Introduction to

prokaryotes and
Eukaryotes
Dr. Sumit Sharma
1. Definition & Basic Difference
Prokaryotes: Single-celled organisms that lacks a true nucleus and membrane-bound
organelles.
• Bacteria (E. coli, Streptococcus, Mycobacterium)
• Archaea (methanogens, halophiles, thermophiles)

Eukaryotes: Organisms (unicellular or multicellular) with a true nucleus enclosed by a

nuclear membrane and membrane-bound organelles.
• Protists (Amoeba, Paramecium)
• Fungi (Yeast, Mushrooms)
• Plants
• Animals
 2. Cell Structure
Feature Prokaryotes Eukaryotes
Present (DNA enclosed in nuclear
Nucleus Absent (DNA in nucleoid region) envelope)
Circular, naked (no histones, except in
DNA Archaea) Linear, complexed with histones

Cell Size Small (0.1–5 µm) Larger (10–100 µm)

Usually present (peptidoglycan in Present in plants (cellulose) & fungi
Cell Wall bacteria, pseudopeptidoglycan in (chitin); absent in animals
archaea)

Many membrane-bound organelles

Organelles No membrane-bound organelles (mitochondria, ER, Golgi, lysosomes,
chloroplasts in plants)

Ribosomes 70S (smaller) 80S (larger; 70S in mitochondria &

chloroplasts)
Contains sterols (e.g., cholesterol in
Plasma Membrane Lacks sterols (except in some) animals)

Flagella Simple Complex

3. Genetic Material & Cell
Division
• Prokaryotes:
• Single circular chromosome.
• May have plasmids (extra-chromosomal DNA).
• Reproduction: Asexual (binary fission).

• Eukaryotes:
• Multiple linear chromosomes inside nucleus.
• DNA wrapped around histones.
• Reproduction: Asexual (mitosis) & sexual (meiosis).
4. Metabolism & Energy
Production
Prokaryotes:
• Wide metabolic diversity: aerobic, anaerobic, photosynthetic,
chemosynthetic.
• Energy production occurs at the plasma membrane.

