Lab #2

Lab #2

Bacteriology & the Archaea

Bacterial Groups

• 1. Proteobacteria: diverse group of gram negative bacteria – 5 well-established groups

• alpha• beta• gamma• delta• epsilon

– newer groups being discovered today – e.g. zeta

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

Bacterial Groups

• 1. Proteobacteria: – a. alpha: live in close association 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

root nodules

Bacterial Groups

• 1. Proteobacteria: – b. beta: nutritionally diverse

• Nitrosomonas – soil bacteria that plays a role in N2 recycling by oxidizing ammonium (NH4) into nitrites (NO2

-)

Bacterial Groups

• 1. Proteobacteria: – c. delta: slime secreting myxobacteria

• when the soil dries out – they form into aggregations called fruiting bodies & release spores into the environment for the establishment of new colonies in better environments

• Bdellovibrio bacteria “charge” at other bacteria at speeds equivalent to 240km/hr

• drills into its prey using its flagella and digestive enzymes

• 1. Proteobacteria: – d. gamma: includes many well-known

strains of gram-negative bacteria• autotrophic & heterotrophic species• include the older classification known as

purple-sulfur bacteria (e.g. Thiomargarita namibiensis)– 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

Bacterial Groups

Vibrio cholerae

• 1. Proteobacteria: – e. epsilon: many are pathenogenic to humans and other animals

• includes Campylobacter = blood poisoning• Helicobacter pylori = stomach ulcers

– f. zeta: relatively new classification

Bacterial Groups

Helicobacter pylori

• 2. Gram positive bacteria: rival proteobacteria in diversity– 2 major subgroups based on cell shape: cocci and bacillus– most decompose organic matter in soil– two strains of Actinomycetes cause leprosy and tuberculosis – Streptomyces used by pharmaceutical companies to produce antibiotics– numerous strains are very pathogenic: Bacillus anthracis, Clostridium

botulinum, Staphylococcus and Streptococcus

Bacterial Groups

GRAM-POSITIVE BACTERIA5

µm

Streptomyces Mycoplasmas covering a human fibroblast cell

• 3. Chlamydias– can only survive in animal cells – depend on their

hosts for ATP– Chlamydia trachomatis – cause of non-

gonococcal urethritis (most common STD)

• 4. Spirochetes– move through rotation provided by internal

flagella-like filaments– Treponema pallidum – causes syphilis– Borrelia burgdorferi – causes Lyme disease– Leptospira – causes leptospirosis (field fever)

Bacterial Groups

5 µm

SPIROCHETES

Leptospira

2.5

µm

CHLAMYDIAS

Chlamydia (arrows)

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

photosynthesis– abundant components of fresh water and marine

phytoplankton

Bacterial Groups

CYANOBACTERIA

Oscillatoria50

µm

Bacterial classification

– colony morphology• bacterial colonies grow from single cells• colony is composed of millions of bacteria• each colony has a characteristic size, shape, 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 or spiral• many bacterial 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 dehydration and 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

Gram Staining

• 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. Decolorize by washing the slide briefly with acetone (2-3 seconds) or 95% ethanol• 7. Wash slide thoroughly with water to remove the acetone/ethanol• 8. Flood slide with safranin counterstain for 2 min. • 9. Wash with water. • 10. Blot excess water and dry

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