General Microbiology Laboratory Manualv1yourspace.minotstateu.edu/paul.lepp/General Microbiology... · 2020-02-25 · used. Laboratory personnel have specific training in the procedures

Biol 302 General Microbiology– Spring 2020

General Microbiology Laboratory Manual

BIOL 302

By Paul W. Lepp

First Edition (v1.0) 2020


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TABLE OF CONTENTS

LABORATORY SAFETY RULES...................................................................................................3

WINOGRADSKY COLUMN............................................................................................................9

ASEPTIC TECHNIQUE AND CULTIVATION.............................................................................19

MICROSCOPY..............................................................................................................................25

STAINING.....................................................................................................................................31

BACTERIAL GROWTH................................................................................................................37

AMES TEST.................................................................................................................................44

KOMBUCHA.................................................................................................................................47


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LABORATORY SAFETY RULES

Biosafety in the laboratory Before beginning to work with microorganisms it is important to understand

the risks of working with potentially dangerous living organisms and working in a laboratory setting in general.

The primary responsibility for your safety rests with you. You must understand and follow the rules outlined below, as well as, those provided by the instructor. The most important rule for staying safe in the laboratory is to use your common sense.

You should become familiar with the location and use of the safety equipment in the laboratory which the instructor will point out. This includes a fire extinguisher, fire blanket, eyewash station, safety shower and gas cutoff. In addition, you should be aware of all exits from the rooom.

During this course you will be working with microorganisms that are classifed as biosafety level I and biosafety level II organisms. Biosafety levels are established by the U.S. Center for Disease Control and Prevention. These biosafety levels are described in the section below and summarized in table 1. Bear in mind that you will be working with biosafety level II agents that have the potential to cause disease. The simplest and most effective way to prevent transmission of potentially harmful microorganisms is handwashing, as we will see in one of the following laboratory exercises. Hands must be washed whenever you leave the laboratory.

BIOSAFETY LEVELS

From: • “Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th

Edition” Center for Disease Control and Prevention (CDC) • www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm

BMBL Section III Laboratory Biosafety Level Criteria The essential elements of the four biosafety levels for activities involving

infectious microorganisms and laboratory animals are summarized in Table 1 of this section and Table 1. Section IV (see pages 52 and 75). The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community.

BIOSAFETY LEVEL 1 (BSL-1)


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Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is neither required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science.

The following standard and special practices, safety equipment and facilities apply to agents assigned to Biosafety Level 1: A. Standard Microbiological Practices 1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress. 2. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory. 3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only. 4. Mouth pipetting is prohibited; mechanical pipetting devices are used. 5. Policies for the safe handling of sharps are instituted. 6. All procedures are performed carefully to minimize the creation of splashes or aerosols. 7. Work surfaces are decontaminated at least once a day and after any spill of viable material. 8. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method


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such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility. 9. A biohazard sign must be posted at the entrance to the laboratory whenever infectious agents are present. The sign must include the name of the agent(s) in use and the name and phone number of the investigator. 10. An insect and rodent control program is in effect . B. Special Practices None C. Safety Equipment (Primary Barriers) 1. Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level 1. 2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes. 3. Gloves should be worn if the skin on the hands is broken or if a rash is present. Alternatives to powdered latex gloves should be available. 4. Protective eyewear should be worn for conduct of procedures in which splashes of microorganisms or other hazardous materials is anticipated. D. Laboratory Facilities (Secondary Barriers) 1. Laboratories should have doors for access control. 2. Each laboratory contains a sink for handwashing. 3. The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate.


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4. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment. 5. Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning. 6. If the laboratory has windows that open to the exterior, they are fitted with fly screens.


Table 1. Biosafety levels

BSL Agents Practices Safety Equipment (Primary Barriers)

Facilities (Secondary Barriers)

1 Not known to consistently cause disease in healthy adults

Standard Microbiological Practices

None required Open bench top sink required

2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure

• BSL-1 practice plus: • Limited access • Biohazard warning signs • "Sharps" precautions • Biosafety manual

defining any needed waste decontamination or medical surveillance policies

Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPEs: laboratory coats; gloves; face protection as needed

• BSL-1 plus: • Autoclave available

3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

• BSL-2 practice plus: • Controlled access • Decontamination of all

waste • Decontamination of lab

clothing before laundering

• Baseline serum

Primary barriers = Class I or II BCSs or other physical containment devices used for all open manipulations of agents; PPEs: protective lab clothing; gloves; respiratory protection as needed

• BSL-2 plus: • Physical separation from

access corridors • Self-closing, double-

door access • Exhausted air not

recirculated • Negative airflow into

laboratory 4 Dangerous/exotic agents

which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

• BSL-3 practices plus: • Clothing change before

entering • Shower on exit • All material

decontaminated on exit from facility

Primary barriers = All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure personnel suit

• BSL-3 plus: • Separate building or

isolated zone • Dedicated supply and

exhaust, vacuum, and decon systems

• Other requirements outlined in the text


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GENERAL MICROBIOLOGY LAB RULES

1. If you are pregnant or possibly immunocompromised please see the instructor immediately.

2. Wash hands before leaving lab.

3. Clean the lab table before and after lab with the 10% bleach solution provided.

4. Because the microorganisms used in this class are potentially harmful (BL II), NO eating or drinking is allowed in the lab.

5. All materials and clothes other than those needed for the laboratory are to be kept away from the work area.

6. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposable items are to be placed in the BIOHAZARD container. Reusable items are to be placed in the designated area for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriate container

7. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided on request.

8. Long hair should be tied back while in lab.

9. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab instructor immediately.

10. It is the responsibility of the student to know the location and use of all safety equipment in the lab (eyewash, fire extinguisher, etc.)

11. Reusable items should have all tape and marks removed by the student before being autoclaved.

12. Read labs before coming to class and be on time. Lab instructions will not be repeated if you are late. Do not forget your lab manual. Wait for a laboratory introduction by the instructor before starting work.

