Labortary Excercises in Microbiology, Exercises of Microbiology

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MICROBIOLOGY

BIO 358

LABORATORY EXERCISES

SPRING 2016

Week 1: Media Preparation, Pure Culture Techniques, and Control of Microbial Growth Media Preparation INTRODUCTION A microbiological medium (media, plural) contains the nutrients necessary to support the growth of bacteria, molds, and other microorganisms. It can exist in three consistencies: liquid, solid, and semisolid. In today’s exercise, we will be preparing solid media. Solid media are made by adding a solidifying agent, such as agar, gelatin, or silica gel, to a liquid medium. A good solidifying agent is one that is not metabolized by microorganisms, does not inhibit bacterial growth, and does not liquefy at room temperature. Agar and silica gel do not liquefy at room temperature and are metabolized by very few organisms. We will be using agar as our solidifying agent, and this will enable us to grow and isolate colonies of bacteria on the surface of the medium. MATERIALS AND METHODS – work in pairs One 1-liter beaker One 1-liter graduated cylinder One stir bar Magnetic stir plate pH meter NaOH Two 500-ml bottles Autoclave tape Scissors Stock solution of 100 mg/ml streptomycin Marker for labeling plates Petri plates 55˚ C water bath Striker to light Bunsen burner Bunsen burner PROCEDURE Begin the laboratory period by preparing LB agar containing 100 μg/ml streptomycin for use in Week 5 when we will be studying phage biology.

1. Place the following into a 1 liter beaker: 10 g Bacto Tryptone 5 g yeast extract 5 g NaCl

MATERIALS AND METHODS – work individually Liquid culture of Escherichia coli strain MC4100 grown at 37˚ C or Liquid culture of Staphylococcus epidermidis grown at 37˚ C or Liquid culture of Bacillus subtilis grown at 26˚ C or Liquid culture of Pseudomonas fluorescens grown at 26˚ C Inoculating loop Bunsen burners Sterile cotton swabs Small tubes of sterile water 4 Nutrient agar plates ( 2 plates will be used on Day 1 for the streak plate and the environmental isolate and 2 plates will be used on Day 2 for the re-streaking of both cultures) PROCEDURE Day 1: The day of your scheduled laboratory period. Streak plate. Each person will perform the streak plate. Firstly, do not use the medium you just prepared for this portion of the laboratory exercise. Do use the media provided by the stockroom. A properly executed streak plate will facilitate good dilution of the sample, and good separation of individual colonies for further culturing. For the organisms we will be culturing, the quadrant streak will be most effective for producing isolated colonies.

1. Remove an inoculating loop full of bacterial culture of one of the four available species, Escherichia coli strain MC4100, Staphylococcus epidermidis , Bacillus subtilis or Psuedomonas fluorescens.
2. Inoculate a Nutrient agar plate using the quadrant streak method as I have illustrated. Incubate the plate in the inverted position at either 37°C or 26˚C for 24 hours. The plates are inverted to prevent condensation from dropping onto the agar, a possible source of contamination. Environmental isolate. Each person will perform this portion of the exercise.
3. Isolate microbes from your surrounding environment by taking a sterile swab, dipping it into sterile water, and swabbing a surface in the lab. Swab your isolates onto Nutrient agar.
4. Incubate at room temperature until sufficient growth allows you to see individual colonies.

