol wash → absorb safranin → appear pink/red.

🔬 1. Brightfield Microscope
Feature Description

Function Basic light microscope—light passes through the specimen.

Image Look Flat, colored or clear background. Stained specimens appear in color or
contrast.

Stains Yes (common)—staining increases contrast.

Used For General microbiology, stained bacteria, tissue samples (histology).

Notes Most common microscope in classrooms and labs.

🌫️ 2. Phase Contrast Microscope

Feature Description

Function Enhances contrast in unstained, transparent specimens by exploiting

differences in refractive index.

Image Grey/black and white with a "halo" or glow around structures. Often looks more
Look 3D than brightfield.

Stains Not required

Used For Live cells, protozoa, cell organelles (without killing or staining them).

Notes Great for viewing living, motile organisms.

✨ 3. Fluorescence Microscope
Feature Description

Function Uses high-intensity light to excite fluorescent dyes or proteins. Emitted light
forms the image.

Image Bright, neon colors on a dark background (black). Often blue, green, red,
Look yellow.

Stains Yes—uses fluorescent dyes like DAPI, FITC, or GFP.

Used For Identifying specific proteins, structures, or pathogens; DNA staining.

Notes Used in medical diagnostics, cell biology, and genetics.

🔬⚫ 4. Transmission Electron Microscope (TEM)

 Feature Description

Function Electrons pass through ultrathin specimens to show internal structures.

Image Very high resolution, black & white, 2D, internal cell structures, organelles
Look visible.

Stains Yes, with heavy metals (e.g., osmium, uranium)

Used For Internal ultrastructure of cells, viruses, organelles.

Notes Up to millions of times magnification; sample must be dead and sliced very
thin.

🌍 5. Scanning Electron Microscope (SEM)

Feature Description

Function Electrons scan the surface of a specimen; creates detailed surface

images.

Image Look 3D-like, black and white, textured surface details.

Stains Yes, coated with metal like gold or platinum.

Used For Surface structures of cells, insects, materials, and micro-organisms.

Notes Specimens must be dried and conductive (or coated).

🔬🧬 6. Confocal Laser Scanning Microscope

Feature Description

Function Uses lasers and optical sectioning to create sharp, 3D reconstructions of

fluorescent-stained specimens.

Image High-resolution, fluorescent, often multi-colored layers—like looking inside a

Look cell slice-by-slice.

Stains Yes—uses fluorescent stains or fluorescent proteins.

Used For 3D imaging of cells, tissues, biofilms.

Notes Excellent for thick samples and fine detail in fluorescence.

👀 7. Stereo Microscope (Dissecting Microscope)
Feature Description

Function Low magnification binocular microscope for viewing larger, 3D objects.

Image Look Realistic, 3D-looking whole organisms or objects. Often in natural color.

Stains Not needed

Used For Dissections, insects, plants, electronics, fossils.

Notes Useful for things visible to the naked eye that still need close inspection.

Basic Structural Components of Viruses

Component Structure Function

1. Nucleic Acid DNA or RNA (never both), Carries the virus’s genetic
(Genome) single- or double-stranded instructions for replication and
making proteins.

2. Capsid Protein shell made of Protects the genetic material, and

capsomeres helps the virus attach to host cells.

3. Envelope (in Lipid bilayer with embedded Helps virus enter host cells; contains
some viruses) viral proteins (from host cell glycoproteins that recognize host
membrane) receptors.

4. Spikes Protein projections from Used to bind to specific receptors on

(Glycoproteins) capsid or envelope the host cell surface—key in infection
and host specificity.

5. Enzymes (in e.g., reverse transcriptase, Help with viral replication (e.g., RNA
some viruses) integrase to DNA in retroviruses like HIV).