13. You may want to wear old clothes to lab. We occasional work with stains that may permanently damage clothing. A limited number of lab coats are available upon request.


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WINOGRADSKY COLUMN

OBJECTIVES

After completing this exercise you should be able to:

1. Describe the anaerobic process occurring in the column 2. Describe the cycles occurring in the column 3. Identify some of the bacteria associated with these processes and

cycles

INTRODUCTION

Sergei Winogradsky discovered in the late 1800s that some bacteria use

light energy to make their own food. Before this time, people believed that only plants and algae did this. He developed these columns as a miniature ecosystem to study soil bacteria. He found that if he added the correct substances in the presence of light, the soil bacteria would form a complex world of microbes. The bacteria at the bottom of the column change the environment throughout the column so that new environmental conditions allow other bacteria to thrive. Microbes thrive on different diets. The variety of diets allows microbes to recycle substances in nature. This is important because it helps eliminate or reduce waste. One building block of natural things is the chemical element sulfur. The rotten egg smell is due to a gas called hydrogen sulfide. It has the element sulfur in it. The gas is made by bacteria in the soil. Other bacteria can use this gas as food. Some of these bacteria are aerobic and grow in the presence of air; others are anaerobic and cannot grow if air is present. Winogradsky Columns allow for the visualization of these different bacteria. Groups of organisms will accumulate into those chemical gradients that they require to live in much the same way they do in nature. A Winogradsky Column is a model of an ecosystem. It is an isolated component of an environment that can be used to study chemical change throughout the soil. The chemicals put into the column cause color changes as microorganisms transform nutrients. The bacteria, when incubated for several weeks to several months, begin to multiply and occupy distinct layers where the environmental conditions favor their growth. As oxygen diffuses downward from the surface (aerobic zone), fermentation causes the breakdown of cellulose (provided by the shredded newspaper) and hydrogen sulfide diffuses upward from the lower aerobic zone. Nutrient cycling occurs by the following processes: the anaerobic bacteria break down the cellulose and produce fermentation products, the fermentation products plus the sulfate (added from calcium sulfate) are used by sulfate-


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reducing bacteria to produce hydrogen sulfide, sulfides are then used by anaerobic photosynthetic bacteria (as seen in the purple and green layers), hydrogen sulfide and oxygen are then used by aerobic bacteria found on the top layer of the mud, algae and other photosynthetic organisms are found in the water area producing oxygen for the aerobic bacteria on the surface of the mud. Isolating these bacteria can be tricky and, like the developing column, many of them, especially the anaerobes, take time to develop. The recipes and isolation techniques given below will give you the basic bacteria that are listed in the chart but these are only the “standard” species and there may be others that develop in the column. There are books listed in the references that will help you to identify other bacteria. Table 1. Physical Properties of Bacteria Bacteria Shape Gram stain Classification Beggiatoa Filamentous, long,

gliding - Non-photosynthetic

Chemolithotropic , sulfur oxidizing

Thiobacillus Rods - Colorless sulfur aerobic, Chemolithotropic

Rhodospirillum Spirals, polarly flagellated

- Purple, non-sulfur, Anoxygenic photosynthetic

Rhodopseudomonas Rods, polarly flagellated; divide by budding

- Purple, non-sulfur, Anoxygenic photosynthetic

Chromatium Ovals or rod, polarly flagellated , Sulfur deposits internally

- Purple sulfur anoxygenic photosynthetic

Chlorobium Straight or curved rods; nonmotile

- Green sulfur anoxygenic photosynthetic

Clostridium Rod + Endospore forming anerobic

Desulfovibrio Vibrio - Sulfur Reducing Anaerobic

Isolation Techniques Unlike many of the bacteria that are used in the classroom, the above eight bacteria, which represent the most common species found in the column, have isolation techniques that are a little more complex. Rhodospirilium and Rhodopseudomonas – both of these organisms are non-sulfur purple bacteria and are cultured using Pfennig’s medium (Brock,


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1988). The concentration of the sulfide should be reduced to .01 to .02% Na2S·9H2O or eliminated and an organic substance added to provide Carbon. Chromatium and Chlorobium – both Chromatium (Kingdom I: Proteobacteria) and Chlorobium (Kingdom VII: Green Sulfur) are both photosynthetic anoxygenic bacteria. Though they are unrelated they have the same function. They each use a specialized medium to isolate them. The basic procedure is to add mud to a jar and enough of the respective medium covers the mud to a depth of .5 cm. It is then incubated for 7 days at room temperature while exposed to light. Then .1ml of the medium is then transferred to enriched agar shake deeps and incubated and additional 4-7 in the same conditions. Slides can then be made from the isolated colonies. The media recipes and more detailed procedure can be found in a number of microbiology laboratory manuals some of which are listed in the reference section. Beggiatoa-(Bej je-ah to’ah) these are extremely interesting organisms. They were the initial bacteria that Winogradsky used to study the role of microorganisms in the cycling of sulfur. Beggiatoa, when isolated under the microscope, can be observed to have sulfur granules stored in its cells. Beggiatoa also produce filaments and can migrate. The organism is cultivated by placing them in a sulfide agar closed tube and overlayed with initially sulfide-free mineral agar. Beggiatoa grow at a well defined interface between O2 and the H2S which migrates upward. Clostridium-these are endospore forming bacteria. They don’t reduce sulfates and are fermentative. They have strong industrial use as producers of ethanol, acetone and butanol. Their main habitat is soil and can have harmful effects on humans causing botulism and gangrene. Thiobacillus-Thiobacillus are chemoautotrophs and require an inorganic source of energy. They are found under aerobic conditions that contain sulfur or sulfides. The predominant method to isolate them is a mixture of Starkey’s Medium, Thiosulfate Medium and a coal dust innoculant. This does take time but is effective. Another method is to take the soil sample and cook it to 80 C. Thiobacillus will sporolate and they can then be isolated and grown. Desulfovibrio-is a sulfate reducer obligate anaerobe. They may also reduce nitrates and just use sulfate in place of the nitrates. Desulfovibrio uses a specialized Desulfovibrio Medium to isolate them.

