NYA_F23-Lab_05-Bacteria_Part_A

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LABORATORY 5 Exploring Bacteria Pre-lab exercises on page 11 can be done to help prepare for the lab OVERVIEW This laboratory will be completed in two parts (Parts A and B) over two lab periods. During the first period, you will practice aseptic technique and set-up bacterial cultures for analysis during the second period. You will also start characterizing bacterial species at station demonstrations. LEARNING OBJECTIVES When you complete this lab, you will be expected to: 1. Describe the difference between broth and agar media and list applications for both types. 2. Use aseptic technique to inoculate an agar plate and describe its importance in culturing bacteria. 3. Describe plating strategies to produce colonies or a lawn on an agar plate. 4. List some of the various tools used to characterize and identify bacteria at the colony, cell, and molecular levels. 5. Discuss the importance of characterizing and identifying bacteria in biology and medicine. 6. Design an experiment to compare the number and types of bacterial cells in two test areas. INTRODUCTION Culturing Bacteria Bacteria are prokaryotic microorganisms capable of growing virtually anywhere on Earth. In optimal growing conditions, one bacterium can divide into 2 daughter cells in roughly 20 minutes using a process called binary fission . At this rate of growth, one bacterium can generate 4 x 10 21 replicas of itself in 24 hours! Although they are present almost everywhere on earth, why isn't the earth covered with a thick film of bacteria? Conditions are rarely optimum for growth of bacteria in the environment. Biologists culture bacteria in ideal conditions in the lab. Culturing bacteria is necessary for research applications and identification of bacteria. Bacteria can be cultured in a liquid medium called a broth or a semi-solid medium made of agar . The agar is a natural product derived from the structural polysaccharides in the cell walls of red algae. Agar can provide an ideal semi-solid support as it is cannot be broken down by most microorganisms. Each type of medium offers an advantage. A liquid broth produces a mixture of cells that can reach very high cell concentrations. On agar, isolated cells can produce colonies that can be inspected or quantified. In other applications, agar can be use to culture a film of bacteria referred to as a lawn . Many different mixtures of nutrients and chemicals can make up a bacterial medium. In addition, bacteria can be cultured under different conditions such as temperature, pH, and oxygen levels. Some bacteria will grow in many different media and conditions while some more finicky bacteria only grow when you get the media and conditions exactly right. How can you identify one bacterial species from another? This task can be quite difficult as they are very small cells and structural differences can be very hard to identify. Luckily, bacteria are very diverse in their metabolic abilities and requirements. The metabolic diversity in bacteria explains why they occupy almost every imaginable environment and ecological role on the planet. For example, some are photosynthetic and are primary producers in ecosystems. The majority of bacterial species, however, are not General Biology I Laboratory Exercises Dawson College Fall 2023 PART A Page 1

Lab 5 - Bacteria (Part A) photosynthetic but fill some extremely important ecological roles as decomposers or mutualists . A minority of species can infect plants and animals and cause disease (pathogenic bacteria). In addition to studying the morphological aspects of cells and colonies, the differences in metabolism can be exploited to identify bacteria. This is done by culturing bacteria in a medium or condition that highlights a particular aspect of their metabolism. Why do we want to identify bacteria? This is obviously essential to biological classification (taxonomy). In addition, this is essential to effectively treat infections. Not all antimicrobial drugs are equally effective against a given bacterium. The type of medication used will depend on the species of bacteria that is infecting you. It is important to note that the susceptibility of a bacterium to an antimicrobial drug can also be used as a tool in its identification. Aseptic Technique The media on which you culture desirable microorganisms will readily grow undesirable microorganisms, especially molds and other types of fungus, and bacteria from your surroundings and on your body. It is therefore essential that you protect your cultures from contamination using aseptic technique: use of sterile tools and appropriate techniques to avoid contamination. Nearly all forms of contamination are carried on microscopic dust particles that make their way onto sterile surfaces when they are carelessly handled. Do not attempt to handle any of the equipment prior to your teacher’s discussion on aseptic technique . Here are some important points regarding aseptic technique:  You will use a sterile equipment to inoculate agar plates. Try not to contaminate your cultures or instruments by allowing cultures to be exposed to the environment for longer than need be and by touching non-test surfaces with your tools.  When it is necessary to open a dish, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the agar surface away from your face (and your germs!).  Always be aware of where your hands are, where your face is, and whether your culture is in a position to be contaminated. If you have long hair, make sure it is tied back and it does not hang into your plate . Hair is full of potential contaminants, and is one of the principle sources of contaminating microorganisms.  Once incubated, keep Petri dishes closed, and always discard them in the provided biohazard waste disposal bags or other designated waste container. Never touch, ingest or inhale bacteria after incubation. General Biology I Laboratory Exercises Dawson College Fall 2023 Page 2