1. Bacterial Cells
Component Composition Function

Cell Wall Peptidoglycan Structural support, prevents lysis

(murein)

Plasma Membrane Phospholipid bilayer Controls entry/exit of substances

Cytoplasm Water, ions, enzymes Site of metabolism

Nucleoid Circular DNA Genetic material (not membrane-bound)

Ribosomes (70S) RNA + protein Protein synthesis

Flagella Protein (flagellin) Motility

Pili/Fimbriae Protein Attachment to surfaces; gene transfer (sex

pili)

Capsule/Slime Polysaccharides Protection, adhesion, evasion of immune

Layer system

Archaeal Cells
Component Composition Function

Cell Wall No peptidoglycan—uses pseudopeptidoglycan Structural support

or protein

Membrane Ether-linked phospholipids (branched chains) Stability in extreme

environments

Cytoplasm, Similar to bacteria (70S ribosomes) Basic cell functions

Ribosomes

Genetic Material Circular DNA (like bacteria), but Genetic info

transcription/translation resemble eukaryotes

Flagella Structurally different from bacterial ones Motility in extreme

environments

Eukaryotic Microbial Cells (Microalgae, Fungi, Protozoa)

Component Present in Composition Function

Nucleus All DNA + membrane Stores genetic

info

Plasma All Phospholipid bilayer Controls entry/exit

Membrane
Cell Wall Fungi (chitin), Algae Structural support
(cellulose, silica), not in
protozoa

Mitochondria All Double membrane Energy (ATP)

production

Chloroplasts Microalgae Thylakoid Photosynthesis

membranes +
pigments

Ribosomes All Protein + rRNA Protein synthesis

(80S)

ER & Golgi All Membrane-bound Protein/lipid

Apparatus sacs processing

Cytoskeleton All Microtubules, actin Structure,

movement

Flagella/Cilia Some Microtubules (9+2) Motility

Specialized Structures
Structure Organisms Composition Function

Gas Vesicles Cyanobacteria, aquatic Protein-based, Buoyancy control

bacteria gas-filled

Endospores Bacillus, Clostridium Keratin-like coating, Dormant,

(bacteria) DNA stress-resistant form

Contractile Freshwater protozoa, Membrane-bound Expels excess water to

Vacuoles algae vacuole prevent lysis

Eyespots Microalgae (e.g., Pigment granules Light detection for

(Stigma) Chlamydomonas) phototaxis

Carboxysomes Cyanobacteria, Protein shell with Carbon fixation

chemoautotrophs Rubisco (concentrates CO₂)

Pellicle Protozoa (e.g., Euglena) Protein strips under Flexibility and shape
membrane

Axostyle Some flagellated Microtubules Structural support and

protozoa motility
Gram-Positive vs Gram-Negative vs Acid-Fast Cells
Feature Gram-Positive Gram-Negative Acid-Fast

Peptidoglycan Layer Thick Thin Moderate (but hidden

under lipid layer)

Outer Membrane ❌ Absent ✅ Present ❌ Absent

Teichoic Acids ✅ Present ❌ Absent ❌ Absent
Lipopolysaccharide ❌ Absent ✅ Present (toxic) ❌ Absent
(LPS)

Mycolic Acid ❌ Absent ❌ Absent ✅ Present (waxy layer)

Stain Result Purple (retains Pink/Red (takes up Pink/red (retains carbol
crystal violet) safranin) fuchsin)

Staining Method Gram Stain Gram Stain Acid-Fast Stain

(Ziehl-Neelsen or
Kinyoun)

Antibiotic More sensitive to More resistant due Resistant to many drugs

Susceptibility penicillin to outer membrane

Example Organisms Staphylococcus E. coli, Salmonella Mycobacterium

aureus, Bacillus tuberculosis, M. leprae

●​ Gram-Positive:​

○​ One membrane​

○​ Thick peptidoglycan (~20–80 nm)​

○​ Teichoic acids strengthen the wall​

●​ Gram-Negative:​

○​ Two membranes (inner + outer)​

○​ Thin peptidoglycan (~2–7 nm)​

○​ Outer membrane contains LPS, making them more pathogenic and

antibiotic-resistant​
 ●​ Acid-Fast:​

○​ Structurally similar to Gram-positive but has a thick waxy layer of mycolic

acids​

○​ Makes them slow-growing, highly resistant to chemicals and desiccation​

Streak Plate Method

●​ Purpose: Isolate pure colonies from a mixed bacterial sample.​

●​ How it works: A loop is used to streak bacteria over four quadrants of an agar plate,
diluting it as you go.​

