Microbiology

Bacteria

• -the most numerous and most ancient organisms on Earth

in 1998, estimated at 5 million trillion http://www.ehow.com/about_4674401_how-many-bacteria-live-earth.html

• -unicellular prokaryotes

phschool.com

Classification

• -2 Kingdoms-based on ribosomal RNA differences

• -Eubacteria : common germs ex: E. coli, Salmonella

• -Archaebacteria: ancient bacteria that live in extreme conditions ex: Thermus aquaticus

Archaebacteria

• differences from eubacteria:– introns in DNA– no carbohydrate in cell wall– extra lipids in cell membranes,– archaebacteria genes are more similar to

eukaryotic genes than eubacteria,– non-pathogenic

Types of archaea• 1. Methanogens

-harvest energy by converting H2 and CO2 into methane gas (CH4)

-O2 poisons them only live in anaerobic conditions such as in sewage, swamps, intestines

microimm.queensu.ca

• 2. Extreme halophiles-use high levels of salt to make ATP-found in Great Salt Lake, Dead Sea

• 3. Thermoacidophiles-live in extremely acidic, hot environments pH

<2, temps up to 230 F- found in volcanic and hydrothermal vents

(p. 469)

Salt flats at Lake Magadi, Kenya. The flats are red due to the proliferation of halobacteria

http://www.daviddarling.info/encyclopedia/H/halophile.html

Making cultures

-Liquefy nutrient agar and pour into sterile petri dishes while warm-Store in refrigerator until ready ~ 1 hour before use-use a sterile cotton swab or inoculating needle and swab your sample-Very gently rub the swab over the agar in a few zigzag strokes and replace the lid on the dish & tape it shut-incubate for 3-7 days around 37 C.

Eubacteria• most bacteria, some are pathogenic• 3 basic shapes bacilli (rods), cocci (spheres), spirilla

(spiral) • types of eubacteria• Cyanobacteria, Spirochetes, Gram +, Proteobacteria• posters, p. 472-3

Gram Stain

• way of classifying bacteria, based on retention of purple stain due to a thick layer of peptidoglycan carbohydrate in their cell wall in gram +

• React to different antibiotics & disinfectants• often the 1st test done on pathogens

• -Gram positive withstands dessication better (so can live in non-moist enviro. like skin) , generally killed by penicillin

• -Gram negative killed by drying, thrive in moist enviro, can withstand chemicals like chlorine, more resistant to antibiotics

How to Gram stain

earlham.edu

1. spread some bacteria on a slide2. fire it by passing the slide through a

flame briefly3. flood the slide with crystal violet

solution for 1 minute 4. add iodine solution for 3 minutes - at

this stage all cells are purple 5. Add alcohol for 20 seconds 6. Stain with safranin

This results in gram positive cells remaining purple and gram negative ones being red or pink.

Bacterial structure• (almost) All have– Cell wall, plasma membrane, cytoplasm, circular

chromosome, ribosomes

• Some have – plasmids– capsules– pili– endospores– flagella

Find & put into a ppt slide

• What they are made of• Their function• How they work• pictures

• Some have – Plasmids: extra, self-replicating DNA

-not crucial for cell survival, can carry genes that help in certain conditions (such as ab resistance)

-can be transferred from bacterium bacterium– capsules: protective outer coverings, protect from

white blood cells– pili: short, hairlike proteins to help w/adhesion– endospores: have protective covering to resist extreme

conditions (like boiling & freezing)• Released from cell, can remain dormant for thousands of

years

– Flagella: whiplike tails for movement

-Cell wall function: provides shape for cell, protects against outside environment, prevents cell from rupturing when there is high water pressure in the cell, anchorage for flagella-made of peptidoglycan

-Gram + have many layers, gram – few-cell membranes of gram- provide a barrier

against antibiotics

Pic of cell wall

Pics of flagella, pili, plasmids

leisureguy.com ict-science-to-society.org

cosmology.net

Bacterial Cell Wall

• It is made of: peptidoglycan and teichoic acid• Their function: it maintains the overall shape

of the bacteria cell. Its an anchor for the phili and flagella. It contains the organelles and helps keep the cell wall from bursting.

• Antibiotics that target the cell wall can get rid of the bacteria.

