Lab 3_Dilutions and Spectrophotometer_INSTRUCTIONS 8
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BMB 370 Introductory Biochemistry Laboratory Instructions
Lab 3 – Dilutions and Introduction to the Spectrophotometer
BMB 370 8.18.23 Claire Vieille
Lab Companion Sections
• Tables, Calculations, Graphing, and Conclusions
• Building a Graph in Excel
• Basic Statistics
• Dilutions
• Linear Regression and R
2
in Excel
• Spectrophotometry
• Quantifying bacteria using a spectrophotometer
Before Starting the Lab
• Complete Table 3-1, 3-2, 3-3, and 3-4
• Calculate the expected absorbance for each concentration in Table 3-5
Objectives
●
To understand and practice making serial dilutions
●
To understand and practice using a modern micrometer-based spectrophotometer
●
To understand the Beer–Lambert law and its application to spectrophotometric
measurements
Materials
●
3 mM Red food dye #40 (
ε
≈
25,900 M
-1
cm
-1
at 500 nm) (4 mL per group)
●
Energy drink containing red food dye #40 (4 mL per group)
●
Saturated E. coli
culture (overnight culture) (2 - 5 mL per group)
●
LB (6 mL per group)
●
2 cuvettes (polystyrene or acrylic) with 1 cm path length
●
2 mL microfuge tubes for dilutions (in drawer)
●
400 mL beaker to fill with water for dilutions (in drawer)
Safety
The only significant safety concern in this lab is the Escherichia coli
culture. The E. coli
strains
used in BMB 370 are non-pathogenic: they will not cause disease if ingested. However, they
may contain genes that confer resistance to certain antibiotics. Because antibiotic resistance
genes are easily transferred between bacterial species, even non-pathogenic strains should not
be released into the environment. All materials that come into contact with E. coli
must be
autoclaved prior to reuse or disposal. E. coli
cultures that have not been autoclaved should
never be poured down the drain. As always, wash your hands before leaving the lab.
°
Methods
Preparing serial dilutions of red dye #40
Serial dilutions are used when an exponential dilution of a solution is needed for specific
measurements or when very large dilutions are needed that cannot be prepared through direct
dilutions.
1. Complete Table 3-1 for serial dilutions of red dye #40 using a series of consecutive 5-fold
dilutions using the following instructions:
a. Each dilution will have a total volume of 1.25 mL (1,250 µ
L)
b. Calculate the concentration and dilution factor of dilutions 1 to 5 starting from the initial
3,000 µ
M (3 mM) solution (i.e., dilution 0). The fold dilution will increase exponentially
from one dilution to the next
c. Calculate the volumes of dilutions and of water and needed to prepare dilutions 1 to 5
Table 3-1: Red dye #40 5-fold serial dilutions
Dilution
#
Red dye
concentration (
µ
M)
Total fold
dilution
Volume
solute (
µ
L)
Volume
water (
µ
L)
Total volume
(
µ
L)
0
3,000
1
1,250
0
1,250
1
5
250
1,250
2
1,250
3
1,250
4
1,250
5
1,250
2. Using five 2 mL microfuge tubes prepare dilutions 1 to 5 in the series by serial dilution
starting with the 3,000 µ
M solution (i.e., dilution 0). Make sure to change pipet tips before
each transfer and do not mix your dilutions with a used pipet tip.
Preparing direct dilutions of an energy drink
You have a sample of an energy drink that contains red dye #40. We want to make three
separate 15-fold direct dilutions of the drink (i.e. three technical replicates). Complete Table 3-2
below and prepare the dilutions in 2 mL microfuge tubes
Table 3-2: Energy drink 15-fold direct dilution
Total fold
dilution
Volume energy
drink (μL)
Volume water
(μL)
Total volume (μL)
15
1,500
°
Preparing serial dilutions of an E. coli
culture
In this exercise, you will prepare serial dilutions of a saturated E. coli
culture grown in Lysis
Broth (LB) medium to study how the behavior of suspensions differ from that of solutions in a
spectrophotometer. Because the culture was grown in LB, you will prepare the dilutions in LB
and use LB medium as the blank in the spectrophotometer.
