EDVO-Kit # Ecological Equilibrium I: Population Studies of ... · a solution that produces no change in cell volume. Osmoregulation is the ho-meostasis mechanism of an organism to

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EDVO-Kit #

958Ecological Equilibrium I:Population Studies of Unicellular Organisms

Storage: Store all componentsat room temperature.

EXPERIMENT OBJECTIVES:

The objective of this experiment is to determine theimpact of varying growth conditions on microbial

growth and measure the number of cells usingthe colony count method.

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EDVO-Kit # 958 Ecological Equilibrium I: Population Studies of Unicellular Organisms

Table of Contents

Page

Experiment Components 3Experiment Requirements 3

Background Information 4

Experiment Procedures Experiment Overview 9 Student Experimental Procedures 10 Study Questions 15

Instructor's Guidelines Notes to the Instructor 17 PreLab Preparation 18 Expected Results 20 Study Questions and Answers 21

Material Safety Data Sheets 22

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• NaOH solution - Component A• HCl solution - Component B• Salt (NaCl) solution - Component C• Yeast Extract - Component D• Tryptone - Component E

• E. coli slant• ReadyPour™ Luria Broth Agar• Nutrient Broth• Petri plates, small• Pipets• Inoculating loops• Sterile swabs• Microcentrifuge tube with attached caps (0.5 ml)• Conical tubes with screw caps (15 ml)• Calibrated transfer pipets.• pH paper

Experiment Components

• Incubation oven• Microwave, hot plate or Bunsen burner (for heating ReadyPour agar)• Waterbath• UV transilluminator• Vortex (optional)• Automatic micropipets (5-50 µl) with tips• Lab glassware• Squeeze bottle• Foil, plastic wrap or parafilm• 95% ethanol• Bleach or disinfectant• Sunscreen• Distilled water• Disposable vinyl or latex gloves• Goggles

Requirements

All components are intended for educa-tional research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.

This experiment is designed for 10 student

groups.

Store all components atroom temperature.

No human or animal blood or blood products are used in this experiment.

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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose

without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved. EVT 090914AM

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nEDVO-Kit # 958 Ecological Equilibrium I: Population Studies of Unicellular Organisms

EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc.

Background Information

A unicellular organism is any life form that consists of just a single cell. Most of life is unicellular, with bacteria serving as the majority. The main groups of unicellular life are bacteria, archaea (prokaryotes), and the eukaryota (eukaryotes). The differences between the prokaryota and eukaryota are significant: eukaryotes possess a nucleus, while prokaryotes lack it, and eukaryotes possess a range of subcellular organs called organelles, while prokaryotes are very minimal.

Unicellular organisms are as diverse as they are ubiquitous. The oldest forms of life, unicellular organisms existed 3.8 billion years ago, if not longer. They pursue a variety of strategies for survival: photosynthesis (cyanobacteria), chemotrophy (many archaea), and heterotrophy (amoeba). Some unicellular organisms have flagella, little tails they use for locomotion, or lobopods, extensions of the cellular skeleton (cytoskeleton), which appear as blob-like arms. The flagellum of our unicellular ancestors is retained all the way up into the animals, where it makes an appearance as flagellated sperm.

Of all the six-eukaryote supergroups, four are exclusively composed of unicellular organisms. Only the opisthokonts, consisting of animals, fungi, and close relatives, and the archaeplastids, consisting of both unicellular and multicellular plants, are exceptions. Unicellular organisms vary in size, with the smallest bacteria measuring only a third of a micron (300 nanometers) across, ranging up to the titantic plasmodial slime molds, which can grow to 20 cm (8 in) across. The largest unicellular organisms may have millions of nuclei scattered throughout the cellular envelope. To observe some of the smallest unicellular organisms requires an expensive electron microscope, while the very largest can be seen with the naked eye.

