USING BACTERIA MORPHOLOGY CHARACTERISTICS TO STUDY BACTERIAL DIVERSITY. 092404 EMJT By Ellie Masserrat Pian Lu John Taucher Bacteria…? Leptospira, causes serious disease in livestock Bacteria are often viewed as the cause of diseases in humans and animals. Some bacteria are useful, for example certain bacteria aids in digestion. Bacteria make up the base of the food web in many environments. Bacteria are of such immense importance because of their extreme flexibility, capacity for rapid growth and reproduction, and great age. They can be photosynthetic, using light, or chemosynthetic, using inorganic chemicals as the source of energy, but most are heterotrophic, absorbing nutrients from the environment. Background Information Prokaryotes Prokaryotes represent two domains, bacteria and archaea. Archaea live in Earth’s extreme environments. Bacteria are the most abundant and diversified organisms on Earth. Bacterial Structure Biochemical processes that normally occur in a chloroplast or mitochondrian of eukaryotes will take place in the inner membrane of prokaryotes. Bacterial DNA is circular and arrayed in a region of the cell known as the nucleotide . Scattered within bacteria’s inner membrane are numerous small loops of DNA known as plasmids . Structure Some bacteria have flagella with a different microtubule structure than the flagella of eukaryotes.. Ribosomes are the structures in cells where proteins are assembled. Bacterial ribosomes have different sized ribosomal subunits than do eukaryotes. Bacteria Have One of Three Cellular Shapes Rods (bacilli) Coccoid-Shaped Spirilla Reproduction Prokaryotic cell division is binary fission. – Single DNA molecule that first replicates. – Attaches each copy to a different part of the cell membrane. – Cell begins to pull apart. – Following cytokinesis, there are then two cells of identical genetic composition. Now…On to our experiment... Purpose: Identify varieties of bacterial colonies and investigate bacterial species diversity, by isolating, culturing, and analyzing bacterial colonies, or species, that inhabit: Air Pond Water Raw Chicken Washed/Unwashed hands Keyboard Soil Sample Hypothesis: Knowing that bacteria can thrive in almost anywhere on our planet, we reason that all of the environments tested will grow bacterial species. We further hypothesize that the thumb print of the washed hand with the anti-bacterial soap, should house less species than any others tested, because the anti-bacterial soap should kill off all bacteria. Methods For chicken, soil, pond water, A and keyboard samples, streak . the plate using the streak plate method to isolate bacterial colonies. Leave agar plate open for air B. sample. For the unwashed hand gently press thumb against agar. Take washed hand and gently C. press thumb against agar. Wrap in Parafilm and incubate the cultures for about one week Figure 1. Streak Plate Method. (a) Streak the plate back and forth across top half of plate. (b) at 22 C. Rotate plate a quarter turn counter clockwise and Observe and Interpret Data streak top right quarter of plate. (c) Rotate plate a quarter turn counter clockwise and streak top right quarter of plate again. Results: Soil SIZE SHAPE MARGIN SURFACE COLOR 4 mm Irregular Lobate Wavy Yellow/white 3 mm Irregular Lobate Wrinkled Brown/yellow 5 mm Filamentous Filamentous Wrinkled Green/white 2 1 3 Results: Pond Water Size Shape Margin Surface Color # 1 2 mm round smooth Smooth grey 2 2 mm round lobate contoured beige 3 1 mm round Smooth Smooth clear 2. 1. 3. Results: Raw Chicken # Size Shape Margin Surface Color 1 2mm Irregular lobate contoured yellow/green 2 3mm irregular lobate wrinkled clear/white 3 1mm round Smooth Smooth Yellow/green 4 2mm Irregular wavy contoured brown 1. 3. 2. 4. Results: Air # Size Shape Margin Surface Color 1 5 mm Irregular Smooth Smooth Yellow/orange 2 5 mm Round smooth contoured Yellow/orange 3 1 mm Irregular Wavy contoured Yellow/white 4 3 mm Irregular Lobate wrinkled Yellow/brown 5 3mm Irregular Lobate smooth White 6 5 mm round Smooth Smooth White/yellow 7 8 mm irregular lobate contoured White/yellow 1 2 1 7 3 6 4 5 5 Results: Washed Hand 1. 