7: Selecting Microbes for Testing
- Page ID
- 124420
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Bacterial Nutrition
Like all organisms, microorganisms must use energy to build new cells, divide, and respond to their environments. The types of organic molecules produced by bacteria are the same as eukaryotic organisms: proteins, nucleic acids, lipids and carbohydrates (Table 1). All cells ultimately capture and store released energy in the form of adenosine triphosphate (ATP), the “energy currency” common to all life, however, the processes by which they obtain this energy can vary.
Bacterial metabolism is very much more diverse than eukaryotic cells. We can classify bacteria based on how they obtain carbon and energy from their environments to make biological molecules. Some organisms, autotrophs or the producers in the food chain, produce organic molecules by obtaining or “fixing” carbon from inorganic sources such as CO2. Some examples are photosynthetic organisms such as plants, algae, and cyanobacteria. On the other hand, heterotrophs obtain carbon from organic molecules formed by other organisms. Animals, protozoa, fungi, as well as many bacteria and archaea are heterotrophs. Because we are providing our bacteria with agar plate/media based food sources, the isolates we are studying are primarily heterotrophic. Another major distinction in bacterial classification relates to their tolerance for oxygen. Most bacteria that are in contact with the air are aerobic, meaning that they need oxygen (O2) to survive. Bacteria that do not require oxygen or find O2 toxic are referred to as anaerobic.
| Organic Molecules | Elemental Components | Macromolecules |
|---|---|---|
| Amino acids | C, H, O, N, S | Proteins |
| Nucleic acids | C, H, O, N, P | DNA and RNA |
| Fatty acids | C, H, O | Lipids |
| Sugars | C, H, O | Carbohydrates |
Media and Culture Conditions
In the lab, microbes are grown on many different types of media that contain mixes of essential nutrients suspended in water (e.g., broths, infusions, etc.). Some media like the ones that we are using, are solidified through the addition of agents such as agar or gelatin. Media ingredients range from dehydrated extracts of plant, animal, or fungal material to synthetically derived compounds. Media can be very nutrient rich, containing large concentrations of proteins, amino acids, simple or complex sugars, and/or salts. Media can also be kept minimal, composed or lower concentrations of simple sugars, salts, and water. In our laboratory, we will have multiple choices of media and you will have the opportunity to select the types you wish to use in your experiments.
Other physical parameters such as light exposure, temperature, pH, and salt concentration can drastically affect the composition of microbes that grow on plate. Bacteria can be quite sensitive to the ions, pH, and temperature that they are exposed to and photosynthetic bacteria will only grow in the light.
Growth on a plate indicates that the bacteria are living within an acceptable range of conditions that allows them to survive and proliferate. It does not mean that all of their needs are met or that they are making all of their possible compounds. In previous semesters we have seen examples where one bacterial species grows at multiple temperatures, but only make a pigment at one of them. We've also seen one bacteria make antibiotics on one type of media, but not another. This fascinating finding is related to induced gene expression, or the production of proteins based on signals received by the cell. Finding the right combination of growth conditions and environmental signals to stimulate both growth and antibiotic production is one of the most challenging parts of this research. There are many variables we can consider when choosing the right growth conditions and the more we experiment with conditions, the more we will be able to observe and learn from our samples—so use your imagination.
Growth Conditions: Solid vs Liquid Cultures
Colony Morphology
As you look at your dilution plates you will notice that the colonies display striking differences in physical appearance such as color, texture, shape, and margin or edges. Entire books, like the
thorough Bergey's Manual of Systematic Bacteriology contain descriptions of these features and help microbiologists identify and characterize bacteria. One of the easiest characteristics to observe is the shape of their colony, or colony morphology (Figure 1).
Figure 1. Diagram illustrating the various shapes, edges, and elevations of bacterial colonies. This is a useful guide for describing our soil microbes and being able tell them apart.
Different types of bacteria tend to demonstrate characteristic morphologies – for example, Lysobacter and Pseudomonas colonies are typically slimy, irregular in shape, and may fluoresce under UV light (Figure 2A). Streptomyces colonies have bold colors on the base of the colony and develop a white fuzzy top, which contains spores (Figure 2B). Bacillus colonies may sprawl thinly over the agar and typically show relatively dull, bland colors.
Unfortunately, we can't rely on visual appearance alone to identify our bacteria as we can get easily misled. However, this provides us a good clue about a possible bacterial identity which we will build on with future tests and experiments. The most precise and accurate way to identify our bacteria is using DNA sequencing, which we will do later in the semester.
Figure 2. Three microbes with distinctive colony morphorphologies, typical of their genus. (A) Serratia marcenscens is red colored and can form colonies with white edges. The colonies are irregular in shape with entire edges. (B) Acinetobacter baylyi forms translucent white or clear colonies. The bacteria is gooey and stringy when lifted up off the plate. (C) Micrococcus luteus forms convex circular yellow colonies with an entire edges. These colonies are often small sized.
