BACTERIA: MORPHOLOGY & GROWTH REQUIREMENTS

Major Phenotypic Categories of Bacteria:

Bacteria are divided into three major phenotype categories:
1. Gram - negative & have a cell wall.
2. Gram – positive & have a cell wall
3. Lack a cell wall (Mycoplasma spp.)

Characteristics of Bacteria:
Characteristics of bacteria used in classification & identification include:
⮚ Cell morphology
⮚ Staining reactions
⮚ Motility
⮚ Colony morphology
⮚ Atmospheric requirements
⮚ Nutritional requirements
⮚ Biochemical & metabolic activities
⮚ Pathogenicity, &
⮚ Genetic composition

Bacterial Morphology: Three basic categories of bacteria based on shape:

1. Cocci (round bacteria)
2. Bacilli (rod-shaped bacteria)
3. Curved & spiral-shaped bacteria

Categories based on the shape of their Cells:

Bacterial Morphology: Cocci may be seen: singly/in

⮚ Pairs (diplococci)
⮚ Chains (streptococci)
⮚ Clusters (staphylococci)
⮚ Packets of 4 (tetrads)
⮚ Packets of 8 (octads)

Average coccus is about 1 µm in diameter. Some cocci have “coccus” in their name.

Morphologic Arrangements of Cocci

Arrangement Description Appearance Example Disease

Diplococci Cocci in pairs Neisseria gonorrhoeae Gonorrhea

 Streptococci Cocci in chains Streptococcus pyogenes Strep throat

Staphylococci Cocci in clusters Staphylococcus aureus Boils

Tetrad Packet of 4 cocci Micrococcus luteus Rarely

pathogenic

Octad Packet of 8 cocci Sarcina ventriculi Rarely

pathogenic

Morphologic Arrangements of Bacilli: Bacilli often refereed to as RODS; may be short/long, thick/thin, &
pointed or w/ curved/blunt ends. May occur:
⮚ Singly, or
⮚ In pairs (diplobacilli) or
⮚ In chains (streptobacilli) or
⮚ Long filaments or
⮚ Branched

Average sized bacillus is 1 x 3 µm. Extremely short bacilli are called COCOBACILLI. E.g., of medically
important bacilli: Escherichia, Klebsiella. Proteus, Pseudomonas, Haemophilus

Arrangements of Bacilli.
a. Single bacillus of Escherichia coli
b. Diplobacilli in a young culture of bacillus cereus
c. Streptobacilli in an older culture of Bacillus cereus.
d. V-shapes & palisades of Corynebacterium diphtheriae

Curved & Spiral – shaped Bacteria: Curved &

spiral shaped bacteria. E.g., of curved bacteria
▪ Vibrio spp.
▪ Campylobacter spp.
▪ Heliobacter spp.
E.g., of spiral-shaped bacteria:
▪ Treponema spp.
▪ Borrelia spp.

Gram – Staining Reactions: Two major groups

based on Gram – staining reactions
1. GRAM - POSITIVE (bacteria end up being blue to purple)
 2. GRAM – NEGATIVE (bacteria end up being pink to red)

Name comes from the Danish bacteriologist HANS CHRISTIAN GRAM, who developed the technique.

Cell Wall of Gram – negative Bacteria: Gram-negative cell walls have thin layer of peptidoglycan. Outer
bilayer membrane composed of two different layers or leaflets:
1. inner leaflet - composed of phospholipids & proteins
2. outer leaflet – composed of lipopolysaccharide (LPS). LPS – responsible for its endotoxin activity

The outer membrane is protective, allowing Gram-negative

bacteria to better survive in harsh environments.
Gram-negative cell walls have: Integral proteins called PORINS
form channels through both leaflets of the outer
membrane, allowing glucose & other monosaccharides to move
across the membrane. Periplasmic space – space between the
outer membrane and the inner plasma
membrane. Contains enzymes for the breakdown of large
molecules intro transportable
ones and enzymes to detoxify and inactivate antibiotics

Cell Wall of Gram – positive Bacteria

Gram-positive cell wall has a thick layer of PEPTIDOGLYCAN &
LIPOTEICHOIC ACIDS that anchor the wall to the cytoplasmic
membrane.