●​ Result: Individual colonies grow in the final quadrant.​

●​ Used with: Solid agar media (like Nutrient Agar, TSA).​

●​ Good for: Identifying colony morphology, isolating single species.​

✅ 2. Liquid Culture (Broth Culture)

●​ Purpose: Grow large amounts of bacteria quickly.​

●​ How it works: Inoculate a liquid medium (e.g., Nutrient Broth) and incubate with shaking
or still.​

●​ Result: Turbidity (cloudiness) indicates growth.​

●​ Used for: Biochemical tests, DNA extraction, or further plating.​

●​ Good for: Bulk growth, fast-growing organisms.​

✅ 3. Agar Culture
●​ Purpose: Grow and visually observe colonies on a solid surface.​
 ●​ Media type: Any medium with agar added (usually ~1.5% agar).​

●​ Result: Visible colonies with different sizes, shapes, colors.​

●​ Used for: Isolation, colony counts, hemolysis detection.​

●​ Good for: Characterizing colony morphology and doing streak/spread/pour plate

methods.​

4. Selective Media

●​ Purpose: Inhibit the growth of some organisms, allow others.​

●​ How: Additives (salts, dyes, antibiotics) suppress unwanted microbes.​

●​ Examples:​

○​ MacConkey Agar: Inhibits Gram-positive, selects for Gram-negative.​

○​ Mannitol Salt Agar (MSA): High salt selects Staphylococcus.​

●​ Good for: Isolating bacteria from mixed samples.​

✅ 5. Differential Media
●​ Purpose: Distinguish bacteria based on metabolic traits (color change, pH change).​

●​ How: Contains indicators that change color based on bacterial activity.​

●​ Examples:​

○​ Blood Agar: Shows hemolysis patterns (alpha, beta, gamma).​

○​ MacConkey Agar: Lactose fermenters turn pink; non-fermenters stay colorless.​

●​ Often combined with selective media.​

🧬 Chemical Composition-Based Media Types
✅ 6. Complex Media (Undefined Media)
●​ Composition: Contains unknown mixtures like yeast extract, beef extract, peptone.​

●​ Examples: Nutrient Broth, TSA, LB Broth​

●​ Good for: Growing a wide variety of non-fastidious bacteria.​

●​ Note: Exact chemical composition not known.​

✅ 7. Defined Media (Synthetic Media)

●​ Composition: Exact chemical composition is known (specific salts, sugars, amino
acids).​

●​ Examples: Minimal media, M9 medium​

●​ Used for: Studying specific metabolic pathways or nutritional requirements.​

●​ Good for: Research where consistency is important.​

1. Plate Count Method (Colony Forming Units – CFUs)

✅ How It Works
●​ Serially dilute your culture.​

●​ Plate a small, known volume (e.g. 0.1 mL) on agar.​

●​ Incubate and count visible colonies.​

●​ Each colony = 1 viable cell (or clump) from the original sample.​

✅ Formula to Calculate Cell Concentration:

CFU/mL=Number of colonies×Dilution factorVolume plated (mL)\text{CFU/mL} =
\frac{\text{Number of colonies} \times \text{Dilution factor}}{\text{Volume plated
(mL)}}CFU/mL=Volume plated (mL)Number of colonies×Dilution factor​

🧮 Example:
●​ 50 colonies from a 1:10,000 dilution, plated 0.1 mL​

CFU/mL=50×1040.1=5×106 CFU/mL\text{CFU/mL} = \frac{50 \times 10^4}{0.1} = 5 \times 10^6

\, \text{CFU/mL}CFU/mL=0.150×104​=5×106CFU/mL

💡 This gives you the number of viable cells per mL.

🔬 2. Optical Density (OD600) Measurement

✅ How It Works
●​ Use a spectrophotometer to measure absorbance at 600 nm (OD600).​

●​ As bacteria grow, they scatter more light—OD increases.​

●​ OD is proportional to cell concentration, but not a direct count.​

✅ Using OD to Estimate Cell Number

●​ First, create a standard curve: Plot CFU/mL vs OD600 using a known culture.​

●​ Once the curve is made, you can estimate CFU/mL from OD.​

Example Relationship:

●​ OD600 = 1.0 ≈ 1 × 10⁹ cells/mL (but depends on the organism and instrument!)​

🔁 OD600 measures both live and dead cells — unlike CFUs which only count
live/viable cells.