Eukaryotes:
• Mostly aerobic respiration.
• Energy production occurs in mitochondria (or chloroplasts in plants/algae).
5. Evolutionary Perspective
• Prokaryotes are older (~3.5 billion years ago) and more primitive.
• Eukaryotes evolved later (~2 billion years ago), likely via
endosymbiotic theory.
• Billions of years ago: A large prokaryotic cell engulfed a smaller free-
living prokaryote (like a bacterium), but didn’t digest it.
• Instead, they formed a mutualistic relationship:
• The small cell got protection and nutrients from the big cell.
• The big cell got extra energy from the small cell’s metabolic abilities.
• Over time, the small cell became a permanent resident inside the big
cell — evolving into an organelle (Mitochondria and chloroplast).
6. Key Similarities
Both have:
• Plasma membrane
• Cytoplasm
• DNA as genetic material
• Ribosomes (for protein synthesis)
• Enzymes for metabolic pathways
 Ultra-Structure of Bacteria
Bacteria are
prokaryotic cells, so
they lack a nucleus
and membrane-
bound organelles,
but they still have a
very organized
internal and
external structure.
1. Cell Envelope (Outer
Structures)
• Glycocalyx (Capsule/Slime layer)
• Gel-like outer coating.
• Capsule = organized & firmly attached (protects against phagocytosis, e.g., Streptococcus
pneumoniae).
• Slime layer = loose & irregular (helps in adhesion, e.g., dental plaque).
• Cell Wall
• Provides shape and rigidity.
• Made of peptidoglycan (murein).
• Two types (basis of Gram staining):
• Gram-positive → thick peptidoglycan, teichoic acids.
• Gram-negative → thin peptidoglycan, outer membrane with lipopolysaccharides (LPS).
• Plasma Membrane
• Phospholipid bilayer with proteins.
• Functions: selective permeability, energy generation (respiratory enzymes), transport.
2. Appendages
1. Flagella (for locomotion)
• Structure: Long, whip-like helical filaments made of flagellin protein.
• Movement: Rotates like a propeller → enables bacteria to swim.
• Function: Motility → chemotaxis (movement toward nutrients, away
from toxins).
• Monotrichous → single flagellum (e.g., Vibrio cholerae).
• Peritrichous → flagella all over (e.g., E. coli).
• Amphitrichous → one flagellum at each pole.
2. Fimbriae (for attachment)
• Structure: Thin, short, hair-like projections (much shorter than
flagella).
• Number: Present in hundreds per cell.
• Function:
• Help bacteria stick to surfaces, tissues, and each other.
• Important in forming biofilms (e.g., dental plaque).
• Example: Neisseria gonorrhoeae uses fimbriae to attach to mucosal
surfaces.
3. Pili (for DNA transfer & adhesion)
• Structure: Longer and fewer than fimbriae, hollow tube-like
structures.
• Types:
• Common pili → help in adhesion to host cells.
• Sex pili (conjugative pili) → special type that allows conjugation (transfer of
plasmids/DNA between bacteria).
• Example: E. coli can exchange antibiotic resistance genes via sex pili.
3. Cytoplasmic Components
• Nucleoid → Irregular region containing circular DNA (chromosome).
• Plasmids → Extra-chromosomal DNA, often carry antibiotic resistance
genes.
• Ribosomes (70S) → Sites of protein synthesis.
• Inclusion bodies → Storage granules (glycogen, phosphate, sulfur).
• Mesosomes (artifact in staining, but described in textbooks) →
Infoldings of plasma membrane, role in respiration/DNA replication.
• Endospores → Dormant, highly resistant structures formed by Bacillus
and Clostridium species.
Morphological Classification of
Bacteria
Bacteria can be classified based on shape
and arrangement seen under microscope.
• 1. Cocci (Spherical)
• Monococcus → single (rare).
• Diplococci → pairs (e.g., Neisseria
gonorrhoeae).
• Streptococci → chains (e.g.,
Streptococcus pyogenes).
• Staphylococci → clusters (grape-like;
e.g., Staphylococcus aureus).
• Tetrads → groups of four.
• Sarcinae → cubical packets of eight.
2. Bacilli (Rod-shaped)
• Single bacilli → e.g., Escherichia
coli.
• Diplobacilli → pairs.
• Streptobacilli → chains.
• Coccobacilli → very short rods
(intermediate between cocci &
bacilli; e.g., Haemophilus
influenzae).
3. Spiral Forms
• Vibrio → comma-shaped (e.g.,
Vibrio cholerae).
• Spirillum → rigid spiral with few
turns.
• Spirochetes → flexible spiral
with many turns (e.g.,
Treponema pallidum → syphilis).
4. Filamentous & Pleomorphic
Forms
• Filamentous bacteria → thread-
like (e.g., Actinomyces).

• Pleomorphic bacteria →
variable shapes, no fixed form
(e.g., Mycoplasma — lacks cell
wall).
Nutritional Requirements of
Bacteria
Bacteria require different nutrients for energy generation,
biosynthesis, and survival. These can be classified as:
1. Macronutrients: Needed in large amounts.
• Carbon (C) → energy source, structural component
• Nitrogen (N) → amino acids, proteins, nucleic acids
• Hydrogen (H), Oxygen (O) → organic compounds, water
• Sulfur (S) → amino acids (cysteine, methionine)
• Phosphorus (P) → nucleic acids, ATP, phospholipids
• Other ions → K, Mg, Ca, Fe
Nutritional Requirements of
Bacteria
2. Micronutrients (Trace Elements): Needed in very small amounts, but essential for
enzyme activity.
• Zinc (Zn), Copper (Cu), Manganese (Mn), Molybdenum (Mo), Nickel (Ni).
3. Growth Factors: Growth factors are organic compounds that some bacteria
cannot synthesize on their own but are essential for their survival and multiplication.
Since these bacteria lack the biosynthetic pathways, they must obtain these
compounds ready-made from the environment or culture medium.
• Examples: Vitamins → act as coenzymes (e.g., Vitamin B₁, B₆, B₁₂, biotin, riboflavin)
• Amino acids → required if the bacteria cannot make certain amino acids
themselves.
• Purines & Pyrimidines → building blocks of DNA & RNA.
Culture Media
• A culture media is a nutrient preparation that provides all the
essential requirements (nutrients + environment) for the growth and
multiplication of microorganisms under laboratory conditions.
• Raw Materials Used
• Peptones → source of amino acids and nitrogen.
• Beef extract / Yeast extract → provide vitamins, minerals, growth factors.
• Carbohydrates (glucose, lactose) → main energy source.
• Agar → solidifying agent, inert (does not provide nutrition).
• Selective agents (antibiotics, bile salts, dyes) → inhibit unwanted organisms.
• Indicators (phenol red, neutral red) → detect pH change during biochemical
reactions.
Types and Examples of Culture
Media
1. Simple (Basal) Media → basic growth needs.
• Example: Nutrient agar, Nutrient broth.

