MICRO LAB MIDTERM

Safety procedures and protocols

1. clean work area with disinfectant each period before and after lab

2. wash hands before and after lab and when you need to step out of lab

3. dont smoke, eat, chew gum, or drink in lab

4. keep hands away from face

5. do not apply lip balm

6. cloes toed shoes

7. lab coats worn. no baggy sleeves, floppy shirttails, neckties etc.

8. long hair tied back

9. fingernails short

10. follow teachers instructions on phone

11. spillage or breakage, inform lab instructor, quarantine area

12. notify instructor of any injury

13. know location of all safety equipment

14. labels include: initials, date, lab section number, medium/test, organism

microscope parts

Microscope objective magnification

total magnification= power of ocular lens x power of objective lens

Lens	Magnification (Objective)	Magnification (Ocular)	Total Magnification
Scanner (red)	4×	10×	40×
Low Power (yellow)	10×	10×	100×
High Power (blue)	40×	10×	400×
Oil Immersion (white)	100×	10×	1000×

Microscope usage

Scope should be stored like this

1. cord folded and secured. Placed atop the stage so the cord does not dangle

2. high power objective (blue) in viewing position

3. stage all the way down

4. rheostat on lowest setting

5. free of oil, stain, and dirt (clean with lens paper)

Things to remember

• never use coarse adjustment when using high dry or oil immersion lenses

• when using oil and cannot focus, do not drag high power lens through the oil. Rotate the nosepiece past the scanning lens to the low power lens

Bacterial cell morphology

Coccus- spheres

Bacillus- rods

Spirillum- squiggles

Categorize microbes into Kingdoms and subdivisions

Kingdom Plantae

Kingdom Animalia

Kingdom Protista

• protozoa

  • amoeboid: (100x) single-celled heterotrophs; pseudopodia

  • ciliated: (100x) single-celled; cilia

  • flagellated: (1000x) flagellated protozoa

• higher algae:(100x)

  • chlorophyta:

  • euglenophyta: single flagellum

  • volvox: thousands of cells linked together. easily be seen with naked eye.

Kingdom Fungi: composed of hyphae arranged in mycelium

• yeast: (100-400x) multicellular do not bear hyphae

• filamentous fungi:(100x) black bread mold; horizontal hyphae

• lichen:

Kingdom Monera (1000x)

• archaea:

• bacteria:

• cyanobacteria:

Aseptic techniques for smearing and culturing

types of stains and stain theory

🔹1. Simple Stains

Purpose: Show cell shape, size, and arrangement
Dye: One basic (positively charged) dye

Examples:

• Methylene blue

• Crystal violet

• Safranin

What you see: Blue/purple/pink cells on a light background
Why it works: Basic dyes bind to negatively charged cell components (DNA, cell wall)

🔹 2. Differential Stains

Used to distinguish between groups of microorganisms based on structural differences.

A. Gram Stain

• Separates Gram+ (purple) vs Gram– (pink)

• Based on peptidoglycan thickness and outer membrane

B. Acid‑Fast Stain

• Identifies Mycobacteria (TB, leprosy)

• Acid‑fast = red

• Non–acid‑fast = blue

• Based on mycolic acids (waxy lipids)

C. Endospore Stain (Schaeffer–Fulton)

• Spores = green

• Vegetative cells = pink

• Detects Bacillus and Clostridium

D. Capsule Stain

• Capsules = clear halos

• Background = dark

• Cells = light

• Uses negative staining (acidic dyes)

E. Flagella Stain

• Coats flagella to make them thick enough to see

🔹 3. Special / Structural Stains

Highlight specific structures.