Growth Phases
Solid cultures are advantageous because culturable cells will grow where they are placed. This will allow us to isolate pure cultures. Liquid media is the opposite - the bacteria move around freely and randomly in the medium. If multiple types are present in liquid media, they are impossible the separate. Therefore, we will focus on using solid media to grow our many types of bacteria.
Bacterial growth typically follows a particular pattern. When put on/in a new media, bacteria typically grow very slowly at first (lag phase), as they become familiar with the new environmental conditions. After a few hours, the growth rate takes off and the cells begin to divide rapidly (log phase) or exponential phase. As nutrients are depleted and waste products accumulate, growth
slows such that the number of cells dividing is approximately equal to those dying (stationary phase). Secondary metabolites such as pigments and antibiotics are normally produced toward the end of the log phase, as cell concentration peaks and nutrients become depleted. Bacteria in the genus Streptomyces have been found to start making their secondary metabolites after they finish making their essential cellular components such as DNA (Bibb, 1996).
Protocols
Protocol 1: “Pick” and “Patch”
Our project this semester is to try to isolate and characterize antibiotic producers from soil found on campus. To do this we want a large variety of microbes to test to increase our chances of finding interesting antibiotic producers. Today, we will look at the growth of bacteria we plated on agar plates last week and try to isolate a large number of distinct single bacterial types to test next week.
You completed the first step last week where you used serial dilution and plating to spread out your bacteria on an agar plate. After allowing them to grow for a week, you will see many individual colonies. Unfortunately, you will also see many instances where colonies grew too close together, cells moved and spread across the plate, and/or cultures got contaminated. As you isolate colonies today from your plates, you will want to select colonies that are distinct and avoid those that are mixed or potentially contaminated.
Sterile conditions in your work environment and proper techniques are very important to isolating a bacterium and starting a pure culture. The isolation approach we will use is aptly called “pick and patch” and involves precisely those things: “picking” bacteria from a mixed culture (e.g., usually a
dilution plate) and “patching” them onto a fresh plate, which may be a pure culture or a “master plate” containing all your unique bacteria of interest for your study. The slight touch of a colony with a sterile toothpick or a metal rod picks up thousands of bacteria that can be transferred and smeared or patched onto a fresh plate. At the end of this procedure you will have a “master plate” that will serve as a bacterial catalog for your experiments.
In choosing which colonies of bacteria to follow up with, there are several things that we will need to consider.
|
1. Only select one species at a time |
Select ones that don't touch & don't pick spreaders. |
|---|---|
|
2. Choose a wide variety of bacteria |
Select ones that look different in size, color, texture |
|
3. Keep the single species isolated on the master plate |
Put each species in a different square & leave space |
|
4. Select enough bacteria to find an interesting one |
Try to get 2 full plates of bacteria |
Figure 3. Master plate layout. This example shows how your plate should look after incubation and allowing the isolates to grow. Patches (in red) were oriented on the plate using a grid containing 21 colonies. Notice how patches do not touch—this is critical to prevent cross-contamination.
- Serial dilution plates from last week
- 2+ media plates of choice
- Square grid template
- Sterile Toothpicks
- Lab marker
Protocol
- Allow serial dilution plates of bacterial isolates or master plate to grow for 3-7 days
- Photograph your plates and describe them fully in your notebook
- Obtain 2 sterile agar plates containing your media of choice. Label the bottom (agar side) with:
- Group Name
- Date
- Media Type
- Temperature
- Plate number
- and mark the orientation of the grids
and draw a small vertical line at the edge of your plate as point of orientation
- Observe and describe the color, size, surface texture, elevation, margin, and shape each colony you select in you lab notebook. Note: You are not limited to using the terms in Figure 1. Once the colony is recorded, use a sterile toothpick to pick the unique colony from the dilution plate (10-3 or 10-4)
Note: Keep both plates covered whenever possible.
- Patch (gently zigzag) the colony smear on the toothpick onto fresh media plate within the boundaries of the square on your grid. Be careful not to puncture the agar as you smear. Also be careful to stay well within your gridlines so that you don’t contaminate another square.
- Continue to pick and patch colonies onto media plate, each time into a new square. Make sure patches do not overlap or touch. Try to find as many morphologically unique colonies (i.e., different textures, colony margins, pigments), but do not go over 21 colonies per plate to avoid overcrowding.
- If possible, try to pick and patch 42 colonies (2 master plates). This collection of colony patches will be your “master plate.”