Lab 5 - Bacteria (Part A) Characterizing Bacteria Bacteria can be characterized at the 1. colony level, 2. cell level, and 3. molecular level. This week and next, we will use various tools to characterize some example species of bacteria: E. coli and B. subtilis . In addition, we will characterize and try to identify some unknown bacterial species! 1. Characterization at the Colony Level: Colony Morphology. A bacterial colony is mass of cells derived from a single isolated cell during inoculation on agar medium. After incubation, that one isolated cell produces millions of clones by binary fission resulting in this macroscopic structure. A colony from a particular bacterial species will have a set of defining characteristics such as shape, surface texture, and color. Figure 1 outlines some the many characteristics of bacterial colonies. These characteristics are referred to as the colony morphology . Observation of colony morphology is often made with the naked eye, but a stereoscopic microscope gives the investigator a better view of the colony’s specific characteristics. Since colony morphology differs between bacterial species, it is an important tool to characterize and identify bacteria. 2. Characterization at the Cell Level: Cell Structure, Shape, and Arrangement. A. Cell Structure The cell wall of bacteria is composed of the structural polymer peptidoglycan . In some species, a relatively thick layer of cross-linked peptidoglycan fibers is located outside the plasma membrane, while in others the layer is thinner and sandwiched between the inner plasma membrane and an outer membrane (Figure 2). These different structures are referred to as Gram- Positive and Gram- Negative based on the Gram stain . When cells are stained using the Gram staining technique, Gram- positive cells: thick peptidoglycan layer appear purple/blue while Gram-negative: thin peptidoglycan layer plus outer membrane cells appear red/pink after the procedure. B. Cell Shape and Arrangement The shape of a bacterial cell under a microscope can be used in characterization. Bacterial cells generally have three (3) basic shapes: Coccus , Bacillus , and Spirillum . Bacterial cells will also General Biology I Laboratory Exercises Dawson College Fall 2023 D i p l o c o c c i ! ( i n p a i r s ) ! S t a p h y l o c o c c i ! ( i n c l u m p s ) ! S t r e p t o c o c c i ! ( i n c h a i n s ) ! ! A . C o c c u s ( p l . , C o c c i ) = " S p h e r e - S h a p e d " A r r a n g e m e n t : " S i n g l e C o c c i ! S t e p t o b a c i l l i ! ( i n c h a i n s ) ! B . B a c i l l u s ( p l . , B a c i l l i ) = " R o d - S h a p e d " A r r a n g e m e n t : " S i n g l e B a c i l l i ! Streptococci (in chains) Staphylococci (in clumps) Figure 1. Diversity in Bacterial Colony Morphologies . Various characteristics of colonies can be used for characterization of bacterial species. Figure 2. Gram staining can identify differences in cell structure. Gram- positive cells stain purple blue and Gram- negative cells stain red/pink reflecting the differences in their cell structure. Page 3

