1. Lab #2 Bacteriology & the Archaea

2. 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 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 µm 2 µm Subgroup: Epsilon Proteobacteria Heliocobacter pylori 5 µm Bdellovibrio bacteriophorus

3. 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

4. Bacterial Groups 1. Proteobacteria: – b. beta: nutritionally diverse Nitrosomonas – soil bacteria that plays a role in N2 recycling by oxidizing ammonium (NH 4 ) into nitrites (NO 2 - )

5. 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

6. 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 H 2 S  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

7. 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

8. 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 BACTERIA 5 µm Streptomyces Mycoplasmas covering a human fibroblast cell

9. 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)

10. 5. Cyanobacteria – photoautotrophs – only prokaryotes with plant-like, oxygen-generating photosynthesis – abundant components of fresh water and marine phytoplankton Bacterial Groups CYANOBACTERIA Oscillatoria 50 µm

11. 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

12. 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

13. 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

14. 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