Microbiology of a Winogradsky Column After a month to six weeks, the column should stabilize into three distinct environments and develop communities of bacteria specific to their environmental requirements and should resemble Figure 1. Aerobic Zone (Oxygen Rich)


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The top of the water column can contain large populations of diverse bacteria. These are aerobic organisms that are found in organic-rich freshwater habitats such as shallow ponds, polluted streams, etc. These are generally flagellated which allows the bacteria to migrate and establish themselves in new areas. In addition, there may be a diverse phototrophic fauna as well from the original water and mud source. At the very top of the zone the mud is characterized by a light brown color. This is the most oxygen rich part of the mud and the most sulfur poor.

Figure 1. Winogradsky column Photosynthetic cyanobacteriacan grow in the upper zones. This area is characterized by a Grass green color These are the only bacteria that have


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photosynthesis like that of plants. In fact, there is very strong evidence that the chloroplasts of plants were originally ancestral cyanobacteria that established themselves as symbionts inside the cells of a primitive eukaryote. Similarly, there is equally strong evidence that the mitochondria of present-day eukaryotes were derived from purple bacteria.

From the mud source below, H2S will diffuse upward into the aerobic zone and can be oxidized to sulfate by the sulfur-oxidizing bacteria such as Beggiatoa and Thiobacillus. These bacteria gain energy from oxidation of H2S, to elemental sulfur and they synthesize their own organic matter from CO2. So they are termed chemoautotrophs. Microaerophillic Zone (Oxygen Scarce) In this zone oxygen diffuses down from the surface but is limited in concentraction. Sulfur from the lower part of the column has begun to move up in the form of H2S. This diffusion of H2S from the sediment into the water column enables anaerobic photosynthetic bacteria to grow. They are seen usually as two narrow, brightly colored bands immediately above the sediment - a zone of green sulfur bacteria, such as Chlorobium, characterized by a green/olive color indicative of growing anaerobic conditions, then a zone of purple sulfur bacteria, such as Rhodospirilum and Rhodopseudomonas, which takes on a red/ orange or rust color. The green and purple sulfur bacteria gain energy from light reactions and produce their cellular materials from CO2 in much the same way as plants do. However, there is one essential difference: they do not generate oxygen during photosynthesis because they do not use water as the reducer; instead they use H2S. The following simplified equations show the parallel processes: 6 CO2 + 6 H20 = C6H12O6 + 6 O2 (plant photosynthesis) 6 CO2 + 6 H2S = C6H12O6 + 6 S (bacterial anoxygenic photosynthesis) Anaerobic Zone (Oxygen Depleted) The only organisms that can grow in anaerobic conditions are those that ferment organic matter and those that perform anaerobic respiration. Fermentation is a process in which organic compounds are degraded incompletely; for example, yeasts ferment sugars to alcohol. Anaerobic respiration is a process in which organic substrates are degraded completely to CO2, but using a substance other than oxygen as the terminal electron acceptor There are three basic levels that form in the lower level of the column. At one level purple sulfur bacteria such as Chromatium, in a Red to Purple layer, are processing Sulfates into Sulfur. At another point Gallionella, a stalked bacteria, processes iron to help create the black layer that forms just below. This level is marked by a strong rust/orange color.


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Some cellulose-degrading Clostridium species start to grow when the oxygen is depleted in the sediment. All Clostridium species are strictly anaerobic because their vegetative cells are killed by exposure to oxygen, but they can survive as spores in aerobic conditions. They degrade the cellulose to glucose and then ferment the glucose to gain energy, producing a range of simple organic compounds (ethanol, acetic acid, etc.) as the fermentation end products. Deeper in the column, the sulfur-reducing bacteria, marked by a deep black layer and typified by Desulfovibrio,canutilize these fermentation products by anaerobic respiration, using either sulfate or other partly oxidized forms of sulfur (e.g. thiosulfate) generating large amounts of H2S by this process. The H2S will react with any iron in the sediment, producing black ferrous sulfide. This is why lake sediments (and our household drains) are frequently black. However, some of the H2S diffuses upwards into the water column, where other organisms utilize it.

Finally, at the bottom, depending on the source of the mud, a pink layer will develop due to purple sulfur bacteria with gas vesicles. A characteristic species is Amoebobacter. This environment is very high in H2S and is more tolerant of air and light.

The Sulfur Cycle The sulfur cycle is a poorly understood process by high school students. In fact, beyond the carbon, nitrogen and oxygen cycle, many students are unaware that other essential elements have a well-defined cycle thoroughout the biosphere. The Winogradsky Column is an excellent way to illustrate this process (Fig. 2)


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Figure 3. Sulfur cycle Any diagram of the sulfur cycle will show that the entire cycle is represented by bacteria species that are present in the Winogradsky Column Each can be isolated and grown to show that, indeed sulfur is cycled through nature just as nitrogen and carbon dioxide. However, the isolation of each species is difficult due to the fact that they are anaerobes and special media are needed as well as time to effectively culture them. Each bacterial species that can be found in the Winogradsky column has a role to play in the cycling of sulfur through the system. Thiobacillus and Beggiatoa are sulfur oxidzers breaking down H2SO4 and H2S So + 3/2 O + H2O H2SO4 They use the energy to fix Carbon Dioxide. If there is a significant source of easily degradable carbon then they may be inhibited. Chromatium and Chlorobium are photoheterotrophs and can oxidize reduced sulfur compounds under anaerobic conditions H2S So SO4 2- Desulfvibrio is anaerobic but grows lithtrophically with H2 as the electron donor, sulfate as the acceptor and CO2 as the sole carbon source. Also nitrate can be substituted as the acceptor. It has a very complex biochemistry. There are far more reactions that occur and this is just a few of the pathways that exist in the column. There are also more species that inhabit the environment and each has its own unique contribution to the sulfur cycle. Iron Chemistry

Sulfur is not the only element being cycled through the column, iron is as well. As the column develops, the mud blackens this is due to the migration of H2S upward and being replaced by FeS .