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Lab 5 - Bacteria (Part A) have a specific arrangement under a microscope. Some common arrangements for the three cell shapes are illustrated in Fig. 3. When characterizing bacterial cell arrangement under a microscope, it is important to realize that not every cell will have the arrangement characteristic of that species. You will have to look at many cells within the field or several fields to see the general arrangement of most cells. In addition, a high density of cells may look like they are arranged in a particular manner but they may be single cells with no particular arrangement. C. Other Cell Features Other structural features can be used to characterize bacterial cells. For instance, one could use the presence or absence of flagella (tail structures used in locomotion) to characterize or identify bacterial species. In addition, flagella can have a particular number (i.e., one, two, or many) and arrangement (i.e., at one end, both ends, or all over). Some genera of bacteria (i.e., Bacillus and Clostridium ) can also produce distinctive endospores (resistant, dormant cell triggered by harsh conditions) within their cells. The presence or absence of endospores is another cell-level tool to use in characterization and identification. 3. Characterization at the Molecular Level: Metabolic Capabilities. All organisms can be classified into one of four (4) nutritional modes . This classification is based on two (2) criteria to assort organisms into groups: 1) Source of carbon . 2) Source of energy . Table 1 summarizes the different modes of nutrition observed in organisms. Of all organisms, only prokaryotes have representatives from each category. Many bacterial species are chemoheterotrophs as are all animals, fungi, and some protists. Chemoheterotrophs must consume live or dead organic material to obtain organic compounds . These organisms breakdown organic compounds as a source of energy and use the carbon skeletons in organic compounds as a starting material to build other organic compounds important for life. Some bacteria are photoautotrophs as are plants and algal protists. These bacteria are able to use light as an energy source to produce all of their organic compounds from inorganic sources of carbon such as CO 2 . While chemoheterotrophy and photoautotrophy are shared by the domains Bacteria and Archaea with the domain Eukarya, chemoautotrophy and photoheterotrophy are unique to the prokaryotes . Within each nutritional mode, there are many metabolic types. For instance, whereas obligate aerobes require oxygen to produce ATP, obligate anaerobes are poisoned by oxygen. The latter organisms use substances (e.g. sulfate (SO 4 2- ) or nitrate (NO 3 - ) ions) as a substitute for oxygen in their energy metabolism. Bacteria, as a group, are very diverse in their metabolic capabilities. Such a wide range of metabolic capabilities allows prokaryotes to flourish in all habitats suitable for life on Earth. The chemicals that they can produce or breakdown can be used for characterization at the molecular level. This diversity stems from different enzymes being produced in different species and correlates with their nutritional mode. General Biology I Laboratory Exercises Dawson College Fall 2023 Figure 3. Bacterial Cell Shape and Arrangement. C . S p i r i l l u m ( p l . , S p i r i l l a ) = " S p i r a l - S h a p e d " A r r a n g e m e n t : " S i n g l e S p i r l l a ! Table 1. Modes of Nutrition in Organisms. Page 4

Lab 5 - Bacteria (Part A) Susceptibility to antibiotics can also be used as a tool in its identification (can characterize at both the cell or metabolic level). The type of cell wall, the presence of substances outside of the cell wall, and the presence of specific enzymes can render a bacterium insensitive to a particular antibiotic. Since bacteria are diverse in their structure and metabolic activities, it is not surprising that not all antimicrobial drugs are equally effective against a given bacterium. The purpose of identifying bacteria goes beyond the essential need of biological classification (taxonomy). For example, correct identification is essential to effectively treat infections. The type of medication used will depend on the species of infectious bacteria. Important Notes on Inoculating Agar Plates In Exercises A and B, you will be inoculating agar plates with bacteria. Please read the following information BEFORE getting started. Your two main concerns when inoculating agar plates is: 1. your safety and 2. contamination of stock cultures or the plate itself. A. Before undertaking any of the exercises, make absolute sure you understand the whole procedure before starting. B. The longer the agar is exposed to air, the greater the chance that you will contaminate your plate . Therefore, try to be as efficient as possible when inoculating your plate to minimize the risk of contamination. The best way to inoculate a plate is to handle a plate upside down . Remove the bottom of the plate (containing the agar) while leaving the lid on the bench. Invert the bottom just enough (roughly 90 o ) so that you can use your other hand to inoculate the agar using your instrument. Once finished, re-join the bottom of the plate to the lid on the bench. Plate will be upside-down and is stored that way. Move the inoculated plate to a safe location. C. Agar is soft and can easily be broken. You want to streak the bacteria on the surface of the agar. You do not have to apply much pressure . Applying too much pressure will break through the agar and ruin the plate. D. The technique used to inoculate a plate will depend on your overall goal. In some cases, we want to isolate individual cells to study colonies after incubation. In other cases, we want to spread bacterial cells over an entire surface of the plate to create a film (lawn) after incubation. Make sure you understand the overall goal before you commence any exercise. Inoculating agar is like drawing where your swab is a pen. Bacteria will grow ONLY where the swab touched the agar. Therefore, if you want to grow a lawn of bacteria then every bit of the agar surface must come into contact with the swab! E. Take extra precautions that you do not contaminate your bacterial stock culture. If you feel like your plate may be contaminated, you can always start over as long as your stock culture is not contaminated. General Biology I Laboratory Exercises Dawson College Fall 2023 Page 5