Days 2 and 3 Streak plate. After sufficient growth of the culture, inspect your streak plate for isolated colonies. Carefully subculture an isolated Escherichia coli strain MC4100, Staphylococcus epidermidis , Bacillus subtilis or Psuedomonas fluorescens colony onto a second Nutrient agar plate using the quadrant streak. Incubate at 37°C or 26˚C for 24 hours and then inspect the plate on Day 2 and/or Day 3. You can toss the initial Nutrient agar plates in the Biohazard Waste after you are convinced you have a pure culture. Environmental isolate. Pick an isolated colony from the Nutrient agar plate, and resteak, or subculture onto a second Nutrient agar plate. Again, incubate at room temperature , until well-formed, isolated colonies are visible. After you are convinced that you have a pure culture, toss the Nutrient agar plates in the Biohazard Waste. Control of Microbial Growth: Evaluating Antiseptic and Disinfectant Susceptibilities of Microorganisms Chemical agents are very useful in controlling microbial growth and survival, and you probably have used a number of them in everyday life. One group of chemicals, classified as disinfectants , is used to control microbes on inanimate objects. A second group, antiseptics , is used for topical application to body surfaces. The third group contains disinfectants that also do some degree of cleaning, and so are referred to as sanitizing agents. Microbial sensitivity to these agents can be tested using the agar disc diffusion technique. In this protocol a culture of the test microbe is spread evenly over an agar plate using a swab. Paper discs containing the agents to be tested for antimicrobial activity are placed directly on the agar plate. The test compounds will diffuse out from the disc creating a gradient. When the plate is incubated, the organism will only be able to grow in parts of the plate without the compound or where the concentration of the compound is low enough so that it does not affect the organism’s ability to grow. The clear area of no growth around the disc is called the zone of inhibition. Microbes that are resistant to the inhibitory affects of a compound will be able to grow right up to the edge of the disc containing the compound. You will use this procedure to answer a question relating to the efficacy of a disinfectant or antiseptic- 10% bleach, Listerine, 10% Ceepryn, a quaternary amine, or Providone Iodine (Betadine). You will develop a hypothesis, design an experiment to test that hypothesis, and collect and analyze data. Efficacy of the disinfectant or antiseptic is determined by the inhibition of growth on nutrient agar plates. Microorganisms that you may choose from include: Escherichia coli strain MC4100, Staphylococcus epidermidis , Bacillus subtilis and Pseudomonas fluorescens. Recall the Gram staining status of these bacteria as this may influence the effectiveness of the antimicrobial agent.

1. Using a marking pen to write on the BOTTOM of each plate, divide your plates into four quadrants. This will allow you to test four different compounds by placing a disc in each quadrant. Each disk should be well separated. Label each quadrant so that you know which disk will be placed into it, and be sure to label the plate with your name, the date and the species of bacterium that it will contain.
2. Aseptically insert a sterile swab into the tube containing the selected liquid nutrient broth culture of bacterium, either Escherichia coli , Staphylococcus epidermidis , Bacillus subtilis or Pseudomonas fluorescens. Make sure to cover the entire plate with the bacterial culture, and to run the swab around the rim of the plate.
3. Sterilize forceps by dipping in alcohol, then passing through the flame of a burner. Allow the flame to extinguish. With the sterile forceps place the filter paper disks soaked in the chemical agents of your choice in each of the previously labeled quadrants. Be sure that the disks are thoroughly soaked but well drained otherwise fluid will flow on the surface of the plate and obscure your results. You can remove excess fluid by carefully dabbing on a Kimwipe. Be sure to sterilize the forceps each time you get a new disk. Carefully record the species of bacteria and the chemical agents used for each quadrant on each plate.
4. Incubate the plates, right side up, at the appropriate temperature for ~24 hours. Day 2
5. Using a metric ruler, measure the zones of inhibition for the four disks on each plate that you inoculated. Remember that the zone of inhibition is the diameter of the region around the disk that the bacteria did not grow, and that the disk itself is ~ 6 mm. Thus if a particular bacterial species is resistant to the chemical agent, then the zone of inhibition will be 6 mm, not 0 mm. Record these results in a well labeled table.
6. For the analysis, use JMP to perform an Analysis of Variance (Anova) on the data to test your null hypothesis that there is no difference between the treatments. If you are not familiar with JMP you can use Excel. In JMP, you can analyze your data by using Fit Y by X. Then from the red arrow select Means/Anova. If there is statistical significance you can then compare all pairs by using Tukey-Kramer HSD (honest significant difference) under the red arrow. Using this post-hoc analysis determine whether you can reject the null hypothesis at the 95% confidence interval. Lab Report Only report on the Evaluating Antiseptic and Disinfectant Susceptibilities of Microorganisms portion of the exercise. Clearly state your hypothesis and experimental design. Present results in a well-labeled graph or table. Present your statistical analysis. What can you conclude from your experimentation? Lab reports are due at the beginning of your next laboratory period.