One can observe the larger unicellular organisms, such as amoebae, by using the higher settings on a light microscope. Bacteria just appear as dots. To gather unicellular organisms for observation, one can place a cover slip on the surface of pond water, and leave it overnight. By the next morning, nu-merous unicellular organisms will have grown entire colonies on the bottom of the slip. Unicellular organisms replicate fast: colonies can double their size in between 30 minutes and a few hours.

Practically every phrase of microbiology requires methods for measuring microbial number. The most common method is the plate or colony count. This method is based on the theoretical relationship of one bacterial cell, or clump of cells, giving rise to one colony and on the assumption that the number of colonies that develop on an agar plate corresponds to the origi-nal bacterial count. To obtain the bacterial count, the sample is diluted and plated, and following the incubation the colonies that develop are counted. To make a tenfold dilution of a sample, one will add 1 ml portion of the original sample to a tube containing 9 ml of sterile media. By continuing this dilution stepwise through additional dilution tubes, one can dilute the sample 10-2, 10-3, 10-4, and more in a manner outlined in Figure 1.

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved. EVT 090914AM

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Figure 1 – Diluting and plating sample for determination of microbial numbers.

Microbes, like all forms of life, are greatly influenced by their surround-ings. Environmental effects fall into three general categories: physical – the effects of temperature or extreme pressure; chemical- the need for food and response to poison; and biological – the influences of coexisting spe-cies. There is a particular optimum set of environmental conditions for each microbial species. The human body, for example, has a rigidly regulated nor-mal temperature of 37°C that must remain constant despite of exposure to extreme heat or cold. The bacterial cell has no regulated body temperature, but assumes the temperature of its environment. Its response to prolonged exposure to extreme cold or heat is merely the stopping of the enzymatic activities; it does not necessarily die as would higher forms of life. Temperature profoundly influences the growth of bacteria. Different types of bacteria have distinct requirements as to the temperature at which they will grow. Between the maximum temperature, above which a culture will not develop, and a minimum temperature, below which a culture will not develop, is a temperature range in which growth will occur. The best growth takes place within a rather limited range called the optimum temperature. The influence of temperature on growth is actually a measure of the influ-ence of temperature on the enzyme actions of the cell. As the temperature is lowered, the enzyme activity, thus the growth of the cell, is lowered. As the temperature is raised above the optimum for growth, metabolic activity is markedly increases, but at the same time, the rate of enzyme and protein breakdown (due to protein denaturation) markedly increases, resulting in damage and death of cell.

1 ml

1 ml 1 ml 1 ml 1 ml 1 ml

Originalinoculum 9 ml broth

in each tube

Dilutions 10-1 10-2 10-3 10-4 10-5

10-1 10-2 10-3 10-4 10-5

Plating

1 ml 1 ml 1 ml 1 ml

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Each species of microbe has its own characteristic range of pH values in which it grows and reproduces best. Most bacteria grow best around neutral pH values (pH 5.5 - 8.5). Bacteria that grow best at this pH range are referred to as neutrophiles. However, some bacteria thrive in very acid conditions (pH 1 - 5.5). They are called acidophiles. Other bacteria can live in very alkaline environment (pH 8.0 - 11.5). Such alkaline loving microbes are called alkaliphiles. Since pH limits the types of microorganisms capable of growing under given conditions, its control has considerable practical importance. For instance, sulfur is sometimes added to soil to inhibit the growth of Streptomyces scabies microbes causing potato scab. Sulfur is converted to acid by certain soil bacteria, and it is this resulting drop in the pH that inhibits the potato pathogen.

Bacterial cells and the media in which they grow have independent osmotic concentrations – a function and relationship of substances in solution. When a bacte-rial cell is placed in a medium, an osmotic pressure is exerted across the semipermeable membrane that surrounds the cell. Osmotic pressure is an important factor affecting cells. When the osmotic concentra-tion of the medium is higher than that of the cell interior, the medium is said to be hypertonic with respect to the cell. In a hypertonic medium, water leaves the cell, and the cytoplasmic membrane shrink

Figure 3 - Growth rate vs. pH for three environmental classes of prokaryotes.