Sterilization
✅ Definition:
Complete destruction or removal of all microorganisms, including bacterial spores and
viruses.

Disinfection
✅ Definition:
Destruction of most microbial life, particularly pathogens (except some spores), on
inanimate objects.

Sanitization
✅ Definition:
Reducing microbial numbers to safe levels (based on public health standards). Does not
guarantee complete removal of pathogens.

Fermentation in Bread Making

🦠 Microbe Involved:
●​ Yeast – Saccharomyces cerevisiae​

⚙️ What It Does:
●​ Ferments sugars in the dough → produces carbon dioxide and ethanol​

●​ CO₂ causes the dough to rise (leavening)​

●​ Ethanol evaporates during baking​

🌟 Why It Matters:
●​ Creates light, airy texture​

●​ Adds flavor​
 ●​ Essential in baking​

🍜 2. Fermentation in Soy Sauce Production

🦠 Microbes Involved:
●​ Mold: Aspergillus oryzae (breaks down soy proteins & starch)​

●​ Yeasts: Zygosaccharomyces rouxii​

●​ Bacteria: Lactobacillus spp. and others​

⚙️ What They Do:

●​ Molds produce enzymes to break down soy and wheat during the koji stage​

●​ Yeast and lactic acid bacteria carry out fermentation (up to 6 months!)​

●​ Produce umami flavor, aroma, acids, and alcohols​

🌟 Why It Matters:
●​ Converts raw soy into a flavorful, shelf-stable sauce​

●​ Complex microbial interactions develop depth and richness​

🥬 3. Fermentation in Sauerkraut Production

🦠 Microbes Involved:
●​ Lactic acid bacteria (LAB):​

○​ Leuconostoc mesenteroides​
 ○​ Lactobacillus plantarum​

⚙️ What They Do:

●​ Ferment natural sugars in cabbage → produce lactic acid​

●​ Acid lowers pH → preserves the cabbage and prevents spoilage​

🌟 Why It Matters:
●​ Lactic acid gives sour flavor and preserves the kraut​

●​ Increases shelf life and adds probiotics​

🌱 4. Photosynthesis in Biofuel Production

🦠 Microbes Involved:
●​ Microalgae – e.g., Chlorella, Nannochloropsis, Spirulina​

●​ Cyanobacteria – e.g., Synechocystis​

⚙️ What They Do:

●​ Use sunlight, CO₂, and water to perform photosynthesis​

●​ Produce biomass rich in oils (lipids), which are converted to biofuels (biodiesel,
ethanol)​

🌟 Why It Matters:
●​ Microbial biofuels are renewable, sustainable, and can reduce fossil fuel use​

●​ Algae can grow in wastewater or saltwater and absorb CO₂​

🌾 5. Nitrogen Fixation in the Rhizosphere
🦠 Microbes Involved:
●​ Nitrogen-fixing bacteria – e.g., Rhizobium (legumes), Azospirillum, Frankia​

⚙️ What They Do:

●​ Convert atmospheric nitrogen (N₂) into ammonia (NH₃), which plants can use​

●​ Rhizobium forms nodules on roots of legumes (symbiosis)​

🌟 Why It Matters:
●​ Provides essential nitrogen to plants in a usable form​

●​ Reduces need for chemical fertilizers​

●​ Vital for soil fertility and sustainable agriculture​

1. Transformation

●​ 💡 Uptake of naked DNA from the environment​

●​ Some bacteria can absorb free DNA from dead cells around them​

●​ Example: Streptococcus pneumoniae can pick up DNA to become more virulent​

2. Transduction

●​ 🦠 DNA is transferred by a virus (bacteriophage)​

●​ A virus infects a bacterium, accidentally picks up bacterial DNA, and transfers it to
another bacterium​

●​ Like a virus “messing up” and carrying some host DNA to a new cell​
3. Conjugation

●​ 🔗 Direct transfer of DNA through cell-to-cell contact​

●​ Involves a pilus (a tube-like structure)​

●​ Usually transfers plasmids (small circular DNA)​

●​ Super important for spreading antibiotic resistance genes​