Week 2: Microbiological Assay: Aerobic Versus Anaerobic Utilization of a Limiting Nutrient INTRODUCTION When one nutrient in a microbiological medium is present in a limiting concentration and other nutrients are present in excess, the yield of the culture, or maximum growth, is a function of the concentration of the limiting nutrient. This is the principle of a microbiological assay. In addition to the concentration of a limiting nutrient, the availability of atmospheric oxygen affects the growth yield, or the maximum absorbance of a bacterial culture. In this experiment we will investigate the relationship between the yield obtained per unit of substrate used in the presence of oxygen (respiration) and the growth per unit of substrate used in the absence of oxygen (anaerobic respiration or fermentation). Adapted from microbiology laboratory exercises developed by Wiltraud Pfeiffer, PhD, UC Davis. MATERIALS AND METHODS – work in pairs Overnight culture of Escherichia coli strain MC4100 grown in LB broth Sterile M9 minimal medium. This medium is provided for you and contains, per liter, 6g Na 2 HPO 4 , 3g KH 2 PO 4 , 0. 5g NaCl , 1g NH 4 Cl. After autoclaving, 10 ml of 0.01 M solution of CaCl 2 , 1 ml of a 1 M MgSO 4 .7H 2 0 solution and 1 ml of a 1 mg/ml solution of thiamine are added. 8 Sterile 250 ml flasks containing 100 ml of M9 minimal medium (aerobic cultures) 100 - ml bottle of sterile M9 minimal medium (for anaerobic cultures and blank) 8 Sterile culture tubes for you to dispense 10 ml of M9 minimal medium (anaerobic cultures) Sterile glucose: 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 1 M, and 2 M solutions Glucose solutions of unknown concentration Disposable Cuvettes Micropipettors Spectrophotometers Computers PROCEDURE Day 0: The day prior to your scheduled laboratory period.

1. For the aerobic cultures, aseptically add 1 ml of the sterile glucose solutions to 7 250 - ml flasks containing 100 ml of M9 minimal medium (see Table 1 ). For example, to achieve a final glucose concentration of 1 mM glucose, add 1 ml of the 100 mM glucose solution, for the 2 mM glucose culture add 1 ml of the 200 mM glucose solution, etc. Make sure to label the flasks with your name, glucose concentration and the date using tape and a sharpie.

minimal medium without glucose. Use this for your spectrophotometer blank because we are measing cell density, and glucose will not alter the absorbance at 600 nm.) For both the aerobic and anaerobic cultures, make sure that the bacteria are evenly distributed in the vessel. For the flasks you may swirl, and invert the sealed tubes a few times prior to taking the sample. MIX GENTLY so that you do not get air bubbles into the culture, which might adversely affect your results.

9. Construct a growth response curve by plotting the absorbance units as the y-axis and the FINAL molar glucose concentration as the x-axis using JMP or Excel. You should have growth response curves for both the aerobic and anaerobic cultures. (Make sure that your group measures the absorbance values for the unknown glucose concentration cultures.)
10. Determine the equation of the line for the linear portion of the dose response curves for both the aerobic and anaerobic cultures.
11. Using the equations describing the linear portions of the aerobic and anaerobic cultures, determine the unknown glucose concentration.
12. Compare the efficiency of utilization of glucose aerobically (shaken 100 ml culture) and anaerobically (static 10 ml culture) by determining the following calculations for each condition. Remember that your calculations will only be valid while the glucose is a limited nutrient, i.e., during the linear portion of the curve. Specific Yield = grams dry weight of bacteria/grams of limiting nutrient (glucose) Molar Yield = grams dry weight of bacteria/mole of limiting nutrient (glucose) Moles ATP = Molar Yield Moles glucose YATP Some information you might find useful: A 600 conversion factor: 1 A 600 = 0.845 mg dry weight cells/ml MW glucose = 180 g/mole YATP (grams of dry weight bacteria/mole of ATP) = 10.5 g/mole