Lab 5 - Bacteria (Part A) EXERCISE A. Setting up Cultures of E. coli and B. subtilis for Characterization Overview In Exercises A1 - A3, you will be inoculating agar plates with either E. coli or B. subtilis using various plating strategies to accomplish certain goals. Next week, we will use these plates to further characterize and compare these two test bacterial species. To complete these exercises, you will be working as a team of 4 students (2 sets of lab partners across from each other). Each team will produce a complete set of FIVE (5) inoculated agar plates. Each pair of lab partners will be in charge of ONE test bacterial species for the team: E. coli or B. subtilis . Decide on which one you will do before you proceed! Exercise A1. Preparing a Plate for the Amylase Test Goal: Inoculating a starch agar plate with a “strip” of your test bacterial species. Each pair of lab partners will be in charge of inoculating the SAME plate with their test bacterial species. In this exercise, you will use aseptic technique to plate your test bacterial species on a STARCH AGAR plate (NOT NUTRIENT AGAR) . This medium is nutrient agar but also contains the polysaccharide, starch . Next week, we will be able determine whether the bacterial species can metabolize starch which would indicate that the species makes the enzyme amylase . Procedure A1 1. Using the marker provided, draw a line on the underside of a starch agar plate that divides the surface area of the plate into two halves ( ). Label the initials of the bacterial species (e.g., EC for E. coli ) to be plated in each half. Also, include the initials of your names and your lab section. Leave the plate facedown. N.B. Read ALL of the instructions BEFORE you start. Notes and Reminder: You want to apply the bacteria on top of the agar. Do not press too hard and break the surface of the agar. BE SAFE but work as quickly as possible. The bacterial stock cultures of E. coli and B. subtilis are not airtight. DO NO INVERT TO MIX. 2. Remove the sterile swab from its packaging making and do not contaminate it. 3. Gently swirl the bacterial stock culture tube to suspend the cells. Remove the cap of the tube and carefully dip the tip of the swab into the culture trying not to contaminate the stock. 4. Remove the swab from the culture and keep it in your hand without contaminating it. Place the cap back on and place the stock culture tube back in the rack. 5. Agar plate should be upside down. With the swab still in one hand, remove the bottom of the plate (containing the agar) while leaving the lid of the plate on the bench. Flip the plate just enough so that you can work with the agar and inoculate the plate. Use the swab to streak a line on the side of the plate designated for your test bacterial species. 6. Once completed, replace your plate onto its lid and put the used swab in the biohazards disposal bag. Store your plate, upside down, on the tray in the middle of your bench. 7. Repeat this procedure for the second test bacterial species. General Biology I Laboratory Exercises Dawson College Fall 2023 Page 6