0°C 20°C 37°C 50°C

Increase

Enzyme destruction

Point of diminishing returns

Enzyme activity

Total work done

Figure 2 - Relationship between temperature and enzyme destruction in a bacterial cell.

GrowthRate

1 2 3 4 5 6 7 8 9 10 11

Acidophiles Neutrophiles Alkaliophiles

pH


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Figure 4 – Effect of osmotic pressure on blood cells.

A microorganism’s growth in a culture medium depends partly upon the amount of available energy. Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. The chemicals and elements of the environment that are utilized for bacterial growth are referred to as nutritional requirements, a source of carbon and other required nutrients. The nutritional requirements vary widely among microorganisms. Autotrophic microorganisms, including those that are pho-tosynthetic, can grow and synthesize their cell materials solely from inorganic compounds; heterotrophic species requires one or more organic nutrients. At an elementary level, the nutritional requirements of a bacterium such as E. coli are revealed by the cell's elemental composition, which consists of C, H, O, N, S. P, K, Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo. These elements are found in the form of water, inorganic ions, and other small molecules.


away from the rigid cell wall – a condition called plamolysis. In contrast, when the osmotic concentration of the medium is lower than that of the cell interior (a hypotonic medium), water flows across the cell membrane, causing the cell to swell. In plant cells, the cell wall restricts the expansion, resulting in pressure on the cell wall from within called turgor pressure. Isotonic is the presence of a solution that produces no change in cell volume. Osmoregulation is the ho-meostasis mechanism of an organism to reach balance in osmotic pressure. The effect of osmotic pressure is of great practical importance in the bacteriology of food. The preservation of certain food (salted meat, jam, condensed milk) is partially or completely due to their high osmotic concentrations.

H2O H2O H2O

Hypertonic Isotonic Hypotonic

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Ultraviolet (UV) light kills cells by damaging their DNA. The light initiates a reaction between two molecules of thymine, one of the bases that make up DNA. UV light at this short wavelength causes adjacent thymine molecules on DNA to dimerize. The resulting thymine dimer is very stable. If enough of these defects accumulate on a microorganism's DNA its replication is inhibited, thereby rendering it harm-less. The longer the exposure to UV light, the more thymine dimmers are formed in the DNA. If cellular processes are disrupted because of DNA damage, the cell cannot carry out its normal function. If the damage is extensive and widespread, the irradiated bacterial cell will die.

The series of exercises in this experiment will illustrate the response of E.coli to various environmental condi-tions – temperature, pH, osmotic potential, energy source, and ultraviolet light.

Figure 5 – Lethal action of Ultraviolet light on DNA.


G C

G C

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Wear gloves and safety goggles

Experiment Overview

BEFORE YOU START THE EXPERIMENT:

1. Read all instructions before starting the experiment.

2. Write a hypothesis that reflects the experiment and predict experimental outcomes.

LABORATORY SAFETY:

1. No human materials are used in this experiment. Gloves and safety goggles should be worn as good laboratory practice.

2. Exercise extreme caution when working with equipment which is used in con-junction with the heating and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS OR BULBS.

4. The bacterium used in this experiment is not considered pathogenic, but all microorganisms have the potential to cause disease in some individuals. Although E. coli is rarely associated with illness in healthy individuals, it is good laboratory practice to follow simple safety guidelines in handling and disposal.

At the completion of the experiment:

A. Wipe down the lab bench with a 10% bleach solution, disinfectant or soapy water.

B. All materials, including petri plates, pipets, transfer pipets, loops and tubes , that come in contact with bacteria should be disinfected before disposal in the garbage. Disinfect materials as soon as possible after use in one of the following two ways:

• Autoclave at 121° C for 20 minutes. Tape several petri plates to-gether and close tube caps before disposal. Collect all contaminated materials in a autoclavable, disposable bag. Seal the bag and place it in a metal tray to prevent any possibility of liquid media or agar from spilling into the sterilizer chamber.