2. Enriched Media → contain additional nutrients for fastidious organisms.

• Example: Blood agar, Chocolate agar.

3. Selective Media → contain agents that inhibit some bacteria while

allowing others.
• Example: MacConkey agar (selective for Gram-negative).
Types and Examples of Culture
Media
4. Enrichment Media → liquid media enhancing growth of desired
organisms.
• Example: Selenite F broth (for Salmonella).

5. Transport Media → maintain viability during transport.

• Example: Stuart’s medium, Amies medium.

6. Differential media → culture media designed to distinguish between

different groups of bacteria growing on the same plate.
Bacterial Growth Curve
• When a bacterial population is
inoculated into a fresh culture
medium and incubated, the cell
number changes in a
characteristic pattern called the
growth curve.
• It can be studied by plotting log
of viable cell count vs. time
Phases of Growth Curve
1. Lag Phase
• Cells metabolically active but not dividing.
• Bacteria are adjusting to the new medium, synthesizing enzymes and
molecules.
• Length depends on inoculum condition & medium.

2. Log (Exponential) Phase

• Rapid cell division at constant rate (maximum growth).
• Population doubles at each generation (binary fission).
3. Stationary Phase
• Nutrients become limited, toxic products accumulate.
• Growth rate = death rate → population stabilizes.

