Identification of Resistant Bacteria and Exercises 1-4 are from the PARE project; SLCC is a participant:
Genné-Bacon EA, Bascom-Slack CA. The PARE Project: A Short Course-Based Research Project for National Surveillance of Antibiotic-Resistant Microbes in Environmental Samples. J Microbiol Biol Educ. 2018;19(3):19.3.97. Published 2018 Oct 31. doi:10.1128/jmbe.v19i3.1603
Identification of Resistant Bacteria
Most of the organisms growing on your plates are from Domain Bacteria. Each colony on a plate potentially represents a genetically unique organism. For over a century, the only strategy available to identify bacterial strains was to grow them in pure culture, then use a variety of biochemical assays, stains, and growth tests (e.g. can it break down urea? Is it Gram-positive? Can it grow in extremely salty conditions?). This is labor-intensive and potentially dangerous if you are working with a pathogen.
Today, we are lucky enough to be able to directly identify colonies using polymerase chain reaction (PCR). In PCR, a stretch of DNA is replicated over and over until you have a workable quantity. We can then use the amplified DNA for sequencing. Since we don’t know the identity of our unknown tetracycline-resistant bacterial strains, we will use PCR primers targeted to a gene found in all organisms – the 16S/18S rRNA gene. This gene encodes one of the rRNA molecules that are used to construct ribosomes, the essential cell machinery that catalyzes the formation of new proteins. The 16S/18S gene is highly conserved across all life on Earth. However, the sequence in different organisms is not 100% identical. Rather, the gene sequence has slowly changed over evolutionary time – in other words, once a group of organisms is split into two populations that no longer exchange DNA, mutations will slowly accumulate in genes such that the two groups will be slightly different in their DNA sequence. The longer the time since divergence of the two groups, the more differences between their DNA sequences.
In today’s lab, you will use PCR to isolate a part of the 16S/18S rRNA gene. We will send your DNA out for sequencing. In a future lab, you will compare your sequence to a database of known sequences to identify your bacterial strain.
Exercise 1: Count Colonies from your dilution plates
- Incubated, plated cultures from your dilution series last lab
- Washable marker (or ones that can be removed with ethanol)
- Hand-held manual counter
- Plate counter (if needed)
- Your data worksheet
- Arrange plates from your dilution series as shown in the figure below. Take a photograph of your entire Plate Set—you will need it for Step 8: Recording Soil and Colony Count Data into National Database. Depending on what your ambitions are for the PARE Project, you may also want to take photographs of individual plates.
2. Scan plates for obvious errors in dilution plating. Each plate should have 10-fold fewer colonies than the plate to its immediate left. If this is not the case, make note of this as you will record this information in the “notes” section of the database. For each row of plates, determine the most “countable” plates (i.e. those that have between 30 – 300 colonies).
3. Each team pair should count the colonies on the most countable plates for their Plate Set, and record these values in the appropriate section of Table 1.5 of your Data and Results Worksheet. Be sure to come to an agreement with your partner on the colony counts for each row of your Plate Set. You may use washable markers to mark on the plates which colonies have already been counted. If the parafilm gets in the way of counting, loosen it slightly, but do not remove it—the parafilm helps reduce personal exposure to the microbes. Indicate in Table 1.5 which plates have too much growth to count (TM), which plates have too few to count (TF), and which plates cannot be counted due to overgrowth of microbes (OG). Note: Plates containing any growth that obscures the visibility of individual colonies, such as fuzz, or slime, cannot be counted and should be recorded as OG.
4. Wipe the marker from your set of plates (but do not remove any of the important labeling information) and then swap plates with the other team pair in your group. Each pair should repeat #3 above with the second Plate Set. Record your count values for the other pair of your team’s plates on Table 1.5 of your data worksheet.
5. Compare the numbers between your plate values in Table 1.5 of your pair’s data worksheet, and the other pair’s values for the plates in Table 1.5 of their data worksheet. If the numbers are similar (for example: 105 colonies vs 108 colonies), you can take the average of the two numbers for that Plate Set. If the numbers are very different (for example: 80 colonies vs 115 colonies), work with your team members to figure out why the numbers are so different.
6. Before performing calculations, ensure that all team members agree on the colony counts entered in Table 1 for both Plate Sets. Your instructor may have you enter these agreed-upon values into an Error-Checking Spreadsheet so you can compare your results to those from different soil samples. After team members are in agreement about Plate Set 1 and 2 counts (i.e. both tables in 1.5 should be identical for all team members at this stage), you are ready to perform calculations.
