Bacteria

Bacteria in the Lab: Growing Bacteria for Studies

• Culture - A bacterial growth (colonies of cells) maintained in the laboratory.

Growth Media:

• Nutrient Broth- a liquid growth medium used in flasks, culture bottles or test tubes.

Agar- a solid-type of culture medium made from seaweed that has a somewhat rubbery or gel-like consistency. It is used in a “culture dish”

Bacteria reproduce by splitting in half-----called Binary Fission.

If this growth pattern continued for several days, bacteria would cover the planet!

This is known as exponential growth. Exponential growth occurs when individuals of a population reproduce at a constant rate.

J- growth

curve

The bacterial culture eventually reaches carrying capacity however and the unchecked growth stops.

Carrying capacity - is the largest numbers of individuals that a given environment can support.

S-growthcurveas resources become less available, population growth slows or stops)

Carrying capacity is related to Environmental Resistance factors: environmental factors such as limited space, limited food, and an abundance of cellular waste products that inhibit the bacterial cell population’s growth, and finally, put an end to the bacterial culture.

Identification of Bacteria based onOXYGEN REQUIREMENTS:

Obligate Aerobe – must live in the presence of O2 (metabolism - cell respiration) Example: Mycobacterium tuberculosis

Obligate Anaerobe – can’t live in the presence of O2 (metabolism - fermentation) Examples: Clostridium tetani – causes tetanus Clostridium botulinum – causes Botulism

Facultative Anaerobe – can live under both conditions (with or without O2) (metabolism – cell respiration or fermentation) Example: Escherichia coli in human intestines

Test tubeculturesbased on

O2

Identification of bacteria based on

shapes and arrangement:

1) single spheres

2) two spheres

3) bunches of spheres

4) four spheres

5) Eight spheres – 4

behind 4

6) chain of spheres

7) rod-shaped

8) two rods

9) chain of rods

10) spiral

11) comma

(spirilla)

Identification by Gram Staining

Developed by Dutch Physician Dr. Hans Christian Gram. He wanted to differentiate bacteria from patient cell nuclei in tissue slides.

Most Species of Eubacteria can be grouped into two categories based on their response to laboratory stain:

GRAM + or GRAM -

GRAM +: The bacteria that retain crystal violet stain

& appear purple. They have a thick cell wall layer known as peptidoglycan that holds the stain. Peptidoglycan is composed of protein and carbohydrate. These cells produce exotoxins.

GRAM - The bacteria that have a thinner peptidoglycan

layer and therefore do not retain the gram stain. They are counterstained with a pink stain called safranin.

An outer layer of lipids and carbohydrates hold the pink color. These cells produce endotoxin.

E.coli

The peptidoglycan layer – a protein & carbohydrate layer within the cell wall.

It is affected by antibiotics. It is not present in archaebacteria and is a determining factor in the Gram staining procedure.

Gram - & Gram +

Gram staining Procedure

Gram staining Procedure

A dying, gram negative bacterium leaks endotoxin after an attack by antibiotics. The body’s response to this endotoxin launches a sequence of events that often results in septic shock. The life-threatening bacteria infection that physicians call sepsis strike two patients every hour in US hospitals.

• The Center for Disease control estimate that 100,000 in the US die of septic shock each year, making it the 13th leading cause of death. Roughly half of all sepsis patients die, even if an antibiotic manages to clear the bacteria from the bloodstream.

• Gram negative bacterial walls are thinner and made of a lipopolysaccharide endotoxin, which brings about the rapid deterioration characteristic of the sepsis syndrome.

• As long as the cell wall is intact, the endotoxin presents little health hazard. But, when the immune system or antibiotics attack gram-negative bacteria, they expose a “toxic region” at the root of the endotoxin molecule, which leads to the high fever and blood pressure drop that can be fatal.

Identification by Colonial shape: Observations of a bacterial colony’s shape and characteristics within a culture dish.

Bacteria identification is also based on nutrition, metabolism, growing conditions & cell structures. (Decomposers = saprophytes)

To Identify bacteria ---go to Bergey’s. (4 vol.s)

Basic Bacterial Cell Structure:

Structures & Functions

• cell wall - protects the bacteria, supports the cell, prevents bursting

• outer membrane – protects the cell from some antibiotics(only present in gram – cells)

• cell membrane

regulates movement of materials in & out of the cell. It also contains enzymes important to cell respiration

• cytoplasm – contains DNA, ribosomes, and organic compounds

• chromosome (bacterial DNA) It is arranged in a single closed loop. It carries genetic information inherited from past generations. (also referred to as a nucleoid)

plasmid – a circular, self-repeating loop of DNA. It contains some genes obtained through genetic

recombination.

• capsule & slime layer – protects the cell and assists in attaching the cell to surfaces (glycocalyx – a fuzzy coat of sticky sugars in the capsule layer enables bacteria to stick to the surface of host cells and tissues.)

• endospore – Some gram + cells can form a thick-coated resistant structure cell when environmental conditions are harsh. The harsh conditions may destroy the original cell, but the endospore and the cell’s DNA survive. When good conditions return, the endospore gives rise to a normal bacterial cell. (pg469)

Endospores seen in gram positive cells

The endospore is the reason that boiling water does not always kill all bacteria. An autoclave is used to sterilize surgical & dental tools which is a device that uses high temperature steam under pressure.

• pilus (pili = plural) – assists the cell in attaching to other surfaces, which is important to recombination (serves as a conjugation bridge to transfer plasmids from one cell to another).

• flagellum – moves the cell – long whip-like extensions. They are made of protein and turn like a propeller. There can be one, a tuft, at one end, at both ends, or completely surrounding the cell.

The “motor” of a bacterial flagellum.

Bacterial Movement• 1) Flagella movement: They turn to propel the

bacteria in an erratic, “run and tumble” motion.

• 2) Some bacterial produce a layer of slime and glide in the slime by wavelike contractions.

• 3) Some spiral shaped bacteria move with a corkscrew- like rotation.

• 4) Other bacteria have flexible cell walls and cell wall filaments that contact to cause the bacteria to turn and move ahead.