Labs
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Labs
Enzymes and Protein Standard Curve - Investigative
Enzymes and Protein Standard Curve
Photosynthesis Using LoggerPro
Determining the Free Chlorine Content of Swimming Pool Water
Enzymes and Protein Standard Curve-Investigative
Objectives
1. Observe enzyme activity and specificity by means of a colorimetric enzyme reaction
2. Determine the effects of temperature on enzymatic activity.
3. Determine the effects of substrate concentration on enzymatic activity.
4. Determine the effect of pH on enzymatic activity.
5. Prepare a protein standard curve.
6. Determine the protein concentration of a sample by means of a total protein assay and use of a standard curve
Background
Enzymes are biological catalysts that are characterized by their ability to rapidly carry out cellular reactions that would otherwise occur only very slowly or under extreme conditions. Enzymes are effective in minute amounts, are not used up in the reaction and are very specific as to the reactions they catalyze. In fact, one of the defining characteristics of a reaction catalyzed by an enzyme is the extraordinary degree of specificity which the enzyme displays for a particular substrate. The change in orientation of a hydroxyl group in the structure of the substrate may be enough to prevent an enzyme catalyzed reaction from occurring because it may no longer "fit" into the active site of the enzyme.
Most biochemical reactions proceed slowly at low temperatures because the reaction molecules do not collide with each other with sufficient energy to form an “activated complex” or “transition state complex”. It is only when such an activated complex is formed that the reaction can go to completion. By raising the temperature, a large proportion of the molecules will achieve this minimal energy and the rate of the reaction will increase accordingly. If the temperature is increased too much though, enzymes will gain so much kinetic energy (and thus will vibrate with such vigor) that they will lose their characteristic structure and no longer will be able to function as an efficient catalyst. Thus, unlike most uncatalyzed chemical reactions, enzymatic reactions in general display a temperature optimum.
Enzyme activity is usually expressed in terms of the rate of the reaction catalyzed by the enzyme. The rate is defined as the amount of substrate transformed, or the amount of product formed, per unit of time. This rate will change depending on the concentration of substrate available for the reaction and the temperature and pH of the reaction mixture.
The particular assay employed in this lab is a quantitative enzymatic determination of glucose used in the diagnosis of disorders associated with abnormal carbohydrate metabolism. The most significant of these diseases is diabetes mellitus, which is characterized by abnormally high concentrations of glucose in physiological fluids. Increased glucose concentration also occurs during hyperactivity of endocrine glands such as the thyroid and adrenals. Hypoglycemia is a condition characterized by low glucose levels that can result from a variety of conditions such as insulin overdose liver diseases, and hypopituitarism (Sigma Diagnostics Bulletin #315)
Enzyme activity is usually expressed in terms of the rate of the reaction catalyzed by the enzyme. The rate is defined as the amount of substrate transformed, or the amount of product formed, per unit of time. This rate will change depending on the concentration of substrate available for the reaction and the temperature and pH of the reaction mixture.
Assignment
Your lab group has been given the assignment to determine some defining characteristics of the reaction catalyzed by enzyme X. You will have the option to concentrate on the effect of one of three different variables: temperature, substrate concentration or pH. Unfortunately the previous researchers on this project have left you only sketchy background information (obviously they didn’t know how to keep a lab notebook!). There are several things you will need to determine this week before you can carry out your actual experiment next week.
You are given the following information:
1. One of the following sugars is the substrate:
- lactose
- galactose
- glucose
- mannose
What you’ll need to determine – which of these four sugars is the substrate
2. This is a colorimetric reaction (the reaction produces a product that is pink in color) so a spectrophotometer can be used to measure the concentration of the product as the reaction proceeds (as more product is produced, the intensity of the color will increase, thus absorbance will increase).
What you’ll need to determine - what wavelength to set the spectrophotometer (see spectrophotometry section that follows this lab section). Do an absorption spectrum of the product from 400-700 nm, taking a reading every 20 nm. The wavelength that gives the highest absorbance reading is the wavelength to use.
3. The volume of the reaction mixture is held constant at 3.0 ml. Of these 3.0 ml., 1.0 ml will always be enzyme.
What you’ll need to determine – what volumes of substrate and dH2O to use (make sure you don’t use so much substrate [concentration or volume] that your final readings go off the spectrophotometer scale). For example, you might try 1.0ml of dH2O, 1.0ml substrate, and 1.0ml enzyme. Remember, the reaction starts as soon as enzyme and substrate come in contact.
4. To get a reaction rate you’ll need to take readings at certain intervals (as soon as the enzyme is added to the substrate/dH2O mixture the reaction begins, this is your time zero)
What you’ll need to determine – what intervals readings should be taken (every 15 sec, 30, 45?). Once you’ve determined the volumes you want to try in #3 above, you will want to start the reaction by adding the substrate, water, and enzyme together. You will need to quickly mix (time zero) and place the tube in to the spectrophotometer. Take readings for whatever intervals seem appropriate based on the rate of the reaction.
5. For experiments done at room temperature, the cuvette may be left in the spectrophotometer for the duration of the experiment. For other temperatures the cuvette will need to be placed back into the waterbath.
What you’ll need to determine – what the duration of the experiment should be (3 minutes, 5 minutes?). As you are doing #4 above note when the absorbance starts to level off. This will probably be a good place to end your experiment.
6. The suggested concentration of stock sugar is 0.2 mg/ml.
What you’ll need to do – make up the sugar solution (next week). The stock solution is 2g substrate/100ml. You will need a substrate solution with a concentration of 0.2mg/ml. Figure out how many ml of the substrate solution you will need for your experiment next week, then determine how you will make up that solution.
7. You have not been given the enzyme concentration.
What you’ll need to determine - although you don’t need this information to do the experiment, for a complete lab report you must determine the enzyme concentration by doing a protein standard curve (see below for procedure) and then a protein assay of the enzyme X solution.
Protein Standard Curve and Assay
In order to determine the amount of protein present in an unknown sample one first needs some way to compare the unknown to known protein values. This is accomplished by setting up a protein standard curve. You will take known amounts of a protein, in this case bovine serum albumin (BSA), and do a standard colorimetric test known as a total protein assay. When the total protein reagent (TPR) is added to a solution containing protein, a chemical reaction occurs that turns the solution blue. The more protein present the deeper blue the resultant product, and thus the higher the absorbancy reading in the spectrophotometer. By graphing the relationship between these absorbancy readings and the known amounts of protein assayed you will then have a standard curve. To determine the protein content of your enzyme X solution you will do the protein assay on it, take an absorbancy reading, and plug that number into the line equation of your protein standard curve. By knowing the volume of enzyme X you assayed you then can find the concentration of protein in the enzyme X solution.
The Standard Curve
1. To obtain data for your standard curve set up six test tubes with the following amounts of protein and deionized water. The protein being used as the standard is bovine serum albumin (BSA) in a concentration of 5 mg/ml.
|
Test Tube #
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ml BSA (5 mg/ml)
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ml dH2O
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1
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0.0
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1.0
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2
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0.1
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0.9
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3
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0.3
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0.7
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4
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0.5
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0.5
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5
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0.7
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0.3
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6
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1.0
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0.0
|
2. To determine the protein content of your unknown (enzyme X) set up 2 tubes each with 1.0 ml of Enzyme X. (you are doing this in duplicate to check your accuracy)
3. Add 3.0 ml of Total Protein Reagent to each of the test tubes (#1-6 and the 2 tubes with 1 ml of enzyme X); mix and wait 10 minutes.
4. Pour the contents of test tube #1 into a spec tube (this is your blank). Set the spec to 540nm, put the blank into the spectrophotometer and zero (set the needle to zero on the absorbency scale).
5. Take the blank out. Sequentially take the readings for tubes 2-6 and then your enzyme X samples making sure you record your data in each case (spec readings in must be done in spec tubes).
6. Plot your protein standard curve - absorbance on the y-axis and protein content (mg) on the x-axis. Plug in your absorbance value for enzyme X into the equation of the line to determine the protein content. The concentration of protein in the enzyme X solution would be the protein content/volume of enzyme X assayed (in this case 1 ml)
Week 1 – What follows is a summary of the information you have been given and what you’ll need to determine this week in order to proceed with next week’s experiment
| Information Given | What you need to determine | |
| The substrate for the rxn catalyzed by enzyme X is a sugar | Which sugar is the substrate– galactose, glucose, mannose, lactose (Hint: you need to do this first in order to proceed) | |
| Reaction is colorimetric, pink product formed, so can use the spectrophotometer | What wavelength to use (you will need to do an absorption spectrum of the completed reaction mixture to determine the optimal wavelength) | |
| Volume of rxn mixture held constant at 3 ml, of which 1 ml is always enzyme X | Volume of dH2O and substrate stock soln to use (concentration(s) of substrate) | |
| Concentration of sugar (substrate) to be used is 0.2 mg/ml | Need to make soln of the substrate from a stock solution of 2g/100ml | |
| For experiments done at room temp the cuvette may be left in the spectrophotometer | The duration of the experiment | |
| As soon as enzyme is added to the substrate (time zero) the rxn starts, readings should be done at intervals | What intervals to take readings (15, 30, 60, or? second intervals) | |
| The enzyme is identified only as enzyme X, know nothing else about it | The protein concentration of the enzyme X solution you are given |
Week 2 – having figured out the background information necessary to conduct an experiment with enzyme X in week 1, you will now carry out an experiment to answer one of the following questions:
1. What happens to the rate of the reaction when the substrate concentration is varied (try at least 5 different substrate concentrations)
2. What happens to the rate of the reaction at different temperatures (besides room temperature (about 22o ), water baths are available at 4, 37, and 60oC) When you test the reaction at other temperatures be sure to equilibrate all components of the reaction mixture to the temperature being tested before adding them together. This takes a minimum of 5-10 minutes. The cuvette needs to be put back into the water bath between readings, (so you are looking at the reaction of the temperature testing and not of somewhere between that temperature and room temperature).
3. What happens to the rate of the reaction at different pHs (you will need to make up solutions of different pHs)
Your Lab Report
Week 1
Hand in a data sheet that will include:
1. The protein standard curve. Be sure to label axes on figure (x-axis is protein content in mg, y-axis is absorbance @ 540nm) and have a title.
2. From the protein std curve you will determine the protein content of the enzyme X soln assayed. From that you should determine the protein concentration of the enzyme X soln.
3. A figure of the absorption spectrum of the pink product formed by reaction of enzyme X with the substrate. Indicate what wavelength you will use for your experiment next week.
4. A figure showing the results of your sample enzyme reaction. Be sure to label x-axis and y-axis, and give appropriate figure legend.
5. State how you will make your substrate solution next week from a stock solution of 2g/100ml. You will need to know the volume of the 0.2mg/ml solution you will need for your experiment next week in order to determine this.
6. State the molarity of both the stock substrate solution and the diluted solution you will make (MW of substrate is 180).
Week 2
--- your lab report should include a methods and a results section. Also include an interpretation of your results (not a formal discussion section) Remember, the results section should include text and the appropriate tables or figures of your data on temperature, substrate concentration, or pH. Think about different ways to display your results (figure of abs vs time, figure of rate of rxn vs temp, sub conc, or pH).