4. Decline (Death) Phase

• Nutrients exhausted, wastes accumulate.
• Death rate exceeds growth rate.
• Some cells die, few may persist in dormant form.
Isolation of Pure Cultures
Pure Cultures: A pure culture is a culture that contains only a single
species of microorganism. Isolation techniques are used to separate
one type of microbe from a mixed population.
Purpose
• For identification and classification of bacteria.
• To study morphology, physiology, and pathogenicity.
• For industrial use (antibiotics, enzymes).
• In clinical microbiology → to identify the causative pathogen.
Streak Plate Method
Definition: A surface-dilution technique in which an inoculating loop
progressively thins the bacterial load across solid agar, producing
isolated colonies in later streak zones.
Principle
• Mechanical dilution on the plate: each new streak drags fewer cells
from the previous zone.
• After incubation, spatially separated cells grow into discrete colonies.
A colony picked from the last zones is considered a pure culture
Materials & Setup
• Media (solid agar): Nutrient agar/Blood agar/MacConkey, etc.
• Inoculating loop: Nichrome/platinum (flame-sterilized) or pre-sterile
plastic loop.
• Bunsen burner/spirit lamp (for aseptic zone & flaming), marker, 70%
alcohol, incubator (usually 35–37 °C for pathogens).
• Inoculum: Colony from a plate/slant or clinical specimen.
• Protective clothing and gear
Procedure
• Flame & cool loop: Heat to red-hot; cool 5–10 s.
• Load inoculum: Touch a colony/immersed loop in specimen
• Quadrant 1 (Q1): Lift lid minimally (≈45°). Streak a tight zig-zag near the plate edge
• Flame & cool loop.
• Quadrant 2 (Q2): Rotate plate ~90°. Start in Q1, make 2–3 gentle crossovers, then streak a
fresh area.
• Flame & cool loop.
• Quadrant 3 (Q3): Rotate; again 2–3 crossovers from Q2, then streak a new sector.
• Flame & cool loop.
• Quadrant 4 (Q4): Final rotation; 2–3 crossovers from Q3, then streak into the largest unused
area. Finish with a few long, well-spaced lines.
Advantages & Limitations
Pros
• Simple, fast, cost-effective.
• Excellent for obtaining pure cultures from mixed specimens.
Cons
• Requires skill and experience in flaming, cooling loop, streaking
pattern.
• Plate is opened multiple times during streaking → higher chance of
airborne contamination
Spread Plate Method
Definition
• The spread plate method is a technique used to isolate and
enumerate microorganisms by spreading a diluted sample evenly
over the surface of solid agar using a sterile spreader.
Principle
• A known volume (usually 0.1 mL) of a diluted microbial suspension is
spread uniformly over the agar surface.
• Each viable cell grows into a discrete colony.
Materials Required
• Sterile Petri plates with nutrient agar or other selective/differential
media.
• Inoculum (serial dilutions of the sample).
• Micropipette (or sterile pipette) to deliver 0.1 mL sample.
• Sterile L-shaped glass rod or glass spreader.
• Alcohol beaker and Bunsen burner (to flame-sterilize spreader).
• Incubator at required temperature (commonly 35–37 °C for
pathogens).
Procedure
• Prepare serial dilutions of the sample to reduce bacterial load.
• Pipette 0.1 mL of the selected dilution onto the agar surface.
• Sterilize the L-shaped glass rod by dipping in alcohol and flaming, then cool.
• Spread the inoculum evenly over the entire agar surface with gentle
circular/rubbing motion while rotating the plate.
• Allow plate to dry for a few minutes to absorb the inoculum.
• Invert the plate and incubate at appropriate temperature (e.g., 37 °C for 24–
48 hrs).
• After incubation, count surface colonies
Example
• You dilute a bacterial culture to 10⁻⁶.
• From this dilution, you pipette 0.1 mL onto an agar plate.
• Here, the volume plated = 0.1 mL.
• After incubation, you count 45 colonies.
Advantages
• Simple and inexpensive.
• Suitable for counting viable organisms (CFU).
• Works well for aerobic bacteria and yeasts.
Disadvantages
• Only aerobic organisms can grow (anaerobes not supported).
• Heat-sensitive organisms may be damaged by flaming spreader if not
cooled.
• Requires serial dilution to get countable plates.
Pour Plate Method
Definition
• The pour plate method is a microbiological technique where a diluted
microbial sample is mixed with molten agar (at ~45 °C) and poured into
sterile Petri plates.
Principle
• A small measured volume (usually 1 mL) of a diluted inoculum is placed in a
sterile Petri plate.
• Molten agar (45 °C to avoid killing cells) is poured over it and gently mixed.
• Bacteria become trapped at different depths of the medium.
• Each viable cell gives rise to a colony, allowing colony count (CFU/mL).
Procedure
• Pipette 1 mL of selected dilution into a sterile empty Petri plate.
• Add 15–20 mL molten agar (cooled to ~45 °C).
• Gently swirl the plate to mix inoculum evenly with agar.
• Allow agar to solidify.
• Invert the plates and incubate at suitable temperature (24–48 hrs).
• Count colonies
Advantages
• Quantitative method → allows CFU count.
• Can detect aerobic and facultative anaerobic organisms
• Useful for organisms that prefer subsurface growth (less oxygen).
Disadvantages
• Heat from molten agar may kill heat-sensitive organisms.
• More time-consuming than spread plate method.
• Requires more glassware/media.
Cultivation of Anaerobes
• Anaerobes are microorganisms that require little or no oxygen for growth.
• Oxygen can be toxic for strict (obligate) anaerobes because they lack enzymes like
catalase, peroxidase, and superoxide dismutase that detoxify reactive oxygen species.
• When oxygen is present inside a medium, it gets converted into toxic by-products such
as: Superoxide radical (O₂⁻), Hydrogen peroxide (H₂O₂), Hydroxyl radicals (·OH). These are
poisonous to the cell because they damage proteins, lipids, and DNA.
• Aerobes (like E. coli) have protective enzymes: Superoxide dismutase (SOD) → converts
O₂⁻ into H₂O₂, Catalase/peroxidase → break H₂O₂ into harmless water and oxygen.
• Obligate anaerobes (like Clostridium) lack these enzymes, so they cannot neutralize the
toxic products. When exposed to oxygen, the toxic radicals accumulate → cell death.
• Hence, special methods are needed to exclude oxygen during their cultivation.
1. Physical Methods to cultivate
anaerobes
Candle jar method
• Inoculated plates are placed in a jar with a candle.
• Candle burns O₂ partially, leaving ~3–5% CO₂.
• Suitable for Neisseria, Haemophilus (not strict anaerobes).