• Soak in a 10% bleach or disinfectant solution. Immerse petri plates, open tubes and other contaminated materials into a tub containing a 10% bleach solution. Soak the materials overnight and then discard. Wear gloves and goggles when working with bleach.

C. Always wash hands thoroughly with soap and water after handling con-taminated materials.

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sEDVO-Kit # 958 Ecological Equilibrium I: Population Studies of Unicellular Organisms

Student Experimental Procedures

VARIOUS ECOLOGICAL CONDITIONS/VARIABLES:

Temperature:

This option requires 4-6 different chambers (incubators, refrigerators, etc.) for incubation of cultures. Suggested temperatures are 4° C, 20° C, 37° C, and 65° C. It is important to maintain accurate temperatures.

1. Dispense 5.0 ml sterile nutrient broth into sterile tubes labeled with the desired temperature growth conditions.

2. Obtain the OD550 0.05 stock culture of E.coli from your lab instructor. Mix the culture in between each aliquot and dispense 10 µl of this culture into each of the tubes containing the nutrient broth from step 1.

3. Cap loosely and place the tubes in the desired incubator.

4. Grow the cultures for no longer than 19 hours.

5. Continue with "General Plating Procedure" on page 13.

pH:

With this option, students will use NaOH (a base which raises pH) and HCl (an acid which lowers pH) to adjust the pH of the bacterial growth medium used for grow-ing the cultures. Suggested pHs are 4.0, 7.0, and 10.0. Use caution when working with NaOH and HCl – wear gloves and safety goggles!!

1. Aseptically dispense 5.0 ml sterile nutrient broth into tubes labeled with the desired pH.

2. Add diluted HCl or diluted NaOH (start by adding one drop at a time) to the tubes containing nutrient broth. Cap, mix, and use the pH paper to check the pH of each tube. Adjust by adding additional NaOH or HCl until the desired pH is obtained.

3. Use the OD550 0.05 stock culture of E.coli previously obtained from your lab instructor.

4. Mix the culture in between each aliquot and dispense 10 µl of this culture into each of the tubes from steps 1 and 2.

5. Cap loosely and place the tubes in a rack in a 37°C incubator.



Loops and other materials used in this experiment should be properly disposed in a designated disinfectant tray.

HINT: Any dilution or plat-ing procedure will be simplified if dilution tubes and petri plates are arranged at the beginning. Mark the tubes and plates ac-cording to the condi-tions and dilutions.


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Osmotic Potential:

By adjusting the salt concentration of the growth medium, students will deter-mine the effect of changing the osmotic potential on the growth of the bacteria. Suggested salt concentrations to try: 0.1 M, 0.3 M, 0.5 M, 1.0 M

1. Use the following formula to calculate the salt concentration:

V (M) ÷ FV = FM

Example:

V = ? M = 5 M NaCl FV = 6 ml FM = 0.5 M NaCl

V (5 M) ÷ 6 ml = 0.5 M V = 0.6 ml of 5 M NaCl to a final volume of 6 ml (with sterile nutrient broth)

to yield 0.5 M NaCl. Label accordingly.

2. Repeat Step 1, using the above formula to calculate the salt concentration for the remaining growth medium tubes (i.e. 0.1M, 0.3M, 1.0M).

3. Use the OD550 0.05 stock culture previously obtained from your lab instructor.

4. Mix the culture in between each aliquot and dispense 10 µl of this culture into each of the tubes from steps 1 and 2.

5. Cap loosely and place the tubes in a rack in a 37°C incubator.



Energy Source:

With this option, students can make adjustments to the nutrient source in the bacterial growth medium used for growing the cultures and decide which combi-nations yield optimum growth.