Lab Report The data tables and answers to questions are to be turned in by the end of the laboratory period. List your absorbance values for the 16 cultures in the table below: Aerobic Culture 1 mM 2 mM 3 mM 4 mM 5 mM 10 mM 20 mM Unkn A 600 Anaerobic Culture 1 mM 2 mM 3 mM 4 mM 5 mM 10 mM 20 mM Unkn A 600 Summarize your calculations in the following table. Include in your analysis a well-labeled graph, with an informative figure legend, of the final molar glucose concentration versus the A 600 for both the aerobic and anaerobic cultures. Use one point on the linear portion of each curve to fill in the table below. Please show calculations on a separate sheet. Include the calculations and the graphs with your report. Aerobic Culture Anaerobic Culture Specific Yield Molar Yield Moles ATP/ Mole Glucose QUESTIONS

1. Which unknown glucose solution did you use, and what was its original concentration? Present the equation of the line for the standard curve used to make this determination.
2. Which is more efficient – aerobic growth or anaerobic growth? Explain your answer. How are the bacteria growing if they are not growing aerobically?
3. What is meant by a “limiting nutrient”? In this experiment, at what concentration is glucose no longer limiting?
4. Were the anaerobic cultures truly anaerobic or were they oxygen-limited? Explain your answer.

Week 3: Microbial Diversity INTRODUCTION The normal bacterial flora, or microbiome plays an important role in human health. The microbiome influences nutrition, resistance to colonization by microbial pathogens, carcinogenesis, obesity and is even thought to affect human behavior. An accurate understanding of these roles, and the nature of the interactions among and between individual members and the host, requires knowledge of the composition of the microbial community. Many studies of environmental microbial communities have demonstrated the limitations of cultivation-dependent methods in determining community composition. Environmental surveys based on acquisition of phylogenetically useful microbial sequences such as that of the 16S rRNA gene (16S rDNA) have revealed a great deal of previously unsuspected bacterial and archaeal diversity. In most instances, the cultivated members represent less than 1% of the total extant population. Broad range small subunit rDNA PCR methods have also revealed cultivation-resistant pathogens in disease settings. Despite the limitations of this approach, most surveys of the human endogenous bacterial flora, historically, have relied on cultivation. Using cultivation techniques, nearly 500 bacterial strains have been recovered from the human subgingival crevice, a particularly well-studied microbial niche. Many of these bacteria are thought to be commensals, and a smaller number, opportunistic pathogens. Local disease, including dental caries, gingivitis, and periodontitis, has prompted most examinations of the oral bacterial flora. These diseases are associated with changes in both local bacterial density and species composition. In order to investigate the bacterial flora of the subgingival crevice, our approach we will use PCR amplification of 16S rDNA sequences in order to identify molecularly these bacterial genera, as well as classical culture techniques. By these methodologies we will initiate characterization of the bacterial diversity within a specimen from the human subgingival crevice. We predict that our results will reveal a significantly broader diversity of bacterial 16S rDNA sequence types (phylotypes) using the cultivation- independent approach, although both methods can identify previously uncharacterized phylotypes and should be viewed as complementary. This laboratory exercise was adapted from Kroes, I., Lepp, P.W. and D.A. Relman (1999) Bacterial diversity within the human subgingival crevice. Proceedings of the National Academy of Sciences USA. 96: 14547 – 14552.

MATERIALS AND METHODS – work in pairs Sterile molecular biology grade water Sterile nutrient broth Microcentrifuge tubes Sterile toothpicks Microcentrifuge racks Inoculating loops Microscopes Microscope kits Stage micrometers Materials for performing Gram stain 2 Thermalcyclers with heated lids Enzyme cold boxes 0.8% agarose in 1 X TAE Buffer, pH 8. 1 X TAE Buffer, pH 8. Electrophoresis apparatuses 6X DNA Gel-Loading Buffer (Sambrook, Frisch and Maniatis) SYBR Safe DNA gel dye 2 - log ladder (NEB) Gel documentation system Cultures of E. coli strain MC4100 and Staphylococcus epidermidis for quality control of LB agar plates containing 100 μg/ml streptomycin prepared in Week 1 PROCEDURE Day 0: The day prior to your schedule laboratory period.