Anaerobic jar (McIntosh and Fildes jar)

• Inoculated plates kept inside a sealed jar.
• Oxygen is removed either chemically or by vacuum.
• Palladium catalyst combines H₂ with residual O₂ → water
• Used for clinical isolates like Clostridium.
2. Chemical Methods
Reducing agents in media
• Chemicals like sodium thioglycolate, cysteine, ascorbic acid, or cooked
meat medium reduce O₂ and maintain anaerobic environment.
• Example: Thioglycolate broth → widely used for cultivating
anaerobes.
• Robertson’s cooked meat medium: supports growth of Clostridium
3. Biological Methods
• Aerobes + anaerobes grown together.
• Aerobes consume O₂, protecting anaerobes.
• Example: Anaerobes growing beneath a surface layer of facultative
anaerobes.
4. Anaerobic Chambers/Glove
Boxes
• Sophisticated method for strict anaerobes.
• Entire work is carried out inside a chamber filled with N₂, H₂, and CO₂
atmosphere.
• Provides a completely oxygen-free environment.
• Expensive but reliable.
Quantitative Measurement of
Bacterial Growth

Bacterial growth can be measured in two main ways:

1. Total Count → counts all cells (living + dead).

2. Viable Count → counts only living cells that can divide and form
colonies.
Total count methods
1. Microscopic Count (Haemocytometer / Neubauer’s Chamber)
• A haemocytometer is just a special glass slide that has tiny squares (grid)
engraved on it.
• It also has a fixed depth (0.1 mm), so the volume of liquid above each
square is known.
• You put a drop of your bacterial suspension on it, cover it with a coverslip.
• Then you look under the microscope → the bacteria sitting in the squares
can be counted directly.
• Because the volume is known, you can calculate how many bacteria are
present in 1 mL of the sample.
Advantages
• Direct and quick.
• You can see the cells
directly (shape, size,
motility).
Disadvantages
• Cannot tell if cells
are alive or dead.
• Not suitable if
bacteria are very few
(dilute sample)
2. Coulter Counter (Electronic Counter)
• The sample is passed through a very narrow aperture between two
electrodes.
• On each side of the hole there’s an electric current.
• Every time a cell passes through, it blocks the current for a moment
→ the machine counts it as one cell.
• In the end, you get the total number of cells (alive + dead).
• Pros: Fast, accurate, works automatically.
• Cons: Expensive machine, and again — can’t tell alive vs. dead.
3. Turbidimetry (Spectrophotometer Method)
• When bacteria grow in a broth, the liquid becomes cloudy (turbid).
• The more bacteria there are → the more light gets scattered.
• A spectrophotometer shines light through the broth.
• Clear broth = light passes easily.
• Cloudy broth = less light passes.
• Pros: Quick, non-destructive, good for monitoring growth in real-time.
• Cons: Cannot distinguish live vs. dead cells, only works well with dense
cultures.
Viable count method
1. Plate count method
• You dilute your bacterial sample.
• Spread or pour it on agar plates.
• After incubation, each living bacterium grows into a colony.
• Count the colonies = number of colony forming units (CFU).
• Example: If you see 150 colonies after plating, that means about 150 living
bacteria were in your diluted sample.
• Dead ones won’t form colonies, so they don’t count.
• Pros: Simple, direct, gives only viable count, widely accepted.
• Cons: Time-consuming (needs incubation), may underestimate if cells clump.
2. Membrane Filter Method
• Pass a large volume of water through a filter with tiny pores (bacteria get
trapped).
• Place the filter on a nutrient agar plate.
• Each living bacterium trapped on the filter grows into a visible colony.
• Count the colonies → gives number of bacteria in the original water.
• Best for water testing (e.g., drinking water, lakes)
• Pros: Good for large volume liquid samples, useful for water testing,
sensitive.
• Cons: Only works with samples that can be filtered
3. Most Probable Number Method
• For liquid samples, especially when bacteria are few.
• Make serial dilutions of your sample.
• Inoculate broth tubes (like test tubes with nutrients).
• After incubation, check which tubes show growth (turbidity or color
change).
• Use a statistical table to estimate the number of bacteria.
• Pros: Useful for low bacterial numbers, works with liquid samples (e.g.,
water, milk).
• Cons: Indirect, only an estimate (not exact), requires statistical tables.