Lab #2

Bacteriology & the Archaea

Bacterial Groups• 1. Proteobacteria: diverse group of gram

negative bacteria – a. alpha: live in close associated with eukaryotes

• Rhizobium – lives in nodules within the roots of legumes – convert atmospheric N2 into compounds that the plants can use (nitrogen fixation)

• some strains can cause tumors in plants – Agrobacterium – used to genetically modify plants

– b. beta: nutritionally diverse• Nitrosomas – soil bacteria that plays a role in N2

recycling by oxidizing NH4 into NO2-

– c. delta: slime secreting myxobacteria• when the soil dries out – they form into aggregations

called fruiting bodies – release spores into the environment

• establishment of new colonies in better environments• bdellovirbio bacteria “charge” at other bacteria at

speeds equivalent to 240km/hr• drills into its prey using its flagella and digestive enzymes

PROTEOBACTERIA

Subgroup: Alpha Proteobacteria

Rhizobium (arrows)

2.5

µm

Subgroup: Beta Proteobacteria

Nitrosomonas

1 µm

Subgroup: Gamma Proteobacteria

Chromatium

0.5

µm

Subgroup: Delta Proteobacteria

Chrondromyces crocatus

10 µ

m2

µm

Subgroup: Epsilon Proteobacteria

Heliocobacter pylori

5 µm

Bdellovibriobacteriophorus

• 1. Proteobacteria: diverse group of gram negative bacteria – d. gamma: autotrophic & hetertrophic species

• include the older classification known as sulfur bacteria (e.g. Thiomargarita namibiensis)

• these obtain energy by oxidizing H2S – producing sulfur as a waste

• many heterotrophic strains are pathenogenic (e.g. Legionella, Salmonella and Vibrio cholerae)

• non pathenogenic strain = E. coli

– e. epsilon: many are pathenogenic to humans and other animals• includes Campylobacter = blood poisoning• Helicobacter pylori = stomach ulcers

– f. zeta: relatively new classification

PROTEOBACTERIASubgroup: Alpha Proteobacteria

Rhizobium (arrows)

2.5

µm

Subgroup: Beta Proteobacteria

Nitrosomonas

1 µm

Subgroup: Gamma Proteobacteria

Chromatium

0.5

µm

Subgroup: Delta Proteobacteria

Chrondromyces crocatus

10 µ

m2

µm

Subgroup: Epsilon Proteobacteria

Heliocobacter pylori

5 µm

Bdellovibriobacteriophorus

• 2. Gram positive bacteria: rival proteobacteria in diversity– 5 major subgroups– two strains of Actinomycetes cause leprosy and tuberculosis –

most decompose organic matter in soil– Streptomyces used by pharmaceutical companies to produce

antibiotics– numerous strains are very pathogenic: Bacillus anthracis,

Clostridium botulinum, Staphylococcus and Streptococcus

• 3. Chlamydias– can only survive in animal cells – depend on their hosts for ATP– Chlamydia trachomatis – cause of nongonococcal urethritis (most

common STD)

• 4. Spirochetes– move through rotation provided by internal flagella-like filaments– Treponema pallidum – causes syphillis– Borrelia burgdorferi – causes Lyme disease

• 5. Cyanobacteria– photoautotrophs– only prokaryotes with plant-like, oxygen-generating

photosynthesis– abundant components of fresh water and marine phytoplankton

Bacterial Groups

CYANOBACTERIA

Oscillatoria

50 µ

m5

µm

SPIROCHETES

Leptospira

2.5

µm

CHLAMYDIAS

Chlamydia (arrows)

GRAM-POSITIVE BACTERIA

5 µm

Streptomyces Mycoplasmas covering a human fibroblast cell

Bacterial classification– colony morphology

• bacterial colonies grow from single cells• colony is composed of millions of bacteria• each colony has a characteristic size, sheep, consistency, texture and

color• common colony shapes:

– punctiform = each colony is less than 1mm– round– filamentous – often confused with fungus (which is more “fuzzy”)– irregular

Bacterial classification– cell morphology• bacilli (rod)• cocci (spherical)• spirilla• many cells adhere to each other and form

clusters or chains• under some environments – many different

species may associate with each other – creating a community called a biofilm• biofilms are usually found where nutrients are

plentiful– soils, water pipes, surface of your teeth

Gram staining

– both Gram-positive and Gram-negative bacteria take up the same amounts of crystal violet (CV) and iodine (I).

– CV-I complex is trapped inside the Gram-positive cell by the washing of the bacteria with 95% ethanol – results in the dehydration and reduced porosity of the thick cell wall – limits the loss of CV-I complex – PURPLE STAIN

– thin peptidoglycan layer of the gram negative bacteria does not impede extraction of the CV-I complex

– plus the outer membrane limits the amount of CV-I complex that can reach the PG layer – CLEAR STAIN

1. Place a slide with a bacterial smear on a staining rack. 2. STAIN the slide with crystal violet for 1-2 min. 3. Pour off the stain and rinse with water thoroughly.4. Flood slide with Gram's iodine for 1-2 min. 5. Pour off the iodine and rinse with water thoroughly.. 6. Decolourize by washing the slide briefly with acetone (2-3 seconds) – alternatively use 95% ethanol7. Wash slide thoroughly with water to remove the acetone8. Flood slide with safranin counterstain for 2 min. 9. Wash with water. 10. Blot excess water and dry by hand over bunsen flame.

http://www.youtube.com/watch?v=OQ6C-gj_UHM