Spectrophotometry
Visible light
Visible light (that which can be seen by the naked eye) is composed of different wavelengths () of light ranging from violet (380 nm) to red (760 nm). A wavelength of light is defined as the distance from one peak to the next in the wave and is measured in nanometers (nm), one nm is equivalent to 10-9 meters.
Colors
Many kinds of molecules interact with or absorb specific types of radiant energy in a predictable fashion. When white light strikes an object, the color the human eye perceives is determined by the wavelengths of light absorbed and the remaining wavelength(s) that are reflected or transmitted. An object that appears red absorbs wavelengths of all colors but red. The red wavelength of light is what reflects back to the eye. If all wavelengths of light are absorbed by an object, the object appears black, if all are reflected, it appears white.
Spectrophotometry –the Spectrophotometer
The perception of color by the eye, as just described, is qualitative. There are instruments called spectrophotometers that electronically quantify the amount and kinds of light that are absorbed by molecules in solution. Spectrophotometry is the measurement of the interaction of radiant energy with matter in the UV and visible portion of the electromagnetic spectrum. In its simplest form a spectrophotometer has a source of white light (for visible spectrophotometry) that is focused on a prism or diffraction grating that separates the white light into individual bands of radiant energy. Each wavelength (color) is then selectively focused through a narrow slit. The width of the slit is important to the precision of the measurement: the narrower the slit the more closely the absorption is related to a specific wavelength of light. Conversely, the broader the slit, the more light of different wavelengths passes through which reduces the precision of the measurement.
The light that passes through the slit, the incident beam (Io), then passes through the sample being measured. The sample, which is dissolved in a suitable solvent, is contained in an optically selected tube called a cuvette. The light that passes through the sample is known as the transmitted beam (It). If the substances in the cuvette have absorbed any of the incident light, the transmitted light will be reduced in total energy content. If the substance in the cuvette does not absorb any of the incident beam, the radiant energy of the transmitted beam will be about the same as the incident beam. When the transmitted beam strikes the photodetector it generates an electrical current proportional to the intensity of light striking it. The photodetector is connected to a galvanometer that directly measures the current, thus the intensity of the transmitted beam. In the Bausch & Lomb Spectronic 21 spectrophotometer the galvanometer has two scales: one indicates the % transmittance (%T) and the other, a logarithmic scale with unequal divisions graduated from 0.0 to 2.0, indicates the absorbance (A). The term optical density (OD) may be used instead of absorbance, especially when experimenting with cell suspensions: however, absorbance is more commonly used.
Transmittance
As mentioned above, the light transmitted (T) is the ratio of the intensity of the light exiting the sample (It) to the intensity of the light entering that entered the sample (Io).
T=It/Io
The percentage of light transmitted (%T) is equal to T x 100.
The amount of light transmitted depends on three factors:
1. if the sample will absorb light at the particular wavelength () tested (this is dependent on the color of the sample – pure H2O transmits all visible wavelengths)
2. the amount of sample the light passes through (cell width)
3. the concentration of the absorbing material
The transmittance of the sample varies logarithmically with the concentration of the absorbing material.
T = 10 -abC
log (1/T) = -log T = abC
where
a= molar absorbtivity
b= path length
c = concentration of the absorbing material
Absorbance, Beer’s Law, and Standard Curves
There is an inverse correlation between transmittance and absorbance. The more light that is absorbed by a sample, the less light will be transmitted.
0 absorbance = 100% transmittance
By substituting absorbance (A) for transmittance, absorbance and concentration become directly proportional.
A = log (1/T)
A = abC this is known as Beer’s Law
This relationship enables us to set up what is known as a standard curve. If we take absorbance readings of at least five dilutions of a standard of known concentration plus a blank we can plot absorbance vs concentration and get a straight line. By then taking an absorbance reading of a sample of the same substance of an unknown concentration and locating where that absorbance reading (Y axis) falls on the standard curve we can find the corresponding concentration on the X axis.
Using the Spectrophotometer
1. Turn the power on (front right side, fig 1). Allow at least a 5-minute warm up period before taking readings.
2. Select the wavelength to be used by turning the wavelength selector knob located on the top right side of the spec. The wavelength selected shows in the window to the left of the knob.
3. Wipe off fingerprints from the reference blank cuvette with a kimwipe. (to assure the spec measurement is due only of the light absorption of the molecules being studied a mechanism for “subtracting” the absorbance of the solvent is necessary. To achieve this a “blank” of the solvent is read first to calibrate the spec)
4. Insert the cuvette into the sample holder lining up the vertical mark on the cuvette with the notch in the sample compartment. Close the sample compartment cover.
5. Set the spec to 0 absorbance (100% transmittance) by turning the 100% T/Zero A control knob (located front left of the spec) until the needle lines up over zero absorbance (you may use the mirror located behind the scale to align the needle).
6. Remove the reference blank cuvette.
7. Wipe fingerprints off of cuvette containing sample and insert into sample compartment. Close the cover and read the meter display. Depending on the experiment you will read either the absorbance scale or the transmittance scale.
8. Whenever changing the wavelength be sure to “reblank” the spec with the reference balnk before putting the sample in to be read.
9. Whenever operating at a fixed wavelength for an extended period of time it is best tp periodically check the absorbance with the blank to be sure it is still zeroed.
Enzymes and Protein Standard Curve
Objectives
1. Observe enzyme activity and specificity by means of a colorimetric enzyme reaction.
2. Determine the effect of temperature on enzymatic activity.
3. Determine the effect of substrate concentration on enzymatic activity.
4. Prepare a protein standard curve.
5. Determine the protein concentration of a sample by means of a total protein assay and use of a standard curve.
Background
Enzymes are biological catalysts that are characterized by their ability to rapidly carry out cellular reactions that would otherwise occur only very slowly or under extreme conditions. Enzymes are effective in minute amounts, are not used up in the reaction and are very specific as to the reactions they catalyze. In fact, one of the defining characteristics of a reaction catalyzed by an enzyme is the extraordinary degree of specificity that the enzyme displays for a particular substrate. The change in orientation of a hydroxyl group in the structure of the substrate may be enough to prevent an enzyme catalyzed reaction from occurring because it may no longer "fit" into the active site of the enzyme.
Most biochemical reactions proceed slowly at low temperatures because the reaction molecules do not collide with each other with sufficient energy to form an “activated complex” or “transition state complex”. It is only when such an activated complex is formed that the reaction can go to completion. By raising the temperature, a large proportion of the molecules will achieve this minimal energy and the rate of the reaction will increase accordingly. If the temperature is increased too much though, enzymes will gain so much kinetic energy (and thus will vibrate with such vigor) that they will lose their characteristic structure and no longer will be able to function as an efficient catalyst. Thus, unlike most uncatalyzed chemical reactions, enzymatic reactions in general display a temperature optimum.
Enzyme activity is usually expressed in terms of the rate of the reaction catalyzed by the enzyme. The rate is defined as the amount of substrate transformed, or the amount of product formed, per unit of time. This rate will change depending on the concentration of substrate available for the reaction and the temperature and pH of the reaction mixture.
The particular assay employed in this lab is a quantitative enzymatic determination of glucose used in the diagnosis of disorders associated with abnormal carbohydrate metabolism. The most significant of these diseases is diabetes mellitus, which is characterized by abnormally high concentrations of glucose in physiological fluids. Increased glucose concentration also occurs during hyperactivity of endocrine glands such as the thyroid and adrenals. Hypoglycemia is a condition characterized by low glucose levels that can result from a variety of conditions such as insulin overdose liver diseases, and hypopituitarism (Sigma Diagnostics Bulletin #315).
The actual reaction that you will be following is a composite of two reactions catalyzed by two different enzymes, so in a sense it is a bit complicated, but it is an easily measurable colorimetric assay which is why we are using it in the intro course. The enzymatic reactions involved are as follows:
Glucose + H2O + O2 Glucose Oxidase -------> Gluconic Acid + H2O2
H2O2 + 4 Aminoantipyrine + p-Hydroxybenzene Sulfonate Peroxidase -------> Quinoneimine Dye + H2O
Glucose is first oxidized to gluconic acid and hydrogen peroxide in the reaction catalyzed by glucose oxidase. The hydrogen peroxide formed reacts with 4-aminoantipyrene and p-hydroxybenzene sulfonate to form a quinoeimine dye which is pink in color. The intensity of the color produced (as measured on the spectrophotometer) is directly proportional to the glucose concentration in the sample. Hence the more glucose present, the darker the pink color in the reaction mixture, and thus the greater the absorbancy.
Experimental Procedure
1. Specificity - One of the defining characteristics of a reaction catalyzed by an enzyme is the extraordinary degree of specificity the enzyme displays for a particular substrate. As an example of this specificity, consider the following sugars, all of which are physiologically important:
Note that galactose and mannose differ from glucose only in the orientation of a single OH group with respect to the plane of the ring. Lactose is made up of a glucose and galactose molecule.
Procedure:
A. Zero the spectrophotometer with a spec. tube containing 4.0 ml dH2O and 1.0 ml enzyme (this will be your blank). The wavelength should be set at 510 nm.
B. To another spec. tube add 3.0 ml dH2O and 1.0 ml glucose (0.2 mg/ml).
*NOTE: In this next step the addition of the enzyme starts the reaction and is therefore time zero. Be sure to keep track of time.
C. Add 1.0 ml enzyme to the spec. tube from step B, quickly mix in the contents of the tube on the vortex mixer and insert into the spec.
D. Read and record absorbance at 15 sec., 30 sec., 45 sec., 1 minute, 2 minutes and 3 minutes after the addition of the enzyme.
E. Repeat steps B-D except, use galactose instead of glucose as the substrate (then mannose and lactose in place of glucose).
F. Plot your data -- Absorbance vs time (time on the X-axis) for all substrates (plot all lines on one graph, using different symbols, colors, etc. to distinguish between them).
2. Temperature - Most biochemical reactions proceed slowly at room temperatures because the reaction molecules do not collide with each other with sufficient energy to form an “activated complex”, or “transition state complex”. It is only when such an activated complex is formed that the reaction can go to completion. By raising the temperature, a large proportion of the molecules will achieve this minimal energy, and the rate of the reaction will increase accordingly. However, if the temperature is increased too much, enzymes will gain so much kinetic energy (and thus will vibrate with such vigor) that they will lose their characteristic structure and no longer will be able to function as an efficient catalyst. Thus, unlike most uncatalyzed chemical reactions, enzymatic reactions in general display a temperature optimum.
Procedure:
A. Set up six test tubes as follows:
| Test Tube | ml dH2O | ml glucose (0.2 mg/ml) | ml enzyme | |||
| 1 | -- | -- | 1.0 | |||
| 2 | 3.0 | 1.0 | -- | |||
| 3 | -- | -- | 1.0 | |||
| 4 | 3.0 | 1.0 | -- | |||
| 5 | -- | -- | 1.0 | |||
| 6 | 3.0 | 1.0 | -- |
B. Place test tubes #1 and 2 into the ice bath (4oC), test tubes #3 and 4 into the 37oC water bath, and test tubes # 5 and 6 into the 65oC water bath, for at least 10 minutes. This is to equilibrate the components of the reactions to the appropriate temperatures.
C. At the end of the equilibration period, mix the components of test tubes 1 and 2 in a spec. tube, this is time zero. Place the spec. tube back into the ice bath. Take readings at 1 minute intervals for 5 minutes. Be sure to wipe all water off spec. tubes before inserting into the spectrophotometer.