Made up of proteins, lipids, and polysaccharides

(1) Acts as a barrier that allows nutrients in and harmful substances out(2) used to attach or adhere to specific surfaces or tissues(3) Enzymes take care of surface reactions important to cell survival(4) protective structures against host defenses(5) antigenic disguises to bypass activation of host immune defenses; (6) Endotoxins that cause an inflammation in the host(7) proteins that can respond to temperature, salinity, light, oxygen, nutrients

Most functions are taken care of by the hydrophobic layer of lipids that allow things in or out of the cell and the polysaccharides on the outer layer

Bacteria Membrane

Pili-Connect a bacterium to another of its species, or to another bacterium of a different species, and build a bridge between the interior of the cells.-Enables the transfer of plasmids between the bacteria.-They are antigenic (any substance that can stimulate the production of antibodies and combine specifically with them. ) [dictionary.com]-Fragile and consistently replaced-Hair-like structure, often many on one bacteria-Shorter than flagella, very thin-made up of a long chain protein or polysaccharide

[pee-lee]

http://en.wikipedia.org/wiki/Pilus#Pili http://www.daviddarling.info/encyclopedia/P/pilus.html

ENDOSPORES

•Endospores consist of the bacterium's dna and part of its’ cytoplasm.•These endospores are released to protect the bacteria.•When the bacteria detects environmental hazards it releases an endospore for safety. •Endospores are resistant to ultra violet light, dessication, high temperature, and many other things.

Plasmids• Are circular self-replicating organelles in the cell, of bacteria, that hold a small amount genetic information.

•Organisms usually have the same amount as the generation before them

•They are the foundation of recombinant DNA

•Bacteria are able to exchange information with one another using these plasmids causing mutations.

•They are separate from the chromosomal DNA; they only hold a few genes. http://askabiologist.asu.edu/plasmids

Made of three main parts:A cloning site or place of insertion

of DNAAn origin of replicationA selectable marker gene

http://escience.ws/b572/L2/L2.htm

(ORI)- origin of replication

Flagella•Prokaryotes: protein flagellin•Allow bacteria to move •Flagella have hooks that can turn and propel the organism•When the flagella rotates clockwise, the bacteria can fall or tumble

Metabolism

• the sum of all chemical reactions in an organism

• Weight of a bacterium= 50% carbon, 14% nitrogen, 4% sulfur & phosphorus

• heterotrophic microbes use organic substances ,esp. glucose to make energy

Metabolism• http://player.discoveryeducation.com/index.cfm?guidAssetId=E7965

B93-1D6D-4740-8C55-D6E80EDB4C49&blnFromSearch=1&productcode=US#

• All go through glycolysis– Availability of oxygen determines next step

• Aerobes cellular respiration = higher ATP yield due to Krebs cycle & ETC, aerobic– oxygen as final e- acceptor, lots of ATP= fast growth

• Anaerobicfermentation= little ATP produced, creates alcohol and lactic acid, anaerobic

-final e- acceptor not oxygen, often nitrate, little ATP= slower growth

• table

• Autotrophs make organic compounds from CO2 or other inorganic molecules– Chemotrophs get energy from electron donors

in their environments (chemicals)-glucose, acetate, etc. or inorganic chemicals H2, H2S, Fe, NH4, etc.

. Phototrophs- convert solar energy into ATP

Measuring Microbes

• Serial dilution• a set of dilutions with several steps in which

each step has the same dilution factor- usually 10

• each time, the sample get 10 times less diluted as the previous one

• http://www.professorcrista.com/bio_205_animations.htm

How to do a serial dilution

• 1. set out a series of tubes with 9mL of buffer water (suitable for bacteria)

• 2. Draw up 1mL from the original sample and put it in the first 9mL buffer. This is a 1 to 10 dilution which makes the sample exactly 10 times less concentrated as the sample.

• 3. Then draw up 1mL from the first dilution tube and put it in the second tube. This is another 1 to 10 dilution which makes the sample exactly 10 times less concentrated as the first tube and 100 times less concentrated as the original sample.

• 4. Draw up 1mL from the 2nd tube and put it in the 3rd tube.This will be a 1: ____________dilution and so on…

Streak plate method• 1. Get a loopful of bacteria from a sample• 2. Streak the top 1/3 of the agar plate horizontally• 3. Sterilize loop• 4. Get some bacteria from 1st streak in loop• 5. Streak vertically ~1/3 of plate on right side• 6. Sterilize loop• 7. Get some bacteria from 2nd streak in loop• 8. Streak the bottom 1/3 of the plate horizontally– This should allow you to isolate colonies of bacteria

psu.edu

Reproduction

• Binary fission• http://

www.textbookofbacteriology.net/growth.html

Counting bacteria

• http://www.microbiologybytes.com/LabWork/bact/bact1.htm

• Viable count= # bacteria or clumps of bacteria per cm3

Evolution

• Transformation-use plasmids to transfer DNA to other bacteria or other organisms

lab

Bacteria & humans

• Useful bacteria– Agriculture (n2 fixation), food (cheese, wine),

biofuels

ice nucleating lab• Disease

• Antibiotics

• A.b. lab

Viruses