1. Aseptically dispense 5.0 ml sterile H2O into tubes.

2. Add 100 µl of NaCl stock to each tube. Label tubes and add varying amounts of tryptone and yeast extract concentrate to the desired concentration. Suggestions include 1 ml yeast extract/1 ml tryptone, 2 ml yeast extract/1 ml tryptone, 1 ml yeast extract/2 ml tryptone, 1 ml yeast extract/no tryptone, no yeast extract/1 ml tryptone, etc.

Use caution when working with NaOH and HCl – wear gloves and safety goggles!!

V = volume of salt to be addedM = molarity of salt stockFV = final volume of total solutionFM = final molarity of salt in total solution

Invert plates (agar side up) during incubation. This prevents condensation from dropping onto the agar surface.

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Energy Source, continued...

3. Use the OD550 0.05 stock culture previously obtained from your lab instruc-tor. Mix the culture in between each aliquot and dispense 10 µl of this culture into each tube from Steps 1 and 2.

4. Cap loosely and place all the tubes in a rack in a 37°C incubator.



Ultraviolet Light:

Students will observe the effects of ultraviolet rays and the effectiveness of sunscreen in protecting the cells from the harmful light. By exposing the cultures to UV light at different time intervals, students will also observe the correlation between UV light exposure and cell death.

1. Obtain the OD550 0.05 stock culture of E.coli. Mix and add 0.1 ml of the culture to a fresh tube containing 5 ml of sterile nutrient broth.

2. Mix well, cap the tube, and allow the culture to incubate for 2 hours (shak-ing or stationary) at 37°C.

3. Set up 3 microcentrifuge tubes as follows: • Tube 1: Cover its body with aluminum foil and label it as "CP" (Complete Protection). • Tube 2: Apply a thin layer of SPF sunscreen around the tube. Label the tube as "SA" (Sunscreen Application). • Tube 3: Leave the tube as-is and label it "CE" (Complete Exposure).

4. After the 2-hour incubation time, remove the culture tube from the incuba-tor. Transfer 500 µl of the culture into each of the tubes in step 3.

5. Starting with the tube labeled "CP", mix and lay it on its side directly on a transilluminator (254 - 302 nm).

6. Set the timer for 20 seconds. Turn on the transilluminator to expose the cells to the light for 20 seconds. Be sure to shield eyes and skin!

7. Turn the light off and remove 50 µl to a tube labeled "20 sec" and place on ice.

8. Repeat the exposure for different time periods until you have collected a sufficient number of data points. Remember to take samples from the same culture tube that has been exposed to increasing time on the UV light source.

9. Repeat steps 6-8 for the "SA" and "CE" tubes.

10. After all the time points have been collected, follow steps 3 - 6 of the "Gen-eral Plating Procedure" on page 13 to inoculate the plates.


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GENERAL PLATING PROCEDURE

1. After the incubation period is complete, remove cultures from the 37°C incu-bator and proceed to diluting (if required) and plating.

Temperature: See Table I on page 14 for dilutions. pH, Osmotic Potential, Energy source: See Table II on page 14 for dilutions.

2. Aseptically dispense 5 µl of each culture onto a labeled agar plate. Using a sterile loop, spread the culture evenly over the entire surface of the plate. Discard the loop in the disinfectant tray.

3. Stack your group's set of plates on top of one another and tape them together. Put your initials or group number on the taped set of plates.

The plates should be left in the upright position to allow the samples to be absorbed by the agar.

4. Place the set of plates in a safe place designated by your instructor.

5. After the samples are completely absorbed by the agar, you or your instructor will place the plates in the inverted position (agar side on top) in a 37°C bacterial incubation oven for overnight incubation (19-24 hours).

If the samples have not been absorbed into the medium, it is best to incubate the plates upright. The plates are inverted to prevent condensation on the lid, which could drip onto the culture and interfere with experimental results.

continued

Spread in one

direction

Same plate -spread again in

opposite direction.