D. Mix test tubes 3 and 4 in a spec. tube (time zero) and quickly place back in 37oC water bath. Take readings at 1 minute intervals for 5 minutes.
E. Mix test tubes #5 and 6 in a spec. tube and place back into the 65oC water bath. Take readings at 1 minute intervals for five minutes.
E. Plot your data--Absorbance vs time for all four temperatures (4oC, 37oC, 65oC and room temperature, approx. 22oC, data for this one from part 1) on one graph.
3. Substrate concentration- In this portion of the experiment you will be looking at the effect of varying substrate concentration on the velocity of the enzyme reaction. Enzyme activity is usually expressed in terms of the rate of the reaction catalyzed by the enzyme. The rate is defined as the amount of substrate transformed, or the amount of product formed, per unit of time.
Procedure:
A. In a spec. tubes set up the following:
| Sample | ml dH2 O | ml glucose (0.2 mg/ml) | ||
| 1 | 3.9 | 0.1 | ||
| 2 | 3.5 | 0.5 | ||
| 3 | 3.0 | 1.0 | ||
| 4 | 2.5 | 1.5 | ||
| 5 | 2.0 | 2.0 |
B. Add 1.0 ml enzyme to test tube #1. Quickly mix (this is time zero), and put into the spec. Take readings at 15, 30, and 45 seconds, 1, 2, and 3 minutes. Record data.
C. Repeat step B for samples 2-5.
D. Plot your data -- Absorbance vs. time for each of the 5 substrate concentrations on one graph.
Protein Standard Curve and Assay
In order to determine the amount of protein present in an unknown sample one first needs some way to compare the unknown to known protein values. This is accomplished by setting up a protein standard curve. You will take known amounts of a protein, in this case bovine serum albumin (BSA), and do a standard colorimetric test known as a total protein assay. When the total protein reagent (TPR) is added to a solution containing protein, a chemical reaction occurs that turns the solution blue. The more protein present the deeper blue the resultant product, and thus the higher the absorbancy reading in the spectrophotometer. By graphing the relationship between these absorbancy readings and the known amounts of protein assayed you will then have a standard curve. To determine the protein content of your enzyme X solution you will do the protein assay on it, take an absorbancy reading, and plug that number into the line equation of your protein standard curve. By knowing the volume of enzyme X you assayed you then can find the concentration of protein in the enzyme X solution.
The Protein Standard Curve
1. To obtain data for your standard curve set up six test tubes with the following amounts of protein and deionized water. The protein being used as the standard is bovine serum albumin (BSA) in a concentration of 5 mg/ml.
| Test Tube # | ml BSA (5 mg/ml) | ml dH2O | mg protein | |||
| 1 | 0.0 | 2.0 | (you fill in) | |||
| 2 | 0.1 | 1.9 | ||||
| 3 | 0.3 | 1.7 | ||||
| 4 | 0.5 | 1.5 | ||||
| 5 | 0.7 | 1.3 | ||||
| 6 | 1.0 | 1.0 |
2. To determine the protein content of your unknown (enzyme X) set up 2 tubes each with 1.0 ml of Enzyme X. (you are doing this in duplicate to check your accuracy)
3. Add 3.0 ml of Total Protein Reagent to each of the test tubes (#1-6 and the 2 tubes with 1 ml of enzyme X); mix and wait 10 minutes.
4. Pour the contents of test tube #1 into a spec tube (this is your blank). Set the spec to 540nm, put the blank into the spectrophotometer and zero (set the needle to zero on the absorbance scale).
5. Take the blank out. Sequentially take the readings for tubes 2-6 and then your enzyme X samples making sure you record your data in each case (spec readings in must be done in spec tubes).
6. Plot your protein standard curve - absorbance on the y-axis and protein content (mg) on the x-axis. Plug in your absorbance value for enzyme X into the equation of the line to determine the protein content. The concentration of protein in the enzyme X solution would be the protein content/volume of enzyme X assayed (in this case 1 ml)
Sample Enzyme Data
Photosynthesis Using LoggerPro
Objectives
1. To determine the absorption spectrum of the photosynthetic pigments of spinach
2. To determine the effect of light intensity on the rate of photosynthesis
3. To determine what effect different wavelengths of light have on photosynthetic rate
4. To determine the wavelengths of light transmitted by various colored filters
Background
Photosynthesis, the metabolic process by which visible light energy is trapped and used to convert inorganic compounds to organic compounds and oxygen, is initiated by the absorption of photons by pigments in the chloroplasts of plants. Ultimately glucose and oxygen are produced from CO2 and water. Glucose is usually then converted to transport and storage molecules. The oxygen produced is utilized for aerobic respiration in the cells of those plants and other organisms.
6 CO2 + 6 H2O -------light energy------- C6H12O6 + 6 O2
The rate of photosynthesis is affected by numerous factors including the amount and the wavelength of light available. One tends to think of sunlight as white, but in reality it is a continuum of wavelengths, each wavelength representing a different color of light.
When light hits a pigmented surface some wavelengths are absorbed while others are reflected or transmitted. An object appears green because it absorbs all wavelengths of light but green which is reflected back to our eye. Plants contain pigments that absorb and reflect various wavelengths of light. Chlorophyll A and B are the predominate pigments in green plants, but there are also accessory pigments such as carotenes and xanthophylls which absorb slightly different wavelengths of light than the chlorophylls giving a wider range of energy that can be absorbed by the plant. This absorption of light as a function of wavelength is known as the absorption spectrum. Plants will be most productive if they receive light of the wavelengths that correspond to where their photosynthetic pigments absorb the photons most strongly. If we vary the wavelength of light reaching the plant we can vary the photosynthetic rate. If we vary the intensity of light we will also vary that rate.
In this experiment you will test to see how the rate of photosynthesis is affected by the amount of light available to the plant by varying the light intensity reaching the spinach and you will also see if some colors (wavelengths) of light are better for photosynthesis than others. If so, then there should be a difference in the photosynthetic rate and amount of oxygen produced/CO2 utilized. You will also do an absorption spectrum of the photosynthetic pigments extracted from spinach and note if there is a correlation between the rates of photosynthesis under the different colored filters, the wavelengths of light that those filters transmit, and the optimum wavelengths of light absorbed by the plant.
To test the amount of CO2 consumed you will use a CO2 probe (Vernier software) inserted into a flask containing spinach leaves (figure 1).
Figure 1. CO2 probe inserted in chamber containing spinach leaves (from Biology with Computers by Scott Holman and David Masterman, Vernier Software and Technology)
Week 1
This first week will allow you to familiarize yourself with the Vernier software and probes that will be used in next week’s experiment and also give you time to collect the necessary data on the absorption spectrum of spinach pigments and transmittance data of the different colored cellophanes.
Absorption spectrum of Pigment Extract:
Obtaining the pigment extract (use one of following methods)
A. Using EtOH
- Obtain a spinach leaf and place it in a 250ml beaker.
- Cover the leaf with 95% EtOH (approximately 50-75 ml)
- Place beaker on hotplate and heat to boiling (pigments will extract out into the EtOH). Remove from heat.
- Fold a piece of filter paper in quarters, place into a funnel and moisten it with a few drops of EtOH
- Put the end of the funnel into a test tube. Pour the pigment extract through the filter paper into the test tube
- Take 0.5 ml of the filtered pigment extract and put it into a cuvette containing 3ml of EtOH
- Pipette 3 ml of EtOH into another cuvette for your blank
B. Using acetone
- Obtain a spinach leaf and place it in a mortar. Add approximately 5 ml of acetone. (when using acetone it is important to use glass pipets, plastic pipets will dissolve in the acetone). Gently grind the leaf with a pestle to release the photosynthetic pigments
- Fold a piece of filter paper in quarters, place into a funnel and moisten it with a few drops of acetone.
- Put the end of the funnel into a test tube. Pour the pigment extract through the filter paper into the test tube.
- Take 0.1 ml of the filtered pigment extract and put it into a cuvette containing 3ml of acetone.
- Pipette 3 ml of acetone into another cuvette for your blank.
Doing the absorption spectrum
- Set the spectrophotometer to 380 nm. Blank the spec, then take a reading of your extract. (note: you may do these readings at the same time as you do your % transmittance spectrum of the color filters, just be sure to use the appropriate blank and read the correct scale).
- Set the spec to 400 nm. Reblank and take an absorbance reading of your extract.
- Continue taking readings at 20 nm intervals up to 700 nm, being sure to reblank at each wavelength before taking the reading of your extract.
% Transmittance of color filters
- Cut pieces of the color filters used in your experiment such that each piece will fit completely around the inside of a cuvette. Place the filters each into a separate cuvette
- Fill those cuvettes with tap water. Fill another cuvette with water for your blank.
- Set the spec to 380 nm.
- Blank the spec then sequentially take the % transmittance reading for each color filter.
- Set the spec to 400nm, reblank, and read all cuvettes containing the color filters. Continue taking readings at 20 nm increments to 700 nm, being sure to reblank at each change of wavelength.
Using Vernier Software/CO2 Probe for Obtaining Photosynthetic Rates
(adapted from Biology with Computers by Scott Holman and David Masterson, Vernier Software and Technology)
- Connect the CO2 Gas Sensor to the Vernier interface, plug into computer and power socket (this should already be set up for you).
- Prepare the computer for data collection by opening applications, then Logger pro 3, then Experiments, then Biology with Computers, and finally double click on Experiment 31B, Photosynthesis-Respiration CO2. The vertical axis has carbon dioxide concentration scaled from 0 to 5 ppt (parts per thousand). The horizontal axis has time scaled from 0 to 10 minutes. The data rate is set to 20 samples/minute.
- Obtain one or two spinach leaves from the back table (if damp, blot them dry between two pieces of paper towel).
- Place the leaves into the respiration chamber, using forceps if necessary. Wrap the respiration chamber in aluminum foil so that no light reaches the leaves.
- Place the CO2 Gas Sensor into the bottle as shown in Figure 1. Gently twist the stopper on the shaft of the CO2 Gas Sensor into the chamber opening. Do not twist the shaft of the CO2 Gas Sensor or you may damage it. Wait 5 - 10 minutes before proceeding to Step 6.
- Click to begin data collection. Data will be collected for 10 minutes.
- When data collection has finished, determine the rate of respiration:
a. Move the mouse pointer to the point where the data values begin to increase. Hold down the left mouse button. Drag the pointer to the point where the data ceases to rise and release the mouse button.
b. Click on the Regression button, , to perform a linear regression. A floating box will appear with the formula for a best fit line.
c. Record the slope of the line, m, as the rate of respiration. - Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu.
- Remove the aluminum foil from around the respiration chamber.
- Place the leaves as close to the lamp as reasonable. Do not let the lamp touch the tissue culture flask. Note the time. The lamp should be on for 5-10 minutes prior to beginning data collection.
- After the five to ten-minute time period is up, click to begin data collection. Data will be collected for 10 minutes.
- When data collection has finished, determine the rate of photosynthesis:
- a. Move the mouse pointer to the point where the data values begin to decrease. Hold down the left mouse button. Drag the pointer to the point where the data ceases to decline and release the mouse button.
b. Click on the Regression button, , to perform a linear regression. A floating box will appear with the formula for a best fit line.
c. Record the slope of the line, m, as the rate of photosynthesis.