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MATERIALS

Soil A clear container

Calcium carbonate (CaCO3) Calcium sulfate (CaSO4) Finely shredded newspaper

Plastic spoon and cup Aluminum foil Lab pen

PROCEDURE

1. Label a clear plastic container with your name and the date. 2. Fill one quarter of the cup with soil. Make sure the soil is fairly loose

(has good tilth). 3. Measure out 2.5 g CaCO3 and 2.5 g CaSO4 on to the weigh paper.

Add the chemicals to the soil. 4. Finely shred some newspaper. Add ½ as much newspaper as soil to

the cup. 5. Add tap water to the cup and mix to make a slurry (aka, mud). 6. Fill 1/3 of the clear plastic container with the mud mixture. Use the

handle of the spoon to remove any air bubbles. 7. Fill another 1/3 of the clear plastic container with soil. 8. Fill the remainder of the container with water leaving approximately ½

inch at the top. Mark the water level. 9. Cover with aluminum foil and place in the window.


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WINOGRADSKY LAB REPORT I.D. NO.: LAB SECTION:

1. What did the column smell like after 2 weeks?

2. What did the column smell like after 8 weeks? 3. Why is the bottom of the column black? 4. Where is the aerobic or oxic zone of the column? 5. What bacteria dominate the oxic zone?


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ASEPTIC TECHNIQUE AND CULTIVATION

APPROXIMATE DURATION: 1 HOUR

OBJECTIVES

After completing this exercise you should be able to: 1. Identify various types of media 2. Isolate bacteria using aseptic technique.

INTRODUCTION

ASEPTIC TECHNIQUE

When working with microorganisms it is desirable to work with a pure culture. A pure culture is composed of only one kind of microorganism. Occasionally a mixed culture is used. In a mixed culture there are two or more organisms that have distinct characteristics and can be separated easily. In either situation the organisms can be identified. When unwanted organisms are introduced into the culture they are known as contaminants.

Aseptic technique is a method that prevents the introduction of unwanted organisms into an environment. When changing wound dressings aseptic technique is used to prevent possible infection. When working with microbial cultures aseptic technique is used to prevent introducing additional organisms into the culture.

Microorganisms are everywhere in the environment. When dealing with microbial cultures it is necessary to handle them in such a way that environmental organisms do not get introduced into the culture. Microorganisms may be found on surfaces and floating in air currents. They may fall from objects suspended over a culture or swim in fluids. Aseptic technique prevents environmental organisms from entering a culture.

Doors and windows are kept closed in the laboratory to prevent air currents which may cause microorganisms from surfaces to become airborne and more likely to get into cultures. Transfer loops and needles are sterilized before and after use in a Bunsen burner to prevent introduction of unwanted organisms. Agar plates are held in a manner that minimizes the exposure of the surface to the environment. When removing lids from tubes they are held in the hand and not placed on the countertop during the transfer of materials from one tube to another. All of these techniques comprise laboratory aseptic technique.


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CULTIVATION

Microorganisms must have a constant nutrient supply if they are to survive. Free-living organisms acquire nutrients from the environment and parasitic organisms acquire nutrients from their host. When trying to grow microbes in the lab adequate nutrition must be provided using artificial media. Media may be liquid (broth) or solid (agar). Any desired nutrients may be incorporated into the broth or agar to grow bacteria.

Agar is the solidifying material used in solid media. It is an extract of seaweed that melts at 100°C and solidifies at about 42°C. Most pathogenic bacteria prefer to grow at 37°C so agar allows for a solid medium at incubator temperatures.

Organisms grown in broth cultures cause turbidity, or cloudiness, in the broth. On agar, masses of cells, known as colonies, appear after a period of incubation. Certain techniques will allow bacterial cells to be widely separated on agar so that as the cell divides and produces a visible mass (colony), the colony will be isolated from other colonies. Since the colony came from a single bacterial cell, all cells in the colony should be the same species. Isolated colonies are assumed to be pure cultures. Colony morphology is described in terms of shape, margin or edge, elevation and color (Fig. 2.1).

You will be using these isolated colonies for next week lab. It behooves you work carefully. Otherwise you will have to repeat the exercise until you have isolated colonies.

Figure 2.1. Bacterial colony morphology descriptions.


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MATERIALS

Mixed culture of: Escherichia coli Staphylococcus aureus 2 Large nutrient agar plate 1 Small nutrient agar plate

Wire Inoculating loop Bunsen burner Striker Sharpie marker

PROCEDURE

1. Label the agar side of the large plates with your name, date and source material using the laboratory marker. This should become a habit and done every time you pick up a new plate.

2. Remove the lid and leave the plate exposed to the air for 2 minutes. 3. Obtain and label another plate. 4. Sterilize the inoculating loop in the inner flame of the Bunsen burner. 5. Obtain a loop of broth from the mixed culture tube using aseptic

technique. Do not set the test tube cap on the benchtop. 6. Lift the agar plate from the lid and streak the first quadrant of the plate as

shown in step one of figure 2.2. Do not set the Petri dish lid on the bench top. The loop should be parallel to the agar surface to prevent digging into or gouging the agar. Return the plate to the lid.

7. Re-sterilize the inoculating loop and return to the agar plate. Streak once through the first quadrant, which you have already inoculated, and continue on into the next quadrant in a zig-zag pattern, as shown in step 2 of figure 2.2. Repeat the process for steps 3 and 4 shown in figure 2.2. As a result of this process you will pick up fewer and fewer bacterial cells with each pass and distribute them farther and farther apart. In the end you should have several well isolated bacterial colonies (Fig. 2.3).