To avoid contamination when plating, do not set the lid down on the lab bench -- Lift the lid of the plate only enough to allow spreading. Be careful to avoid gouging the loop into the agar.

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TABLE I: DILUTIONS FOR TEMPERATURE CONDITION

4° C incubated tube: Dilute the original culture to a 10-1 dilution.

20° C incubated tube: Dilute the original culture to a 10-3, 10-4, and 10-5 dilution.

37° C incubated tube: Dilute the original culture to a 10-3, 10-4, and 10-5 dilution.

65° C incubated tube: No dilution required.

TABLE II: DILUTIONS FOR PH, OSMOTIC POTENTIAL, ENERGY SOURCE CONDITIONS

Serially dilute the original culture to a 10-3, 10-4, and 10-5 dilution.

GENERAL PLATING PROCEDURE, CONTINUED

6. Store all plates inverted at 37°C. After 19-24 hour incubation, remove plates from incubator, count and record the number of colonies on each plate. Calculate the CFU/ml based on the following formula.

CFU/ml original sample = CFU/plate x (1/ml aliquot plated) x dilution factor

For Example:

89 colonies counted on plate x 1/0.005 ml (aliquot) x 104 (dilution factor) =1.78 x 108 CFU/ml


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Answer the following study questions in your laboratory notebook or on a separate worksheet.

1. Name some of the environmental factors that profoundly influence the growth of a microorganism.

2. Compare and contrast acidophiles and alkaliphiles. Describe one ex-ample to illustrate practical applications of such microorganism in the environment.

3. What force does a bacterial cell experience as it is placed in a medium. What determines whether a cell is hypertonic or hypotonic?

4. In what specific way does ultraviolet light damage bacterial cell?

Study Questions

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17EDVO-Kit # 958 Ecological Equilibrium I: Population Studies of Unicellular Organisms

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Notes to the Instructor

This experiment module contains biologicals and reagents for 10 Lab Groups.

Prelab preparations require approximately 45-60 minutes.

Agar plates can be prepared up to a week in advance.

FAX: (301) 340-0582web: www.edvotek.com

email: [email protected]

Technical ServiceDepartment

Please have the following information:

• The experiment number and title• Kit Lot number on box or tube • The literature version number (in lower right corner)• Approximate purchase date

Mon - Fri9:00 am to 6:00 pm ET


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Pre-Lab Preparations

PREPARING INITIAL CULTURE (INOCULUM)To be done the afternoon before the lab.

1. Use a sterile loop to inoculate 35 ml sterile nutrient broth with a small loopful of cells scraped from the E.coli cell slant.

2. Add the cells to the nutrient broth and cap – vortex or shake well to resuspend cells.

3. Discard the loop in the disinfectant tray.

4. Grow cells @ 37°C overnight (~19 hours). The next day, check OD550 and dilute the cells to 0.05 with additional sterile nutrient broth.

5. Dispense 2 ml of the culture into tubes for each of the 10 groups.

6. The inoculum is now ready for the experiment.

Wear Hot Gloves and Goggles during all steps involving heating.

• Use a sterile 10 ml pipet with a pipet pump to transfer the

designated volume of medium to each petri plate. Pipet care-fully to avoid forming bubbles.

• Rock the petri plate back and forth to obtain full coverage.

• If the molten medium contains bubbles, they can be removed by passing a flame across the surface of the medium.

• Cover the petri plate and al-low the medium to solidify.

Quick Reference: Pouring Agar Plates

PLATING

Pouring LB plates (Prior to the Lab experiment)

Heat to Melt the ReadyPour™ Medium

1. Equilibrate a water bath at 60°C for step 5 below.

2. Loosen, but do not remove, the cap on the ReadyPour™ medium bottle to allow for the venting of steam during heating.

Caution: Failure to loosen the cap prior to heating or microwaving may cause the ReadyPour™ medium bottle to break or explode.