- Repeat above except this time wrap the bottle with one of the cellophanes used in % transmittance spectra above
- Print a graph showing your photosynthesis and respiration data (1/group) to pass in today.
- Label each curve by choosing Text from the Insert menu. Enter “Photosynthesis” in the edit box. Repeat to create an annotation for the “Respiration” data. Drag each box to a position near its respective curve. Print a copy of the Graph window, with all sets displayed.
- Remove the plant leaves from the respiration chamber, using forceps if necessary. Clean and dry the respiration chamber.
- Use no more than 2 spinach leaves
- Once the level of CO2 reaches 5 ppt you will have reached the maximum level of the probe, take the probe out for a minute or so to let out excess CO2 , then restart experiment
- Turn the dial to the electric lamp position.
- Hold the sensor so that the sensor top surface is horizontal and directly under the light source (be careful not to shade the sensor with your hand or any other object).
- Read the PPF off the display (units are µmol photons m-2 s-1).
- Be sure to turn the meter off when done.
- Graph of absorption spectrum of spinach pigments – note the peaks of maximum absorption.
- Graph of % transmittance of colored cellophanes – note areas of maximum transmittance for each color.
- A statement hypothesizing under which cellophane the spinach will have the greatest/least photosynthetic rate (compare figures from #1 & #2) and why.
- Bar graph of preliminary data of the effect of light on photosynthetic rate and a statement as to what those data are showing.
- A methods section.
- A results section. State your results and include figures showing what happened at different light intensities or at different wavelengths of light.
- A data analysis section (not a formal discussion). Interpret and tie together your results.
- a. Set the wavelength at 660 nm.
- b. “Blank” the spectrophotometer with a cuvette containing nutrient broth (why this?)
- c. Read the absorbance of the culture. Record. Discard the material in the cuvette in the appropriate container.
- a. Remove all unnecessary items from workspace.
- b. Be sure your hair is tied back
- a. Wash your hands
- b. Disinfect your area
- c. Sterilize instruments
- d. Dispose of all contaminated materials in appropriate containers
- a. Do not talk
- b. Never lay caps or covers on the bench tops
- c. Open petri dishes only when adding and/or spreading bacteria. Tilt the petri dish lid to form a barrier between the culture and you
- d. Work quickly
- e. Use sterile pipets right out of the package (don’t use one that has been on the lab bench)
- f. Only take the caps off the dilution tubes or bacterial cultures when pipeting material either into or out of containers. Do not leave caps off for any longer than necessary
-
Photosynthesis With Disks
Objectives
1. To determine the absorption spectrum of the photosynthetic pigments of spinach.
2. To determine the effect of light intensity on the rate of photosynthesis.
3. To determine what effect different wavelengths of light have on photosynthetic rate.
4. To determine the wavelengths of light transmitted by various colored filters.Background
Photosynthesis, the metabolic process by which visible light energy is trapped and used to convert inorganic compounds to organic compounds and oxygen, is initiated by the absorption of photons by pigments in the chloroplasts of plants. Ultimately glucose and oxygen are produced from CO2 and water. Glucose is usually then converted to transport and storage molecules. The oxygen produced is utilized for aerobic respiration in the cells of those plants and other organisms.
6 CO2 + 6 H2O -------light energy------- C6H12O6 + 6 O2
The rate of photosynthesis is affected by numerous factors including the amount and the wavelength of light available. One tends to think of sunlight as white, but in reality it is a continuum of wavelengths, each wavelength representing a different color of light.When light hits a pigmented surface some wavelengths are absorbed while others are reflected or transmitted. An object appears green because it absorbs all wavelengths of light but green which is reflected back to our eye. Plants contain pigments which absorb and reflect various wavelengths of light. Chlorophyll A and B are the predominate pigments in green plants, but there are also accessory pigments such as carotenes and xanthophylls which absorb slightly different wavelengths of light than the chlorophylls giving a wider range of energy that can be absorbed by the plant. This absorption of light as a function of wavelength is known as the absorption spectrum. Plants will be most productive if they receive light of the wavelengths that correspond to where their photosynthetic pigments absorb the photons most strongly. If we vary the wavelength of light reaching the plant we can vary the photosynthetic rate. If we vary the intensity of light we will also vary that rate.
In this experiment you will use disks of spinach leaves to look at the rate of photosynthesis at various conditions. Since oxygen is a product of photosynthesis, measuring the rate of its production would enable us to measure the rate of photosynthesis. Leaf tissue has gas-filled intercellular spaces so disks cut from a leaf will float. If we remove the air from those intercellular spaces and replace it with liquid the disks will sink. As photosynthesis occurs the oxygen that is produced will diffuse into those intercellular spaces and once enough gas has accumulated in those spaces the disks will once again float. By observing the number of those leaf disks that start to float over time we will be indirectly measuring oxygen production as an indication of photosynthetic activity.
In this lab you will test to see how the rate of photosynthesis is affected by the amount of light available to the plant by varying the light intensity reaching the spinach disks and you will also see if some colors (wavelengths) of light are better for photosynthesis than others. If so, then there should be a difference in the photosynthetic rate and amount of oxygen produced and thus a difference in the rate the disks float to the surface. You will also do an absorption spectrum of the photosynthetic pigments extracted from spinach and note if there is a correlation between the rates of photosynthesis under the different colored filters, the wavelengths of light that those filters transmit, and the optimum wavelengths of light absorbed by the plant.Procedure
Light intensity and color variations:
1. 1. Decide at what distances from the light source you want to run your light intensity experiment (do a minimum of three distances). Place books or whatever else is handy under the lights to get the distances desired. Measure from the light to where the plant disks will be, then refer to fig 2 to determine the PAR value (photosynthetically active radiation - µmol photons/m2/sec) for each distance.
2. For your experiment with the color filters, pick one of the distances used for the light intensity experiment (the distance closest to the light might be best) and use that distance for all color filters used (use a minimum of three colors).
3. On a cutting board cut out disks from spinach leaves using a cork borer (try not to get any of the central leaf vein). To prevent drying, place the disks into a beaker containing 20 ml of 0.2% NaHCO3. (sodium bicarbonate). Think about how many disks should be used. Too few disks will not give an accurate representation, too many will crowd others from the light. Be sure to keep the number of disks used consistent from one treatment to the next.
4. Fill small petri dishes with approximately 20 ml of 0.2% NaHCO3 solution (one dish for each condition being tested). Don’t forget to set up controls. What should your controls be?
5. Pull vacuum on disks – choose from one of two methodsa. Take your spinach disks to the vacuum pump and place them in the side-arm flask containing 0.2% sodium bicarbonate. Stopper the flask and turn the vacuum pump on. Pull a vacuum for about 30-45 seconds (you will be drawing the air out of the intercellular spaces of the leaf disks and replacing it with sodium bicarbonate). Turn the pump off. Gently release the vacuum by slowly pulling the stopper out of the flask. The disks should sink to the bottom.
b. Pull the plunger out of a 30ml syringe. Hold finger over the end of syringe and pour sodium bicarbonate solution containing disks into the barrel of the syringe. Put plunger back in and push out all air. Place finger over the end of syringe and pull down on plunger to create a vacuum. Hold for 10-15 seconds, release finger. Disks should sink, if not repeat pulling vacuum.6. Transfer the disks in the liquid to a beaker, keeping them as much out of the light as possible.
7. Transfer the appropriate number of disks carefully with forceps to the small petri dishes.
8. Place the petri dishes on a piece of black construction paper under the light at the distances you have decided. For those dishes that will have a color filter over them, place those filters on now as well. Be sure to have your controls set up.
9. The data you collect will be of the number of disks floating. You will need to decide when you will collect your data. Are you going to collect your data at certain intervals (how long will those intervals be and how many), or will you wait until all disks have floated and compare times between conditions, or will it be some combination of the two. You also need to decide what you consider to be floating disk – one that totally risen to the surface or one that has come up off the bottom and is sort of sideways, just be consistent
10. Turn on the lights, start timing. Record your data.Absorption spectrum of Pigment Extract
Obtaining the pigment extract (use one of following methods)
A. Using EtOH
1. Obtain a spinach leaf and place it in a 250ml beaker.
2. Cover the leaf with 95% EtOH (approximately 50-75 ml).
3. Place beaker on hotplate and heat to boiling (pigments will extract out into the EtOH). Remove from heat.
4. Fold a piece of filter paper in quarters, place into a funnel and moisten it with a few drops of EtOH.
5. Put the end of the funnel into a test tube. Pour the pigment extract through the filter paper into the test tube.
6. Take 0.5 ml of the filtered pigment extract and put it into a cuvette containing 5ml of EtOH.
7. Pipette 5 ml of EtOH into another cuvette for your blank.B. Using acetone
1. Obtain a spinach leaf and place it in a mortar. Add approximately 5 ml of acetone. (when using acetone it is important to use glass pipets, plastic pipets will dissolve in the acetone). Gently grind the leaf with a pestle to release the photosynthetic pigments.
2. Fold a piece of filter paper in quarters, place into a funnel and moisten it with a few drops of acetone.
3. Put the end of the funnel into a test tube. Pour the pigment extract through the filter paper into the test tube.
4. Take 0.1 ml of the filtered pigment extract and put it into a cuvette containing 5ml of acetone.
5. Pipette 5 ml of acetone into another cuvette for your blank.Doing the absorption spectrum
1. Set the spectrophotometer to 380 nm. Blank the spec, then take a reading of your extract. (note: you may do these readings at the same time as you do your % transmittance spectrum of the color filters, just be sure to use the appropriate blank and read the correct scale).
2. Set the spec to 400 nm. Reblank and take an absorbance reading of your extract.
3. Continue taking readings at 20 nm intervals up to 700 nm, being sure to reblank at each wavelength before taking the reading of your extract.Transmittance of color filters
1. Cut a piece of one of the color filters used in your experiment such that the piece will fit completely around the inside of a cuvette. Fill the cuvette with tap water. Do this for each color filter you used.
2. Fill a cuvette with water for your blank. Set the spec to 380 nm.
3. Blank the spec with the water blank then sequentially take the % transmittance reading for each color filter.
4. Set the spec to 400nm, reblank with water, and read all cuvettes containing the color filters. Continue taking readings at 20 nm increments to 700 nm, being sure to reblank at each change of wavelength.Your lab report
1. Include a methods section.
2. Your results section should state your results and include figures showing what happened at different light intensities, at different wavelengths of light, an absorption spectrum, and the % transmittance of the various color filters at different wavelengths.
3. Your discussion should interpret your results; be sure to tie together your results of the absorption spectrum and % transmittance of color filters with your data of the leaf disks under different color filters.
Pigment Extract Data
Bacterial Growth
Objectives
1.Measure growth of a bacterial culture by spectrophotometry
2.Quantitate viable cells in a bacterial culture by standard plate count
3.Observe differentially stained bacteria using light microscopy
4.Determine the effectiveness of some common chemical disinfectants and antibiotics
Background
A. Growth measurements
When attempting to study the basic processes of life, biologists often turn to “simpler” organisms to make their observations and develop working theories. Observations are then made on more complex biological systems to determine if the information obtained from the simpler organisms can be extrapolated to the higher forms. For this lab we shall use prokaryotic organisms, bacteria, to examine the growth process.
