User:Portia P. Ofori/Notebook/Biology 210 at AU

1/28/16

Header: Identification and characterization of bacteria.

Purpose: Identify species of bacteria based on motility, gram stain, colony morphology and sequencing of 16s ribosomal subunit gene.

Taking one last look at my hay infusion, I observed that the level of water has reduced, its color changed to brownish and had a pungent smell. Hypothesis: The hay infusion will become cloudier and have a more pungent smell from week to week because bacteria in the hay infusion is going to decompose. Archaea that can grow in moderate or room temperatures might grow on the agar plates but the Archaea organisms that thrive in extremely high or low temperatures might not grow.

Table 1: 100 fold serial dilutions results

Dilution	Agar type	# Colonies on plate	Conversion factor	Colonies/ml
10-3	nutrient	1040.00	x 103	1040000
10-5	nutrient	60	x 105	6000000
10-7	nutrient	2	x 107	20000000
10-9	nutrient	3	x 109	3000000000
10-3	Nutrient + Tetracycline	121	x 103	121000
10-5	Nutrient + Tetracycline	1	x 105	100000
10-7	Nutrient + Tetracycline	1	x 107	10000000
10-9	Nutrient + Tetracycline	0	x 109	0

Header: Quantifying and observing Microorganisms

Purpose: Observe and quantify organism in serial dilution grown on nutrient agar and nutrient agar with tetracycline.

Generally, Nutrient Agar plates grew more colonies compared to nutrient agar with tetracycline. Addition of tetracycline makes the agar selective with the kind of bacteria that can grow on the agar. Certain organisms will be inhibited from growing. The bacteria that can grow in the presence of tetracycline indicates the organism’s resistance to the effect of antibiotic but those that could not grow are susceptible or sensitive to the antibiotic.

Conclusion: Tetracycline reduces the number of bacteria that grows on the agar plate. It is important to also note that growth rate decreased with increase serial dilution.

Header: Tetracycline’s mechanism of action.

Purpose: Determine the mechanism of action for tetracycline and the types of bacteria that are sensitive to it.

Tetracycline’s mechanism of action is through penetration of bacterial cells by passive diffusion and the inhibition of bacterial growth by interference with protein synthesis or by membrane destruction (Schnappinger and Hillen). As indicated by Chopra and Roberts, tetracycline inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. Tetracycline is used as a broad spectrum antibiotic for a wide variety of gram positive and gram negative bacteria. Organisms such as mycoplasmas, chlamydia, rickettsia and protozoan parasites are sensitive to it.

Works cited.

Schnappinger D, Hillen W. June 1996. Tetracyclines: antibiotic action, uptake, and resistance mechanisms. Archives of Microbiology, Volume 165, Issue 6, pp 359-369.

Chopra I and Roberts M. June 2001. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. The American society for microbiology. Microbiology and molecular biology reviews. Volume 65, Issue 2, pp 232-260.

Header: Bacteria characterization

Purpose: prepare a wet mount, stain and observe under a microscope to characterize the bacteria according to color, shape texture and gram stain reaction.

Materials: loop, microscope slide, coverslip, Kim wipe, staining tray.

Reagents needed: crystal violet stain, grams iodine, 95% alcohol, safranin stain, water.

Wet mount Procedure:

Sterilize a loop over a flame until it is bright red and let it cool before scrapping a tiny portion of growth from the surface of the agar.

Mix with a drop of water and cover with a cover slip.

Observe the wet mount using 10X and then 40X objective lens.

Gram stain procedure: Sterilize a loop over a flame and make a wet mount without covering with a cover slip.

Label the slide and heat fix after it has air dried.

Place the prepared slides on the staining tray and cover the smear with crystal violet for 1 minute and rinse with water

Cover the smear with grams iodine mordant for 1 minute and rinse with water.

Flood the smear with 95% alcohol for 10-20 seconds to decolorize and rinse gently with water.

Counter stain the smear with safranin stain for 20-30 seconds and rinse with water.

Blot excess water with Kim wipes, air dry and observe under the microscope.

Table 2: Characterization of Bacteria.

Colony Label	Plate atype	Colony Description	Cell Description	Gram + or Gram -
10-3	Nutrient	0.1-0.3 (cm)in diameter,cream in color	cocci and rods	gram -
10-3	Nutrient + Tetracycline	0.5-1.2(cm)in diameter,yellow,raised and glistening surface	cocci	gram +
10-5	Nutrient	1(cm), pale yellow and round colonies	Rod shaped	gram -
10-5	Nutrient + Tetracycline	0.6(cm) pale yeloow and smooth surface	Rods and coocci	gram -

Organisms observed after Gram stain.

nutrient agar 10^-3 Nutrient agar plus tetracyclin 10^-3 nutrient agar 10^-5

nutrient agar plus tetracycline 10^-5

Pictures of colony growth of bacteria.

Nutrient agar 10^-3 Nutrient agar plus tetracycline 10^-3 Nutrient agar 10^-5

Nutrient agar plus tetracycline 10^-5

1/21/16

Header: Hay Infusion culture observations.

Purpose: To observe the invisible world of unicellular eukaryotic organisms under a microscope and use the dichotomous key to identify protest and algae.

Hypothesis: Organisms in the various niches of the hay infusion will exhibit slight differences between them.