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Lab 5 - Bacteria (Part A) Exercise A2. Preparing Plates for the Antibiotic Susceptibility Test Goal: Plating a lawn of your test bacterial species on nutrient agar and adding antibiotic disks. Each pair of lab partners will inoculate their OWN plate with their test bacterial species and add the antibiotic discs. In this exercise, you will use aseptic technique to plate your test bacterial species on a regular nutrient agar plate. In this case, we want to culture a lawn of bacteria on the plate. After incubation, the layer of bacteria will look like a film on top of the plate. In order to accomplish this task, every bit of the plate has to be contacted by the swab. This will take some time. If you do not inoculate the whole plate, there will be areas that are clear and the plate will not be ideal for its application. Once the plate is inoculated, you will add antibiotic discs. These discs are made of paper, which contain a certain amount of the antibiotic. The antibiotic will diffuse into the agar as the bacterial cells are dividing. If the test bacterial species is sensitive to the antibiotic, then it will interfere with its growth and results in clearings in the lawn see Fig. 3. These clearings are referred to as zones of inhibition. Procedure A2 Part 1. Plating a lawn of bacteria. 1. Using the marker provided, label the underside of a nutrient agar plate with the initials of the bacterial species to be plated. Also include the initials of your names and your lab section. Leave the plate facedown. 2. Remove the sterile swab from its packaging and do not contaminate it. 3. Gently swirl the bacterial stock culture tube to suspend the cells. Remove the cap of the tube and carefully dip the tip of the swab into the culture trying not to contaminate the stock. 4. Remove the swab from the culture and keep it in your hand without contaminating it. Place the cap back on and place the stock culture tube back in the rack. 5. Agar plate should be upside down. With the swab still in one hand, remove the bottom of the plate (containing the agar) while leaving the lid of the plate on the bench. Flip the plate just enough so that you can work with the agar and inoculate the plate. To inoculate, streak the swab over the ENTIRE surface of the agar . Note: this might take some time, but it is necessary to culture a lawn . 6. Once completed, replace your plate onto its lid and put the used swab in the biohazards disposal bag. Complete part 2. Part 2. Adding the antibiotic disks. In this step, you will add one disk of each type of antibiotic to your plate. The antibiotics available are: P: Penicillin , NOR: Norfloxacin , and S: Streptomycin . 1. Sterilize the tweezers by dipping the tips in ethanol . Remove the excess alcohol by tapping on the edge of the beaker for 10 seconds. 2. Use the sterile tweezers to take one antibiotic disc from the container. Make sure to close the container immediately after. General Biology I Laboratory Exercises Dawson College Fall 2023 Figure 3. Zones of inhibition. Page 7

Lab 5 - Bacteria (Part A) 3. Turn each plate at around 90° angle, or slightly more to facilitate application of the disc and open the cover. 4. Note: You do not want your discs too close to the edge (or each other) as you want to leave enough space to observe a distinct zone of inhibition after incubation. Gently place the disc in your inoculated plate in one of the areas shown in Fig. 4. Do not scratch the agar with the tweezers. Gently tap the disc to make sure that the disc adheres to the agar and will not detach when incubated upside-down. Make sure you do not move the disc once in its place. 5. Repeat steps 1-4 to add the other two antibiotic discs to the plate. See Fig. 4 for the ideal placement of the three discs. 6. Store your plate, upside down, on the tray in the middle of your bench. Exercise A3. Preparing Plates for to Observe Colony Morphology Goal: Isolating individual cells on the surface of the agar to grow colonies. You will be using inoculation loops for this streak plate technique NOT swabs. You will take a sample of the stock culture and streak it (zig-zag pattern) in one quadrant (1/4 area) of the plate. You will repeat this 3 times with new sterile loops but taking a sample from the previous streak. The strategy is to decrease cell density gradually to isolate cells from one another by the time you get to the fourth and final quadrant. The general procedure for the streak plate method is outlined below in Figure 5. 1. Using the marker provided, label the underside of a nutrient agar plate with the initials of the bacterial species to be plated. Also include the initials of your names and your lab section. Leave the plate facedown. 2. Remove the sterile loop (yellow color) from its packaging and do not contaminate it. Gently swirl the bacterial stock culture tube to suspend the cells. Remove the cap of the tube and carefully dip the tip of the loop into the culture trying not to contaminate the stock. 3. Remove the loop from the culture and keep it in your hand without contaminating it. The loop should carry a film of the liquid in its circle (think blowing bubbles!). Place the cap back on and place the stock culture tube back in the rack. General Biology I Laboratory Exercises Dawson College Fall 2023 Figure 4. Placement of antibiotic discs. Incubation 18 hrs Loop inoculation Quadrant 1 Quadrant 2 Quadrant 3 Quadrant 4 Colonies Figure 5. Streak Plate Technique to Produce Colonies. Four streaks are carried out to gradually decreases cell density and isolate cells. Each cell will develop into a colony after incubation. Page 8