Bacteria are a diverse group of small, single-celled organisms in the kingdoms Eubacteria and Archaebacteria. Found in virtually every extreme of all habitats, they have existed on earth longer and are more widely distributed than any other group of organisms. Bacteria have their genetic material organized in a circular DNA molecule that is not surrounded by a nuclear membrane. Reproduction is by binary fission with the formation of two equal size progeny. During active bacterial growth the size of the population continuously doubles, one cell becomes 2, 2 become 4, etc. in a geometric progression. When bacteria are inoculated into a fresh medium, the resulting culture exhibits a characteristic growth curve of four distinct phases (Fig. 1). During the lag phase there is no increase in cell number, but a time when the cells prepare for synthesis of DNA and enzymes needed for cell division. This is followed by the log phase where the culture reaches its maximum rate of growth for specific conditions. The time required to achieve doubling of the population is known as the generation time. The generation time will vary from organism to organism and will vary in different environmental conditions. The graphical determination of doubling time can be made by extrapolation (Fig. 2). As the bacteria multiply, nutrients are exhausted and inhibitory metabolic end products accumulate. These conditions give rise to the stationary phase which represents no net increase in numbers (growth rate equals death rate). Given enough time there will be a total decline in cell number-the death phase.
Figure 1. Bacterial Growth CurveFigure 2. Determination of Generation TimeIn this lab you will quantitate bacteria by the two most widely used methods: viable plate count and spectrophotometric analysis. In liquid culture, the medium appears more and more cloudy as the bacteria increase in number by division. A tube of bacteria will tend to reflect light so that less light is transmitted through the tube. A spectrophotometer can measure the amount of light passing through the tube, or conversely the amount of light absorbed. These measurements of turbidity or optical density (OD) are not direct measurements of bacterial numbers, but an indirect measurement of cell biomass which includes both living and dead cells. As the bacterial cell population increases, the amount of transmitted light decreases, increasing the absorbance reading on the spectrophotometer. (Fig. 3). If one takes readings of the same culture over time, the absorbance readings will increase as the cell number increases. This can then be graphed to show the growth curve for the particular conditions being tested. There are some limitations with this method, though. A growth curve that includes the lag,log, and stationary phase will take several hours to complete and the relationship between cell number and absorbance will begin to deviate from linearity at high cell densities. Generally an absorbance reading or O.D. of 0.8 is about as high as one should try to measure. To give you an idea of how the turbidity measurements correspond to actual numbers, more than a million cells /ml need be present in order to get even a trace of a measurement on the spectrophotometer.
Figure 3. Spectrophotometric determination of cell densities
To quantitate viable cells a plate count is done. A sample of bacteria is diluted in a sterile medium until the numbers are very low. This diluted sample of bacteria is then transferred onto an agar plate and spread out evenly so that each cell is separate from the others. Each viable cell will continue to divide into a discrete colony of millions of bacterial cells which can now be seen with the naked eye. These colonies can then be counted. Keeping in mind that each colony arose from a single cell that was plated onto the agar, the number of colonies can be used to determine the number of bacterial cells present in the original culture.
For this portion of the lab you will use Escherichia coli, a bacterium which is found by the hundreds of grams in the human lower digestive track. Of all microbes, E. coli is probably the most utilized by biologists and biochemists. It has fairly simple growth requirements and has a fairly rapid growth rate which makes it useful for a one laboratory period experiment.
B. Gram Stain
Most bacteria are characterized by having not only a cell membrane but also a cell wall which lies outside of the cell membrane. This cell wall is composed mostly of peptidogycan and helps to maintain osmotic pressure and the cell’s characteristic shape. Some taxonomic groups of bacteria also have an outer membrane that is attached to the peptidoglycan by small lipoprotein molecules (Fig. 4). This difference in outermost cell structure is the basis for classification of bacteria by a differential staining technique known as the Gram stain. Gram-positive cells (those without an outer membrane) stain purple in the procedure, gram-negative cells (which have the outer membrane) stain red or pink. The usual first step in any bacterial identification is the determination of whether or not it is a G+ or G- bacterium. A sample of the bacteria in question is first stained with the primary cationic dye crystal violet. Since most bacteria carry a net negative charge at pH 7 they pick up the dye. At this point morphological features such as relative size, shape, and characteristic arrangement of cell groups can be seen. A mordant (in this case Gram's iodine) is then added to form a tighter complex between the stain and the cells . To remove any excess stain or stain that has not adhered to the cell, a decolorizing agent is then added(ethanol). At this point gram-positive cells are purple and gram-negative cells are colorless. Cells are then stained with a counterstain (safranin). Gram-negative cells will pick up the counterstain and appear red or pink. In this lab you will be given an unknown bacterium that you will be asked to identify as either gram positive or negative.
Figure 4. Schematic diagram of the cross section of bacterial cell walls. Note that although both gram-positive and gram-negative cells have a layer of peptidoglycan that gives the cell its rigidity and strength, the peptidoglycan layer in gram-positive cells tends to be thicker. Gram-negative cells have an outer membrane that gram-positive cell do not.
C. Antimicrobials
A variety of substances are used to control the growth of unwanted bacteria. These chemicals may be divided into three main subdivisions: disinfectants (used on inanimate objects such as tabletops to reduce the level of bacterial contaminants), antiseptics (used on the surface of living tissue) and antibiotics (absorbed or taken internally). No single antimicrobial substance is ideal in all situations. Antimicrobial agents must be matched to specific organisms and environmental conditions as they all have different modes of action. Lysol, for example, is a disinfectant made of 50% cresol and 50% vegetable oil. Its germicidal effect is due to the fact it causes proteins to denature. Hexachlorophene, a chemical added to soaps and lotions has similar germicidal activity. Some antibiotics inhibit protein synthesis, others inhibit cell wall synthesis (Appendix 1). A simple way of determining the susceptibility of microorganisms to a particular antimicrobial is to inoculate an agar plate with the bacteria to be tested. A filter disk impregnated with the antimicrobial substance is then placed onto the same plate. The antimicrobial substance will diffuse into the agar medium, the concentration decreasing the further away from the disk. If the antimicrobial has an effect on the bacteria, a clear zone of inhibition will form around the disk. The larger the zone, the more effective the chemical is at preventing bacterial growth. This is known as the Kirby-Bauer method of anti-microbial testing. In this lab you will test the antimircrobial effect of a number of chemicals on a gram-negative and a gram-positive microbe.
D. Sterile Technique
The handling of bacterial cultures requires aseptic (sterile) techniques in order to avoid contamination of your experimental bacterial cultures from the millions of microorganisms present in the surrounding environment and to prevent contamination of you and your lab space by the culture you are using. These procedures (Appendix 2) ideally should be followed in a bacteriological hood, but in this lab, since we are starting out with a huge inoculum for the growth curve, the experiment should be fine if the procedure is closely followed, without the use of a hood.
Laboratory Procedure
A. Growth Curve
1. Each group of students will receive a 125 ml flask containing 50 ml of nutrient broth (prewarmed to 37oC--why?). The lab instructor will then add E. coli which is in “log phase” (exponential growth phase) to each flask.
2. Swirl the flask so there is an even suspension of bacteria. Pipette 3.0 ml out of the flask into a cuvette (this will be your 0 time point, be sure to record the time). Replace the cap and put flask back in 37oC incubator (the culture is shaken to keep it mixed and aerated). Dispose of pipets in the appropriate container.
3. Read absorbance of 0 time point –
4. In order to obtain a good growth curve you should take 4 more turbidity (absorbance) measurements, roughly one every 20 minutes following steps 3a-c above. Try to do them as quickly as possible. To avoid cooling the culture take out the 3 ml needed for an absorbency reading and immediately return the stoppered stoppered flask to the 37oC incubator, then take reading.
B. Plate Count
1. At each table will be a set of four dilution tubes along with a flask of sterile saline (0.85% NaCl). Using one pipette tip for the series, add 9.9 ml of saline to tubes #1 and # 2, and 9.0 ml to tube #3 and #4.
2. Label the bottom of 3 petri dishes with the date, some identifying name, and A,B or C.
3. At one of the time points for the turbidity measurements (in this case where the O.D. is between 0.08 and 0.1) remove 0.1 ml from your growing culture and add it to the first tube of your dilution series. Thoroughly mix.
4. Remove 0.1 ml from dilution tube #1 and deposit it into tube #2. Mix thoroughly.
5. Remove 1.0 ml from tube #2 and deposit the entire 1.0 ml into tube #3. Thoroughly mix. With the same pipette tip deposit 0.1 ml from tube #2 onto the appropriately marked agar dish. Spread the bacteria evenly over the entire surface of the agar with a sterile loop and replace the cover. Dispose of loops in designated container.
6. Remove 1.0 ml from tube #3 and deposit the entire 1.0 ml into tube #4. Mix. With the same pipette tip deposit 0.1 ml from tube #3 onto another petri dish. Spread as directed above.
7. Deposit 0.1 ml from tube #4 onto a third plate. Spread. Dispose of all pipets and loops in appropriate containers.
8. If reading through this has confused you, look at the flow sheet - Appendix 3.
9. After approximately 10 minutes turn the petri dishes upside down to prevent condensation from falling on the agar. Why? Place plates in 37oC incubator.
10. The following day come to lab and count the bacterial colonies on the one plate with 30-300 colonies. Do NOT open plates. Dispose of plates in appropriate container.C. Gram Stain
1. Obtain a clean glass slide
2. Prepare a smear of the organisms to be stained by taking a loopful of the bacteria (with a sterile yellow loop) and spreading it over a small area in the center of the slide. Be sure you keep track of which organisms you used.
3. Allow the smear to air dry and then heat fix by passing the slide quickly through a flame.
4. Place the slide on paper towels and add a drop or two of crystal violet to the smear, let set 1 minute.
5. Gently wash the stain off with tap water, being careful not to wash off bacteria.
6. Apply Gram’s iodine, let set 1 minute.
7. Gently wash the iodine off with tap water and then add the decolorizing agent (EtOH) drop by drop until it runs clear.
8. Wash off the decolorizing reagent with tap water.
9. Counterstain with safranin by adding 1-2 drops and let it set for 45 seconds.
10. Rinse with tap water and look at under the microscope. Determine if bacterium is Gram + or-.
D. Antimicrobials
1. Obtain two agar plates. Label bottoms with date, name, and type of bacteria (use the same two types of bacteria as you used for the Gram stain).
2. Add 0.5 ml of bacteria to the agar plate and spread with a sterile loop. Be sure to dispose of pipet tip and loop in the appropriate container.
3. Determine which antimicrobials you want to test. There are disks that have already been saturated with antibiotics. If you want to use antiseptics and disinfectants you will need to soak sterile disks in the material you want to test (pick up a disk with forceps and place into liquid to be tested until it is saturated). Use the same antimicrobials for both types of bacteria so you may do a comparison between gram positive and gram negative bacteria.
4. Number either on the bottom of the plate or the side 1-6.
5. Place saturated disks onto agar plate (fig 5).
6. Place plates into 37o incubator.
7. Come in tomorrow and record the zones of inhibition for each substance tested (fig 6). Do NOT open plates. When all data are collected dispose of plates in appropriate container.