1. Complete Table 3-3. Calculate the volumes of E. coli
culture dilutions and LB medium
needed for a series of 3-fold dilutions. Each dilution will have a total volume of 1.5 mL (1,500
µ
L). You will estimate the bacterial concentration of the initial undiluted culture based on the
optical densities
(OD) you measure of the dilutions
Table 3-3: E. coli
culture 3 fold-serial dilutions
Dilution
#
Total fold
dilution
Volume bacterial
suspension (
µ
L)
Volume LB
(
µ
L)
Total volume
(
µ
L
)
0
1
1,500
0
1,500
1
3
1,500
2
1,500
3
1,500
4
1,500
5
1,500
2. You have a test tube containing a saturated E. coli
culture on your bench. Immediately
before starting with your dilutions, mix your culture by gently vortexing. E. coli
cultures are
suspensions, not solutions, and bacteria tend to settle at the bottom of the tube
3. In five 2 mL microfuge tubes, prepare your dilutions according to Table 3-4 above. Make
sure to use LB rather than water for your dilutions. Mix E. coli in each test tube by inverting
the tube a few times before transferring the calculated volume with a fresh pipette tip to the
next dilution tube. Discard the pipette tips in a biohazard waste container
Determining the wavelength (
λ
) of maximum absorbance
of red dye #40
1. Set the spectrophotometer to do a wavelength scan to determine the wavelength of
maximum absorbance (
λ
max
) of red dye #40. Use the following settings:
On the Jasco choose the “Spectra Measurements” program then use the
“Parameters” icon at the top right to adjust the settings
On the Persee: First choose the “Spectrum” window then the “Measure” menu,
then “Parameter Settings” to adjust the settings
a. Set the spectrum to scan from 400 nm to 700 nm. This range is from blue to red and
is roughly the spectrum the human eye can see
b. Set the UV/vis or spectral bandwidth to 1 nm
c. Set the data interval to 1 nm with a UV/vis response of 0.24 s and a scan speed of
200 nm/min on the Jasco or “Fast” on the Persee
d. On the Persee you will need to set the “Display Range” to 0 to 1 Abs units
2. Transfer 1 mL of deionized H
2
O into a spectrophotometer cuvette and blank the
spectrophotometer in the wavelength scan mode
3. Empty the cuvette in the sink and fill it with your serial dilution #4. You can do this by
carefully pouring the dilution directly from the 2 mL microfuge tube into the cuvette.
4. Perform the wavelength scan with dilution #4. Pour dilution #4 back into the microfuge tube
when you are done. You will use that dilution again
5. When you are done, save your scan on a USB drive or email it to yourself and your lab
partner so it can be graphed later.
On the Jasco, data are autosaved as a *.csv in a folder called Jasco files on the
computer desktop
On the Persee, click on the “File” menu and choose “Export”. Export your data as
a .csv file, not an Excel file which will cause glitches
Determining the wavelength (
λ
) of maximum absorbance
of an energy drink
1. Use your energy drink dilution to perform a wavelength scan as described above to verify
that the energy drink contains a single dye. Make sure that the scan of the energy drink has
a single maximum absorbance peak around 500 nm.
2. Save your data as a csv file on a USB drive or email it to yourself.
3. When the wavelength scan is done, pour the dilution back into the 2 mL microfuge tube
Measuring the absorbances of the red dye serial and
direct dilutions
1. Set the spectrophotometer to end point or direct assay mode:
On the Jasco, use “Fixed wavelength measurement”. Click on the “Parameters”
icon to input settings for your assay
On the Persee, use the “Photometry” window. Click on the “Measurements”
menu at the top, then choose “Parameters”
a. Set the wavelength to 500 nm (cyan-colored light)
b. Set the UV/vis or spectral bandwidth to 1 nm
c. Leave all other settings at their default values
2. Rinse the cuvette you were using before 2–3 times with deionized water
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- Instruction: answers must be in numbers. You are culturing bacteria using a petri dish, the bacteria grow very well on this plate you have. However, your microbiology instructor wants to know how many bacteria per milliliter (bacteria/mL) are on your plate. And as you have remembered during the first day of inoculating this bacteria you used a imL aliquots sample of a 1:10,000 dilution, and the total colony you counted on this plate is 170. How many bacterial per milliliters would that be?arrow_forwardTopic: Isolation of Crude Ovalbumin from Egg White by Ammonium Sulfate Precipitation (Salting Out) The computed amount of powdered ammonium sulfate was added to the egg white sample portion byportion with constant stirring while submerged in an ice bath. The solution is expected to become moreturbid, and a white precipitate is expected to form. The resulting mixture was then filtered using a cheesecloth. The residue was discarded, and 30.0 mL of thefiltrate was brought from 40% to 60% saturation by adding the required amount of powdered ammoniumsulfate in the same manner as the previous addition. After adding ammonium sulfate, the mixture was allowed to stand with occasional stirring for 30 minutes inan ice bath. The formation of a white precipitate is expected to happen QUESTION: Explain the significance of allowing the mixture to be submerged in an ice bath for a given time.arrow_forwardTopic: Reducing and Non Reducing Carbohydrates Among the Benedict's, Reagent, and Iodine tests, which is most highly applicable in biomedical field? Explainarrow_forward
