Prokaryotic Structure o All prokaryotes are unicellular, lack a nucleus and membranebound

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1 Microbiology is a subdivision of cellular biology that focuses on microorganisms and their effect on other living things. Some microbes are pathogenic, but most are harmless. Microorganisms occur in a variety of shapes and sizes. Microbes can be divided into groups bacteria, archaebacteria, viruses, fungi, protist. Archaebacteria live in extreme environments including places too acidic, too hot, too cold or too salty for most organisms. These bacteria are more stable in extreme conditions and can live in many of the most hostile environments on Earth. These extremophiles have cell walls that differ in structure from other bacteria lack peptidoglycan. Bacteria are found in every habitat on Earth some live in or on other organisms. Some bacteria live in the soil or on dead plant matter where they play an important role in cycling of nutrients. Relatively few in this group are pathogenic. These bacteria have cell walls that contain peptidoglycan. Prokaryotic Structure o All prokaryotes are unicellular, lack a nucleus and membranebound organelles. o Capsule sticky protective layer that helps evade immune system and adhere to substances o Plasmids - accessory rings of DNA; can be used as vectors to carry foreign DNA into bacteria during genetic engineering procedures o Endospore thick internal wall that encloses the DNA; formed so bacteria can remain dormant during harsh environmental conditions. o Pili allow bacteria to stick to surfaces o Some bacteria have specialized membranes to help carry out metabolic functions respiratory & thylakoid membranes. o Typical prokaryotic genome is a ring of DNA in the nucleoid region in the cytoplasm no nuclear membrane. o Prokaryotic cells have a variety of shapes -- cocci (spherical), bacillus (rod-shaped) and spiral. o Prokaryotic cells are arranged in certain ways when they grow staphylo (clusters), strepto (chains) and diplo (pairs). o Most motile bacteria have flagella to propel themselves.

2 Metabolic Adaptations Major nutritional roles - Heterotrophic (use endocytosis/exocytosis) OR Autotrophic (photosynthesis use light energy or chemosynthesis use inorganic compounds) O 2 requirements obligate anaerobe (can t survive in O 2 ) OR obligate aerobes (must have O 2 to survive) OR facultative anaerobe (live with or without O 2 ) Gram Stain lab test Bacterial cell walls are made of a peptidoglycan layer. It is composed of alternating sugars: N- acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). Scientist can classify many bacterial species into two groups based on cell wall composition. Gram positive (+) bacteria have a cell wall with large amount of peptidoglycan that traps the violet dye in the cytoplasm. The alcohol rinse does not remove the violet dye, which masks the added red dye. (Turns purple, more peptidoglycan) Gram negative ( ) bacteria have less peptidoglycan and is located in a layer between the plasma membrane and the outer membrane. The violet dye is easily rinsed from the cytoplasm, and the cell appears pink or red after the red dye is added. (Turns pink, less peptidoglycan) Reproduction o Bacteria reproduce quickly by binary fission a single cell divides into two identical daughter cells. Binary fission begins when the bacterial DNA replicates. The bacterial cell elongates and splits into two daughter cells with identical DNA to the parent cell asexual reproduction. Bacterial Uses Food production cheese, yogurt, etc. Protein production recombinant protein production to produce hormones, etc. Decomposers breaking down dead matter and recycling nutrients Treatment Penicillin-based antibiotics prevent bacterial growth by disrupting the cell wall formation. They do this by inhibiting protein cross-linking between these sugar chains. Some antibiotics kill by inhibiting protein synthesis. Bacterial Life Cycle 1. Lag Phase: Bacteria do not grow they adjust to their environment and metabolize. Bacteria begin making copies of DNA. This phase is short if the environment is nutrient rich. 2. Exponential Phase: Bacteria reproduce rapidly by binary fission the fastest bacteria can double in about 20 minutes. 3. Stationary Phase: Bacteria growth slows due to accumulating waste and lack of space. If bacteria are moved to another culture rapid growth resumes. 4. Death Phase: Bacteria lose all ability to reproduce.

3 o Viruses are nonliving and do NOT have cellular structure. Viruses are composed of proteins and nucleic acid molecules that become active once they are within a suitable host cell. o Virus size ranges from 25 to 250 nm. Viruses are classified into main categories based on the type of cell they attack bacterial (bacteriophages), plant and animal. o Viruses have a thick protein coat (capsid) surrounding a nucleic acid core of either DNA or RNA. Viral nucleic acid contains genes for making new viruses. Capsid contains special sites on protein surface that allow the virus to attach and penetrate the host cells membrane. o Nonpathogenic viruses or virus particles are often used in biotechnology research as vectors to carry DNA between cells. Viral Replication Within a host cell, the viral nucleic acid is released the viral genes are read by the host cell s enzymes, decoded into viral mrna and translated into viral proteins. New viruses assemble and release from the host cells to infect other cells these viruses are lytic viruses. Some viruses incorporate their DNA into the host chromosomes when released in the host cell these viruses are lysogenic viruses. o Fungi are mostly multicellular, heterotrophic, contain a nucleus and have cell walls composed of chitin. o Hyphae secrete enzymes into the growth medium and absorb the soluble products of external digestion. o Fungi are the major decomposers of organic matter. The diverse metabolic pathways of fungi generate many commercial products such as ethanol, organic acids, enzymes, antibiotics, etc. o Fungi typically reproduce using spores reproductive cells. Spores are released and develop into a thin filament called hyphae. The hyphae grow in masses called mycelium. o Fungi can produce spores sexually less common. Two mating cells from hyphae of different strains of fungi can fuse and form a spore stalk. Spore caps at the end of the stalk fully mature and burst spores are released. Model organisms used in biotechnology research -- Mammalian models: mouse, rat Non-mammalian models: Arabidopis (plant), C. elegans (roundworm), D. melanogaster (fruit fly), E.coli (bacteria), S. cerevisiae (budding yeast), S. pombe (fission yeast)