Negative Stain

• Uses acidic dyes (eosin, nigrosin)

• Background stains, cells remain clear

• Great for capsules and morphology

Endospore Stain

(Already listed under differential)

Flagella Stain

(Already listed)

Metachromatic Granule Stain

• Shows storage granules (e.g., Corynebacterium)

Lactophenol Cotton Blue

• Used for fungi

🧬 II. Stain Theory (Why Stains Work)⭐ 1. Charge Interactions

• Basic dyes (crystal violet, safranin, methylene blue)
  → Positively charged
  → Bind to negatively charged bacterial surfaces

• Acidic dyes (nigrosin, eosin)
  → Negatively charged
  → Repelled by bacteria → stain the background

⭐ 2. Cell Wall Chemistry

Different stains exploit structural differences:

Gram Stain Theory

• Gram+ = thick peptidoglycan traps CV‑I complex

• Gram– = outer membrane dissolves, thin PG loses dye

Acid‑Fast Theory

• Mycolic acids are waxy, hydrophobic

• Carbol fuchsin + heat penetrates

• Acid‑alcohol cannot remove it

Endospore Theory

• Spores are highly resistant

• Malachite green + heat forces dye in

• Safranin stains vegetative cells afterward

⭐ 3. Solubility & Permeability

• Some dyes require heat to penetrate (acid‑fast, endospore)

• Some stains rely on lipid solubility (carbol fuchsin)

⭐ 4. Contrast Creation

Stains increase contrast between:

• Cell vs background

• Cell types vs each other

• Cell structures vs cell body

Microbiological stains fall into simple, differential, and special categories. All stains rely on charge interactions, cell wall chemistry, and dye solubility to make invisible cells visible and distinguishable.

gram stain

Type of Stain

• A differential stain that separates bacteria into Gram‑positive and Gram‑negative based on cell wall structure.

Name of Methods

• Gram Staining Method (developed by Hans Christian Gram)

Procedure:

1. Primary stain: Crystal violet

2. Mordant: Iodine (forms CV‑I complex)

3. Decolorizer: Alcohol or acetone‑alcohol

4. Counterstain: Safranin

Chemicals:

• Crystal violet (primary stain)

• Gram’s iodine (mordant)

• Alcohol/acetone (decolorizer)

• Safranin (counterstain)

Appearance and results:

• Gram‑positive:

  • Purple

  • Thick peptidoglycan retains CV‑I complex

• Gram‑negative:

  • Pink/red

  • Alcohol dissolves outer membrane → thin peptidoglycan loses CV‑I → takes up safranin

acid fast stain

Type of Stain:

• Differential stain

• Separates acid‑fast organisms (with mycolic acids) from non–acid‑fast organisms.

Name of Methods:

• Ziehl–Neelsen Method (hot method)

• Kinyoun Method (cold method)

Procedure:

1. Prepare smear

• Air‑dry and heat‑fix.

2. Primary stain

• Flood with carbol fuchsin.

• Ziehl–Neelsen: heat gently to steam (helps dye penetrate waxy wall).

• Kinyoun: no heat; uses higher phenol concentration.

3. Decolorize

• Rinse with acid‑alcohol (3% HCl in ethanol).

• Acid‑fast cells retain carbol fuchsin.

4. Counterstain

• Apply methylene blue (or brilliant green).

• Non–acid‑fast cells take up counterstain.

Chemicals:

• Carbol fuchsin (primary stain)

• Acid‑alcohol (decolorizer)

• Methylene blue or brilliant green (counterstain)

• Ziehl–Neelsen only: heat source (steam)

Appearance and results:

• Acid‑fast bacteria

  • Bright red/fuchsia

  • Due to retention of carbol fuchsin

  • Thick mycolic acid layer prevents decolorization

• Non–acid‑fast bacteria

  • Blue or green

  • Take up counterstain after losing primary dye

Common microbial genera that are acid fast or form endospores:

• Mycobacterium

  • M. tuberculosis, M. leprae

• Nocardia (partially acid‑fast)

Capsule Stain

Type of Stain:

• Special / structural stain

• Negative stain technique

• Designed to visualize capsules, which do not take up most dyes.