- Discuss the principles and applications of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in molecular diagnostics.arrow_forwardAvailable LEGO Quadrant streak plate Pure culture T-streak plate Zig-zag streak plate Environment sample Pure culture Once you have viewed these results, please complete Data Sheet 1-3 on pages 41-42. For question 2, be the critiquing lab partnie For questions 3-6, assume the first person role as if the results were your own. Question 7 requires deep thinking.arrow_forward3) Define gel electrophoresis, including its theory and application. Describe the steps of running gel electrophoresis using the following image. More detailed reading: https://www.sciencedirect.com/topics/medicine-and-dentistry/agar-gel-electrophoresis POWER SUPPLY CATHODE ELECTROPHORETIC BUFFER ANODE WELL SAMPLE AGAROSE GEL POWER SUPPLY CATHODE ANOCE HIGH MOLECULAR WEIGHT SPECIES LOW MOLECULAR WEIGHT ANALYTESarrow_forward
- A senior medtech in clinical chemistry section runs Quality Control on a morning shift. The testing was smooth and there were no problems encountered. Later in the afternoon shift, some of the analytes were found to be out of range. The machine was fine, the junior medtech tried running it again but still having an invalid results. Define the problem, what is/are the 3 possible reasons of the problem encountered, and identify the 3 best possible solution/s ?arrow_forwardSeparation of Amino Acids by Thin Layer Chromatography Lab Questions 1. Describe in detail Thin layer chromatographic experiment. Example: the theory behind it, how youwould prepare the materials to spot on the plate with different mobile amino acids and unknown andhow TLC Plate is developed and the reasoning behind which solvent/ solvent mixture should be used,along how to correctly identify of the unknown. 2. Calculate the Rf value if a solute travelled 5 cm from the base spot and the solvent front is 10 cmfrom the origin? 3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted thedevelopment of spots in the origin, what can you suggest about this observation?arrow_forwardnsert Format Arrange View Share Window Help Biology Lab Exam 1 Sp 21 目 125% v Collaborate Insert Table Chart Тext Shape Media Comment View Zoom Add Page 3. (1 point) Show the answer to the following question in correct significant figures. 10.219 + 3.12 = 4. (7 pts) True or False. Circle the correct answer. True or False. A blank used to zero of spectrophotometer always contains just distilled water. True or False. The mode is the most commonly occurring number in a data set. True or False. One milliliter (mL) equals 1,000,000 microliters (µL). True or False. Peroxidase activity increases when the enzyme is boiled True or False. Enzymes are carbohydrates. True or False. Denaturing involves an enzyme breaking down into individual amino acids. True or False. Hydroxlyamine is a competitive inhibitor of peroxidase. 5. (1 point) How many µL (microliters) are in 2.32 L? Show your work for full credit. 6. (1 pts) Convert 1,745 mm tọ Km. Show your work for full credit. MAR étv 9. MacBook Airarrow_forward
- Topic: Isolation of Crude Ovalbumin from Egg White by Ammonium Sulfate Precipitation(Salting Out) The computed amount of powdered ammonium sulfate was added to the egg white sample portion byportion with constant stirring while submerged in an ice bath. The solution is expected to become moreturbid, and a white precipitate is expected to form. Explain the purpose of using powdered ammonium sulfate in the isolation of ovalbumin from egg whites.arrow_forward1) Dosage calculation with same units of measure. EX: MD orders 1000mg Ancef. You have available 2000mg in 10mls. How many mls you give?arrow_forwardAnswer this ff question: 1. Explain the importance of system suitability on HPLC method of analysis. 2.In UV and visible spectrophotometry, the specimen is generally dissolved in a solvent, and determinations are made at room temperature using a path length of 1 cm. Give the most common solvents suitable for UV or visible spectrophotometry.arrow_forward
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