4 Bacterial Cell Cultures To grow bacteria, scientist must provide an environment or medium that the cells like. Some bacteria grow well in a liquid medium (broth) and some prefer a solid medium (agar). Agar medium is a mixture of water and protein molecules. To prep agar: 1. Mix powdered agar in water and heat until the agar is completely suspended. The agar is sterilized at high temperatures (121 o C or higher) for a minimum of 15 minutes. 2. The agar is allowed to cool to about 65 o C and is poured under sterile conditions into sterile Petri dishes. The agar cools and solidified within 15 minutes. 3. The poured plates may be used after 24 hours. Liquid or broth (water and protein molecules) cultures grow as suspensions of millions of floating cells. Under sterile conditions, a colony of cells is introduced to the sterile broth. The cells grow, divide and spread throughout the liquid. Broth cell cultures reproduce quickly since they have better access to nutrients than colonies growing on solid media oxygen and food diffuse into these cells easily. Broth culture cells may replicate every 20 minutes. Temperature, ph, oxygen and nutrients call all influence cell growth in a lab and all cells have optimal culture conditions that promote the desired growth. Some media contains additives, such as antibiotics, that allow only certain cells to grow in the presence of such additives. Often genetically engineered bacteria contain enzymes that allow them to live if they have been engineered correctly. Other additives create changes in bacteria that allow identification. Important part of all media preparation is sterilizing the medium. The medium must be free of any unwanted bacteria or fungi before it is used. An autoclave or pressure cooker/sterilizer is used to heat the medium to over 121 o C for a minimum of 15 minutes to destroy any cells or spores. If the medium is to be used in other flasks or plates, it must be transferred under sterile conditions to the new sterile vessel. Mammalian Cell Culture Growing mammalian cell culture is more difficult than growing bacterial cells this is because mammalian cells grow within a multicellular organism. Mammalian cells depend on other cells for several products and stimuli. Need to provide an environment that is an adequate substitute for the normal environment. On a small scale, mammalian cells are typically grown in broth culture in special tubes and bottles with a bottom surface to which the cells can stick. In production facilities, large-scale mammalian cell cultures are grown in suspension broth cultures in fermenters. The media are specially designed to have all the special nutrients that each cell type may require. Special indicators may be added to monitor the culture. Example: Phenol red changes from red to gold as the solution becomes more acidic from cell overcrowding. Eukaryotic Cell Cultures Primary cells are taken directly from animals and are used to determine how normal cells function. They last only a short amount of time. Cell Lines Cells that have changed in culture so that they grow indefinitely or are immortal. Most eukaryotic cells need to adhere to a surface in order to grow and they stop growing when they touch each other (contact inhibition). Some cells have lost the requirement for adhesion and can grow in suspension suspension cultures.

5 Review: Review the information about the ingredients required by E. coli bacteria cells verses human cells in culture. Explain why mammalian cells, such as human cells, have so many more required ingredients in their growth media. Proteins Produced and Their Host Cells Protein Used in the treatment of Cell Production Insulin Diabetes E. coli Human growth hormone Growth disorders E. coli Erythropoietin Anemia CHO cells Tissue plasminogen activator Heart attack CHO cells Hepatitis B virus vaccine Vaccination Yeast Human papillomavirus vaccine Vaccination Yeast Microbiological Tools Inoculating loops and needles used to transfer liquid cultures, stab deep tubes or move a colony from one petri plate to another. Aseptic Techniques Sterile technique is the process of doing something without contamination by unwanted microorganisms or their spores prevents the introduction of unwanted organisms. Key Practices: Wash hands. Wipe down work surfaces with 70% alcohol or antimicrobial cleanser. Change gloves frequently. Air is a source of contamination so how containers are opened should be noted. Do not set lids on a surface that is not sterile. Labeling Petri Dishes Label the side that contains the agar. Place at outermost edge. Note type of agar, who made it, date poured. Once inoculated, write microorganism name and date of inoculation.

6 Streak Plate The lid of the agar plate has to be opened just sufficiently enough to streak the plate with the inoculation loop. Minimize the amount of agar and the length of time the agar is exposed to the environment during the streak process. Drag the loop across the agar in a zig-zag pattern streak lightly, do not gouge the agar. Serial Dilution and Plate Count Diluting an unknown concentration of cells by a known dilution factor which is often a series of tenfold dilutions Cells are plated onto agar media and grown to determine how many cells or colony forming units (CFU) are present. Calculations are made to determine the number of CFU in the undiluted culture Review: A biotech student performs a tenfold serial dilution of a bacterial culture six times to achieve a 1 million-fold dilution, and if 1 ml of that final dilution yielded 45 colony forming units (CFU) when plated, what is the concentration of bacteria in the original culture of CFU/mL? MICROBIOLOGY In Biotechnology Standards: HS-EB-6: Compare and contrast common organisms used in biotechnology and relate the manipulation of living organisms to product and procedure development. 6.2 Distinguish between prokaryotic cells, eukaryotic cells, and non-living entities, such as viruses. 6.3 Describe the characteristics and life cycles of model organisms used in biotechnology, including bacteria (e.g., E. coli and insulin), fungi (e.g., yeasts and Aspergillus), and animals (e.g., C. elegans, fruit flies, and rodents). 6.4 Monitor how environmental factors affect the growth of cells and model organisms in the laboratory. 6.5 Apply the basic concepts of cell growth to manipulate cultures under aseptic conditions in the laboratory and demonstrate proficiency in gram staining, streaking culture plates and Stock bacterial cultures. 6.6 Identify bacteria using morphology and metabolic analysis.