Gram – Staining Reactions:

Gram – staining procedure
1. Methanol – fix specimen to slide. Flood slide w/ crystal violet solution; allow to act for 1 min.
2. Rinse the slide, then flood w/ iodine solution; allow iodine to act for 1 min. Before ethanol
decolorization (next step), all organisms appear purple, that is, Gram – positive.
3. Rinse off excess iodine. Decolorize w/ ethanol, approximately 5 secs. (time depends on density
of specimen)
4. Wash slide immediately in water. After ethanol decolorization, those organisms that are Gram –
negative are no longer visible.
5. Apply safranin counterstain for 30 secs.
6. Wash in water, blot, & dry in air. Gram – negative organisms are visualized after application of
the counterstain.

Key:
Gram – positive violet
Gram – negative red
Colorless

Gram Staining Reactions: Final Gram reaction (positive/ negative) depends on the organism’s cell wall
structure. Cell walls of Gram-positive bacteria have a thick layer of peptidoglycan, making it difficult to
remove the crystal violet–iodine complex.
Gram-negative organisms have a thin layer of peptidoglycan, making it easier to remove the crystal
violet; the cells are subsequently stained with safranin.
Staining Reactions: Gram Stain
Reagent Function Gram-positive Gram-negative

Crystal Violet Primary stain Purple/ blue Purple/ blue

Gram’s iodine Mordant (enhances uptake Purple/ blue Purple/ blue
of primary stain)
Acetone/ 95% alcohol Decolorizer Purple/ blue Colorless
Safranin Counterstain/ secondary Purple/ blue Red/pink
stain

Acid – fast Bacteria: Some additional chemicals are associated w/ the walls of some Gram-positive
bacteria. e.g., species of Mycobacterium have walls with up to
60% mycolic acid, a waxy lipid. Mycolic acid helps these cells survive desiccation (drying out) and makes
them difficult to stain w/ regular water-based dyes. Special staining procedure called the acid-fast stain
is used to stain these Gram-positive cells that contain large
amounts of waxy lipids

Staining Reactions: Gram Stain

Reagent Function Result
Ziehl–Neelsen Kinyoun Acid-fast Non-acid-fast

Carbol-fuchsin Carbol-fuchsin Primary Stain Red/pink Red/pink

Acid alcohol Acid alcohol Decolorizer Red Colorless

Methylene blue Malachite green Counter stain/ Ziehl–Neelsen: red Ziehl–Neelsen: blue
secondary stain organism/ blue organism/ blue
background background

Kinyoun:red organism/ Kinyoun: green

green background organism/ green
background

Bacteria Motility: A bacterium is able to “swim,” it is said to be motile. Most

spiral-shaped bacteria and about 50% of bacilli are motile; cocci are generally
nonmotile.

Projecting structures:
Flagella - thread like structures made up of protein subunits called flagellin.
Project from the capsule. Serve as organs for motility

Endoflagella (Axial elements ) - composed of bundles of fibrils. Arise from the

ends of bacterial cell; spiral around the cell. Rotate producing movement that
propels the organism forward.
Pili/ fimbriae - Made up of subunits called pilins. Function for adherence to cell
surface or for attachment to another bacterium.
Bacterial Growth Requirements: Growth encompasses an orderly increase in the sum of all components
of the organism, entailing replication of cellular structures &
components. Microbial growth involves increase in the number of cells and not in the size of organism.
Growth of organisms in a culture is implied by formation of colonies which are composed of billions of
cells.

Atmospheric Requirements: Bacteria can be classified on the basis of their atmospheric requirements,
including their relationship to O2 (Oxygen) & CO2 (Carbon Dioxide). W/ respect to O2, bacterial isolates
can be classified as:
⮚ Obligate aerobes
⮚ Microaerophilic aerobes
⮚ Facultative anaerobes
⮚ Aerotolerant anaerobes
⮚ Obligate anaerobes

Capnophilic organisms grow best in the presence of increased concentrations of CO2 (usually 5%–10%)

Culturing Microorganisms in Thioglycollate Broth (THIO): Thioglycolate broth is a multipurpose, enriched,

differential medium used primarily to determine the oxygen requirements of microorganisms. Sodium
thioglycolate in the medium consumes oxygen & permits the growth of obligate anaerobes. Combined w/
the diffusion of oxygen from the top of the broth, produces a range of oxygen concentrations in the
medium along its depth.
Aerobic and anaerobic bacteria can be identified by growing them in test tubes of thioglycolate broth

1. Obligate aerobes: need oxygen because they cannot ferment/respire anaerobically. They gather
at the top of the tube where the oxygen concentration is highest.

2. Obligate anaerobes: are poisoned by oxygen, so they gather at the bottom of the tube where the
oxygen concentration is lowest.

