Lab assignment 3

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Brooklyn College, CUNY *

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3004

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Biology

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Jan 9, 2024

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docx

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Lab 3: Assignment Practice Cell Enumeration 1. Do the direct and viable counts of a suspension agree? If not, why might this be? No. The direct and viable counts of a suspension do not agree because the direct count includes cells that are both alive and dead but the viable count accounts only for alive cells. 2. You dilute an original sample at 1:30. You then count the number of yeast in the 1:30 dilution. You count an average of 7 cells in the 1/25 mm2 sized boxes. What is the concentration, in cells/ml, of the original sample? 1/25 mm^2= 4x10^-6 mL Stock Concentration (cells/ml)= (Average # of cells/5 boxes)/(dilution * 4x10^-6 mL) = (7 cells)/(1/30 *4x10^-6 mL)= 5.25 x 10^7 cells/mL 3. You count the number of bacteria in 5 of the 1/400 mm2 small boxes of the central grid on the hemocytometer. Your results are 12 cells in box #1, 17 cells in box #2, 17 cells in box #3, 14 cells in box #4, and 16 cells in box #5. You are counting a 1:10 dilution of the original sample. What is the concentration of the original culture (in cells/ml)? 1/400 mm^2= 2.5 x 10^-7 mL Stock Concentration= (Ave # of cells/ 5 boxes)/(dilution* 2.5 x10^-8 mL) = ([12+17+17+14+16]/5)= 15.2 x 10 (bc we are counting 1:10 of the original sample)= 152 cells = (152 cells)/(2.5 x10^-7 mL) = 6.08 x 10^8 cells/mL 4. You count 47 cfu on a spread plate. The plate was prepared by spreading 0.2 ml of a 1:10,000 dilution of the original sample. What is was the concentration of the original culture (in cells/ml)? CFU= (Ave # of colonies)/(Vol in mL plated*dilution) CFU = 47 colonies/(0.2 mL* 1/10,000) CFU= 2.35 x 10^6 cells/mL 5. You have a culture of yeast that is at a concentration of 6.74 x 10^6 cells/ml. You dilute the sample 1:100, and then 1:100 again, and finally you dilute the sample an additional 1:3. You add 0.1 ml of the final dilution to a spread plate. Assuming that most of the cells in the original culture were living, how many CFUs do you expect to count on your spread plate the next day? I’d expect to count 22 cells on my spread plate the next day.

Serial dilution= (6.74*10^6)(1/100)(1/100)(⅓)= 225 cells/mL CFU= (2.22*10^2 cells/mL)(0.1mL)= 22 cells 6. What is the average size of a yeast cell? The average size of a yeast cell is 3-4 um. Yeast are fungi classified as Eukaryotes; these organisms typically fall between 5 and 50 um. Urban Microbiome 7. In your own words, briefly and succinctly list the steps the Urban Microbiome project and give a description of each step. The Urban Microbiome project has 8 main steps: 1. Experimental setup and sample collection. In this step, a sample of soil is acquired, and placed in a collection tube, GPS coordinates are noted, and the sample is stored in a cool and dry place. 2. Initial Viability of bacteria. A 1:10 and 1:100 dilutions are made. 100 ul of 1:100 dilution is plated on a Tryptic Soy Agar (TSA plate) and stored for the next lab session. 3. DNA extraction from soil sample. DNA is extracted from the soil sample through a series of steps. 4. DNA qualification by Nanodrop. DNA is qualified through the use of the NanoDrop instrument, a pipette, an elution buffer, and control lambda DNA of known concentration. The results are recorded along with the concentration and OD 260/280 ratio. 5. Sample sequencing. The soil sample is sent to a DNA sequencing facility for Illumina sequencing. PCR procedures and gel electrophoresis are performed (in the next steps) for validation. 6. PCR amplification. A variable region from the 16S rRNA gene is amplified using the PCR primers. This is performed in 2 PCR reactions. 7. Gel electrophoresis. This step is done to verify if the PCR product was amplified. A fraction of the PCR reactions is placed on a gel to make DNA bands in the 2 PCR reactions displayed visually. 8. Microbiome data analysis. The instructor will provide information that will guide further data analysis. Micropipettor Usage 8. When using a micropipettor, you should go to which stop prior to taking in liquid? While using a micropipettor, you should go to the first stop prior to taking in a liquid. The second stop is only for dispensing a liquid. 9. When trying to keep the samples sterile, when is it appropriate to change the tip of the micropipette?

It is appropriate to change the tip of the micropipette after each sample. It is also essential to change the tip when it leaves the sterile field or if contamination is suspected. 10. Convert 2.0mL to uL. 1ml=1000uL so, 2mL=2000uL Cytological Techniques: Special Cell Stains 11. List the primary stain, counterstain, and mordant for the Special Cell Stains – Endospore Stain Primary stain: Malachite green Counter stain: 0.5% Safranin Mordant/Fixer= Heat/Steam Decolorizer = water 12. Which microorganism is associated with this stain? Bacillus cereus 13. What is an endospore? An endospore is often defined as a dormant and heat resistant spore or seed like structure that forms in bacteria. Vegetative cells give rise to endospores to ensure the survival of bacteria in stressful or unfavorable conditions such as overpopulated or nutrient poor environments. Endospores allow bacteria to become inactive for months, years, and sometimes even centuries until more favorable conditions arise.

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