1. Using good aseptic technique, each person will dispense 1-ml of nutrient broth into a microcentrifuge tube.
2. Collect a subgingival specimen from the surface of a tooth using a sterile toothpick as demonstrated in class. Rub the end of the toothpick on the inside of the centrifuge tube containing the nutrient broth.
3. Label the tube with your name and date, and place it in the 37˚ C incubator in a rack. Only one sample will be used for PCR, but both samples can be subjected to Gram staining. Day 1: The day of your scheduled laboratory period.
4. Set up three PCR mixtures using broadly conserved bacterial 16S rDNA primers.

7. Gently mix the above reaction mixture by flicking the tube with your finger or gently vortexing.
8. Taq Polymerase, the enzyme, is always added last. Using a micropipettor, pipette 1. μl of Taq Polymerase enzyme into each tube.
9. Gently mix by flicking or by gently aspirating up and down with a P-100 pipettor and a yellow tip. Do not vortex!
10. Program the Thermalcyclers as follows: For Reactions 1- 3 Step 1 Hot Start 94°C 3 minutes Step 2 Denaturation 94°C 30 seconds Step 3 Annealing 55°C 30 seconds Step 4 Extension 72°C 30 seconds Step 5 Return to Step 2 twenty-nine times for a total of 30 cycles Step 6 End
11. Start the thermalcycler by selecting your program and press . Allow the cycler to reach 94°C before you place the tubes in the holes.
12. When it reaches 94°C, put in the tubes and let ‘em rip.
13. While the PCR is running pour a 0.8% agarose gel. Start by melting the agarose in the microwave- the setting works well for this. Stop the microwave after a few seconds, and swirl the bottles gently to make sure the agarose is completely melted. Wear a hot mitt and be careful!
14. Allow the agarose to cool slightly on the bench top so that you can pick the bottle up without the hot mitt.
15. Before the agarose begins to solidify, pour ~25 ml into a gel apparatus containing the appropriate comb.
16. Immediately place 3 μl of SYBR Safe DNA gel dye directly into the agarose. Spread thoroughly with the fat end of a yellow pipette tip.
17. Allow agarose to solidify before loading samples.

Microscopy While you are waiting for the PCRs perform a Gram stain on the microbes that you sampled from your subgingival crevice, and grew in an overnight nutrient broth culture. Observe the specimens under brightfield microscopy. Recall that in 1884 the Danish bacteriologist Christian Gram developed a staining technique that separates bacteria into two groups: those that are Gram-positive and those that are Gram-negative. The procedure is based on the ability of microorganisms to retain the purple color of crystal violet during decolorization with alcohol. Gram-positive bacteria are not decolorized and remain purple. After decolorization, safranin, a red counterstain, is used to impart a pink color to the decolorized Gram-negative bacteria. Follow the Gram staining procedure illustrated on the handout provided. Of all the staining techniques you will use in the identification of bacterial specimens, Gram staining is the most important tool. Although this technique seems quite simple, performing it with a high degree of reliability requires some practice and experience. Here are two suggestions that can be helpful: first, don’t make you smears too thick. Second, pay particular attention to the comments in the decolorization step. Using the Gram stain you can determine a microbe’s cell shape, arrangement of the cells and Gram stain status. Obtain a stage micrometer from the front bench and use this along with the ocular micrometer in your microscope to determine the size of the microbes observed in the Gram stain. Agarose Gel Electrophoresis

1. Remove 20 μl of your completed PCR mixtures and combine with 4 μl 6X DNA Gel- Loading Buffer (1:5 or 1/6 of the total sample volume).
2. Flick the tubes to mix, and load samples into a wells in a 0.8% agarose gel in 1X TAE buffer, pH 8.0. If the well will not hold the full 24 μl, simply fill it to a maximum volume. Don't overload the well so that the sample spills into other wells; this will confuse your results.
3. Prepare the molecular weight marker by combining 1 μl of 2-log DNA ladder (NEB) with 4 μl of sterile water. Add 1 μl of DNA Gel-Loading Buffer and mix. Load the 6 μl of the molecular weight marker into one of the wells in the gel.
4. Turn on the power supply, approximately 15 volts per cm (distance is measured between the two electrodes). You can run most gels at around 120 volts.
5. After the lower dye front is about two-thirds down the gel, turn off the current.