Materials: Microscope slides, cover slips, microscope, plastic pipette.

Method:

-Obtain three clean microscope slide and put a drop of the specimen from each niche.

-Cover with a cover slip and observe with a microscope at 4X and 10X.

-Use the 40X for resolution. Record the sizes using ocular micrometer

-Obtain a dichotomous key and use it to determine organism present.

Observations: The hay infusion culture appeared cloudy with a pungent smell. The hay infusion had changed color to light brown with three distinct layers. Molds were present on top of the liquid. There were no green shoots present. Fine granular-like sediments were seen at the bottom layer.

Organisms differ close to verses away from plant matter because they have different carrying capacities (the maximum number of individuals of species that can survive in a particular niche)

Using the dichotomous key to identify 3 different organisms from three niches in the hay infusion.

Top niche:

Paranema: Cell elongated, colorless with a broad or truncate posterior during locomotion. Highly plastic when stationary, often appears to vibrate when in motion. Amoeba: Small, creeps using pseudopodia and has a single disc shaped nucleus. Actinosphaerium: Colorless organism sliding slowing without apparent motion, spherical shaped with radiating spines.

Middle niche:

Colpidium: Small body, oval shaped and covered in cilia. Fast swimmer. Paranema: Cell elongated, colorless with a broad or truncate posterior during locomotion. Highly plastic when stationary, often appears to vibrate when in motion. Pelomyxa: large, creeps using pseudopodia, many (100s) of small nuclei.

Bottom niche:

Chilomonas (algae genus): Colorless, cells organ of locomotion is a long whip-like flagella (no cilia), single motile cell, 2 observed locomotor flagella at one end, cell elongated with narrowed posterior. The cell measures 20 ocular spaces using 40X objective lens. Paranema (protozoa): Cell elongated, colorless with a broad or truncate posterior during locomotion. Highly plastic when stationary, often appears to vibrate when in motion. Chlamydomonas (Algae): Cell is greenish, a single motile cell, two observed locomotor with an oval shaped body. The green color suggest photosynthetic activity.it measures around 10μm.

Description of how Chlamydomonas meets all the needs of life as described on page 2 of the freeman text. Chlamydomonas has photosynthetic activity therefore absorbs sunlight as its source of energy. It has a cell membrane that regulates the passage of materials in and out of the cell. Chlamydomonas has a flagella which aids in movement. It can undergo both sexual and asexual reproduction.

If the hay infusion grew for another two months my prediction will be a reduction in the number of organisms present. Struggle for resources through which organisms survive and pass on genes to the next generation will be very evident. One selective pressure that will affect the community of my sample will be limitation of nutritional resources leading to competition for food and energy among microorganisms. Therefore if certain organisms have a lower chance of survival, they cannot stay alive to reproduce.

Diagrams of organisms observed in the different niches.

Header: Serial Dilutions.

Purpose: To prepare and plate serial dilutions of organisms in the hay infusion nutrient agar and enriched agar.

Materials: Four test tubes containing 10ml of sterile broth, micropipette set at 100μl, micropipette tips, four nutrient agar petri dishes, four nutrient agar with tetracycline petri dishes, test tube rack, inoculation spreader, Bunsen burner.

Method:

-Swirl hay infusion to mix up organisms.

-Aliquot 100μl of sample from hay infusion and add to 10ml of sterile broth (10-2 dilution)

-Swirl inoculated tube to mix and add 100μl from 10-2 dilution to 10-4 tube.

-Repeat above step to make the 10-6 and 10-8 serial dilutions.

-Pipette 100μl and inoculate on both nutrient and nutrient agar with tetracycline for each of the serial dilutions

-Place agar plate in a rack with the agar side facing up.

-Incubate at room temperature for one week.

Diagram of serial dilution procedure

1/14/16 Observation of a 20/20 transect

Location: The transect is located in an area behind the AU library close to the student health center. It is a 20 by 20 foot dimension marked with four Popsicle sticks with a pond at the center.

Topography: The transect had Cornus florida commonly called flowering dogwood, Parrotia persica treesome trees and some budding flowers as well. The soil was moist and covered mostly with fallen leaves. The sides of the pond was riprapped with rocks to protect it from wind, wave action and erosion. There were grass grown around the pond probably to prevent erosion.

Biotic Components: 1. Pond 2. Two big trees without leaves 3. Three short trees with green leaves and budding flowers 4. Mushrooms 5. insects

Abiotic component of transect: 1. Bamboo grid used to cover the pond 2. Net used to cover the pond. 3. Classroom block. 4. Lights form the classroom wall 5. Two lovers seats 6. Two statues ( Duck statue in the pond and a woman with three birds at her feet on land) 7. Irrigation control valve just above the pond. 8. Deflated balloon on land 9. Paper on land 10. Wiring that goes into the pond 11. Large and small rocks outlining the pond

Characteristics	Chlamydomonas	Gonium	Volvox
Number of cells	1	4	1(containing many smaller cells)
Colony size	4os / 10μm	8os /22μm	22os / 220μm
Specialization of cells	Unicellular, egg-shaped, cup-shaped chloroplast	Gelatinous matrix	Multicellular, presence of spiked cells
Mechanism of motility	Flagella	Flagella	Flagella
Isogamous or Oogamous	Isogamous	Isogamous	Oogamous

Pictures from the transect.

Aerial diagram of the transect