2. Size Shape Margin Surface Color # 1 4 mm Irregular lobate Smooth yellow 2 1 mm filamentous filamentous smooth white Results: Unwashed Hand Size Shape Margin Surface Color # 1 2 mm Irregular lobate smooth yellow 2 2 mm round Smooth smooth yellow 3 1 mm round Smooth Smooth white 1 2 3 Results: Keyboard Size Shape Margin Surface Color # 1 4 mm Irregular lobate Smooth yellow 2 1 mm filamentous filamentous smooth white # Size Shape Margin Surface Color 1 2 mm Irregular lobate wrinkled yellow 2 1 mm round smooth smooth greenish 2. 1. Control An unopened agar nutrient plate, which ruled out agar contamination, had no bacteria species present. Species vs. Environments Number of Bacterial Species on Agar Plate 8 6 # of Species 4 2 0 Air Keyb Unw Was Pond Chic Soil Series1 7 2 2 3 3 4 3 Environments Conclusions/Observations The results supported our hypothesis since bacteria grew in all of our samples. The results did not support our hypothesis concerning the hand washed with anti-bacterial soap since it did not house less species than the other environments tested. We were surprised to learn that the air not only housed the most bacteria, but housed the most bacterial diversity of species as well. Further Investigations… Further studies can be conducted by using TEM microscopy, SEM microscopy, and gram staining, to specifically identify what type of bacterial species were present in each environment. Research can also be conducted to figure out as to why the unwashed hand contained more bacteria than the washed hand. Further research can be done to determine if any of the bacteria found in our samples are harmful to humans. Questions to Ponder… Do all bacteria grow at the same rate, and what factors in the environment contribute to determining their “carrying capacity?” What research can be done to determine whether bacterial species and fungus compete with each other for nutrients and space in selected environments? References Coccoid-shaped Bacterium (causes skin infections), Enterococcus faecium (SEM x33,370). This image is copyright Dennis Kunkel at www.davidkunkel.com, used with permission. Morgan, I.G. and Brown Carter, M. E., Investigating Biology: A Laboratory Manual for Biology. California: Benjamin/Cummings Publishing Co., Inc. 1993. Rod-Shaped Bacterium, hemorrhagic E. coli, strain 0157:H7 (division) (SEM x22,810). This image is copyright Dennis Kunkel at www.davidkunkel.com, used with permission. Spirilla- shaped Bacterium (SEM x33,370). This image is copyright Dennis Kunkel at www.davidkunkel.com , used with permission. Serratia marcescens Gram stain Negative. Motility Motile. Habitat Occurs naturally in soil and water as well as the intestine. Pathogenicity: Associated with urinary and respiratory tract infections, endocarditis, wound infections, and eye infections. Gram-positive and gram-negative bacteria Difference Between Gram-Negative and Gram-Positive Bacteria Gram-Negative Bacteria Gram-Positive Bacteria More complex cell wall. Simple cell wall. Thin peptidoglycan celll wall layer. Thick peptidoglycan celll wall layer. Outer lipopolysaccharide wall layer. No outer lipopolysaccharide wall layer. Retain safranin. Retain crystal violet/iodine. Appear pink/red. Appear blue/purple. Antibiotic Sensitivity Test Antibiotic Sensitivity Test P10 TE30 Ø10 E15 C30 Ø14 Ø20 K30 NA30 Ø14 Ø8 S10 NB30 Hypothesis: Kanamycin is one of the most sensitive antibiotics because infections treated include respiratory tract, urinary tract, skin, soft- tissue and abdominal infections. Prediction: The size of the zone of inhibition is the largest. Results: The size of the zone of inhibition is the 2nd largest Antibiotic Resistance • Some bacteria have developed resistance to antibiotics naturally. • Bacteria can become resistant to drugs in a number of ways. - Mutation. - Exchange genes with other bacteria. - Resistant traits spread to future generations quickly because of rapid reproducing. Mechanism Antibiotics kill or stop the growth of harmful bacteria. Limitations Reason unknown why S. marcescens is sensitive to certain medications. Further research needed. Future Work Develop new drugs to confront bacteria resistance.
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