Lab Report Clearly state the objective of the laboratory exercise. Present an image of the gel, including the molecular weight of bands of the DNA ladder (available at the instructor’s bench) and present a well-written figure legend to accompany the image. Also draw and describe what you observed by Gram stain using brightfield microscopy. Describe the microbes’ size , shape, arrangement and Gram stain status. Stage micrometers are on the instructor’s bench as well. QUESTIONS

1. Did you observe mostly prokaryotic or eukaryotic organisms by the Gram staining procedure? How do you know this?
2. Starting from the DNA bands on the gel, how would identify the community of bacterial species residing on your tooth surface? Remember that each band represents a population of DNA fragments.
3. Assume that you sequenced all of the 16S rDNAs from organisms found in the tooth scraping. If you searched long enough, could you eventually find all the corresponding organisms by culturing them? In other words, would you expect the number of unique organisms identified molecularly and those identified by culturing to be the same? Lab reports are due at the beginning of your next laboratory period.

Week 4: Transposon mutagenesis in Vibrio fischeri INTRODUCTION Transposon mutagenesis is a powerful genetic tool for studying biological problems in bacteria. In this procedure, random mutations are generated in the chromosome or plasmid of the bacterium and researchers screen for the loss of a particular function. Transposons commonly carry selectable markers, most often antibiotic resistance genes. In this procedure a collection of mutants is generated, each containing a single transposon insertion. One distinct advantage of transposon mutagenesis is that the selectable marker can be used to locate the site of insertion and can be used to clone the gene of interest. Transposon mutagenesis is often facilitated by the transfer of a conjugative plasmid containing a transposon to a recipient strain. The donor for this experiment, E. coli strain S17-1 contains the genes necessary to assemble the sex pilus and transfer the plasmid pLOFKm containing the mini Tn 10 Km transposon. The origin of replication of pLOFKm is recognized by the π protein, encoded by the pir gene, in strain S17-1 that is not found in the Vibrio fischeri recipient. Thus the plasmid pLOFKm will not replicate inside the recipient strain and acquisition of the selectable marker will only occur if the transposon inserts into the chromosome of Vibrio fischeri. pLOFKm contains an IPTG-inducible promoter upstream of a transposase. The transposon itself contains a kanamycin resistance gene. On the same plasmid, but not in the transposon, there is an ampicillin resistance gene. The biological problem we will address using transposon mutagenesis is: What genes are necessary for the production of light by the marine bacterium Vibrio fischeri? This particular wild type Vibrio species was isolated out of the gut of a squid from the Oregon Coast. Vibrio fischeri is a Gram-negative bent rod, or vibrio, which is a symbiotic bacterium found in the intestines of many marine organisms. It colonizes the light organs of many fish as well as the bobtailed squid Euprymna scolopes. We will conjugate the plasmid pLOFKm into the Vibrio , selecting for kanamycin resistance conferred from the insertion of the transposon. We will then screen for loss of bioluminescence, or light production. A couple of things to keep in mind while working on this lab— V. fischeri is heat and cold sensitive. Do not refrigerate any of the plates or samples. Also, use only the 30˚ C incubator. Since V. fischeri is a relatively slow growing bacterium, contamination is a big problem— always use good aseptic technique. References

1. Herrero et al ., 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. Journal of Bacteriology 172 : 6557-6567.
2. Bassler BL, 1999. How bacteria talk to each other: regulation of gene expression by quorum sensing. Current Opinion in Microbiology 2 : 582-587.