3. Squeeze and vigorously shake the plastic bottle to break up the solid agar into chunks

4. Heat the bottle of ReadyPour™ medium by one of the methods outlined below. When completely melted, the amber-colored solu-tion should appear free of small particles.

A. Microwave method: • Heat the bottle on High for two 30 second intervals. • Using a hot glove, swirl and heat on High for an additional 25

seconds, or until all the ReadyPour™ medium is dissolved. • Using a hot glove, occasionally swirl to expedite melting.

B. Hot plate or burner method: • Place the bottle in a beaker partially filled with water. • Heat the beaker to boiling over a hot plate or burner. • Using a hot glove, occasionally swirl to expedite melting.


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5. Allow the melted ReadyPour™ medium to cool slightly. Placing the bottle in a 60°C water bath will allow the agar to cool, while preventing it from prematurely solidifying.

When the ReadyPour™ reaches approximately 60°C, the bottle will be warm to the touch but not burning hot.

6. Pour small LB plates • Use a pipet pump and 10 ml pipet to dispense 5 ml of ReadyPour™

medium to each plate. You will need a minimum of 50 plates. • Replace the lid onto each plate after the medium has been poured. • Stack the plates and allow them to cool & solidify • Refrigerate plates (wrapped in plastic sleeves) inverted.

PREPARING COMMON SOLUTIONS FOR LAB STATIONS

Instructor will set up various lab stations that will allow the students to study different growth conditions. Student groups are encouraged to explore themselves to these lab stations based on their own interests to determine the impact of varying growth conditions on microbial growth.

1. pH-based Lab Station

a. NaOH Solution: Add all of the concentrated NaOH solution (Com-ponent A) to 45 ml of distilled water. Mix. Label with "diluted NaOH solution".

b. HCl Solution: Add all of the concentrated HCl solution (component B) to 45 ml of distilled water. Mix Label this "diluted HCl solution".

2. Osmotic Potential - based Lab Station

a. Remove 3 ml of the provided Salt Solution (Component C) into a tube. Save this tube for the Energy Source-based Lab Station.

b. Use the remaining Salt Solution to provide to this lab station. Stu-dents will use this for their experiment.

3. Energy Source-based Lab Station

a. Salt Solution: provide this lab station the tube containing 3 ml of the 5M Salt Solution. Students will use this for their experiment.

b. Yeast Extract and Tryptone Solutions: provide this lab station the Yeast Extract (Component D) and Tryptone (Component E) solutions that are included in this kit.

Pre-Lab Preparations


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Expected Results

1. The more diluted the culture is, the fewer colonies are seen when cul-ture is plated.

(i.e. 10-5 dilution yield fewer colonies than 10-3 dilution does).

2. When a microorganism is placed out of its optimal growth conditions (temperature, pH, etc.), it will perform less effectively.

(i.e. Higher temperature or higher pH cultures yield fewer colonies than 37°C culture and pH 7.0 colonies.


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Study Questions and Answers

1. Name some of the environmental factors that profoundly influence the growth of a microorganism.

Factors influence bacterial growth are temperature, pH, osmotic pres-sure, energy source and ultraviolet light.

2. Compare and contrast acidophiles and alkaliphiles. Describe one ex-ample to illustrate practical applications of such microorganism in the environment.

Both acidophiles and alkaliphiles greatly depend on the pH of their external environment to live and reproduce. While acidophiles live at pH around 1-5.5, alkaliphiles prefer pH around 8.0 - 11.5.

The S. scabies, a microorganism that can survive indefinitely in slightly alkaline soil but is relatively scarce in highly acid soils, is known to cause potato scab. Sulfur is added to the soil and then converted to acid by certain soil bacteria, and it is this resulting drop in the pH that inhibits the potato pathogen.

3. What force does a bacterial cell experience as it is placed in a medium. What determines whether a cell is hypertonic or hypotonic?