Figure 5. Place 6 disks (each containing a different antimicrobial) evenly spaced on the agar as above.
Figure 6. Collecting data from Kirby-Bauer antimicrobial experiment
E. Data Work-Up
For this lab you will need to write only a data sheet. Be sure to include the following, appropriately labeled:
1. Growth curve of E. coli. Use Excel or another computer graphing program (plot the absorbance on the y-axis vs. time on the x-axis, this needs to be a semi-log plot to get a straight line, see instructions in graphing section of lab manual).
2. Doubling time of E. coli. Determine the doubling time from your graph.
3. Plate count of E. coli. . Be sure to state which plate in the dilution series you counted.
4. Determine the concentration of bacteria in your culture flask at the time your sample for the serial dilution was taken.
5. Gram stain results.
6. Results of your antimicrobial tests on E. coli and B. cereus - include a table or figure and briefly state your results.Appendix 1.
Agents Used to Control Microbial Growth
A. Antiseptics and Disinfectants
1. Phenols and phenolics - these compounds inactivate proteins, denature enzymes, and injure plasma membranes and should only be used on surfaces. Examples include Lysol, hexachlorophene, and pHisoHex.
2. Halogens – may be used on surfaces either alone or as components of organic or inorganic solutions to inactivate enzymes and other cellular proteins. Tend to be strong oxidizing agents. Iodine combines with the amino acid tyrosine, chlorine when added to water forms hypochlorous acid. Betadine is another example often used instead of iodine.
3. Alcohols – denature proteins and dissolve lipids. Examples include ethanol and isopropanol.
4. Heavy metals – such as silver, mercury, copper, and zinc exert their influence through oligo-dynamic action such as combining with the sulfhydryl (-SH) groups and denaturing proteins. Examples include silver nitrate, mercurochrome, and copper sulfate.
5. Surface active agents – soaps and detergents decrease the tension between molecules that lie on the surface of a liquid.
6. Quaternary ammonium compounds (quats) –cationic detergents attached to NH4+ disrupt plasma membranes, denature proteins, and inhibit enzymes. Examples include Cepacol and Zephran.
7. Organic acids – used in the food and cosmetic industry to prevent growth of microorganisms. Examples include sorbic acid, benzoic acid, and propionic acid.
8. Aldehydes – formaldehyde and glutaraldehyde attach methyl or ethyl groups to DNA and proteins making them nonfunctional.B. Antibiotics
1. Inhibition of cell wall synthesis – may inhibit synthesis of petidogylcan. Include penicillins, cephalosporins, vancomycin, bacitracin, oxacillin, and nafcillin.
2. Damage to plasma membrane – polymyxin B, nystatin, and amphotericin B.
3. Inhibition of protein synthesis – streptomycin (causes misreading of codons on mRNA), chloramphenicol (prevents peptide bond formation between amino acids), tetracyclines (prevents hydrogen bonding between anticodon on tRNA-aa complex and codon on mRNA), kanamycin, erythromycin, and gentamicin.
4. Inhibition of nucleic acid synthesis – rifamycin, actinomycin D, nalidxic acid, ciprofloxacin, and norflaxacin.
5. Structural analogs – such as sulfonamides that are structurally similar to cellular metabolites and compete with these in enzymatic reactions.Appendix 2.
Aseptic Techniques to be Used in this Lab
1. Before handling of cultures:
2. Before and after handling cultures:
3. While working with cultures:
Appendix 3 - Serial Dilution.
Determining the Free Chlorine Content of Swimming Pool Water
Beaker Number
1. Obtain swimming pool water sample (25 mL is needed for each experiment)
2. Make up a 10mg/L free-chlorine standard in a 100ml beaker from your chlorine ampule standard.
3. Obtain six 100ml beakers and label 1 through 6.
4. Make up the solutions for you free-chlorine standard:
Beaker Number Free-chlorine standard (10mg/L) (mL) Distilled H2O (mL) Free-chlorine concentration (µg/mL) 1 1.00 24.00 0.40 2 2.00 23.00 0.80 3 3.00 22.00 1.20 4 4.00 1.00 1.50 5 5.00 20.00 2.00
5. Add 25ml of your swimming pool water sample to beaker 6.
6. Add one DPD free-chlorine powder pillow to each of the six labeled beakers.
7. Mix beakers thoroughly into the sample using a stirring rod.
8. Obtain and label 7 spec. tubes.
9. Labels one blank and fill with 5ml dH2O.
10. Label the remaining tubes 1 through 6 and fill each with 5m of solution from the matching beaker.
11. Set the wavelength at 565nm on your spec and blank.
12. Record absorbance for tubes 1-6.
13. Graph your standard curve with points 1 through 5.
14. Solve for your unknown concentration using the equation for your standard curve line.
15. If your unknown absorbance was too high (greater than that of point 5) dilute the pool water sample and so analysis again.
Standard Curve: Food Dyes
Make up Red Food Dye Stock Solution:
Stock = 0.1mL red food dye/100mL H2OFor Standard Curve:
1. Make up solutions to be used to determine standard curve:
A = 10mL Red Stock Solution + 10mL H2O B = 10mL A + 10mL H2O C = 10mL B + 10mL H2O D = 10mL + 10mL H2O E = 10mL D + 10mL H2O
2. Read absorbance at 500nm for tubes A-E.
3. Determine the concentration of red dye in mL/100mL.
4. Convert to mL red dye/100mL x 10-3.
5. Make standard curve. Plot absorbance on y axis, concentration of red dye (mL/100mL x 10-3) on x axis.
6. Take absorbance readings of unknowns.
7. Using standard curve, determine concentration of unknowns.
Solutions Absorbance Concentration of red dye(mL/100mL H2O) Concentration of red dye (mL/100mL H2O x 10-3) A B C D E unknown1 unknown2
Trouble shooting
Measuring Light Intensity
To measure the light intensity reaching the plant material you will use a quantum meter to measure the number of photons emitted from the light source from between 400-700 nm (the photosynthetic photon flux -PPF). PPF is measured in µmol m-2 s-1 (micromoles of photons per square meter per second).
- Turn the dial to the electric lamp position.
- Hold the sensor so that the sensor top surface is horizontal and directly under the light source (be careful not to shade the sensor with your hand or any other object).
- Read the PPF off the display (units are µmol photons m-2 s-1).
- Be sure to turn the meter off when done.
Week 2
Set up/perform your photosynthetic rate experiment. You may choose to do an experiment on the effect of light intensity on photosynthetic rate, the effect of wavelength (color) of light on photosynthetic rate, photosynthetic rate differences between different plants, or some other experiment of your choosing. If comparing different plants you will need to keep surface area and mass consistent. There are corers available to cut leaf disks (4-6 sections are probably best).
Your Lab Report
Week 1
A data sheet with the following
- Graph of absorption spectrum of spinach pigments – note the peaks of maximum absorption.
- Graph of % transmittance of colored cellophanes – note areas of maximum transmittance for each color.
- A statement hypothesizing under which cellophane the spinach will have the greatest/least photosynthetic rate (compare figures from #1 & #2) and why.
- Bar graph of preliminary data of the effect of light on photosynthetic rate and a statement as to what those data are showing.
Week 2
A lab report to include:
- A methods section.
- A results section. State your results and include figures showing what happened at different light intensities or at different wavelengths of light.
- A data analysis section (not a formal discussion). Interpret and tie together your results.
- a. Set the wavelength at 660 nm.
- b. “Blank” the spectrophotometer with a cuvette containing nutrient broth (why this?)
- c. Read the absorbance of the culture. Record. Discard the material in the cuvette in the appropriate container.
- a. Remove all unnecessary items from workspace.
- b. Be sure your hair is tied back
- a. Wash your hands
- b. Disinfect your area
- c. Sterilize instruments
- d. Dispose of all contaminated materials in appropriate containers
- a. Do not talk
- b. Never lay caps or covers on the bench tops
- c. Open petri dishes only when adding and/or spreading bacteria. Tilt the petri dish lid to form a barrier between the culture and you
- d. Work quickly
- e. Use sterile pipets right out of the package (don’t use one that has been on the lab bench)
- f. Only take the caps off the dilution tubes or bacterial cultures when pipeting material either into or out of containers. Do not leave caps off for any longer than necessary
-
Photosynthesis With Disks
Objectives
1. To determine the absorption spectrum of the photosynthetic pigments of spinach.
2. To determine the effect of light intensity on the rate of photosynthesis.
3. To determine what effect different wavelengths of light have on photosynthetic rate.
4. To determine the wavelengths of light transmitted by various colored filters.Background
Photosynthesis, the metabolic process by which visible light energy is trapped and used to convert inorganic compounds to organic compounds and oxygen, is initiated by the absorption of photons by pigments in the chloroplasts of plants. Ultimately glucose and oxygen are produced from CO2 and water. Glucose is usually then converted to transport and storage molecules. The oxygen produced is utilized for aerobic respiration in the cells of those plants and other organisms.
6 CO2 + 6 H2O -------light energy------- C6H12O6 + 6 O2
The rate of photosynthesis is affected by numerous factors including the amount and the wavelength of light available. One tends to think of sunlight as white, but in reality it is a continuum of wavelengths, each wavelength representing a different color of light.When light hits a pigmented surface some wavelengths are absorbed while others are reflected or transmitted. An object appears green because it absorbs all wavelengths of light but green which is reflected back to our eye. Plants contain pigments which absorb and reflect various wavelengths of light. Chlorophyll A and B are the predominate pigments in green plants, but there are also accessory pigments such as carotenes and xanthophylls which absorb slightly different wavelengths of light than the chlorophylls giving a wider range of energy that can be absorbed by the plant. This absorption of light as a function of wavelength is known as the absorption spectrum. Plants will be most productive if they receive light of the wavelengths that correspond to where their photosynthetic pigments absorb the photons most strongly. If we vary the wavelength of light reaching the plant we can vary the photosynthetic rate. If we vary the intensity of light we will also vary that rate.
In this experiment you will use disks of spinach leaves to look at the rate of photosynthesis at various conditions. Since oxygen is a product of photosynthesis, measuring the rate of its production would enable us to measure the rate of photosynthesis. Leaf tissue has gas-filled intercellular spaces so disks cut from a leaf will float. If we remove the air from those intercellular spaces and replace it with liquid the disks will sink. As photosynthesis occurs the oxygen that is produced will diffuse into those intercellular spaces and once enough gas has accumulated in those spaces the disks will once again float. By observing the number of those leaf disks that start to float over time we will be indirectly measuring oxygen production as an indication of photosynthetic activity.
In this lab you will test to see how the rate of photosynthesis is affected by the amount of light available to the plant by varying the light intensity reaching the spinach disks and you will also see if some colors (wavelengths) of light are better for photosynthesis than others. If so, then there should be a difference in the photosynthetic rate and amount of oxygen produced and thus a difference in the rate the disks float to the surface. You will also do an absorption spectrum of the photosynthetic pigments extracted from spinach and note if there is a correlation between the rates of photosynthesis under the different colored filters, the wavelengths of light that those filters transmit, and the optimum wavelengths of light absorbed by the plant.Procedure
Light intensity and color variations:
1. 1. Decide at what distances from the light source you want to run your light intensity experiment (do a minimum of three distances). Place books or whatever else is handy under the lights to get the distances desired. Measure from the light to where the plant disks will be, then refer to fig 2 to determine the PAR value (photosynthetically active radiation - µmol photons/m2/sec) for each distance.