8. Place the plate in a 37°C incubator for 24-48 hours. Check your cultures the next day. If you do not have isolated colonies you will need to repeat the exercise immediately. You must have isolated colonies for next weeks lab exercise.

9. Use a lab marker to divide the back of the small nutrient agar plate in half. Mark it with your name, date and source on each half of the plate.

10. Lightly press your finger tips onto one half of the plate. 11. Use a sterile cotton swab to swab an area from the environment such as a

door knob or the bottom of your shoe. Use this swab to lightly swab the other half of the small nutrient agar plate.

12. Place the plate in a 37°C incubator for 24-48 hours. Examine you plates


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during the next lab session. 13. During the next lab period record your findings in terms of whether you

obtained pure (axenic) cultures. Use the terms given above to describe the colony morphologies for both your isolation plate and your environmental sample plate. 2

3 4

1

Figure 2.2. Streak isolation pattern of bacteria.

Figure 2.3. Streak isolation of bacteria.


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CULTIVATION LAB REPORT NAME:

1. Microbes from the air Time (min) No. of colonies Avg. No. of colonies

1 2

5 10

15

Time (min)

Avg.

No.

of c

olon

ies


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2. Describe the shape, edge, elevation and color of the colonies from the air culture using the terms given in figure 2.1:

3. Where may microorganisms be found?

4. Why should you never set the lid on the Petri dish or a test tube cap on the bench top?


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MICROSCOPY

APPROXIMATE DURATION: 2 HOURS

note: this is typically the longest and most frustrating lab for new students.

OBJECTIVES

After completing this exercise you should be able to: 1. Demonstrate the correct use of the compound light microscope. 2. Name the major parts of the microscope. 3. Determine the diameter of a field of view. 4. Prepare a wet mount.

INTRODUCTION

Microorganisms are too small to be seen with the naked eye so a microscope must be used to visualize these organisms. While a microscope is not difficult to use it does require some practice to develop the skills necessary to use the microscope to its maximum capabilities. Bacteria and other cellular microorganisms are measured in micrometers (µm) or 1 x 10-6 meters.

There microscopes used in an introductory microbiology laboratory is a compound light or bright-field microscope. All light microscopes have the same basic features shown in figure 1.1.

A compound microscope consists of at least two magnifying lenses. One magnifying lens is in the ocular and one is in the objective. Each contributes to the magnification of the object on the stage. The total magnification of any set of lenses is determined by multiplying the magnification of the objective by the magnification of the ocular. The turret rotates allowing the objectives to change and thus change the magnification of the microscope. An iris diaphragm below the stage should be used to control the amount of light passing through a specimen. Less light is need at low magnification than at higher magnification. Too much light at low magnification may mask the specimen, particularly something as small as a bacterial cell.


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The distance between the specimen on the stage and the objective is known as the working distance. The coarse adjustment knob will cause the working distance to visibly change while the fine adjustment knob is for final, fine focusing.

The ability to see things using a microscope is limited by the resolving power of the microscope. The resolving power of a microscope is the distance two objects must be apart and still be seen as separate and distinct. For the light microscope this is approximately 0.2 µm. Objects closer together than 0.2 µm will

Figure 1.1. Components of a typical compound light microscope.

Oculars

Turret

Coarse Adjustment Fine Adjustment

objectives

Stage

Arm

Light Source

Condenser

Base


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not be distinctly seen. Increasing the magnification will not make the objects more distinct, just bigger.

Each objective has the magnification of the objective written on the objective. The magnification of the ocular is also inscribed on the ocular. Low magnifications are used for quickly examining the slide to find an appropriate area to examine. Higher magnifications allow the examination of a particular object on the slide. Examine your microscope and complete the table below.

When you look through the ocular you will see a lighted circle. This is known as the field of view or the field. While looking through the microscope move the iris diaphragm lever and notice how the brightness of the light changes. As you move the objectives to provide increased magnification you will look at a smaller section of the slide. Be sure you move the object you want to view into the center of the field before moving to the next objective.

These microscopes are parfocal. Once you have focused on an object using one objective the object will be approximately in focus on the next objective. Use of the fine focus knob will sharpen the focus.

MATERIALS

Microscope Newsprint Stage micrometer Slides Coverslips

Transfer pipettes Prepared slides of bacteria Hay infusion Immersion oil Lens paper


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PROCEDURE

1. Place a piece of newsprint on a microscope slide and cover with a coverslip. ALWAYS USE A COVERSLIP!

2. Turn the microscope on and set the light source on its highest setting. 3. Use the coarse adjustment knob to obtain maximum working distance. 4. Place the slide on the stage. The slide should fit into the slide holder but is not

placed under the slide holder. Use the stage adjustment knob to move the slide the edge of the coverslip bisects the hole in the stage.

5. Rotate the scanning objective (4X) into place. 6. Use the coarse adjustment knob to obtain the minimum working distance.

Develop the habit of watching this process to be sure the objective does not crash into the slide.

7. Look through the oculars. Adjust the light with the iris diaphragm lever on the condenser if necessary. Slowly turn the coarse adjustment knob until the edge of the coverslip comes into focus. Use the fine adjustment knob to sharpen the focus.

8. Use the stage adjustment knob to locate the letter “e” in the newsprint. Note the orientation of the letter “e” in the newsprint.

9. Rotate a higher power objective (10X) into place. Use the fine adjustment knob to sharpen the focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm lever if necessary. Draw the letter “e” as it appears in the microscope on the lab report sheet.

10. When finished viewing the slide use the coarse adjustment knob to maximize the working distance and remove the slide from the stage. Dispose of the slide in the broken glass box.

11. Place a stage micrometer on the stage and determine the diameter of the field of view for all four objectives. Record the distances on the lab report sheets.