Name of Methods:

• Anthony’s Capsule Stain

• Negative Stain Method (India ink or nigrosin

Procedure:

1. Prepare smear (NO heat‑fixing)

• Mix bacteria with India ink or nigrosin on slide

• Spread into thin film

• Air‑dry only — heat destroys capsules

2. Apply counterstain

• Flood with crystal violet (Anthony method)
  or safranin (alternative)

3. Rinse gently and air‑dry

• Do not blot — blotting can remove capsules

Chemicals:

• India ink or nigrosin (acidic dye → stains background)

• Crystal violet or safranin (basic dye → stains cells)

Appearance and results:

Capsule‑positive bacteria

• Clear halo around the cell

• Background = dark

• Cell body = purple/pink (depending on counterstain)

Capsule‑negative bacteria

• No halo

• Only stained cell + dark background

Structure and Function of Endospores and Capsules:

Structure

• Thick, gelatinous layer outside cell wall

• Made of polysaccharides or polypeptides

Function

• Anti‑phagocytic (major virulence factor)

• Adherence to surfaces

• Prevents desiccation

• Helps form biofilms

Endospore Stain

Type of Stain:

• Differential stain

• Distinguishes endospores from vegetative cells

Name of Methods:

• Schaeffer–Fulton Method (most common)

• Dorner Method (less common)

Procedure:

1. Prepare smear

• Air‑dry and heat‑fix.

2. Primary stain

• Flood smear with malachite green.

• Steam over heat for ~5 minutes (keeps dye penetrating the spore coat).

3. Rinse

• Rinse gently with water (removes dye from vegetative cells but NOT spores).

4. Counterstain

• Apply safranin for 1 minute.

5. Rinse and blot dry

Chemicals:

• Malachite green (primary stain)

• Heat/steam (mordant-like function)

• Water (decolorizer)

• Safranin (counterstain)

Appearance and results:

Endospores

• Green

• Because malachite green is forced into spores by heat and retained

Vegetative cells

• Pink/red

• Take up safranin after malachite green washes out

Structure and Function of Endospores and Capsules:

Structure

• Core: DNA, ribosomes, dipicolinic acid + Ca²⁺

• Cortex: thick peptidoglycan

• Spore coat: protein layers

• Exosporium: outermost layer

Function

• Survival structure

• Resistant to:

  • Heat

  • UV radiation

  • Chemicals

  • Desiccation

• Allows bacteria to persist for decades

Common microbial genera that are acid fast or form endospores:

• Bacillus (aerobic)

• Clostridium (anaerobic)

Colonial Morphology and Broth/Slant Growth Characteristics

Quorum sensing

• this serratia marsenscens culture shows evidence of a pheomenon known as quorum sensing, in which production of pigment is density dependent

• it only produces pigment when there are enough bacteria present in a colony

Swarming

• this is a pure culture of a highly motile bacteria

• ONE drop of inoculum was placed at the center of the plate

• after 24 hour incubation the “swarm” has covered the entire plate with a thin, transparent film of growth

Too Thick

• the bacteria on this plate are too thick to discern colony morphology

• this can be caused by:

  • too large an inoculation; multiple loops from stock broth or too much inoculum from a plate

  • failure to flame between loops

Pigment Production

• some bacterial colonies produce pigment

• S.marcesens, K.rosea, K.-rhizo, S.aurantiaca

Contaminant

• can come from a “drop-in: which settled onto the agar when the plate was opened during inoculation

• rim is a very common “hiding place”

• growth can be clear/ very tiny

Pure Cultures

Colony shape

• round: circle

• rhizoid: snowflake

• irregular: amoeba

Colony margin (edge)

• entire: circle

• lobate: pokey

• undulate: flowery

Colony size

• punctiform: 1mm<

Colony Color

Other Characteristics

• surface rough, smooth, mucoid, etc.

Broth growth: unagitated

• turbid: cloudy

• sediment: bottom

• pellicle: top

Slant growth characteristics: stroke slant

• filiform: line

• spreading: all around

• beaded: dots

Staphylococcus epidermis: punctiform, entire, white

Escherichia coli: irregular, entire, white

Bacillus subtillis- irregular, undulate, off-white

Broth-to-Broth theory

Broth‑to‑Broth Transfer (Steps Only)

1. Label tubes

2. Mix culture

3. Sterilize loop

4. Remove caps

5. Flame tube mouths

6. Insert loop into culture

7. Transfer inoculum to sterile broth

8. Flame tube mouths again

9. Replace caps

10. Sterilize loop

11. Incubate

Broth‑to‑Broth Theory (List Only)

• Broth‑to‑broth transfer is used to propagate a culture into fresh nutrients.