Lab 5 - Bacteria (Part A) 4. Agar plate should be upside down. With the loop still in one hand, remove the bottom of the plate (containing the agar) while leaving the lid of the plate on the bench. Flip the plate just enough so that you can work with the agar and inoculate the plate. To inoculate, steak the first quadrant of your plate. Replace your plate onto its lid and put the used loop in the biohazards disposal bag. 5. To inoculate the second quadrant, take a sterile loop, pass it over the END of the streak in the first quadrant (to collect some cells) and streak in the untouched area of quadrant 2. Replace your plate onto its lid and put the used loop in the biohazards disposal bag. 6. Repeat step 5 for quadrants 3 and 4. Note your culture source is quadrant 2 for quadrant 3 and quadrant 3 for quadrant 4. EXERCISE B. Plating Bacterial Communities for Ecosystem Comparison Goal: Here, you will carry out an experiment as a team of four. To carry out your experiment, you will collect samples from two test ecosystems and inoculate nutrient agar plates with the bacteria collected. You will also carry out a control to identify contamination (contamination control). Next week, this plate will act as a readout to allow you to compare the number of bacteria cells and the number of bacterial species collected from your test ecosystems. Note: Three separate culture samples will be produced in the water tubes and each of these samples will be plated on agar to produce a colony readout of the bacteria present. Try to spread the work out evenly among group members. Procedure B 1. Answer the questions in the Lab Report section (exercise B) to build your experiment. 2. Prepare the plates: Turn the nutrient agar plates upside down and indicate which ecosystem they will correspond to: A. Aquatic or B. Terrestrial or C: Contamination Control . Include the initials of your names and your lab section. Leave the plate upside-down. For EACH test ecosystem inoculation: 3. Label a sterile water aliquot tube with the sample it will correspond to (e.g., A). 4. Bring a sterile water aliquot (in your rack) and swab (still in packaging) to the ecosystems provided on the side bench. Carefully remove a sterile swab from its packaging and do not contaminate it. 5. Uncap the tube and moisten the cotton tip of the swab in the water. Replace the cap on the water tube and place in a rack. 6. Dip the swab in the corresponding ecosystem and plunge it back into the water tube and spin the swab between your fingers to mix. 7. Agar plate should be upside down. With the swab still in one hand, remove the bottom of the plate (containing the agar) while leaving the lid. Flip the plate just enough so that you can work with the agar and inoculate the plate. Make sure that the swab is thoroughly spread out over the entire surface of the agar (like what you did when plating for a lawn). You will want to rotate it as well so that all area of the swab is used to transfer the bacterial cell to the plate. 8. Once completed, replace your plate onto its cap and put the used swab in an appropriate waste container. For the contamination control inoculation: 9. Repeat all steps but do not collect bacterial from an ecosystem . 10. Store your inoculated plate, upside down, on the tray in the middle of your bench. General Biology I Laboratory Exercises Dawson College Fall 2023 Page 9

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Lab 5 - Bacteria (Part A) EXERCISE C. Exploring Bacterial Cell Characteristics One microscope per team of two is required to complete this exercise. The prepared slide you will view has three (3) separate bacterial species specimens in distinct areas of the slide (see Fig. 6). Each of these species has one of the three general cell shapes observed in bacterial cells: coccus , bacillus , spirillum and the specimens have been Gram stained. 1. View each species at 400X total magnification. Remember don’t skip straight to high magnification! Gradually magnify. Following proper microscope technique will be essential here to find and get a good view of these tiny bacterial cells. 2. Complete the questions in the Lab Report section . 3. Put away your microscope correctly and clean your bench using the following checklist. Task Done ( √ ) Light intensity knob turned all the way down and light power switched off. Sample stage brought down to lowest position. Slide removed and placed in appropriate location. Active slot with no objective put in place (or shortest objective). Stages centered. Microscope observed for spilled liquid or other mess and cleaned if necessary. Wound up cord placed over ONE ocular. Plastic bag replaced. Microscope placed in correct area of storage cabinet. Microscope placed in cabinet with objectives facing the back of the cabinet. Material used placed back in tray and bench area cleaned. General Biology I Laboratory Exercises Dawson College Fall 2023 Figure 6. Exercise slide set-up. Slide contains three (3) prepared specimens: Species 1 , Species 2 , and Species 3 in distinct areas. These specimens have also been Gram stained. 1. 2. 3. Page 10