12. When using the high power objective (100X) use the following procedure. Rotate the turret halfway between the 40X and 100X objective. Place a drop of immersion oil on the slide and rotate the oil immersion objective (100X) into place. The objective should be immersed in the oil on the slide. Use the fine adjustment knob to sharpen the focus. Adjust the light using the iris diaphragm lever if necessary. Never use the coarse adjustment knob with high power.

13. Place a drop of water from the hay infusion on a microscope slide. Cover with a coverslip and view under all four objectives. Sketch three (3) of the organisms.

14. Obtain a prepared slide for each three bacterial species. View slides under the 100X objective and sketch the bacteria. Don’t forget the immersion oil!

15. When you are finished with the microscope clean the microscope, as described


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below, and return it to storage.

PROCEDURE FOR CLEANING A MICROSCOPE

1. Turn off the light and unplug the cord. Store the cord appropriately. 2. Using the coarse adjustment knob to obtain maximum working distance and

remove the slide from the stage. 3. Using lens paper clean all the lenses starting with the cleanest first—oculars, 4X

through 100X objectives. 4. Clean any oil off of the stage using Kimwipes or paper towels. 5. Rotate the scanning objective into place. Use the coarse adjustment knob to

obtain minimum working distance. 6. Return the microscope to the appropriate storage area.


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MICROSCOPY LAB REPORT NAME:

1. Sketch the orientation of the letter “e” as viewed through the microscope.

2. Fill-in the table below. Magnification Field of

Objective Objective Ocular Total view (µm)

Scanning Low Power High Power Oil Immersion

3. Sketch two different microorganisms from an environmental sample and give

their approximate sizes in micrometers in the space below.

4. Sketch the bacteria from two prepared slides under 1,000X magnification and give their approximate sizes in micrometers in the space below.


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STAINING

APPROXIMATE DURATION: 1½ HOURS

OBJECTIVES

After completing this exercise you should be able to: 1. Describe the major division between bacteria 2. Describe and conduct the Gram Stain procedure 3. Describe the purpose of the malachite green stain

INTRODUCTION

Bacteria have almost the same refractive index as water. This means when you try to view them using a microscope they appear as faint, gray shapes and are difficult to visualize. Staining is one method for making microbial cells easier to visualize.

Simple stains use only one dye that stains the cell wall of bacteria much like dying eggs at Easter. Differential stains use two or more stains and categorize cells into groups. Both staining techniques allow the detection of cell morphology, or shape, but the differential stain provides additional information concerning the cell. The most common differential stain used in microbiology is the Gram stain.

The Gram stain uses four different reagents and the results are based on differences in the cell wall of bacteria. Some bacteria have relatively thick cell walls composed primarily of a carbohydrate known as peptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycan and lipopolysaccharides. Peptidoglycan is not soluble in organic solvents such as alcohol or acetone, but lipopolysaccharides are non-polar and will dissolve in nonpolar organic solvents.

Crystal violet acts as the primary stain. This stain can also be used as a simple stain because it colors the cell wall of any bacteria. Gram’s iodine acts as a mordant. This reagent reacts with the crystal violet to make a large crystal that is not easily washed out of the cell. At this point all cells will be the same color. The difference in the cell walls is displayed by the use of the decolorizer. A solution of acetone and alcohol is used on the cells. The decolorizer does not affect those cell walls composed primarily of peptidoglycan but those with the lipid component will have large holes develop in the cell wall where the lipid is dissolved away by the acetone and alcohol. These large holes will allow the


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crystal violet-iodine complex to be washed out of the cell leaving the cell colorless. A counterstain, safranin, is applied to the cells which will dye the colorless cells.

The cells that retain the primary stain will appear blue or purple and are known as Gram positive. Cells that stain with the counterstain will appear pink or red and are known as Gram negative. The lipopolysaccharide of the Gram negative cell not only accounts for the staining reaction of the cell but also acts as an endotoxin. This endotoxin is released when the cell dies and is responsible for the fever and general feeling of malaise that accompanies a Gram negative infection.

When reporting a Gram stain you must indicate the stain used, the reaction, and the morphology of the cell. Round, purple (blue) cells would be reported as Gram positive cocci and rod-shaped, purple (blue) cells would be reported as Gram positive bacilli.

In order to survive some bacteria produce endospores that are highly resistant to harsh environmental conditions. The malachite green staining procedure is a differential staining that is used to distinguish between vegetative cells and endospores.

MATERIALS

Microscope slides Coverslips Immersion oil Clothes pin Gram Stain kit

Lens paper Transfer loop Mixed culture of: Escherichia coli Staphylococcus aureus

PROCEDURE

1. Place a drop of distilled water on a slide. 2. Flame sterilize a loop and transfer some material from an isolated

colony to the drop of water. 3. Using a clothes pin to hold the slide, heat fix the sample by passing

it through the flame until all of the water has evaporated. This may take several minutes. Be patient. You do not want to cook your bacteria.

4. Place the slide on the staining rack and flood with crystal violet for 1 minute.


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5. Rinse the slide with distilled water, tilting the slide slightly to rinse all the stain from the slide.

6. With the slide slightly tilted, drop a few drops of Gram’s iodine on the slide to rinse off the last of the rinse water. Place the slide flat and flood with Gram’s iodine for 1 minute.

7. Rinse the slide with water as in step 5. 8. With the slide tilted, slowly drop acetone-alcohol decolorizer on the

slide. Blue color will run from the smear. Continue to apply decolorizer drop-by-drop until the blue stops running from the smear. This should take approximately 15 seconds.

9. Immediately rinse with water. 10. With the slide slightly tilted add safranin to the slide to replace the

rinse water then lay the slide flat and flood the slide with safranin for 30 seconds.

11. Rinse safranin from the slide with distilled water. Gently tap the slide to remove excess water.

12. Place a piece of bibulous paper or paper towel on the lab table and put the slide on it. Fold the paper over the slide and gently blot the slide to remove the water.

13. If the slide is still damp place a coverslip on it. Otherwise, place a drop of water on the slide and place a coverslip on top.