Once a bacterial cell is placed in a medium, it experiences osmotic pres-sure, the movement of water molecules from an area of high concentra-tion to an area of low concentration.

The movement of water into or out of a cell determines whether the cell is hypertonic or hypotonic to its medium. If a cell is hypertonic to its environment (water is in higher concentration inside the cell), water will move by osmosis, to the outside in an attempt to equalize the concentra-tions. Water leaving the cell will cause shrinkage of the cell. If a cell is hypotonic to its environment (water is in higher concentration outside of the cell), water will move, by osmosis, to the inside of the cell, causing the cell to swell.

4. In what specific way does ultraviolet light damage bacterial cell?

Ultraviolet light damages bacterial cell by create chemical bonding between adjacent thymine bases on the same DNA strand, or sometimes between DNA strands, that interferes with the proper replication and function of the DNA molecule. As a result, the irradiated bacterial cell cannot reproduce and will die.

Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

Nutrient Broth

No data

No data

No data

No sata

No data

No data

soluble

light golden colour

NA

CO2, extinguishing powder or water spray.

Use NIOSH approved SCBA and full protective equipment.

Product is not Flammable. Product does not present an explosion hazard.

StabilitySection V - Reactivity Data

Unstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

Causes no irritation, no sensitizing effects.

Swallowed - wash out mouth with water. Skin/eye contact - flush with water Inhalation - remove to fresh air

Yes Yes Yes

No special safety precautions are required.

NIOSH/MSHA approved respirator

N/A N/AN/A N/A

Yes Yes

None known

None known

Smaller quantities can be disposed of with household waste. Disposal must by made according toofficial regulations. Recommended cleansing agent: water, if necessary, with cleansing agents.

gloves, safety goggles

Non Hazardous ingredients

11/13/08

EDVOTEK® X

Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

Ready Pour Agar

1413°C

1.0 @ 865°C

N/A

2.16 g/cm3

804°C

N/A

35.7 g/100g at 0ºC

Colorless crystals or white powder. Characteristic odor.

N.D. = No data

N/A N/A N/A

Suitable extinguishing agents CO2, extinguishing powder or water spray.

Fight larger fires with water or alcohol resistant foam. Firefightersshould wear protective equipment and self-contained breathing apparatus.

During heating or in case of fire, poisonous gases are produced.

StabilitySection V - Reactivity Data

Unstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

Proper disposable gloves Chem. resistant goggles

Impervious clothing to prevent skin contact

Keep away from food and beverages. Immediately remove all soiled or contaminated cloting.

X Stable under ordinary conditions of use and storage.

Reacts with acids, alkalis, oxidizing agents, Lithium and bromine trifluoride.

X Incompatible materials

Yes Yes Yes

Irritant. Irratating to eyes, reapiratory system and skin.

Irritant to skin and mucous membranes. Irritating to eyes.

No sensitizing effects known. If large doses, can cause vomiting diarrhea, and prostration.

If swallowed, seek medical attention. If inhaled, supply fresh air or oxygen. Seek medical attention.If eye contact, rinse open eye for 15 min. Seek medical attention. If Skin, wash with soap and water.

Dispose of in accordance with all applicable federal, state and local environmental regulations.

Keep container tightly closed. Protect from moisture. Suitable for any general chemical storage area. Store away from oxidizing agents.

Prevent formation of dust. Ensure good ventilation. Avoid prolonged exposure.

None required where adequate ventilation conditions exist.

Sodium Chloride, NaCl, Common salt N/A N/A

Emits toxic fumes of chloride and sodium oxide when heated to 801°C.

Ventilate spill area. Wear suitable protective clothing. Wipe up with damp sponge or mop.

50-100 CFM

N/A

11/13/08

EDVOTEK®

Documents

EDVO-Kit # Ecological Equilibrium I: Population Studies of ... · a solution that produces no change in cell volume. Osmoregulation is the ho-meostasis mechanism of an organism to