Lab 5 - Bacteria (Part A) LABORATORY 5 (Part A) Exploring Bacteria Name: ___________________________________________ Lab Section: _____ Introduction 1. What are the 2 main types of growth media used to culture bacteria described in this lab? i) ___________________ ( liquid ) ii) ___________________ ( semi-solid ) 2. What is the main use of each type in culturing bacteria? i) _________________________________________________________________________________ _________________________________________________________________________________ ii) _________________________________________________________________________________ _________________________________________________________________________________ 3. What is aseptic technique and why is it important when culturing bacteria? ____________________________________________________________________________________ ____________________________________________________________________________________ 4. Some culturing techniques on semi-solid medium aim to: a) Isolate colonies . i) What is a bacterial colony? ____________________________________________________________________________________ ii) What is the general STRATEGY used when inoculating semi-solid medium to produce COLONIES? ____________________________________________________________________________________ b) Create a lawn . i) What is a bacterial lawn? ____________________________________________________________________________________ ii) What is the general STRATEGY used when inoculating semi-solid medium to produce a LAWN? ____________________________________________________________________________________ General Biology I Laboratory Exercises Dawson College Fall 2023 Page 11

Lab 5 - Bacteria (Part A) LABORATORY 5 (Part A) Exploring Bacteria Name: ___________________________________________ Lab Section: _____ Lab Report Exercise B: Comparing Bacterial Communities Design of your experiment 1. Which ecosystems will you compare? ________________________ vs. _______________________ 2. What is the question you would like to answer? Be specific. Are you interested in comparing species richness (species numbers) or cell density (number of cells per unit volume) in the two ecosystems? ____________________________________________________________________________________ ____________________________________________________________________________________ 3. What is your hypothesis? ____________________________________________________________________________________ ____________________________________________________________________________________ 4. What is the rationale behind your hypothesis? ____________________________________________________________________________________ ____________________________________________________________________________________ 5. What will be the READOUT for your dependent variable? Be specific. ____________________________________________________________________________________ ____________________________________________________________________________________ 6. Briefly describe what you expect to see in the contamination control section of your agar plate after incubation if your procedure was successful in eliminating contamination. Be specific. ____________________________________________________________________________________ ____________________________________________________________________________________ 7. If you observe the result above in your contamination control, what information does this give you about the bacterial colonies growing in your ecosystem culture plates? Be specific. ____________________________________________________________________________________ ____________________________________________________________________________________ General Biology I Laboratory Exercises Dawson College Fall 2023 Page 12

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Lab 5 - Bacteria (Part A) Exercise C: Exploring Bacterial Cell Characteristics Record your data from your observations. Species 1 Species 2 Species 3 Cell Shape [ coccus / bacillus/spirillum ] Gram Stain [ positive / negative ] Integrating the Concepts Next week (hopefully) we will have many different types of colonies on our test plates that represent the community of bacteria living in different ecosystems. Let’s say that you observe a colony from the terrestrial ecosystem that has a similar COLONY MORPHOLOGY to B. subtills . How would you further characterize this species of bacteria to identify it as B. subtilis? Briefly describe ONE characterization at the CELL level (e.g., cell structure) and ONE characterization at the MOLECULAR level (e.g., metabolic capabilities) that could be done to identify the unknown species as B. subtilis. Cell level: _________________________________________________________________________________ _________________________________________________________________________________ Molecular level: _________________________________________________________________________________ _________________________________________________________________________________ General Biology I Laboratory Exercises Dawson College Fall 2023 Page 13

NYA_F23-Lab_05-Bacteria_Part_A

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