14. Examine the stained smear with the microscope and record your results below.


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STAINING LABORATORY REPORT I.D. NO.: LAB SECTION:

1. What color did the E. coli stain? Is it Gram positive or negative? How would you describe the shape of the cells?

2. What color did the Staphylococcus stain? Is it Gram positive or negative? How would you describe the shape of the cells?

3. What is the purpose of staining bacteria?

4. What is the most common differential staining procedure used in microbiology?

5. What difference in structure between bacteria results in the differential staining observed in the Gram stain procedure? Be detailed and specific.


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BACTERIAL GROWTH

APPROXIMATE DURATION: 2 HOURS

OBJECTIVES

After completing this exercise you should be able to: 1. Define a CFU 2. Describe the mathematical equation for bacterial growth 3. Determine the cell density of a starting culture

INTRODUCTION

Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosomes, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission.

Methods for measurement of the cell mass involve both direct and indirect techniques.

1. Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation.

2. Direct chemical measurement of some chemical component of the cells

such as total N, total protein, or total DNA content.

3. Indirect measurement of chemical activity such as rate of O2 production or consumption, CO2 production or consumption, etc.

4. Turbidity measurements employ a variety of instruments to determine

the amount of light scattered by a suspension of cells. Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number, after construction and calibration of a standard curve. The method is simple and nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria.


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THE BACTERIAL GROWTH CURVE

In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23…......2n (where n = the number of generations). This is called exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature.

When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve (Fig. 6.1).

Four characteristic phases of the growth cycle are recognized.

1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physiacal damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 20 40 60 80 100 120 140

Time (min)

O.D

. (6

00

nm

)

Figure 6.1. An idealized bacterial growth curve.


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required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

1. Exponential (log) Phase. The exponential phase of growth is a

pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time (below) derive.

2. Stationary Phase. Exponential growth cannot be continued forever in

a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of “biological space”. During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It si during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process.

3. Death Phase. If incubation continues after the population reaches

stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or 39icroscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.

GROWTH RATE AND GENERATION TIME

As mentioned above, bacterial growth rates during the phase of exponential growth, under standard nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium’s generation time. Generation times for bacteria vary from about 15 minutes to 24 hours or more. The generation time for E. coli in the laboratory is 15-20 minutes, but in the intestinal tract, the


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coliform’s generation time is estimated to be 12-24 hours.

PLATE COUNTS

Often it is desirable to know the concentration of a bacterial population in or on a source material. For example, the EPA freshwater standards require that lakes and streams contain fewer than 200 E. coli per 100 ml of water. One method of determining whether a freshwater lake meets this standard is to culture a small amount of water on an agar plate and count the number of colonies that form. Thus, one obtains a count of colony forming units (CFUs). Particularly high concentrations of the bacteria may require the dilution of the starting material before culturing on an agar plate. This reduces the number of CFUs to a more manageable number, ideally some where between 30 and 300 colonies.

MATERIALS

Broth Culture of E. coli 3 test tubes nutrient broth 3 Nutrient Agar plates 4 one milliliter pipettes Pipette aid

beaker of Alcohol Hockey Stick Spectrophotometer Test tube rack

PROCEDURE

TURBIDIMETRIC MEASUREMENT OF GROWTH

2. Turn on the spectrophotometer using the knob on the left and allow it to warm up for 15 min.

3. Set the wavelength dial on the top to 600 nm. 4. Adjust the zero control (left knob) so that the meter reads zero.

Mind the parallax. The sample compartment should be empty. 5. Blank the spectrophotometer by placing one of the small test tubes

of nutrient broth in the tube holder on the top and adjusting the dial on the right until the needle reads zero.

6. Add 3 ml of E. coli culture to the remaining test tube and gently mix. 7. Place new E. coli culture in the spectrophotometer and record the

absorbance. 8. Place the culture in the 37°C incubator. 9. Record the absorbance at 15 min, 30 min, 45 min, 1 h and 1.5 h.

Place the culture back in the 37°C incubator between readings.


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DILUTION CULTURING

1. Label the four test tubes 1 through 4 2. Label the back of the four plates 1 through 4, as well as, with your

name and the date. 3. Using aseptic technique transfer 0.1 ml of the E. coli culture to the

test tube labeled #1. Gently mix the test tube. 4. Using a new pipette transfer 0.1 ml of test tube #1 to test tube #2.

Gently mix the test tube. 5. Using a new pipette transfer 0.1 ml of test tube #2 to test tube #3.

Gently mix the test tube. 6. Using a new pipette transfer 0.1 ml of test tube #3 to test tube #4.

Gently mix the test tube. 7. Using aseptic technique and a new pipette transfer 0.1 ml of the E.

coli culture in test tube #4 to the center of the plate labeled #4. Flame a hockey stick to remove the alcohol and spread the culture around the plate.

8. Using the same pipette and aseptic technique transfer 0.1 ml of the E. coli culture in test tube #3 to the center of the plate labeled #3. Flame a hockey stick to remove the alcohol and spread the culture around the plate.



11. Invert the plates and incubate at 37°C for 24-36 h. 12. Count the number of colony forming units (CFUs) on each plate

and back calculate to the cell concentration in the starting culture.


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BACTERIAL GROWTH LAB REPORT NAME:

1. What is a CFU?

2. Record the number of CFUs for each plate in the table below. If you have more than 300 CFUs on a plate record TMTC (too many to count). If you record TMTC in a row you do not need to calculate the stock concentration for that row. Record “NA”, for “not applicable”, in the row in which you recorded TMTC. Record the dilution factor for each plate. Calculate the concentration of cells/ml in the starting stock culture.

Plate No. CFU Dilution Factor (DF) Stock conc. (CFU x DF)

1 2 3

3. Record the absorbance of E. coli at 600 nm at each of the following time

points:

Time (min) Abs. 600 nm Time (min) Abs. 600 nm 0 15 30 45 60 90


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BACTERIAL GROWTH LAB REPORT NAME:

Plan B

1. Using Excel create an XY scatter plot the ABS of the E. coli grown in Luria Bertani broth + glucose + amino acids versus time.