3. Facultative anaerobes: can grow w/ or w/out oxygen because they can metabolize energy
aerobically/ anaerobically. They gather mostly at the top because aerobic respiration generates
more ATP than either fermentation/ anaerobic respiration.

4. Microaerophiles: need oxygen because they cannot ferment/ respire anaerobically. However,
they are poisoned by high concentrations of oxygen. They gather in the upper part of the test
tube, but not the very top.

5. Aerotolerant organisms: do not require oxygen as they metabolize energy anaerobically. They
are NOT poisoned by oxygen. They can be found evenly spread throughout the test tube
Nutritional Requirements: All bacteria require some form of the elements such as carbon, hydrogen,
oxygen, sulfur, phosphorus, & nitrogen for growth. Some bacteria require special elements (e.g., calcium,
iron/ zinc). Organisms w/ especially demanding nutritional requirements are said to be fastidious
(“fussy”). The nutritional needs of a particular organism are usually
characteristic for that species & are sometimes important
clues to its identity.

Organisms w/ especially demanding nutritional requirements are said to be fastidious (“fussy”). E.g.,
Neisseria gonorrhoeae - requires blood or hemoglobin & several amino acids and vitamins to grow.
Campylobacter spp. & Helicobacter spp. - are capnophilic – require elevated CO2. They are difficult to
culture simply because it is difficult to accurately simulate their natural milieu in a culture medium.

Based on their CARBON source, bacteria may be classified as:

⮚ Autotrophs (lithotrophs) – use inorganic compounds (e.g., carbon dioxide) as their sole carbon
source
⮚ Organotrophs (heterotrophs) – use organic compounds (like glucose) as their carbon source

Both autotrophs and organotrophs, may derive their energy from either:
⮚ Light (photolithotorphs & photoorganotrophs), or
⮚ Oxidation of inoroganic substances (chemolithotrophs & chemoorganotrophs)

Most medically important bacteria are chemoorgnotrophs.

Nitrogen, sulfur & phosphorus: Important for synthesis of proteins (N,S), nuclei acids (N, P) & ATP (N, P).
The dry weight of bacteria is ≈ 14% nitrogen and ≈ 4% sulfur & phosphorus

Inorganic ions:
⮚ Magnesium - stabilizes ribosomes, cell membranes & nucleic acid, serves as cofactor of many
enzymes
⮚ Potassium – required for ribosomal functions, and for some enzymatic activities
⮚ Calcium – important component of Gram-positive cell wall, & protective of bacterial endospores
⮚ Iron – component of cytochrome of the electron transport chain & acts as cofactor of some
enzymes
⮚ Trace elements -Manganese, zinc, copper, cobalt

Physical Requirements:
Moisture and water: Bacterial cell is mainly made up of water. Serves as medium from which bacteria
acquire nutrients.

Temperature:
⮚ Thermophiles – grow best at T > 40oC
⮚ Mesophiles – grow best at T 20oC- 40oC
⮚ Psychrophiles – grow best at T 10oC- 20oC
Most medically important bacteria are mesophiles

pH
 ⮚ Alkalophiles – grow best at pH 8.4 – 9.0
⮚ Neutropohiles – grow beast at pH 6.5 – 7.5
⮚ Acidophiles – grow best at pH <6.0
Most medically important bacteria are neutrophiles

Bacterial Growth Curve: The bacterial growth curve represents the number of live cells in a bacterial
population over a period of time.

Lag Phase: Initial phase is characterized by cellular activity but not

growth. Allows the bacteria to synthesize proteins & other
molecules necessary for replication. These cells increase in size,
but no cell division occurs in the phase. This may last for 1-4 hours

Exponential (log) Phase: Time when the cells are dividing by binary
fission & doubling in numbers after each generation time.
Metabolic activity is high as DNA, RNA, cell wall components, &
other substances necessary for growth are generated for division.
It is in this phase that antibiotics and disinfectants are most effective targeting bacteria cell walls or the
protein synthesis processes of DNA transcription and RNA translation. Duration is about 8 hours.

Stationary phase: Phase when bacterial cell growth reaches a plateau, where the number of dividing cells
equal the number of dying cells, as the available nutrients become depleted & waste products start to
accumulate. This results in no overall population growth. Phase when spore forming bacteria produce
endospores & pathogenic bacteria begin to generate substances (virulence factors).

Death or decline phase: Number of living cells decreases exponentially & population growth experiences
a sharp decline. Due to continuous depletion of nutrients & accumulation of waste products. Sporulation
continues during this stage. Spores are able to survive the harsh conditions of the death phase & become
growing bacteria when placed in an environment that supports life.