• It demonstrates aseptic technique, preventing contamination from the environment or other cultures.

• Only a small inoculum is needed because bacteria reproduce rapidly in nutrient broth.

• The goal is to maintain a pure culture while allowing cells to enter log phase growth.

Streak Plate Process and theory

Streak Plate Process (Steps Only)

1. Label plate

2. Sterilize loop

3. Obtain inoculum

4. Streak quadrant 1

5. Sterilize loop

6. Streak quadrant 2

7. Sterilize loop

8. Streak quadrant 3

9. Sterilize loop

10. Streak quadrant 4

11. Invert plate

12. Incubate

Streak Plate Theory (List Only)

1. The streak plate method uses mechanical dilution: each quadrant spreads fewer cells.

2. By the final quadrant, individual cells land far enough apart to grow into isolated colonies.

3. Each colony arises from a single cell (or genetically identical cluster), making it a pure culture.

4. This method is essential for identifying bacteria, performing biochemical tests, and maintaining uncontaminated stocks.

Vocab Lab 1 and 2

• Compound light microscope: A microscope that uses visible light and multiple lenses to magnify specimens.

• Monocular: A microscope with one ocular (eyepiece) lens.

• Binocular: A microscope with two ocular lenses for more comfortable viewing.

• Resolution: The ability to distinguish two close points as separate, determining image clarity.

• Numerical aperture: A measure of a lens’s light‑gathering ability that directly affects resolution.

• Rheostat: The control that adjusts the brightness of the microscope’s light source.

• Parfocaling: A feature where a specimen stays nearly in focus when switching between objectives.

Vocab Lab 3

• Binomial Nomenclature: A two‑name scientific naming system using genus and species.

• Kingdoms: The broadest major categories of life, such as Animalia, Plantae, Fungi, Protista, and Bacteria.

• Ubiquity/Ubiquitous: The concept that microorganisms are found everywhere in the environment.

• Culturing bacteria: Growing microorganisms under controlled conditions in the lab.

• Growth medium: A nutrient-rich substance that supports microbial growth.

• Broth: A liquid growth medium used to culture microorganisms.

• Cultures: Populations of microorganisms grown in a medium.

• Contamination: The accidental introduction of unwanted microorganisms into a culture or environment.

• Sterile: Completely free of all living organisms, including spores.

• Inoculate: To intentionally introduce microorganisms into a sterile medium.

• Incubate: To place cultures in controlled conditions (temperature, time) to promote growth.

• Aseptic Technique: Procedures used to prevent contamination of cultures, media, and the environment.

• Pure Culture: A culture containing only one microbial species.

• Mixed Culture: A culture containing two or more microbial species.

• Growth Medium: A nutrient source that supports microbial growth (solid or liquid).

• Agar: A solidifying agent derived from red algae used to create firm surfaces for microbial growth.

Vocab Lab 4

• Simple stain: A staining method that uses one basic dye to show cell shape, size, and arrangement.

• Negative stain: A stain using acidic dyes that color the background while leaving cells unstained for clearer morphology.

• Differential stain: A staining technique that uses multiple dyes to distinguish between different types of bacteria or structures.

• Mordant: A chemical that intensifies or fixes a dye to a structure, helping it bind more strongly.

• Decolorizer: A chemical (often alcohol or acid‑alcohol) that removes primary stain from some cells but not others during differential staining.

• Over decolorizing: Removing too much primary stain, causing cells to appear falsely negative (too light or wrong color).

• Under decolorizing: Removing too little primary stain, causing cells to appear falsely positive (too dark or wrong color).

Vocab Lab 5

• Acid‑fast stain: A differential stain that identifies bacteria with waxy mycolic acids in their cell walls, causing acid‑fast cells to remain red after acid‑alcohol decolorization.

• Endospores: Highly resistant, dormant bacterial structures formed by genera like Bacillus and Clostridium that survive extreme heat, chemicals, and drying.

• Capsules: Thick, gelatinous outer layers surrounding some bacteria that help with protection, adhesion, and evading phagocytosis, appearing as clear halos in capsule stains