2. For your experiment with the color filters, pick one of the distances used for the light intensity experiment (the distance closest to the light might be best) and use that distance for all color filters used (use a minimum of three colors).
3. On a cutting board cut out disks from spinach leaves using a cork borer (try not to get any of the central leaf vein). To prevent drying, place the disks into a beaker containing 20 ml of 0.2% NaHCO3. (sodium bicarbonate). Think about how many disks should be used. Too few disks will not give an accurate representation, too many will crowd others from the light. Be sure to keep the number of disks used consistent from one treatment to the next.
4. Fill small petri dishes with approximately 20 ml of 0.2% NaHCO3 solution (one dish for each condition being tested). Don’t forget to set up controls. What should your controls be?
5. Pull vacuum on disks – choose from one of two methodsa. Take your spinach disks to the vacuum pump and place them in the side-arm flask containing 0.2% sodium bicarbonate. Stopper the flask and turn the vacuum pump on. Pull a vacuum for about 30-45 seconds (you will be drawing the air out of the intercellular spaces of the leaf disks and replacing it with sodium bicarbonate). Turn the pump off. Gently release the vacuum by slowly pulling the stopper out of the flask. The disks should sink to the bottom.
b. Pull the plunger out of a 30ml syringe. Hold finger over the end of syringe and pour sodium bicarbonate solution containing disks into the barrel of the syringe. Put plunger back in and push out all air. Place finger over the end of syringe and pull down on plunger to create a vacuum. Hold for 10-15 seconds, release finger. Disks should sink, if not repeat pulling vacuum.6. Transfer the disks in the liquid to a beaker, keeping them as much out of the light as possible.
7. Transfer the appropriate number of disks carefully with forceps to the small petri dishes.
8. Place the petri dishes on a piece of black construction paper under the light at the distances you have decided. For those dishes that will have a color filter over them, place those filters on now as well. Be sure to have your controls set up.
9. The data you collect will be of the number of disks floating. You will need to decide when you will collect your data. Are you going to collect your data at certain intervals (how long will those intervals be and how many), or will you wait until all disks have floated and compare times between conditions, or will it be some combination of the two. You also need to decide what you consider to be floating disk – one that totally risen to the surface or one that has come up off the bottom and is sort of sideways, just be consistent
10. Turn on the lights, start timing. Record your data.Absorption spectrum of Pigment Extract
Obtaining the pigment extract (use one of following methods)
A. Using EtOH
1. Obtain a spinach leaf and place it in a 250ml beaker.
2. Cover the leaf with 95% EtOH (approximately 50-75 ml).
3. Place beaker on hotplate and heat to boiling (pigments will extract out into the EtOH). Remove from heat.
4. Fold a piece of filter paper in quarters, place into a funnel and moisten it with a few drops of EtOH.
5. Put the end of the funnel into a test tube. Pour the pigment extract through the filter paper into the test tube.
6. Take 0.5 ml of the filtered pigment extract and put it into a cuvette containing 5ml of EtOH.
7. Pipette 5 ml of EtOH into another cuvette for your blank.B. Using acetone
1. Obtain a spinach leaf and place it in a mortar. Add approximately 5 ml of acetone. (when using acetone it is important to use glass pipets, plastic pipets will dissolve in the acetone). Gently grind the leaf with a pestle to release the photosynthetic pigments.
2. Fold a piece of filter paper in quarters, place into a funnel and moisten it with a few drops of acetone.
3. Put the end of the funnel into a test tube. Pour the pigment extract through the filter paper into the test tube.
4. Take 0.1 ml of the filtered pigment extract and put it into a cuvette containing 5ml of acetone.
5. Pipette 5 ml of acetone into another cuvette for your blank.Doing the absorption spectrum
1. Set the spectrophotometer to 380 nm. Blank the spec, then take a reading of your extract. (note: you may do these readings at the same time as you do your % transmittance spectrum of the color filters, just be sure to use the appropriate blank and read the correct scale).
2. Set the spec to 400 nm. Reblank and take an absorbance reading of your extract.
3. Continue taking readings at 20 nm intervals up to 700 nm, being sure to reblank at each wavelength before taking the reading of your extract.Transmittance of color filters
1. Cut a piece of one of the color filters used in your experiment such that the piece will fit completely around the inside of a cuvette. Fill the cuvette with tap water. Do this for each color filter you used.
2. Fill a cuvette with water for your blank. Set the spec to 380 nm.
3. Blank the spec with the water blank then sequentially take the % transmittance reading for each color filter.
4. Set the spec to 400nm, reblank with water, and read all cuvettes containing the color filters. Continue taking readings at 20 nm increments to 700 nm, being sure to reblank at each change of wavelength.Your lab report
1. Include a methods section.
2. Your results section should state your results and include figures showing what happened at different light intensities, at different wavelengths of light, an absorption spectrum, and the % transmittance of the various color filters at different wavelengths.
3. Your discussion should interpret your results; be sure to tie together your results of the absorption spectrum and % transmittance of color filters with your data of the leaf disks under different color filters.
Pigment Extract Data
Bacterial Growth
Objectives
1.Measure growth of a bacterial culture by spectrophotometry
2.Quantitate viable cells in a bacterial culture by standard plate count
3.Observe differentially stained bacteria using light microscopy
4.Determine the effectiveness of some common chemical disinfectants and antibiotics
Background
A. Growth measurements
When attempting to study the basic processes of life, biologists often turn to “simpler” organisms to make their observations and develop working theories. Observations are then made on more complex biological systems to determine if the information obtained from the simpler organisms can be extrapolated to the higher forms. For this lab we shall use prokaryotic organisms, bacteria, to examine the growth process.
Bacteria are a diverse group of small, single-celled organisms in the kingdoms Eubacteria and Archaebacteria. Found in virtually every extreme of all habitats, they have existed on earth longer and are more widely distributed than any other group of organisms. Bacteria have their genetic material organized in a circular DNA molecule that is not surrounded by a nuclear membrane. Reproduction is by binary fission with the formation of two equal size progeny. During active bacterial growth the size of the population continuously doubles, one cell becomes 2, 2 become 4, etc. in a geometric progression. When bacteria are inoculated into a fresh medium, the resulting culture exhibits a characteristic growth curve of four distinct phases (Fig. 1). During the lag phase there is no increase in cell number, but a time when the cells prepare for synthesis of DNA and enzymes needed for cell division. This is followed by the log phase where the culture reaches its maximum rate of growth for specific conditions. The time required to achieve doubling of the population is known as the generation time. The generation time will vary from organism to organism and will vary in different environmental conditions. The graphical determination of doubling time can be made by extrapolation (Fig. 2). As the bacteria multiply, nutrients are exhausted and inhibitory metabolic end products accumulate. These conditions give rise to the stationary phase which represents no net increase in numbers (growth rate equals death rate). Given enough time there will be a total decline in cell number-the death phase.
Figure 1. Bacterial Growth CurveFigure 2. Determination of Generation TimeIn this lab you will quantitate bacteria by the two most widely used methods: viable plate count and spectrophotometric analysis. In liquid culture, the medium appears more and more cloudy as the bacteria increase in number by division. A tube of bacteria will tend to reflect light so that less light is transmitted through the tube. A spectrophotometer can measure the amount of light passing through the tube, or conversely the amount of light absorbed. These measurements of turbidity or optical density (OD) are not direct measurements of bacterial numbers, but an indirect measurement of cell biomass which includes both living and dead cells. As the bacterial cell population increases, the amount of transmitted light decreases, increasing the absorbance reading on the spectrophotometer. (Fig. 3). If one takes readings of the same culture over time, the absorbance readings will increase as the cell number increases. This can then be graphed to show the growth curve for the particular conditions being tested. There are some limitations with this method, though. A growth curve that includes the lag,log, and stationary phase will take several hours to complete and the relationship between cell number and absorbance will begin to deviate from linearity at high cell densities. Generally an absorbance reading or O.D. of 0.8 is about as high as one should try to measure. To give you an idea of how the turbidity measurements correspond to actual numbers, more than a million cells /ml need be present in order to get even a trace of a measurement on the spectrophotometer.
Figure 3. Spectrophotometric determination of cell densities
To quantitate viable cells a plate count is done. A sample of bacteria is diluted in a sterile medium until the numbers are very low. This diluted sample of bacteria is then transferred onto an agar plate and spread out evenly so that each cell is separate from the others. Each viable cell will continue to divide into a discrete colony of millions of bacterial cells which can now be seen with the naked eye. These colonies can then be counted. Keeping in mind that each colony arose from a single cell that was plated onto the agar, the number of colonies can be used to determine the number of bacterial cells present in the original culture.
For this portion of the lab you will use Escherichia coli, a bacterium which is found by the hundreds of grams in the human lower digestive track. Of all microbes, E. coli is probably the most utilized by biologists and biochemists. It has fairly simple growth requirements and has a fairly rapid growth rate which makes it useful for a one laboratory period experiment.
B. Gram Stain
Most bacteria are characterized by having not only a cell membrane but also a cell wall which lies outside of the cell membrane. This cell wall is composed mostly of peptidogycan and helps to maintain osmotic pressure and the cell’s characteristic shape. Some taxonomic groups of bacteria also have an outer membrane that is attached to the peptidoglycan by small lipoprotein molecules (Fig. 4). This difference in outermost cell structure is the basis for classification of bacteria by a differential staining technique known as the Gram stain. Gram-positive cells (those without an outer membrane) stain purple in the procedure, gram-negative cells (which have the outer membrane) stain red or pink. The usual first step in any bacterial identification is the determination of whether or not it is a G+ or G- bacterium. A sample of the bacteria in question is first stained with the primary cationic dye crystal violet. Since most bacteria carry a net negative charge at pH 7 they pick up the dye. At this point morphological features such as relative size, shape, and characteristic arrangement of cell groups can be seen. A mordant (in this case Gram's iodine) is then added to form a tighter complex between the stain and the cells . To remove any excess stain or stain that has not adhered to the cell, a decolorizing agent is then added(ethanol). At this point gram-positive cells are purple and gram-negative cells are colorless. Cells are then stained with a counterstain (safranin). Gram-negative cells will pick up the counterstain and appear red or pink. In this lab you will be given an unknown bacterium that you will be asked to identify as either gram positive or negative.
Figure 4. Schematic diagram of the cross section of bacterial cell walls. Note that although both gram-positive and gram-negative cells have a layer of peptidoglycan that gives the cell its rigidity and strength, the peptidoglycan layer in gram-positive cells tends to be thicker. Gram-negative cells have an outer membrane that gram-positive cell do not.