Exercise 2: Calculate the Number of CFU’s per Gram of Soil
- Your data worksheet
- Once your plate values have been accurately determined, you will each be working separately on your calculations. This will reduce error in calculations.
- For each Plate Set, refer to the number of colonies on the countable no-antibiotic (NA) plates (Table 1, Row 1 of your data sheet). Copy these numbers down in the first row of Table 2.
- The second row of Table 2 indicates the volume of plated cells—in this lab, we plated 200μL, but convert this over to milliliters (i.e. you plated 0.2ml onto each plate).
- In the third row of Table 2, write the dilution of the NA plate that was counted. Determine the dilution factor for the countable plates for row 4. The 1/101 dilution was diluted by a factor of 10, so the dilution factor is 10. The 1/102 dilution was diluted by a factor of 100 (102), so the dilution factor is 100 and so on. If the most countable plate for a Plate Set resulted from the 1/102 dilution, the dilution factor for that plate would be 100.
- Using the following formula, calculate the total number of colonies per gram of soil and enter into Table 2, Row 5 for both Plate Sets. After you have finished calculating, check your answers with your teammates and make corrections as necessary.
CFU’s/g soil = (colony count) x (plate dilution factor) x (volume correction)
Note: This formula is based on 1g = 1ml; we must correct the volume used by multiplying by 5 because we plated a volume 0.2mL and we need the value for 1ml (i.e. 5 x 0.2ml = 1ml) to arrive at the number of cells per ml. Also, the volume plated was diluted relative to the original soil sample, so we must also multiply by the dilution factor.
Example: If there are 198 colonies on the 1/103 dilution plate:
(198) x (103) x (5) = 9.90 x 105 CFU’s/gram soil
Note: You may use scientific notation (9.90 x 105) on your data worksheet, but not when entering the information into the database (you must enter the entire number of 990000).
Exercise 3: Calculate the Frequency of Tetracycline-Resistant Colonies
- Your data worksheet
- Using the same logic as Step 6, fill out Table 3 of your data worksheet. First, transfer values for the Tet3 and Tet30 plates in Table 1 to Row 2 of Table 3. If no colonies appear on these plates, then you can say that there are no detectable tetracycline-resistant colonies present under our testing conditions. Tetracycline-resistant bacteria may still be present, but perhaps not enough cells were plated to where they could be detected (i.e. low frequency), or they simply could not grow under our growth conditions (e.g. critical nutrient was missing).
For each concentration of tetracycline, fill in rows 3-5 of Table 3 and calculate the TetR CFU’s per gram of soil using the same equation as above: (CFU’s on plate) x (5) x (dilution factor)
Example: If there are 58 colonies on the 1/102 dilution of the Tet3 plate:
(58) x (102) x (5) = 2.90 x 104 Tet3R CFU’s/gram soil
In Table 3, row 6, record the total tetracycline-resistant CFUs per gram soil for each concentration of tetracycline for both Plate Sets.
- Calculate the relative frequency (%) of tetracycline-resistant cells as a function of the total CFU’s per gram of soil calculated in Step 6, by dividing the total number of TetR CFU’s per gram of soil (Table 3, Row 6) by the total CFU’s on the NA plate (Table 2, Row 5), then multiplying by 100 to get the percent of resistant cells.
Record percent values in Row 7 of Table 3 for each Plate Set.
- Compare your results with other team members. Re-count and re-calculate until the team is in agreement. Compare the results to those of your classmates.
Exercise 4: Extract DNA for sequencing
- Obtain 2 microfuge tubes with 100 ml sterile H2O each.
- Label the tops of the tubes with your group name.
- Use a sterile disposable loop to pick a single tetracycline-resistant colony into each tube. Make sure the colony is well-isolated on the plate from other colonies.
- Transfer the colony to the sterile water and rub the loop along the inside of the tube to get as much of the colony as possible into the water. Dispose of the loop in the biobucket.
- Repeat steps 2 and 3 for a second well-isolated colony.
- Cap the tubes and vortex them to suspend the bacterial cells in the water.
- Incubate the tubes at 90°C for 10 minutes, then in ice water for 10 minutes. Tap the tubes on the bench to get all of the water off the lid. Vortex the tubes.
- Repeat step 7 two more times.
- Spin the tubes in the microcentrifuge at 200 rpm for 5 minutes, then place the tubes in a rack. Make sure the centrifuge is balanced. You MUST have one tube directly across from the other for balance.