2. Select the data points by clicking on one of them and then select “add trend line” under the chart options

3. You want to choose “exponential” trend line. 4. You also want to choose the ‘add trend line’ options

button and then check the ‘display equation’ and ‘display R-squared value’ boxes.

5. Print this out and turn it in with the above sheet from pg. 14 (your raw data).

6. Pick two points on the y-axis. The second point should be twice the first point. It doesn’t matter if the second point is higher than your actual raw data points.

7. Calculate the two values for x using the equation and the two y values that you chose.

8. Subtract the smaller x value from the larger. This is your calculated doubling time.

9. Record this and hand it in with your other work. 10. Repeat these steps for the E. coli grown in M9 salts +

glucose. 11. This assignment is due next Tuesday. DO NOT come

to me Tuesday morning asking how to complete this lab. You have a whole week before hand in which you may ask for help if you are having problems.

12. If you need a primer on using excel there is one on the course website.


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AMES TEST

OBJECTIVES


1. Describe the purpose of the Ames test 2. Define a revertant 3. Define auxotroph and prototroph

INTRODUCTION

Every day we are exposed to a variety of chemicals, some of which are carcinogens; chemicals that can induce cancer. Many chemical carcinogens induce cancer because they are mutagens that alter the nucleotide base sequence of DNA.

The Ames test uses an auxotroph of Salmonella typhimurium which cannot synthesize the amino acid histidine (his-). The Salmonella typhimurium starin TA 1535 carries a point mutation at nucleotide 46 in the hisG gene; the first enzyme in histidine biosynthesis. This point mutation changes the DNA sequence from CTC, which encodes for the amino acid leucine, to the DNA sequence CCC, which encodes for the amino acid proline. Additionally this strain contains the rfa mutation which causes partial loss of the LPS, making the cell more permeable, and a defect in one of its DNA repair mechanisms (uvrB). The addition of a mutagen alters the DNA sequence in a small number of these bacteria and restores the ability to make histidine. These restored bacteria are called revertants. The more revertants that are produced the powerful the mutagen.

Thus, this assay provides a quick, initial, inexpensive means to survey the mutagenic potential of a compound. This assay would be followed-up testing in animal models.


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MATERIALS

3 plates 2% glucose-minimal salts agar 3 minimal salts soft agar tubes 1 Salmonella TA 1535 (ATCC 29629) culture 1 ml pipettes

Sterile filter disks Forceps Sterile saline Sodium azide (1ug/10 ul) Suspected mutagen

PROCEDURE

1. Label one glucose-minimal salts agar plate “negative control”, one plate “positive control” and the last plate “test”.

2. Aseptically add 0.1 ml of Salmonella to one of the soft agar tubes, mix gently between palms for 5 sec and pour onto a plate. Repeat for the other two plates.

3. Dip the forceps in alcohol and flame to sterilize. Place one sterile disks on each plate using the forceps.

4. Place a drop of sterile saline on the disk on the negative control plate. Place a drop of sodium azide on the disk on the positive control plate. Place a drop of potential mutagen on the disk on the test plate. Incubate at 37C.

5. Count and record the number of revertants on each plate.

Caution: These chemicals are potential carcinogens. Salmonella typhimurium can cause gastroenteritis.


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AMES TEST LAB REPORT NAME:

1. Define a revertant: Table 1. Revertant counts

Plate No. Revertants Negative control Positive control Test

Table 2. Class Means Plate Mean No. Revertants ± SD

Negative control Positive control Test: Test: Test:

In order to be considered a potential mutagen the test compound must produce twice as many revertants as the negative control. Which compounds were potential mutagens?


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KOMBUCHA

OBJECTIVES


1. Describe the microbial community present in Kambucha 2. Define a symbiotic relationship 3. Describe the nature of the products produced by this symbiosis

INTRODUCTION

Long before the beginning of recorded history man was intentionally or unwittingly using microbes to create or preserve food. Before the advent of refrigeration many cultures relied on microorganisms to produce, flavor and preserve foods such as cheese, bread, beer, wine, yogurt, soy sauce, sauerkraut and many others. Today food and industrial microbiology is a billion dollar industry producing not only edible foodstuffs but everything from industrial solvents to biodegradable plastics. Kambucha is a fermented tea that has been drunk in Asia for thousands of years. It is a microbial community composed of a several species of yeast (a fungus) and a bacterial species belonging to the genus Acetobacter. The fermented tea is repudiated to have health benefits ranging from improving skin tone and aiding weight loss to curing cancer and AIDS. Although there is no scientific evidence to support these claims the drink has recently been gaining in popularity in the West.

MATERIALS

Kombucha SCOBY One tea bag

Table sugar

Clean glass jar

PROCEDURE


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10. Label a clear, clean glass jar with your name and the date. 11. Boil tap water in a 500 ml beaker and steep a tea bag in it for 5 min. 12. Remove the tea bag and add sugar to your liking. 13. Allow the tea to cool until you can hold it in your hands for 30 s. 14. Add a small piece of the Kombucha SCOBY and 1 ml of the liquor. 15. Cover the jar with a paper towel held in place with a rubber band.

16. Incubate the tea for 10 days or more. 17. Remove the SCOBY, pour the fermented tea into a 500 ml bottle, add

flavoring and seal bottle. Allow the bottle to continue to ferment at room temperature for at least 24.

18. Refrigerate bottle for 24 h before opening!


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Kombucha lab report Fall 2011

1. Why is the Kombucha fizzy?

2. What organic compound gives Kombucha its astringent taste?

Documents

General Microbiology Laboratory Manualv1yourspace.minotstateu.edu/paul.lepp/General Microbiology... · 2020-02-25 · used. Laboratory personnel have specific training in the procedures