C. Antimicrobials
A variety of substances are used to control the growth of unwanted bacteria. These chemicals may be divided into three main subdivisions: disinfectants (used on inanimate objects such as tabletops to reduce the level of bacterial contaminants), antiseptics (used on the surface of living tissue) and antibiotics (absorbed or taken internally). No single antimicrobial substance is ideal in all situations. Antimicrobial agents must be matched to specific organisms and environmental conditions as they all have different modes of action. Lysol, for example, is a disinfectant made of 50% cresol and 50% vegetable oil. Its germicidal effect is due to the fact it causes proteins to denature. Hexachlorophene, a chemical added to soaps and lotions has similar germicidal activity. Some antibiotics inhibit protein synthesis, others inhibit cell wall synthesis (Appendix 1). A simple way of determining the susceptibility of microorganisms to a particular antimicrobial is to inoculate an agar plate with the bacteria to be tested. A filter disk impregnated with the antimicrobial substance is then placed onto the same plate. The antimicrobial substance will diffuse into the agar medium, the concentration decreasing the further away from the disk. If the antimicrobial has an effect on the bacteria, a clear zone of inhibition will form around the disk. The larger the zone, the more effective the chemical is at preventing bacterial growth. This is known as the Kirby-Bauer method of anti-microbial testing. In this lab you will test the antimircrobial effect of a number of chemicals on a gram-negative and a gram-positive microbe.
D. Sterile Technique
The handling of bacterial cultures requires aseptic (sterile) techniques in order to avoid contamination of your experimental bacterial cultures from the millions of microorganisms present in the surrounding environment and to prevent contamination of you and your lab space by the culture you are using. These procedures (Appendix 2) ideally should be followed in a bacteriological hood, but in this lab, since we are starting out with a huge inoculum for the growth curve, the experiment should be fine if the procedure is closely followed, without the use of a hood.
Laboratory Procedure
A. Growth Curve
1. Each group of students will receive a 125 ml flask containing 50 ml of nutrient broth (prewarmed to 37oC--why?). The lab instructor will then add E. coli which is in “log phase” (exponential growth phase) to each flask.
2. Swirl the flask so there is an even suspension of bacteria. Pipette 3.0 ml out of the flask into a cuvette (this will be your 0 time point, be sure to record the time). Replace the cap and put flask back in 37oC incubator (the culture is shaken to keep it mixed and aerated). Dispose of pipets in the appropriate container.
3. Read absorbance of 0 time point –
4. In order to obtain a good growth curve you should take 4 more turbidity (absorbance) measurements, roughly one every 20 minutes following steps 3a-c above. Try to do them as quickly as possible. To avoid cooling the culture take out the 3 ml needed for an absorbency reading and immediately return the stoppered stoppered flask to the 37oC incubator, then take reading.
B. Plate Count
1. At each table will be a set of four dilution tubes along with a flask of sterile saline (0.85% NaCl). Using one pipette tip for the series, add 9.9 ml of saline to tubes #1 and # 2, and 9.0 ml to tube #3 and #4.
2. Label the bottom of 3 petri dishes with the date, some identifying name, and A,B or C.
3. At one of the time points for the turbidity measurements (in this case where the O.D. is between 0.08 and 0.1) remove 0.1 ml from your growing culture and add it to the first tube of your dilution series. Thoroughly mix.
4. Remove 0.1 ml from dilution tube #1 and deposit it into tube #2. Mix thoroughly.
5. Remove 1.0 ml from tube #2 and deposit the entire 1.0 ml into tube #3. Thoroughly mix. With the same pipette tip deposit 0.1 ml from tube #2 onto the appropriately marked agar dish. Spread the bacteria evenly over the entire surface of the agar with a sterile loop and replace the cover. Dispose of loops in designated container.
6. Remove 1.0 ml from tube #3 and deposit the entire 1.0 ml into tube #4. Mix. With the same pipette tip deposit 0.1 ml from tube #3 onto another petri dish. Spread as directed above.
7. Deposit 0.1 ml from tube #4 onto a third plate. Spread. Dispose of all pipets and loops in appropriate containers.
8. If reading through this has confused you, look at the flow sheet - Appendix 3.
9. After approximately 10 minutes turn the petri dishes upside down to prevent condensation from falling on the agar. Why? Place plates in 37oC incubator.
10. The following day come to lab and count the bacterial colonies on the one plate with 30-300 colonies. Do NOT open plates. Dispose of plates in appropriate container.C. Gram Stain
1. Obtain a clean glass slide
2. Prepare a smear of the organisms to be stained by taking a loopful of the bacteria (with a sterile yellow loop) and spreading it over a small area in the center of the slide. Be sure you keep track of which organisms you used.
3. Allow the smear to air dry and then heat fix by passing the slide quickly through a flame.
4. Place the slide on paper towels and add a drop or two of crystal violet to the smear, let set 1 minute.
5. Gently wash the stain off with tap water, being careful not to wash off bacteria.
6. Apply Gram’s iodine, let set 1 minute.
7. Gently wash the iodine off with tap water and then add the decolorizing agent (EtOH) drop by drop until it runs clear.
8. Wash off the decolorizing reagent with tap water.
9. Counterstain with safranin by adding 1-2 drops and let it set for 45 seconds.
10. Rinse with tap water and look at under the microscope. Determine if bacterium is Gram + or-.
D. Antimicrobials
1. Obtain two agar plates. Label bottoms with date, name, and type of bacteria (use the same two types of bacteria as you used for the Gram stain).
2. Add 0.5 ml of bacteria to the agar plate and spread with a sterile loop. Be sure to dispose of pipet tip and loop in the appropriate container.
3. Determine which antimicrobials you want to test. There are disks that have already been saturated with antibiotics. If you want to use antiseptics and disinfectants you will need to soak sterile disks in the material you want to test (pick up a disk with forceps and place into liquid to be tested until it is saturated). Use the same antimicrobials for both types of bacteria so you may do a comparison between gram positive and gram negative bacteria.
4. Number either on the bottom of the plate or the side 1-6.
5. Place saturated disks onto agar plate (fig 5).
6. Place plates into 37o incubator.
7. Come in tomorrow and record the zones of inhibition for each substance tested (fig 6). Do NOT open plates. When all data are collected dispose of plates in appropriate container.Figure 5. Place 6 disks (each containing a different antimicrobial) evenly spaced on the agar as above.
Figure 6. Collecting data from Kirby-Bauer antimicrobial experiment
E. Data Work-Up
For this lab you will need to write only a data sheet. Be sure to include the following, appropriately labeled:
1. Growth curve of E. coli. Use Excel or another computer graphing program (plot the absorbance on the y-axis vs. time on the x-axis, this needs to be a semi-log plot to get a straight line, see instructions in graphing section of lab manual).
2. Doubling time of E. coli. Determine the doubling time from your graph.
3. Plate count of E. coli. . Be sure to state which plate in the dilution series you counted.
4. Determine the concentration of bacteria in your culture flask at the time your sample for the serial dilution was taken.
5. Gram stain results.
6. Results of your antimicrobial tests on E. coli and B. cereus - include a table or figure and briefly state your results.Appendix 1.
Agents Used to Control Microbial Growth
A. Antiseptics and Disinfectants
1. Phenols and phenolics - these compounds inactivate proteins, denature enzymes, and injure plasma membranes and should only be used on surfaces. Examples include Lysol, hexachlorophene, and pHisoHex.
2. Halogens – may be used on surfaces either alone or as components of organic or inorganic solutions to inactivate enzymes and other cellular proteins. Tend to be strong oxidizing agents. Iodine combines with the amino acid tyrosine, chlorine when added to water forms hypochlorous acid. Betadine is another example often used instead of iodine.
3. Alcohols – denature proteins and dissolve lipids. Examples include ethanol and isopropanol.
4. Heavy metals – such as silver, mercury, copper, and zinc exert their influence through oligo-dynamic action such as combining with the sulfhydryl (-SH) groups and denaturing proteins. Examples include silver nitrate, mercurochrome, and copper sulfate.
5. Surface active agents – soaps and detergents decrease the tension between molecules that lie on the surface of a liquid.
6. Quaternary ammonium compounds (quats) –cationic detergents attached to NH4+ disrupt plasma membranes, denature proteins, and inhibit enzymes. Examples include Cepacol and Zephran.
7. Organic acids – used in the food and cosmetic industry to prevent growth of microorganisms. Examples include sorbic acid, benzoic acid, and propionic acid.
8. Aldehydes – formaldehyde and glutaraldehyde attach methyl or ethyl groups to DNA and proteins making them nonfunctional.B. Antibiotics
1. Inhibition of cell wall synthesis – may inhibit synthesis of petidogylcan. Include penicillins, cephalosporins, vancomycin, bacitracin, oxacillin, and nafcillin.
2. Damage to plasma membrane – polymyxin B, nystatin, and amphotericin B.
3. Inhibition of protein synthesis – streptomycin (causes misreading of codons on mRNA), chloramphenicol (prevents peptide bond formation between amino acids), tetracyclines (prevents hydrogen bonding between anticodon on tRNA-aa complex and codon on mRNA), kanamycin, erythromycin, and gentamicin.
4. Inhibition of nucleic acid synthesis – rifamycin, actinomycin D, nalidxic acid, ciprofloxacin, and norflaxacin.
5. Structural analogs – such as sulfonamides that are structurally similar to cellular metabolites and compete with these in enzymatic reactions.Appendix 2.
Aseptic Techniques to be Used in this Lab
1. Before handling of cultures:
2. Before and after handling cultures:
3. While working with cultures:
Appendix 3 - Serial Dilution.
Determining the Free Chlorine Content of Swimming Pool Water
Beaker Number
1. Obtain swimming pool water sample (25 mL is needed for each experiment)
2. Make up a 10mg/L free-chlorine standard in a 100ml beaker from your chlorine ampule standard.
3. Obtain six 100ml beakers and label 1 through 6.
4. Make up the solutions for you free-chlorine standard:
Beaker Number Free-chlorine standard (10mg/L) (mL) Distilled H2O (mL) Free-chlorine concentration (µg/mL) 1 1.00 24.00 0.40 2 2.00 23.00 0.80 3 3.00 22.00 1.20 4 4.00 1.00 1.50 5 5.00 20.00 2.00
5. Add 25ml of your swimming pool water sample to beaker 6.
6. Add one DPD free-chlorine powder pillow to each of the six labeled beakers.
7. Mix beakers thoroughly into the sample using a stirring rod.
8. Obtain and label 7 spec. tubes.
9. Labels one blank and fill with 5ml dH2O.
10. Label the remaining tubes 1 through 6 and fill each with 5m of solution from the matching beaker.
11. Set the wavelength at 565nm on your spec and blank.
12. Record absorbance for tubes 1-6.
13. Graph your standard curve with points 1 through 5.
14. Solve for your unknown concentration using the equation for your standard curve line.
15. If your unknown absorbance was too high (greater than that of point 5) dilute the pool water sample and so analysis again.
Standard Curve: Food Dyes
Make up Red Food Dye Stock Solution:
Stock = 0.1mL red food dye/100mL H2OFor Standard Curve:
1. Make up solutions to be used to determine standard curve:
A = 10mL Red Stock Solution + 10mL H2O B = 10mL A + 10mL H2O C = 10mL B + 10mL H2O D = 10mL + 10mL H2O E = 10mL D + 10mL H2O
2. Read absorbance at 500nm for tubes A-E.
3. Determine the concentration of red dye in mL/100mL.
4. Convert to mL red dye/100mL x 10-3.
5. Make standard curve. Plot absorbance on y axis, concentration of red dye (mL/100mL x 10-3) on x axis.
6. Take absorbance readings of unknowns.
7. Using standard curve, determine concentration of unknowns.
Solutions Absorbance Concentration of red dye(mL/100mL H2O) Concentration of red dye (mL/100mL H2O x 10-3) A B C D E unknown1 unknown2