Patching Video
When you are finished patching your samples, please put your new plates side up at the appropriate temperature. Stake your serial dilution plates and put them in the plate bag provided by your instructor. Label the correctly colored tape provided by your instructor with your group name and the date. These plates will be stored in the fridge in case you need them next week. The used sticks should go in the appropriate container to be sterilized for reuse. Any tubes or tips that you used can be thrown in the red biohazard bin. Please return all reagents tools to the place that you got them.
Using the 70% ethanol sprayers on your bench, spray down your bench to disinfect it and wipe it clean with a paper towel.
Meet the ESKAPE Pathogens
Understanding Infectious Disease
For centuries, people thought that disease was due to “bad air” or miasmas and as a result had little understanding of infectious disease. John Snow, a London physician, made a huge leap forward and traced the epidemic and transmission of cholera to a contaminated communal water pump.
The water source had been contaminated by the sewage thrown into the street and backyards of sick individuals. As a result of this discovery, the pump was closed, saving hundreds of lives, but more importantly began to connect disease to a physical source. Around the same time, Louis Pasteur, a French chemist and microbiologist demonstrated that sterile broth (like the media we use in lab) needs contact from the air or another external source to become contaminated. This began to draw attention to the microbes and resulted in the pasteurization process now used to make milk, juice, and beer safe to drink and increase their shelf-life.
Another 19th century scientist, the German physician Robert Koch developed four criteria (postulates) that we use to identify pathogens, or disease-causing microorganisms.
- The microbe is always present in diseased individuals but not healthy individuals.
- The microbe is isolated from the infected individual and grown in a pure culture.
- The microbe in pure culture is introduced into a healthy individual and the same disease occurs.
- The microbe is re-isolated and shown to be the same as the original.
These ideas might seem obvious, but were revolutionary to 1800s doctors and helped Koch identify the bacteria causing anthrax (Bacillus anthracis) and tuberculosis (Mycobacterium tuberculosis) and earn the Nobel Prize in 1905.
Unfortunately, many pathogens that were easily treated with antibiotics are now emerging in deadly and drug resistant infections. As a result, we are rapidly running out of treatments especially for hospital acquired "superbugs" and need to both slow the rate of the development of antibiotic resistance as well as discover new antibiotic treatments. Our research focuses on the discovery portion. As you isolate individual microbes, hopefully some will produce antibiotics. Initially, for practical reasons, researchers assay these microbes for the ability to inhibit the growth of only a few test organisms. Next week, we will describe experiments to screen our isolates for their ability to produce antibiotics. But first, we must decide which microbe to test our isolates against.
Choosing Test Organisms - The ESKAPE Pathogens
Since our goal is to fight human pathogens, it makes sense to select a test organism that is clinically relevant. There are six organisms that are today considered to be major threats, because they comprise the majority of antibiotic-resistant infections seen in health care settings. They are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and several species of Enterobacter (ESKAPE).
Because prokaryotes differ in their anatomical and physiological traits, different organisms may be more or less susceptible to the effects of any particular antibiotic. One anatomical feature that plays a significant role in the susceptibility of a microbe to a particular antibiotic is its cell wall composition. Gram Positive bacteria have one layer of membrane and are surrounded by a thick layer of the cell wall made of peptidoglycan. Gram Negative bacteria, on the other hand, have two layers of cell membrane with a thinner layer of cell wall in between. As a result of this additional cell membrane, it is harder for antibiotics to get into Gram Negative cells and they tend to be more antibiotic resistant. We can't use the ESKAPE pathogens in lab because of safety concerns and worries that we might sicken ourselves or others. Instead, we will use their safe relatives in our tests: closely related species that do not cause disease. The names of the ESKAPE species, their safe relatives, and whether they are Gram Positive or Negative are listed in Table 2.
ESKAPE Pathogen |
Safe Relative |
Gram |
|---|---|---|
|
Enterococcus faecium |
Bacillus cereus |
Positive |
|
Staphylococcus aureus |
Staphylococcus cohnii |
Positive |
|
Klebsiella pneumonia |
Escherichia coli |
Negative |
|
Acinetobacter baumannii |
Acinetobacter baylyi |
Negative |
|
Pseudomonas aeruginosa |
Pseudomonas putida or fluoresence |
Negative |
|
Enterobacter species |
Enterobacter aerogenes |
Negative |
References
Bibb, M. (1996). The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology, 142:1335–1344.
Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert, D., Rice, L. B., Scheld, M., Spellberg, B., & Bartlett, J. (2009). Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 48(1):1–12.
Hernandez, S., Tsang, T., Bascom-Slack, C., Broderick, N., & Handelsman, J. (2018). Tiny Earth: A Research Guide for Student Sourcing Antibiotic Discovery. XanEdu Publishing Inc. Print
Slonczewski, J., & Foster, J. W. (2011). Microbiology: An Evolving Science (2nd ed.). New York: W.W. Norton & Co.
Wessner, D. R., Dupont, C., & Charles, T. (2013). Microbiology. Print.