- Transfer 5.0 ml of each DNA sample to a 0.2 mL PCR tube. Label the tubes and then give the tubes to your instructor.
Your instructor will place the tubes in the thermocycler for PCR with the following protocol:
1 cycle of 98°C for 10 seconds
30 cycles of: 98°C for 1 second (to denature DNA)
48°C for 5 seconds (to anneal primers)
72°C for 30 seconds (to synthesize DNA)
1 cycle of 72°C for 5 minutes
Exercise 5: Culture bacteria from your environment
- Nutrient agar plate
- Sterile swab
- Decide on a surface to sample (e.g., floor, shoe, drinking fountain, phone, skin, etc.)
- Wipe your swab across your chosen surface.
- Open the lid of your agar plate and lightly streak your swab across the agar.
- Close the lid and turn the plate upside down.
- Label the bottom with your name, date, and the surface that you sampled.
- Wrap the petri dish in parafilm.
- You will examine these plates for bacterial growth next week.
What do you predict about the bacteria present on your chosen surface?
- Do you think you will be able to culture bacteria from this surface?
- Compare the surface you chose to at least two of your classmate’s chosen surfaces. Which do you think will grow the most bacteria?
Your surface: ____________________________
Classmate #1: ____________________________
Classmate #2: ____________________________
Your prediction for the surface that will grow the most colonies: ___________________
Exercise 6: Gram Staining
Bacteria can be classified as Gram-positive or Gram-negative according to their response to Gram staining. The Gram staining method is named after its inventor, Danish scientist Hans Christian Gram (1853–1938). The different bacterial responses to the staining procedure are due to cell wall structure. Up to 90 percent of the cell wall in Gram-positive bacteria is composed of peptidoglycan. Gram-negative bacteria have a relatively thin cell wall composed of a few layers of peptidoglycan (only 10 percent of the total cell wall), surrounded by an outer envelope containing lipopolysaccharides (LPS) and lipoproteins. The crystal violet stain is retained in the peptidoglycan layer of the cell wall of Gram-positive bacteria, but is washed away during the destaining step in Gram-negative bacteria.
You may work in a group. Each person should complete at least one complete Gram stain from prepared culture and one Gram stain from their mouth sample. Each group should complete a Gram stain from each of the five prepared cultures and every person in the group should observe each sample under the microscope. Each person should take one picture of one Gram stain (your choice).
- Eye protection
- Inoculation loop
- Bunsen burner
- Crystal violet (primary stain)
- Iodine (fixative)
- Safranin (secondary stain)
Exercise 6.1: Gram Staining from prepared cultures
- Place a drop of water on a slide.
- Dip the inoculation loop in the bacterial culture and apply to the drop of water on your slide. If needed, spread the drop to make a thin layer over the slide.
- Air dry the slide completely
- Heat-fix the slide over an open flame by holding the slide with a clothespin and slowly passing it back and forth over the flame.
Caution: Do NOT hold the slide with your fingers.
Caution: Do NOT hold the slide in the flame for more than a second or two. It will shatter.
- Make sure the slide is completely dry before beginning the stain. Read through the staining steps before starting.
- Add crystal violet to the slide and let stand for 20 seconds.
- Rinse the slide gently with water (2 seconds).
- Add Gram’s iodine to the slide and let stand for 1 minute
- Decolorize with ethanol by adding drops of ethanol and letting it trickle off, until no purple is seen in the ethanol running off the slide. This should take about 3-5 seconds. Further decolorization will remove too much of the purple stain.
- Rinse with water (2 seconds).
- Add safranin and let stand for 20 seconds.
- Gently rinse with water (2 seconds).
- Allow the slide to air dry. You may also blot gently with bibulous paper. Do NOT rub the slide. Make sure the slide is completely dry before observing under the microscope.
- Observe under the microscope using the oil immersion (100x) objective. Do not use a coverslip.
- Record the size, shape, and gram stain for each of the samples.
|Specimen||Shape||Length||Width||Gram status (+/-)|
Exercise 6.2: Gram staining from mouth bacteria
- Place a drop of water on a slide
- Scrape your teeth near the gums with a toothpick.
- Mix the toothpick in the drop of water on the slide.
- Follow the protocol in Exercise 2.1 starting with number 3.
Record your observations. Do you see more Gram positive or Gram negative cells?
Take a pictures of ONE of your Gram stained culture – either a prepared culture OR your mouth bacteria and submit it on Canvas.